http://www1.lsbu.ac.uk/water/enztech/hfcs.

html

High-fructose corn syrups (HFCS)

With the development of glucoamylase in the 1940s and 1950s it became

a straightforward matter to produce high DE glucose syrups. However,
these have shortcomings as objects of commerce: D-glucose has only
about 70% of the sweetness of sucrose, on a weight basis, and is
comparatively insoluble. Batches of 97 DE glucose syrup at the final
commercial concentration (71% (w/w)) must be kept warm to prevent
crystallisation or diluted to concentrations that are microbiologically
insecure. Fructose is 30% sweeter than sucrose, on a weight basis, and
twice as soluble as glucose at low temperatures so a 50% conversion of
glucose to fructose overcomes both problems giving a stable syrup that is
as sweet as a sucrose solution of the same concentration (see Table 4.3).
The isomerisation is possible by chemical means but not economical,
giving tiny yields and many by-products (e.g. 0.1 M glucose 'isomerised'
with 1.22 M KOH at 5�C under nitrogen for 3.5 months gives a 5% yield
of fructose but only 7% of the glucose remains unchanged, the majority
being converted to various hydroxy acids).

One of the triumphs of enzyme technology so far has been the

development of 'glucose isomerase'. Glucose is normally isomerised to
fructose during glycolysis but both sugars are phosphorylated. The use of
this phosphohexose isomerase may be ruled out as a commercial
enzyme because of the cost of the ATP needed to activate the glucose
and because two other enzymes (hexokinase and fructose-6-
phosphatase) would be needed to complete the conversion. Only an
isomerase that would use underivatised glucose as its substrate would be
commercially useful but, until the late 1950s, the existence of such an
enzyme was not suspected. At about this time, enzymes were found that
catalyse the conversion of D-xylose to an equilibrium mixture of D-
xylulose and D-xylose in bacteria. When supplied with cobalt ions, these
xylose isomerases were found to isomerise -D-glucopyranose to -D
-fructofuranose (see reaction scheme [1.5]), equilibration from the more
abundant -D-glucopyranose and to the major product -D-
fructopyranose occurring naturally and non-enzymically. Now it is known
that several genera of microbes, mainly bacteria, can produce such
glucose isomerases: The commercial enzymes are produced
by Actinoplanes missouriensis, Bacillus coagulans and
various Streptomyces species; as they have specificities for glucose and
fructose which are not much different from that for xylose and ways are
being found to avoid the necessity of xylose as inducer, these should
perhaps now no longer be considered as xylose isomerases. They are
remarkably amenable enzymes in that they are resistant to thermal
denaturation and will act at very high substrate concentrations, which
have the additional benefit of substantially stabilising the enzymes at
higher operational temperatures. The vast majority of glucose isomerases
are retained within the cells that produce them but need not be separated
and purified before use.

All glucose isomerases are used in immobilised forms. Although

differerent immobilisation methods have been used for enzymes from
differerent organisms, the principles of use are very similar.
Immobilisation is generally by cross-linking with glutaraldehyde, plus in
some cases a protein diluent, after cell lysis or homogenisation.

Originally, immobilised glucose isomerase was used in a batch process.

This proved to be costly as the relative reactivity of fructose during the
long residence times gave rise to significant by -product production. Also,
difficulties were encountered in the removal of the added Mg2+ and
Co2+ and the recovery of the catalyst. Nowadays most isomerisation is
performed in PBRs (Table 5.2). They are used with high substrate
concentration (35-45% dry solids, 93-97% glucose) at 55-60�C. The pH
is adjusted to 7.5-8.0 using sodium carbonate and magnesium sulphate is
added to maintain enzyme activity (Mg2+ and Co2+ are cofactors). The
Ca2+ concentration of the glucose feedstock is usually about 25 m, left
from previous processing, and this presents a problem. Ca2+ competes
successfully for the Mg2+ binding site on the enzyme, causing inhibition. At
this level the substrate stream is normally made 3 mM with respect to
Mg2+. At higher concentrations of calcium a Mg2+ : Ca2+ ratio of 12 is
recommended. Excess Mg2+ is uneconomic as it adds to the purification
as well as the isomerisation costs. The need for Co2+ has not been
eliminated altogether, but the immobilisation methods now used fix the
cobalt ions so that none needs to be added to the substrate streams.

Table 5.2. Comparison of glucose isomerisation methods

Batch Batch Continuous

Parameter
(soluble GI) (immobilised Gl) (PBR)
Reactor volume (m3) 1100 1100 15
 Enzyme consumption (tonnes) 180 11 2
Activity, half-life (h) 30 300 1500
Active life, half-lives  0.7 2 3
Residence time (h) 20 20 0.5
Co2+ (tonnes) 2 1 0
Mg2+ (tonnes) 40 40 7
Temperature (�C) 65 65 60
pH 6.8 6.8 7.6
Colour formation (A420) 0.7 0.2 < 0.1
Filtration - -
C-treatmenta C-treatment C -treatment
Product refining
Cation exchange Cation exchange -
Anion exchange Anion exchange  -
Capital, labour and
5 5 1
energy costs, � tonne-1
Conversion cost, � tonne-1 500 30 5

All processes start with 45% (w/w) glucose syrup DE 97 and produce
10000 tonnes per month of 42% fructose dry syrup. Some of the
improvement that may be seen for PBR productivity is due to the
substantial development of this process. 
a
 Treatment with activated carbon.

It is essential for efficient use of immobilised glucose isomerase that the

substrate solution is adequately purified so that it is free of insoluble
material and other impurities that might inactivate the enzyme by
chemical (inhibitory) or physical (pore-blocking) means. In effect, this
means that glucose produced by acid hydrolysis cannot be used, as its
low quality necessitates extensive and costly purification. Insoluble
material is removed by filtration, sometimes after treatment with
flocculants, and soluble materials are removed by ion exchange resins
and activated carbon beads. This done, there still remains the possibility
of inhibition due to oxidised by-products caused by molecular oxygen.
This may be removed by vacuum de-aeration of the substrate at the
isomerisation temperature or by the addition of low concentrations (< 50
ppm) of sulphite.
At equilibrium at 60�C about 51 % of the glucose in the reaction mixture
is converted to fructose (see Chapter 1 for a full discussion of such
reversible reactions). However, because of the excessive time taken for
equilibrium to be attained and the presence of oligosaccharides in the
substrate stream, most manufacturers adjust flow rates so as to produce
42-46% (w/w) fructose (leaving 47-51 % (w/w) glucose). To produce 100
tonnes (dry substance) of 42% HFCS per day, an enzyme bed volume of
about 4 m3 is needed. Activity decreases, following a first-order decay
equation. The half-life of most enzyme preparations is between 50 and
100 days at 55�C. Typically a batch of enzyme is discarded when the
activity has fallen to an eighth of the initial value (i.e. after three half
-lives). To maintain a constant fructose content in the product, the feed
flow rate is adjusted according to the enzyme activity. Several reactors
containing enzyme preparations of different ages are needed to maintain
overall uniform production by the plant (Figure 5.10). In its lifetime 1 kg of
immobilised glucose isomerase (exemplified by Novo's Sweetzyme T) will
produce 10 -11 tonnes of 42% fructose syrup (dry substance).

Figure 5.10. Diagram showing the production rate of a seven-column

PBR facility on start -up, assuming exponential decay of reactor activity.
The columns are brought into use one at a time. At any time a maximum
of six PBRs are operating in parallel, whilst the seventh, exhausted,
reactor is being refilled with fresh biocatalyst. ��� PBR activities
allowed to decay through three half-lives (to 12.5% initial activity) before
replacement. The final average productivity is 2.51 times the initial
productivity of one column. - - -- - -  -  PBR activities allowed to decay
through two half-lives (to 25% initial activity) before replacement. The final
average productivity is 3.23 times the initial productivity of one column. It
may be seen that the final average production rate is higher when the
PBRs are individually operated for shorter periods but this 29% increase
in productivity is achieved at a cost of 50% more enzyme, due to the
more rapid replacement of the biocatalyst in the PBRs. A shorter PBR
operating time also results in a briefer start-up period and a more uniform
productivity.

After isomerisation, the pH of the syrup is lowered to 4 - 5 and it is

purified by ion-exchange chromatography and treatment with activated
carbon. Then, it is normally concentrated by evaporation to about 70%
dry solids.

For many purposes a 42% fructose syrup is perfectly satisfactory for use
but it does not match the exacting criteria of the quality soft drink
manufacturers as a replacement for sucrose in acidic soft drinks. For use
in the better colas, 55% fructose is required. This is produced by using
vast chromatographic columns of zeolites or the calcium salts of cation
exchange resins to adsorb and separate the fructose from the other
components. The fractionation process, although basically very simple, is
only economic if run continuously. The fructose stream (90% (w/w)
fructose, 9% glucose) is blended with 42% fructose syrups to give the
55% fructose (42% glucose) product required. The glucose-rich 'raffinate'
stream may be recycled but if this is done undesirable oligosaccharides
build up in the system. Immobilised glucoamylase is used in some plants
to hydrolyse oligosaccharides in the raffinate; here the substrate
concentration is comparatively low (around 20% dry solids) so the
formation of isomaltose by the enzyme is insignificant.

Clearly the need for a second large fructose enrichment plant in addition
to the glucose isomerase plant is undesirable and attention is being paid
to means of producing 55% fructose syrups using only the enzyme. The
thermodynamics of the system favour fructose production at higher
temperatures and 55% fructose syrups could be produced directly if the
enzyme reactors were operated at around 95�C. The use of miscible
organic co -solvents may also produce the desired effect. Both these
alternatives present a more than considerable challenge to enzyme
technology!

The present world market for HFCS is over 5 million tonnes of which
about 60% is for 55% fructose syrup with most of the remainder for 42%
fructose syrup. This market is still expanding and ensures that HFCS
production is the major application for immobilised-enzyme technology.

The high-fructose syrups can be used to replace sucrose where sucrose

is used in solution but they are inadequate to replace crystalline sucrose.
Another ambition of the corn syrup industry is to produce sucrose from
starch. This can be done using a combination of the enzymes
phosphorylase (EC 2.4.1.1), glucose isomerase and sucrose
phosphorylase (EC 2.4.1.7), but the thermodynamics do not favour the
conversion so means must be found of removing sucrose from the
system as soon as it is formed. This will not be easy but is achievable if
the commercial pull (i.e. money available) is sufficient:

phosphorylase                    
starch (Gn) + orthophosphate   starch (Gn-1) + -glucose-1-
phosphate        [5.1]

glucose isomerase        
glucose   fructose        [5.2]

sucrose phosphorylase
-glucose-1 -phosphate + fructose   sucrose +
orthophosphate        [5.3]

A further possible approach to producing sucrose from glucose is to

supply glucose at high concentrations to microbes whose response to
osmotic stress is to accumulate sucrose intracellularly. Provided they are
able to release sucrose without hydrolysis when the stress is released,
such microbes may be the basis of totally novel processes.

http://www.viagusta.com/2012/02/high-fructose-corn-syrup-hfcs/
 High fructose corn syrup (HFCS)
Posted by Corey on Feb 4, 2012 | 0 comments

Disclaimer: I lifted all this straight from Wikipedia. I thought it was actually a really
great scientific summary of what the heck we’ve been eating all these years, and I
expect my scientist brother to tell me if any of it is wrong. I especially appreciate how
much effort we put into turning the syrup into fructose, considering how harmful we
now know that it is…
HFCS was first introduced by Richard O. Marshall and Earl R. Kooi in 1957. They
were, however, unsuccessful in making it viable for mass production. The industrial
production process and creation was made by Dr. Y. Takasaki at the Agency of
Industrial Science and Technology of Ministry of International Trade and Industry of
Japan in 1965–1970. Dr. Y. Takasaki is known to many as the creator of HFCS. HFCS
was rapidly introduced to many processed foods and soft drinks in the U.S. from about
1975 to 1985.
High-fructose corn syrup is produced by milling corn to produce corn starch, then
processing that starch to yield corn syrup, which is almost entirely glucose, and then
adding enzymes that change some of the glucose into fructose. The resulting syrup
(after enzyme conversion) contains approximately 42% fructose and is HFCS 42.
Some of the 42% fructose is then purified to 90% fructose, HFCS 90. To make HFCS
55, the HFCS 90 is mixed with HFCS 42 in the appropriate ratios to form the desired
HFCS 55. The enzyme process that changes the 100% glucose corn syrup into HFCS
42 is as follows:
1. Cornstarch is treated with alpha-amylase to produce shorter chains of sugars
called oligosaccharides.
2. Glucoamylase - which is produced by Aspergillus, species of mold, in a
fermentation vat — breaks the sugar chains down even further to yield the simple
sugar glucose.
3. Xylose isomerase (aka glucose isomerase) converts glucose to a mixture of
about 42% fructose and 50–52% glucose with some other sugars mixed in.
While inexpensive alpha-amylase and glucoamylase are added directly to the slurry
and used only once, the more costly xylose-isomerase is packed into columns and the
sugar mixture is then passed over it, allowing it to be used repeatedly until it loses its
activity. This 42–43% fructose glucose mixture is then subjected to a
liquid chromatography step, where the fructose is enriched to about 90%. The 90%
fructose is then back-blended with 42% fructose to achieve a 55% fructose final
product. Most manufacturers use carbon adsorption for impurity removal. Numerous
filtration, ion-exchange and evaporation steps are also part of the overall process.
The units of measurement for sucrose is degrees Brix (symbol °Bx). Brix is a
measurement of the mass ratio of dissolved sucrose to water in a liquid. A 25 °Bx
solution has 25 grams of sucrose per 100 grams of solution (25% w/w). Or, to put it
another way, there are 25 grams of sucrose and 75 grams of water in the 100 grams of
solution. The Brix measurement was introduced by Antoine Brix.
A more universal measurement of sugars, including HFCS, is called dry solids. Dry
solids is defined as the mass ratio of dry sugars to the total weight of the sugar
solution. Since Brix is based on the refractive index of light against a sucrose molecule
it is not accurate when measuring other sugars such as glucose, maltose, and
fructose.
When an infrared Brix sensor is used, it measures the vibrational frequency of the
sucrose molecules, giving a Brix degrees measurement. This will not be the same
measurement as Brix degrees using a density or refractive index measurement,
because it will specifically measure dissolved sugar concentration instead of all
dissolved solids. When a refractometer is used, it is correct to report the result as
“refractometric dried substance” (RDS). One might speak of a liquid as being 20 °Bx
RDS. This is a measure of percent by weight of total dried solids and, although not
technically the same as Brix degrees determined through an infrared method, renders
an accurate measurement of sucrose content, since the majority of dried solids are in
fact sucrose.
Recently, an isotopic method for quantifying sweeteners derived from corn and sugar
cane was developed which permits measurement of corn syrup- and cane sugar-
derived sweeteners in humans, thus allowing dietary assessment of the intake of these
substances relative to total intake.
http://aklyosov.home.comcast.net/~aklyosov/Volume4.htm

VOLUME IV

INDUSTRIAL PRODUCTION WITH IMMOBILIZED ENZYMES: HIGH-

FRUCTOSE CORN SYRUPS AND AMINO ACIDS FOR FOOD AND FEED

(Flowsheets of the processes and other drawings are not shown on this site.
Please contact the author, if needed) 

INDUSTRIAL ENZYME ENGINEERING: PRESENT STATUS AND FUTURE 377-472

PROSPECTS
A.     INTRODUCTION 378-391
B.     PRODUCTION OF HIGH-FRUCTOSE SYRUPS BY IMMOBILIZED GLUCOSE 392-449
ISOMERASE
        1.     Background 395-403
        2.     Commercial preparations of immobilized glucose isomerase 404-417
        3.     Technological characteristics of the processes 418-436
        4.     Economic estimates 437-441
        5.      Scale of the processes 442-449
C.     OPTICAL RESOLUTION OF AMINO ACIDS BY IMMOBILIZED AMINOACYLASE 450-472
        1.     Background 455-456
        2.     Commercial preparations of immobilized aminoacylase 457-460
        3.     Technological characteristics of the processes 461-469
        4.     Economic estimates 470
        5.      Scale of the processes 471-472
REFERENCES

INDUSTRIAL ENZYME ENGINEERING: 

PRESENT STATUS AND FUTURE PROSPECTS

377. This part of the report describes the state-of-the-art of enzyme engineering
processes on the industrial level. Some of them could be coupled with the
enzymatic production of glucose from cellulose, described in the first three volumes
of this report, and/or with the subsequent microbiological conversion of glucose
into nutritional or other useful products. Others can serve as examples of
contemporary industrial or semi-industrial processes based on practical application
of principles of enzymology and biochemical engineering. 

A.     INTRODUCTION 

378. Enzyme engineering is a relatively new field of science and technology

dealing with the development of useful products or processes based on the
catalytic action of enzymes when either isolated from their source of biological
synthesis or when intact within cells that are immobilized and usually not growing.
This definition (Wingard & Klyosov, 1980) omits fermentation and tissue culture
systems, in which living cells are growing, but does not omit multi-enzymes in sub-
cellular particles. 

379. From this definition it is apparent that enzyme engineering is an applied area
of research and development. However, it may not be apparent that enzyme
engineering so strongly depends on the results of fundamental research in
chemical enzymology, physical chemistry, microbiology, biochemistry, and polymer
chemistry and that often it is not possible to differentiate clearly between enzyme
engineering and these fundamental sciences. Thus, the immobilization of
enzymes, the chemical modification of enzymes, studies into the mechanism of
specific enzymes, and genetic manipulations to improve enzyme availability or
process characteristics all are fundamental scientific topics that become part of
enzyme engineering when considered in the light of a specific enzyme-based
process or product. Similarly, the fundamental topics of heat and mass transfer,
diffusional effects on enzyme-catalyzed reaction rates and mathematical modelling
of enzyme-catalyzed reaction kinetics or overall process economics become part of
enzyme engineering primarily when applied to a specific enzyme-based process or
product. 

380. In the final analysis an enzyme-based process or product must provide

something unique if it is to find practical acceptance. Examples of suitable unique
contributions include: 
       (i) production of a new and useful product or the achievement of a new and
useful chemical
           transformation or of an old transformation in a new environment, e.g. in
vivo, 
       (ii) production of a better quality product, and 
       (iii) production of an existing product more economically. 

381. Traditionally, enzymes have been extracted from plants and animals. Rennin,
for instance, the enzyme employed to curdle milk for cheese production, is
extracted from the stomach of unweaned calves. But the production of enzymes
from bacteria, yeasts and fungi has rapidly become more common. As a rough
estimate more than 50 enzymes are used exclusively in the food industry
throughout the world: in brewing, cheese making, the production of fruit juices and
wines, starch and sugar processing and as meat tenderizers (Yanchinski, 1984;
Barker, 1987). Just 16 enzymes of the thousands produced in nature rank as
industrial catalysts, accounting for over 90% of the total market (Biotechnology
Bulletin, 1986; Linhart, 1987). Three enzymes, for instance, form the basis for an
industry manufacturing liquid sweeteners from cornstarch (see below). 

382. The use of enzymes as catalysts is growing remarkably. Frost & Sullivan
reported recently that consumption of industrial enzymes in Western Europe will
approximately double in quantity over 10 years in real terms from the 1986 sales of
about $160 million (Biotechnology Bulletin, 1986). In 1983 enzyme sales worldwide
were reported to be about $390 million, up nearly 25 percent from 1979 (Maugh,
1984), and in 1985 sales in the USA and Western Europe alone reached $500
million (Chaplin, 1984). A 1982 report from the Office of Technology Assessment
predicted that, within 20 years, enzymes would be used in the production of $15
billion worth of chemicals and pharmaceuticals. On a dollar basis, about 80 percent
of the enzymes are attributable to the food industry (Genet. Eng. Biotechnol. Mon.,
1984). On a volume basis, starch conversion enzymes account for about 40
percent of sales and proteolytic enzymes used in laundry detergents account for
another 30 percent. Europe dominates the world in sales, with Novo Industries in
Denmark and Gist Brocades in the Netherlands controlling 60 per cent of the world
market (Yanchinski, 1984). According to 1991 data, in China alone there are over
40 enzyme manufacturing factories with an annual output of over 2,000 tons (Han
Ying-Shan, 1991). 

383. Immobilized enzymes (a similar definition may be given in relation to

immobilized cells) may be defined as enzymes whose free movement has been
restricted in some manner. One of the major advantages of immobilization is to
"fix" the enzyme so as to retain it in a continuous process. Use of soluble enzymes
in processing has been limited in part by the cost of the enzymes due to the
difficulty and expense of isolation, instability, and the fact that in a freely soluble
form they usually can be used only once. 

384. The use of immobilized enzymes has several advantages. An immobilized

enzyme, in solid or sometimes semisolid form, is readily separable from the
product solution and can be reused, thus increasing utility by a large factor.
Additionally, the enzymic reaction can be terminated easily simply by removing the
enzyme, allowing more precise control of the reaction. Finally, it has turned out that
enzymes (or cells) are often much more stable when in fixed form than in solution,
allowing an enzymatic conversion to occur over a much longer period without
having to replenish the biocatalyst as compared with intact enzymes or cells.
Specific examples will be given in the subsequent sections. 

385. Enzymes (or cells) may be immobilized by several techniques. These

include: 
       (1) Physical adsorption; 
       (2) Covalent attachment to an inert support; 
       (3) Entrapment within a gel matrix; 
       (4) Containment behind a semipermeable membrane (hollow fibers,
microcapsules, etc.); 
       (5) Intermolecular crosslinking of enzyme molecules. 
386. Either "live" or "dead" cells can be immobilized. In dead cells most of the life
processes have been destroyed more or less selectively while the enzyme of
interest is allowed to survive. 

387. Materials used as supports (carriers) for immobilization have included both
organic and inorganic materials (Linhart, 1987; Komori et al., 1987; Bisping &
Rehm, 1988). Cellulose, dextran (Sephadex), agarose (Sepharose), nylon,
acrylamide-based polymers, and styrene-based polymers are typical examples of
organic supports. Inorganic supports studied include porous and solid glass,
diatomaceous earth, silica aluminas, nickel screening, stainless steel balls, sand,
and titania. They often have better flow properties, and some are very inexpensive.
Generally, the specific type of immobilization procedure and the support used are
determined by the application of a given enzyme system. 

388. A combination of the unique catalytic properties of enzymes when insoluble in

an immobilized form has been used for the development of novel technological
processes. At present these processes are related mainly to food and
pharmaceuticals. The restriction of immobilized enzymes to these areas of
technological applications could be explained in part by the existence of respective
industrial chemical and microbiological processes with thoroughly worked-out
technology. In many cases enzymes are produced industrially in relatively small
quantities as yet and are still rather expensive for industrial applications; the cost
for immobilization and for appropriate carriers and chemicals is often too high. 

389. In the near future, immobilized enzymes will only be used industrially when
the product of a process is impossible to obtain without enzymes, or if the cost of
the product is so high compared with the cost of the initial materials (chemicals)
that the difference can cover the expenses for the immobilization of the enzyme.
That is why an interest is growing for the immobilization of enzymes by simple
adsorption onto carriers and for using immobilized cells without isolation of
intracellular enzymes. 

390. Up to the present time, only eight processes with immobilized enzymes or
microbial cells have found industrial application: 

       (1) Production of high-fructose syrups by immobilized glucose isomerase. 

       (ii) Optical resolution of amino acids by immobilized aminoacylase. 

       (iii) Production of optically active D-amino acids by immobilized hydantoinase. 

       (iv) Production of L-aspartic acid by immobilized aspartase. 

       (v) Production of L-malic acid by immobilized fumarase. 

       (vi) Deacylation of penicillin G and cephalosporin derivatives by immobilized

penicillin
            amidase. 

       (vii) Hydrolysis of lactose by immobilized lactase in dairy processing. 

       (viii) Production of glucose-fructose syrups from saccharose by immobilized

invertase. 

391. Aside from these, there is the well-known process, which has already been
worked out on a pilot plant scale, and is apparently not far from larger-scale
applications: 

       (ix) Production of glucose from starch hydrolysates by immobilized

glucoamylase. 

B.     PRODUCTION OF HIGH-FRUCTOSE SYRUPS BY IMMOBILIZED

GLUCOSE ISOMERASE 

392. The production of high-fructose corn syrup (HFCS) by the isomerization of

glucose to fructose, which is catalyzed by glucose isomerase of microbial origin,
has been the major industrial application of immobilized enzyme technology during
the past 20 years (Hagen & Pederson, 1990). The isomerization process converts
about 45% of the glucose (dextrose) in a corn starch hydrolysate, which has been
prepared by enzymatic hydrolysis, into fructose. 

393. Clinton Corn Processing, a division of Standard Brands, now Nabisco Brands,
was first to achieve a successful commercial production of HFCS from starch
hydrolysate using immobilized glucose isomerase in 1973. The process created
the largest new market developed in the ensuing decade, including $40
million/year for immobilized glucose isomerase (for further economic estimates see
paragraphs 437-441). 

394. The use of glucose isomerase began under a unique set of conditions: in
1974, the price of sugar in the U.S.A. increased sharply, making the cost of this
conversion process competitive. By the time the cost of sugar fell, the enzyme
manufacturers were able to reduce costs sufficiently to remain competitive, and
presently immobilized glucose isomerase is used to produce more than 3 million
tons of HFCS yearly (Maugh, 1984). 

1.     Background 

395. Today's typical process for production of fructose syrups uses alpha-amylase
to liquefy starch followed by glucoamylase to saccharify the hydrolyzed starch to
the required 94% dextrose content for the isomerization, which results in a mixture
of glucose and fructose (Quax et al., 1991). The process is as follows (Hultin,
1983): a starch slurry of about 33% dry solids is liquefied with a bacterial α-
amylase at a temperature ranging from 80-110°C, generally for 2-4 hrs. Since α-
amylase usually requires calcium, the pH of the starch slurry is adjusted to 6-7 with
calcium hydroxide. Acid hydrolysis is occasionally employed, but the glucose
concentration produced after saccharification is higher if enzymic hydrolysis is
utilized. The hot slurry is then cooled to about 60°C at which point the liquefied
starch is saccharified by treatment with a fungal glucoamylase that produces
glucose from liquefied starch. 

396. This process is generally carried out at pH 4-4.5 with a "holding time" of 24-90
hr depending on conditions and the amount of enzyme used. On a dry basis this
generally converts starch to 96-97% glucose. This crude syrup, high in glucose, is
then decolorized with carbon and deionized with both acidic and basic resins. It is
important to remove the calcium ions, which inactivate glucose isomerase. 

397. The crude glucose solution may then be concentrated or blended to bring its
content of solids to 40-50%. Magnesium ions are required and generally added for
almost all of the glucose isomerases used presently. The pH of this crude glucose
solution is made slightly alkaline (up to pH 8.5), with the exact pH depending on
the enzyme and the type of reactor. The glucose content of the starting syrup for
the isomerase treatment is minimally 94% on a dry basis. The conditions produce a
fructose syrup containing a minimum of 42% fructose (usually 43%) and about
51% glucose with no more than 6% disaccharides or higher polymers (Hultin,
1983). 

398. The syrup is then treated with activated carbon and deionized with a strong
acid-cation exchange resin in the hydrogen form and a weak base-anion exchange
resin in the free base form. This process should be carried out as rapidly as
possible since the pH values reached with the ion exchange resins can range from
1.5 to 8, and this can induce chemical decomposition of the syrup components.
The pH of the final product is generally adjusted to 4-4.5 which results in maximal
stability. Finally the syrup is concentrated to 71% solids by evaporation during
which process the pH is carefully controlled (Hultin, 1983). 

399. The sweetness of the resultant high fructose is comparable to that of common
sugar (sucrose) or the invert sugar that is produced by acid or enzymatic hydrolysis
of sucrose.

SWEETNESS OF SYRUPS (COMPARISON FOR 15% SOLUTION AT 15 oC)

Sugar (sucrose) 100

Glucose 75

Fructose 165
High fructose syrup (42%) 100

Maltose 30-50

Lactose 20

Source:Okada, 1978; Food Technology, 1986a.

400. The resultant HFCS is the functional equivalent of sucrose for many
applications. It is widely used as a nutritional sweetener in the food industry,
particularly in soft drinks and other beverages, as well as in desserts, baked goods,
canning, and many other packaged products. For certain uses (e.g., cola
beverages), HFCS with 55% or 90% fructose is preferred, made from the 42%-
fructose HFCS by a process of separation analogous to liquid chromatography
(Carasik & Carroll, 1983). 

401. The rapid growth in demand for fructose in the last decade may be attributed
to the following: 
       - it can be used as an alternative to sugar since both are equally sweet, 
       - it can be produced at a cost lower than sugar, 
       - it is produced from starch, which is a common and widely available food
material, 
       - its taste is more refreshing than that of sugar, 
       - consumption of fructose is said to be of lower risk for diabetics and some
other metabolic
         disorders. 

402. The amount of fructose produced by the isomerase reaction is determined by

the equilibrium value of the glucose to fructose isomerization which results in
roughly equal amounts of the two sugars. In order to obtain higher fructose content
of the syrups, large-scale columns of strong acid-ion exchange in the form of
calcium salts are often used as the stationary phase. Oligosaccharides come off
the columns first, owing to a molecular exclusion effect. These are then followed by
glucose and finally fructose. 

403. Another alternative is to concentrate the syrup to a high enough content of

solids to allow crystallization of glucose from the concentrate. The remaining liquid
is then enriched in fructose. In this manner it has been possible to make syrups of
up to 70% fructose (on a dry basis). The isolated glucose is recycled in an
isomerization reaction. Finally, another approach is to complex fructose with borate
compounds. This removes fructose from solutions and shifts the apparent
equilibrium of the reaction. However, the cost of removal and recovery of the
borate has so far prevented commercial adaptation of this process (Hultin, 1983). 

2.     Commercial preparations of immobilized glucose isomerase 

404. Clinton Corn Processing Co. developed the first industrial process HFCS
production, which used the microorganisms Streptomyces sp. as the source of
glucose isomerase. In contrast, Novo Laboratories Inc. selected the thermophilic
microorganism Bacillus coagulans, which produces glucose isomerase without
requiring the addition of an expensive inducer such as xylose in the mixture
(Carasik & Carroll, 1983), and can be grown in continuous culture without
contamination and with high enzyme yields. In spite of the large number of
organisms that can produce glucose isomerase (Antrim et al., 1979), only a few
high-producing strains are actually being utilized on a commercial basis.

MICROBIAL SOURCES OF GLUCOSE ISOMERASE PRESENTLY BEING

USED FOR THE COMMERCIAL PREPARATION OF IMMOBILIZED ENZYMES,
AND THE COMPANIES PRODUCING THEM

    Manufacturer Enzyme source

Clinton Corn Processing Co. Streptomyces rubigenosus

Streptomyces wedmorensis
Novo Industry Bacillus coagulans
Streptomyces murinus
Gist Brocades Actinoplanes missouriensis

ICI Americas, Inc. Arthrobacter

Miles Laboratories, Inc. Streptomyces olivaceus

Flavobacterium arborescens
Mi-Car Int. Streptomyces olivaceus

Corning Glass Works Streptomyces olivochromogenes

Nagase Streptomyces phaeochromogenes

Miles Kali Chemie Streptomyces rubiginosus

Sanmatsu Streptomyces

Snamprogetti Streptomyces

Finnsugar Streptomyces rubiginosus

Godo Shusei Streptomyces griseofuscus

UOP Streptomyces olivochromogenes

Source:Adapted from Antrim et al., 1979; Jensen & Rugh, 1987; Houng et al., 1993.
405. Glucose isomerase typically is an intracellular enzyme. The enzyme is generally obtained by
scaling up a submerged aerated fermentation in several stages. The three stages of development
for such a production are described in U.S. Patent 3,666,628: (a) slant development, (b) culture
development - two substages, (c) final fermentation stage. Glucose isomerase can be recovered in
two ways: (i) recovery of the microorganisms with the enzyme entrapped in the cellular mass, and
(ii) recovery of the enzyme in the soluble form after lysing the cells (Antrim et al., 1979). To prevent
the loss of the enzyme from cells in the first case (as the result of the autolysis of cells during
isomerization) and to allow extended use of cells in fixed-bed reactors, the enzyme is bound to the
cellular matrix by heat or chemical fixation (Antrim et al., 1979). 

406. Commercial immobilized glucose isomerase preparations are generally produced by various
companies in a granular form and a fibrous or amorphous form. Clinton Corn Processing Company,
for example, produces both a fibrous form and a granular form of glucose isomerase. Each form is
designed for a particular reactor technology. The fibrous form of Clinton's enzyme preparation has a
large surface area with very high enzyme potency and is designed for use in shallow-bed reactors.
The granular enzyme with lower potency is designed for deep-bed reactors. Novo Enzyme
Corporation produces a granular isomerase suitable for deep-bed reactors, batch isomerization,
and fluidized-bed operations. The immobilized enzyme preparation from Gist Brocades
(Maxazyme®) utilizes the organism A. missouriensis entrapped in cross-linked gelatin. This
produces a softer particulate enzyme which can also be used in deep-bed reactors. 

407. ICI produces an immobilized enzyme preparation whereby Arthrobacter cells are flocculated by

polyionic reagents. This also produces a rather soft granular particle that can be used in deep-bed
reactors. Sanmatsu (Japan) produces a glucose isomerase by adsorption of the enzyme on an
anion-exchange resin. This yields a high-potency, particulate enzyme suitable for deep-bed
reactors. Denki-Kagaku - Nagase Sangyo (Japan) has produced a polymer isomerase (Sweetase ®)
entrapped in hard granules for use in deep-bed reactors. The catalyst particles swell to about
double their original sizes (0.4-0.8 mm) during the isomerization reaction (Okada, 1978). 

408. Miles and Miles-Kali Chemie have produced a glutaraldehyde cross-linked preparation and a
heat-fixed cellular preparation, respectively. Snamprogetti (Italy) produces glucose isomerase,
extracted and partially purified from Streptomyces sp., and entrapped in cellulose triacetate fibers.
This is carried out according to Snamprogetti's modification of the well-known technology of fiber
wet spinning: an organic solution of fiber-forming polymer is emulsified with an aqueous solution of
the enzyme, and the resulting emulsion is then extruded into a coagulation bath through the holes
of a spinneret (Marconi & Morisi, 1979).

COMMERCIALLY AVAILABLE IMMOBILIZED GLUCOSE ISOMERASE PREPARATIONS

Company and patents Immobilization procedure

Mi-Car Int. Granular particles containing whole cells,

    U.S. 3,625,828 cross-linked with glutaraldehyde
            3,654,081
            3,779,869
Clinton Corn Processing Enzymes adsorbed on DEAE-cellulose
    U.S. 3,623,953 via ion-exchange
            3,788,945
            4,376,824
Corning Glass Works Enzymes adsorbed on controlled pore
    U.S. 3,847,740 alumina carrier (0.46 mm D; APD 140-220 A); 
            3,850,751 carrier can be regenerated
            3,868,304
            3,982,997
            3,992,329
Gist Brocades Whole cells entrapped in gelatin
    U.S. 3,834,848 followed by cross-linking with glutaraldehyde
            3,838,007 (Maxazyme®
ICI Americas, Inc. Flocculating agent used to immobilize 
    U.S. 3,645,848 withing cells; paste then extruded and dried
            3,821,068 into cylindrical pellets
            3,935,068
NOVO Industry (a) Enzymes mixed with inorganic diluent 
    BR   1,362,365 and formed into solid spheres
           1,381,387 (b) Lysed cells cross-linked with glutaraldehyde; 
  U.S.   4,025,389 cylindrical, extruded particles (Sweetzyme ®)
Miles Laboratories, Inc. Spheronized extrudate of polyamine 
flocculated glutaraldehyde cross-linked 
whole cells (Takasweet ®)
CPC International, Inc. Adsorption of alumina (porous) or other 
    U.S. 4,343,902 ceramic carriers or ion-exchange resins
    Canada 998,344
Miles-Kali Chemie (a) Heat-fixed cells cross-linked with glutaraldehyde;
(b) Spherical SiO2 particles with adsorbed,
glutaraldehyde cross-linked purified enzyme
(Optisweet 22)
Sanmatsu Adsorption on anion exchange resins

Snamprogetti Enzyme entrapped in cellulose triacetate fibers by

    U.S. 3,964,970 means of fiber wet spinning
Mitsubishi Chemical Industries Adsorption on undisclosed synthetic
anion exchange resin
Agency of Industrial Science and Heat-fixed cells
Technology (Japan)
Denki Kagaku Kogyo Enzyme adsorbed on cation-exchange resin
having quaternary pyridine ring
R.J. Reynolds Tobacco Co. Cells coagulated with flocculent

Kyowa Hakko Kogyo Enzyme adsorbed on phenol formaldehyde

resin Duolite A7
Finnsugar Pure enzyme adsorbed on DEAE-cellulose
embedded in polystyrene with TiO2 (Spezyme ®)
UOP PEI treated ceramic alumina with glutaraldehyde
cross-linked enzyme (Ketomax GI-100)
Godo Shusei Granulate of chitosan-treated glutaraldehyde
cross-linked cells (Godo-AGI ®)
Denki Kagaku-Nagase Whole heat-treated cell entrapment into a high
polymer, and granulated up to 0.4-0.8 mm 
particle sizes (Sweetase ®)

Source:Adapted from Antrim et al., 1979; Marconi & Morisi, 1979; Sweigart, 1979; Ishikawa,
1977; Jensen & Rugh, 1987.

409. The main conclusion from the above table is that there are actually no covalently bonded
enzymes among commercial preparations of immobilized glucose isomerase. Glucose isomerase is
either adsorbed to ion exchange resins or to porous inorganic carriers (alumina, ceramics), or it is
inside whole cells entrapped in a polymer matrix, usually cross-linked with glutaraldehyde or other
bifunctional reagents. Even Corning Glass Works, which has developed a well-known procedure for
covalent immobilization of enzymes on inorganic carriers, uses immobilization by adsorption in this
particular case. The main reason for this is that immobilized glucose isomerases that have been
produced by adsorption of soluble enzyme onto a solid carrier can generally be regenerated and
reloaded with fresh enzyme after much of the initial activity is depleted. It is usually economically
advantageous to do so, especially when the carrier used is expensive. 

410. In many cases whole cells (heat-fixed or entrapped into polymer matrix) are used instead of
the isolated enzyme. This can be explained both by decreased stability of isolated (extracellular)
glucose isomerase (Ishikawa, 1977) and by the higher cost of isolation and immobilization of
enzymes in comparison with the use of whole microbial cells. 

411. These two points (par. 409 and 410) are illustrated below in the form of a comparison of the
general procedures of enzyme immobilization with respect to several important commercial
properties (Antrim et al., 1979). 

EXPECTED PROPERTIES OF IMMOBILIZED GLUCOSE ISOMERASE

PREPARED BY VARIOUS PROCEDURES

Immobilization Expressed activity Potency Carrier Reusability of

procedure (observed/bound) (activity/weight) cost carrier

Immobilized in High Average Low No

bacterial cells
Adsorption on High to low High High Yes
insoluble carrier
Entrapped in Low Average to low High No
insoluble matrix
Covalently bound to Low Low High No
insoluble carrier

Source: Antrim et al., 1979.

412. The increase in HFCS penetration of the world sweetener market has been made possible in
part by the evolution of immobilized glucose isomerase technology and improvements in enzyme
characteristics. The dynamics can be traced using the example of Novo Laboratories Inc., which
subdivided various types of immobilized glucose isomerase preparations developed by the
company into the preparation of three generations of enzymes (Carasik & Carroll, 1983). These do
not include the first of a series of commercial glucose isomerases marketed under the brand name
Sweetzyme®, which was a spray-dried soluble product introduced in 1973. It had a high production
cost and was used commercially for only a short time. When Novo's first immobilized form of the
enzyme was introduced in 1974, its advantages became readily apparent and use of the spray-
dried soluble form was subsequently discontinued (Carasik & Carroll, 1983). 

413. The steps in the production of immobilized glucose isomerase of "three generations" (and for
three types of reactors) are shown in Figure 1 and described below (Carasik & Carroll, 1983). 

414. For production of the first generation enzyme (1974), in the form of a powder, the cross-linking
agent selected is glutaraldehyde, which immobilized the cell protein. The initial product of cross-
linking is a semigelatinous mass that has the consistency of pudding. After drying, grinding and
screening, the final product has a particle size range of 100-350 microns. The product is designed
for use (and re-use) in a batch reactor (Fig. 2). 

415. The first-generation immobilized enzyme has several limitations. The particles gradually
decrease in size as a result of the shearing effect of the agitator blades. This leads to an overall
decrease in settling rate and thereby to a slowing of the process. According to Novo's data,
decrease in the size of the particles of the immobilized enzyme from 0.35 to 0.25 mm and further to
0.10 mm leads to a decrease in the terminal velocities of settling particles from 9.5 to 5.0 m/hr and
further to 0.8 m/hr. In addition to the degradation of the particle, there is thermal denaturation of the
enzyme, decreasing its activity. Prolongation of the reaction time or addition of new enzyme to the
isomerization reaction tank prior to the next cycle can compensate for this. Normal make-up is
about 5% per reuse. Finally, the long holding time required for the isomerization with the first
generation enzyme in turn requires operation at a relatively low pH to avoid excessive color and
formation of by-products. At this pH, the addition of cobalt salts is necessary to activate the
immobilized enzyme. These drawbacks of the powder form of the enzyme and the batch reactor led
to the development of immobilized enzymes of the second generation, which are suitable for
continuous column reactors, where the reaction can be run at a neutral to slightly alkaline pH, since
the syrup contact time is reduced drastically, with no cobalt addition required. 

416. The second-generation immobilized enzyme for column use was prepared by the extrusion of
the wet cross-linked enzyme mass through a small orifice (< 1 mm diameter) cut into short
cylindrical particles, which are then dried and screened, as shown in Figure 1. This product does
not exhibit pressure-drop characteristics; upon continued operation the particles deform and the
pressure drops dramatically. This is overcome by modifying the immobilized enzyme preparation
through the incorporation of powdered alumina to increase the density of the particles and allow
higher flow rates in the upflow mode (Carasik & Carroll, 1983). However, industrial performance is
entirely satisfactory, and the deviation from laboratory data is ascribed to variations in enzyme
particle size and to problems of density and flow-distribution problems. 

417. For the preparation of the third-generation immobilized glucose isomerase, which should be
able to resist deformation at constant syrup flow for fixed-bed operation, an important change in the
immobilization process is made. The cell slurry is homogenized prior to cross-linking as shown in
Figure 1. Homogenization disrupts the cell membranes and allows more of the protein surface to
react with the cross-linking agent. The result is an enzyme with stronger particles that resist
deformation. The improved enzyme preparation also contains additives such as magnesium oxide
and dextrose, which are incorporated after crosslinking but prior to extrusion. Magnesium oxide
helps to minimize the decrease in pH during isomerization, and the dextrose dissolves during
operations, resulting in a more porous matrix and lowering diffusion resistance (Carasik & Carroll,
1983). The enzyme is shipped to the user in a dry granular form which, prior to use, must be
hydrated in syrup. During hydration, the size of the enzyme particles expands by a factor of about
two. Under operating conditions (pH 7.5-8.0, temperature 55-60°) the half-life of the enzyme is
usually greater than 75 days. 

3.     Technological characteristics of the processes 

418. The literature contains limited information on commercially available glucose isomerase
technologies. Listed below are some data on reactor configurations normally employed, and basic
performance data where available. Although each system listed employs a different enzyme source
(see above), a different level of enzyme purity, and a different immobilization technique, there
appears to be relatively little difference in the performance of the systems for which data are
available (Sweigart, 1978). 

419. Major commercial manufacturers of immobilized glucose isomerase describe the application
for deep-bed reactors. A deep-bed reactor is usually simpler in design than a shallow-bed model
and may therefore require a lower capital expenditure. Flow may be "up" or "down" through the
reactor. Column reactors operating in the "down" mode appear to be more popular. Heights up to
five meters appear to be feasible. Upflow in a column is reported to be less productive, probably
due to wider distribution of the "syrup residence time." Precautions must be taken with upflow to
prevent loss of immobilized enzyme through the top of the reactor, and flow control is considered to
be more critical than for downflow (Antrim et al., 1979). The temperature and pH recommended are
almost identical (60-65°C and 7.0-8.5 respectively). Further, the initial substrate of 40-50% dry
solids (92-94% dextrose, dry basis, 6-8% dry basis polysaccharides) is also much the same.

TECHNOLOGICAL CHARACTERISTICS OF THE IMMOBILIZED GLUCOSE ISOMERASE

SYSTEMS

    Company Reactor configuration and basic performance data

Mi-Car Int. Column reactor

Clinton Corn Shallow bed reactors; the immobilized enzyme constitutes a thin bed of 2.5-
Processing 12.5 cm depth. Several are staged to form a multibed processing unit. The
multistage filter leaves in either a vertical or horizontal tank are packed with
beds at a bed-depth:bed-width ratio of about 0.02-0.05.
Corning Glass Column reactor. Half-life of the enzyme is 40 days.
Works
Gist Brocades Stainless steel column reactor to 6 m bed depth, 1.5 m diameter.
Half-life of the enzyme is 21 days.
Sanmatsu Column reactor. Half-life is 30-50 days. Glucose concentration 45-50%, temp
55-70oC, pH 6.5-8.5.
ICI Columns to 5 m bed depth.

NOVO Columns to 4.5 m bed depth. Conditions: 60oC, pH 7.5-8.0.

Half-life is 75 days.
Snamprogetti Tubular and radial reactors. Fibers containing glucose isomerase (entrapped)
are placed in an ordered arrangement parallel to the long axis of the column
(tubular retire, 0.2-0.25 kg dry fibers per liter reactor volume) or are rolled
around a perforated pipe in an ordered way, as on a bobbin (radial reactor,
 0.35 kg dry fibers/l). Half-life is 70 days.
Denki Kagaku A battery column reactor. Raw material (dextrose, 50 wt% solids) -
Kogyo pH 4.0-4.5, product (at the reaction column outlet) - pH 7.5.

Source:Adapted from Okada, 1978; Marconi & Morisi, 1979; Sweigart, 1978.

420. In continuous production of isomerized sugar by means of the immobilized enzyme process,
raw material purity influences catalyst activity and therefore productivity. Thus, raw material
(dextrose syrups) should be refined as much as possible. Impurities include calcium ions, peptides,
oxygen, oxidation products, etc. According to the data of the Chiba Plant, Denki Kagaku Kogyo K.K.
(Okada, 1978), when high purity substrates such as crystalline glucose are used as the initial
substrate, the productivity of the reactor (i.e., the yield of solid high fructose syrup per unit weight of
catalyst until catalyst activity decreases to a quarter of the original level) reaches 4000 kg/kg of
immobilized enzyme in 100 days, and the half-life for the catalyst is 50 days. However, when
purified dextrose of lower grade is used, the half-life decreases to 20 days, and productivity of the
reactor to 1,500 kg fructose/kg of immobilized enzyme. In general, productivity for immobilized
glucose isomerases used commercially, described by various enzyme manufacturers and used
under the operating conditions recommended by them, varies from 1 to 9 tons of HFCS (dry basis)
per kg of the immobilized enzyme.

PRODUCTIVITY OF SOME COMMERCIAL IMMOBILIZED GLUCOSE ISOMERASES

    Manufacturer Productivity
(tons 42% HFCS/kg enzyme)

Clinton Corn Processing Co. 7.2-9.0

Gist Brocades N.V. 1.78

ICI Americas, Inc. 2.0

Miles Kali Chemie 1.0

Novo Industry A/S 1.0-1.6

Novo Laboratories, Inc. 4-5

Snamprogetti 5-6

Mi-Car Int. 2.0

Source:Carasik & Carroll, 1983; Antrim et al., 1979; Sweigart, 1978; Jensen & Rugh, 1987.

421. A multiplicity of reactor designs has been described for use with immobilized glucose
isomerase (six different design types, according to one particular classification, i.e. batch, packed-
bed, continuous-flow stirred-tank, continuous-flow stirred-tank/ultrafiltration membrane, and others,
including recycle reactors and tubular reactors with enzymatically active walls) (Antrim et al., 1979).
However, most glucose isomerase reactors now in commercial operation are of the packed-bed
type. Novo Industri has described batch reuse of Type A, a glutaraldehyde cross-linked
homogenate of B. coagulans. Although they subsequently found performance advantages with a
continuous system, in 1976 they reported that Type A had been used commercially in large-scale
batch reuse since 1974 [cit. in Antrim et al. (1979)]. Gist-Brocades has also described conditions for
reuse of their Maxazyme GI-Immob in a batch reactor. 

422. Numerous investigators have compared performance and economics of batch versus
continuous reactor systems. Usually in a packed-bed reactor the concentration of active glucose
isomerase is high compared to a batch reactor and contact time between substrate and enzyme is
relatively short, usually 2-4 hr as compared with 20-60 hr for a batch reactor (Antrim et al., 1979).
The short contact time helps to minimize formation of colored materials and nonfructose
isomerization compounds. Additionally, enzyme usage (comparative amounts of enzyme activity), is
found to be considerably higher (1.4-4.0 times) for batch versus continuous packed-bed reactors,
primarily due to loss of active enzyme through multiple batch recovery operations (Antrim et al.,
1979). 

423. U.S. Patents 3,847,740 and 3,847,741 disclose a process for regulating production by means
of temperature control as well as for increasing the productivity of the enzyme. One example
demonstrates that by increasing the temperature in the reactor by 2°C increments from 60° to 70°C
over a period of 14 days, the enzyme productivity increases 42% over a 14-day isothermal run at
60°C. 

424. Novo Laboratories Inc. used the 'first-generation' immobilized glucose isomerase (see par.
413) in a batch reactor (Fig. 2). Typically the enzyme is added to a tank containing purified high-
dextrose syrup (> 93%, dry basis) adjusted to certain conditions (pH 7.0, temperature 60-65°C,
Co+2 3.5x10-4M, MgSO4x7H2O 0.1-1.0 g/liter). The typical reaction time is 20-24 hr, after which the
enzyme is recovered by allowing the particles to settle and the supernatant liquor drawn off. The
entire process is then restarted. The limitations of this mode of process operation are described in
paragraph 415. 

425. Glucose isomerase of the second generation, i.e. crosslinked with glutaraldehyde and
extruded (see par. 416), has led to non-satisfactory pressure-drop characteristics of the column
reactor in the downflow operation mode. Ultimately, the entire enzyme bed will compress to a point
where flow is reduced drastically and the operation is stopped. Therefore, Novo Labs at that stage
of development (1975) attempted to use an expanded- or fluidized-bed reactor, in which the
dextrose syrup flows into the bottom of the column through a flow distributor and the immobilized
enzyme is suspended in the flowing syrup (Carasik & Carroll, 1983). In the fluidized-bed operation,
the substrate solution passes upward through the enzyme bed. In this mode the most critical factor
is uniform flow distribution. In commercial reactors this is achieved with a shallow bed of heavy inert
material (alumina) on a screen. 

426. With third-generation immobilized glucose isomerases, which have stronger particles that
resist deformation (par. 417), after the hydration the enzyme slurry is transferred to the column
reactor, which runs in an upflow (fluid-bed) mode for about 12-24 hr. This permits the enzyme to
come into equilibrium with syrup at column operating conditions without compacting. After this
"fluid" period, the bed is allowed to settle briefly, then fluid is run in the downflow mode. This
process is currently in wide industrial use in many parts of the world (Carasik & Carroll, 1983). The
enzyme bed height at optimum operating conditions is 5 m maximum, diameter 1.5 m maximum
(height:diameter ratio is 3:1 minimum), temperature 55-60°C at inlet, pH 7.5-8.0 at 25°C, dextrose
concentration at inlet 94% minimum (dry basis), feed solids 45%, Mg +2 content equals 20x Ca+2.
Half-lives are usually more than 75 days. 

427. To achieve high enzyme productivity (in this particular case above 4,000 kg of dry solids/kg of
enzyme, at 45% conversion of dextrose to fructose, see also paragraph 420) on a continuing basis,
Novo recommends (Carasik & Carroll, 1983) that careful attention be paid to details of plant design
and operation. Thus, in reactor design, flow distribution is important for operation over a wide range
of flow rates. The enzyme is often used until the flow rate (activity) falls to 10% of the initial flow.
Feed purity is of great importance to avoid cumulative enzyme poisoning (cf. paragraph 420).
Filtration, decolorization (active carbon treatment), deionization, and evaporation must be designed
and operated carefully. It is important to provide a purified feed of constant composition with
consistent control of parameters. 

428. Data on the isomerization process are also available from Kyowa Hakko Kogyo Co., Ltd
(Yokote et al., 1975). Glucose isomerase used in their process was immobilized on a phenol
formaldehyde resin Duolite A7. The flow chart of the process is shown in Figure 3. A 40% (w/v)
glucose solution at 60°C is fed continuously through the immobilized enzyme columns in series at
60°C, pH 8.2. Before feeding, the substrate solution is passed through pretreatment columns that
contain Duolite A7 (buffered) to eliminate some impurities and air bubbles. The effluent solution
from the immobilized enzyme column is passed successively through a cation exchange (Diaion
SK-1A, H-type) and an anion exchange (Diaion WA20, OH-type) column in order to eliminate salts.
Microbial contamination was not observed throughout the whole operation period due most likely to
the high glucose concentration (40%) and the high operating temperature (60°C). The half-life of the
enzyme catalyst under their operating conditions is 34-40 days, depending on the flow rates.
According to the company's data, 1 liter of the immobilized enzyme (an adsorption type) isomerizes
288 kg of glucose within 30 days. On the other hand, 1 liter of a covalently immobilized enzyme
(with triazinyl chloride as a coupling agent) isomerizes 576 kg of glucose in the same period. 

429. In Clinton Corn Processing's approach (flow sheet shown in Figure 4), the immobilized enzyme
constitutes a thin bed of 2.5-12.5 cm depth (Davis, 1974). Several of these beds are staged to form
a multibed processing unit resembling a pressure leaf filter (see also paragraph 419). A major
advantage of having more than one bed is to minimize the effect of channeling that easily occurs in
shallow beds. For bed dimensions, Clinton Corn recommends a depth-to-width ratio of about 0.02 to
0.05 (Antrim et al., 1979; Davis, 1974). Single beds can be removed for regeneration during
processing without interruption of the process flow. Carbon treatment and ion exchange are used to
purify the 42%-fructose effluent, and it is concentrated to syrup by evaporation. Enzyme half-life,
according to Clinton Corn, is "several hundred hours" (Davis, 1974). 

430. In the mid 1970s Sanmatsu Kogyo was the world's first manufacturer of sugar obtained by
isomerization with soluble glucose isomerase. Mitsubishi Chemical Industries Ltd and Seikagaku
Kogyo jointly developed an ion exchange resin that selectively adsorbs glucose isomerase "without
affecting its activity" (Ishikawa, 1977). These three companies signed a license contract for this
process that led to a commercial test plant that was put into operation in 1975. On the basis of
satisfactory test results, Sanmatsu Kogyo decided to switch its whole production process to this
system in 1976. Later on that year Sanmatsu factories in Chiba and Fukuoka were remodelled to
utilize Mitsubishi's technology. Since then these factories have operated commercially. The
production process is summarized as follows (Ishikawa, 1977). The microbial product of glucose
isomerase is filtered for culture liquor separation, repulped in water and treated with additives for
several hours to extract the enzyme. After separation of the residual cells, the enzyme liquor is
allowed to contact the ion exchange resin, which adsorbs almost all the glucose isomerase within
several hours. Since the carrier selectively adsorbs glucose isomerase, refining the enzyme liquor is
unnecessary. The isomerization itself takes place in a simple column packed with the immobilized
enzyme, glucose concentration of 45-50%, reaction temperature 55-70°C, pH 6.5-8.5, space
velocity (initial) 1.5-4 hr-1. The glucose conversion level is 45%. At these operating conditions the
half-life of the immobilized enzyme is 50 days if crystalline glucose is used as raw material, but as
low as 30 days if the raw material is a saccharified glucose solution subjected to conventional
purification. The isomerized glucose syrup is almost colorless and has few byproducts. Simple
treatment of the syrup with ion exchange resin completely desalts and decolorizes the product. After
being used the immobilized enzyme is washed away from its carrier in the reactor through a
regeneration step employing "some chemical solutions". The regenerated carrier is reused for
isomerization after adsorbing new, active enzyme. 

431. According to Zhang (1982), Chinese biotechnologists used glucose isomerase

from Streptomyces roseoruber, adsorbed on a strong basic anion exchange resin 290 (made by
Nankai University, China), for making HFCS. Pilot plant scale experiments have been carried out
with 1.25-2.2 tons of HFCS produced per kg of dry immobilized enzyme. A general overview of
biotechnology programs in China is given in Mang (1991) and Han Ying-Shan (1991). 

432. In the Snamprogetti (Italy) process glucose isomerase entrapped in fibers is used for the
isomerization of industrial glucose solutions. Under operating conditions (65°C, 50% w/w glucose
syrup) the half-life of the immobilized enzyme preparations is about 70 days. The product is
colorless, and activated carbon treatment is not required. The best process performances were
achieved with tubular and radial reactors (Marconi & Morisi, 1979). In the tubular reactor the
packing is obtained with fibers placed in an ordered manner parallel to the long axis of the column,
a packing degree in the range of 0.2-0.25 kg of dry fibers per liter of reactor volume results in a
stable catalyst bed practically incompressible and with negligible channelling and relatively low
pressure drop. On the other hand, the radial reactor fits very well with the filamentous structure of
the fibers. It is prepared by rolling the fibers around a perforated pipe in an ordered manner, as on a
bobbin; the reactor mixture flows from the holes of the central pipe, passes through the fiber layer
where the enzymatic reaction takes place, and exits. Packing degrees up to 0.35 kg of dry fibers per
liter of reactor volume are reached, making it possible to increase the efficiency of the fiber-
entrapped glucose isomerase and to decrease the residence time. According to Snamprogetti, the
radial reactors have the great advantage that they can be easily prepared with standard equipment
of the textile industry, such as winding machines. The total productivity during two half-lives of the
immobilized enzyme in the reactors is around 5-6 tons of HFCS (dry mass) per kilogram of fibers
(Marconi & Morisi, 1979). 

433. Figure 5 shows a flow sheet for the Hungarian industrial process with a daily processing
capacity of 400 tons of native corn, or 120,000 tons/year (Hollo et al., 1984). The plant based on the
wet milling of maize was built in 1983 in Szabadegyhaza, Hungary, and has been operating since
then. The first step of the process is the continuous enzymatic liquefaction of the starch slurry,
based on the technology of Miles/DDS-Kroer and using thermostable alpha-amylase
Optitherm® from Miles Kali-Chemie. The hydrolysate leaves the dextrinizing tank with a DE value of
15. Liquefaction is followed by the enzymatic saccharification at 60°C and pH 4.5 in batch mixing
tanks of 200 m3. For this step, the Aspergillus niger glucoamylase of Miles Kali Chemie (Optidex®) is
used. After a saccharification time of 60 hr, the DE value of the resulting glucose solution is 96-98.
The glucose solution is purified by separating the germ oil by rotary drum filtration in the presence
of active carbon and by full ionization with ion exchange resins. Following the evaporation step, pH
and temperature correction, and the addition of magnesium ions the solution is pumped for
continuous isomerization into the columns, packed with immobilized glucose isomerase
Takasweet®, Miles-USA. A recent general overview of biotechnology in Hungary is given in Dibner
& Burrill (1988) and Wagner & Groo (1992). 

434. In the Hungarian process two columns are fed by down-flow and operated in parallel; new
columns are put into operation at the necessary time, when the activity of the immobilized glucose
isomerase decreases below the control level. The reaction process proceeds at a temperature of
60-62°C, pH 7.8, and yields 2.5 tons of the syrup (dry mass) per 1 kg of the immobilized enzyme;
the enzyme half-life is 40 days. The bed volume of enzyme catalyst in the column is 20 m 3, the
overall consumption of the immobilized enzyme equals 35 tons/year (Hollo et al., 1983). The
isomerization is followed by ion exchange, filtration with active carbon and finally concentration in a
4-stage falling film evaporator to 71% DS with a standard composition of 42% fructose, 52%
glucose and 6% malto-oligosaccharides. 

435. Novo's plant-scale design recommendations (Hemmingsen, 1979), based on the criteria for
their Sweetzyme® such as initial enzyme activity 200 IU/gm, half-life 825 hr, running time 2 half-
lives, and wet bulk density 0.3 gm/cm3, are as follows:
Plant capacity 400 tons dry solids/day

Syrup concentration 40% w/w

Syrup flow 35.6 m3/hr (average)

Bed volume 50 m3

Number of reactors 6

Reactor internal diameter 1.45 m

Reactor bed height 5m

Average linear flow velocity 3.6 m/hr

Initial linear flow velocity 6.5 m/hr

Enzyme consumption 220 kg/day

Operating cycle 275 hr

Flow variation 11% of average

As Novo indicates, similar data have been employed as a basis for the design and engineering of a
number of fructose syrup plants in Europe, the Far East, the United States, Canada, and Latin
America (Hemmingsen, 1979). 

436. In 1977-82 Cetus Corporation, the U.S. R&D company headquartered in Berkeley, California,
developed a patented two-step process for making 100% pure fructose from glucose syrups (U.S.
Patents 4,247,641; 4,284,723), which is "ready for scale-up" (McGraw-Hill's Biotechnology
Newswatch, 1982a). The project had funding of some $8 million from the Standard Oil Company of
California (Socal), which, having retained its 17% corporate investment in Cetus, had left the latter
with full title to all patents and know-how generated. Cetus' proprietary new technology converts the
glucose to 100% fructose in two steps - one enzymatic (the oxidation of glucose to D-
gluconolactone by means of immobilized glucose oxidase derived from Polyporus obtusus), the
other chemical (the reduction of the gluconolactone to fructose by hydrogen with palladium as a
catalyst). The hydrogen peroxide that appears as a by-product of the enzymatic glucose oxidation is
utilized to transform ethylene or propylene into the corresponding oxide by the consecutive action of
the two other enzymes, haloperoxidase and halohydrin epoxidase (Katchalski-Katzir & Freeman,
1982; Borglum & Marshall, 1984). A Cetus representative termed the process developed for the
transformation of glucose as "the third-generation technology for fructose production" (McGraw
Hill's Biotechnology Newswatch, 1982a). Cetus expected to develop the process to a full-scale
facility of 250,000 to 500,000 tons annual capacity (Borglum & Marshall, 1984). Apparently the
method was never scaled up since the enzyme has not become available in large quantities
(Jensen & Rugh, 1987), and Cetus seems to have abandoned the concept. 

4.       Economic estimates 

437. The increase in high fructose syrup penetration of the world sweetener market was made
possible in part by the evolution of immobilized glucose isomerase technology and improvement in
enzyme characteristics and process conditions. Each improvement enhanced cost effectiveness.
Shown below, for example, are the dynamics of decreasing the relative enzyme cost for producing
high fructose corn syrup by Novo (Carasik & Carroll, 1983).
 RELATIVE COST OF IMMOBILIZED ENZYME FOR PRODICING HFCS

 Year first used Type of reactor Relative enzyme cost

        1974 Batch 3-4

        1975 Fluid-bed 2
        1975 Fluid-bed 2
        1976 Fixed-bed 1.1
        1978 Fixed-bed 1.0

Source:Carasik & Carroll, 1983.

438. Economic estimates are available for the Kyowa Hakko process of isomerization of glucose by
means of glucose isomerase immobilized on phenol-formaldehyde resin (see paragraph 428),
which can be compared with data obtained from the use of native (soluble) glucose isomerase
(Yokote et al., 1975). The cost estimate is based on a process in which 50 tons of glucose are
converted monthly to an isomerized sugar mixture containing 45% fructose. In order to isomerize
1,000 kg of glucose, 21 kg of glucose isomerase containing fungal mycelia are required in the batch
process with native enzyme. In the case of the immobilized enzyme systems 9.8 liters of
immobilized enzyme can be obtained from the same amount of mycelia. Being adsorbed on Duolite
A7, 9.8 liters of the immobilized enzyme can isomerize 2,822 kg of glucose within 30 days when
operated at 60°C with 40% glucose solution as substrate. It is also presumed that an immobilized
enzyme system is operated with two columns in series and that older columns are replaced with
fresh columns every 30 days. MgSO4 and CaCl2 requirements in the immobilized glucose
isomerase systems are 10% and 5% respectively of the necessary amounts of MgSO 4 and
CaCl2 with the native enzyme systems, thereby decreasing the volume of ion exchange resins
required for deionization of the isomerized sugar mixture. Coloration of the isomerized sugar
mixture in the immobilized enzyme system is much smaller, i.e. about 1/100, compared with that of
the native enzyme system, thus also decreasing the cost for decolorization. Labor costs are also
reduced in the immobilized enzyme system compared with the batch system. Taking into account
all of these factors, the relative projected costs of these two systems are calculated (Figure 6). The
cost of the Duolite A7 system is 61.5% of the cost of the batch system. 

439. Purdue University presented another set of economic estimates (Emery et al., 1976)
concerning an investigation of the optimum cycle time and relative costs of operation for soluble and
immobilized glucose isomerase in which the latter is covalently bonded "to an expensive but good
carrier", i.e., agarose activated by cyanogen bromide. The goals of the investigation were to bind
the enzyme to the carrier, measure activity and stability of the preparation, and design a plant to
process 450 tons/year of dry HFCS. Optimum cycle time is calculated to give results at minimum
annual cost of the reaction. One-year carrier life is assumed. A batch process (soluble enzyme) is
also designed for comparison purposes. For the batch process, the enzyme is the major cost, as
shown below, while for the immobilized-enzyme process, the expensive carrier and reagents are
the major costs. Even though these latter costs are high, the immobilized enzyme process is less
costly because the expensive enzyme is used in the continuous process many times more
frequently than in the batch. It is noteworthy that the actual isomerization step, including cost of the
enzyme, usually accounts for less than a quarter of the total production cost, with pretreatment of
feedstock and product clean up accounting for the remainder.

ECONOMIC ESTIMATES FOR PRODUCING 450 TONS/YEAR (DRY MASS) 

OF HIGH FRUCTOSE SYRUPS

Glucose isomerase was bound to agarose by cyanogen bromide. 

One-year carrier life was assumed.

  Batch, soluble enzyme Continuous, immobilized enzyme

Reaction time 20 hr -
(arbitrary set)
Optimum running time, days - 29
Annual costs:

    Enzyme $1,000 $37

    Carrier - 1.040
    Reagent - 1.580
    Reactor shell 300 185
    Reaction cost,  1.3 0.28
  cents per pound

Source:Emery et al., 1976.

440. According to Jensen & Rugh (1987), the cost of conversion of glucose to HFS is roughly 10-20
cents per 100 lb of HFS dry substance. 

441. Economic estimates produced in Hungary in the early 1980's indicate that although the sugar
yield of beets per kg was much higher than that of maize, the total monetary value of maize
products should be at least 25% higher. The raw material cost of producing sugar from maize was
3.53 Hung. forint (1986 U.S. $0.28) per kg, whereas beet sugar cost 5.40 Hung. forint (1986 U.S.
$0.44) per kg. As a result, the new maize processing plant was built in Szabadegyhaza, Hungary
(see paragraphs 433 and 447) with a daily processing capacity of 400 tons of native corn (120,000
tons/year). The investment costs for the plant were reported as about 2.1 billion Hung. forint (1986
U.S. $169 mil.), and payback time based on the standards of the national economy was calculated
as 7.3 years (Hollo et al., 1984). 

5.       Scale of the processes 

442. For the industrial production of high fructose syrup, dry solid capacities ranging from 30-100
tons (Okada, 1978) to 400 tons (Hemmingsen, 1979) a day are considered optimum. Owing to the
rapid development of the new technology using immobilized glucose isomerase, high fructose syrup
from corn starch has become a rapidly expanding business and will undoubtedly represent the most
successful use of an immobilized enzyme in food chemistry. Currently, the United States accounts
for most of the world's HFCS production utilizing immobilized glucose isomerase, with Japan as the
second-largest producer. The growing utilization of HFCS in these two countries is shown below.

ANNUAL PRODUCTION OF HIGH FRUCTOSE CORN SYRUP

IN THE USA AND JAPAN

        Year U.S.A. Japan

(thousands of tons) (thousands of tons)

        1972 - -
        1973 136 30
        1974 363 -
        1975 645 75
        1976 908 130
        1977 1135 216
        1978 1585 -
        1979 1680 -
        1980 2300 300
        1981 2680 -
        1983 3250 -

Source:Ishikawa, 1977; Okada, 1978; Samejima & Kimura, 1980; Katchalski-Katzir & Freeman,
1982; Carasik & Carroll, 1983.

443. By the end of the 1970's, high fructose syrup filled 10% of the demand for sugar consumption
in Japan, which was estimated between 2.4 and 3 million tons a year (Okada, 1978). In the U.S.,
usage of HFCS in 1978 reached 6 kg (dry basis) per person (12% of sugar consumption) (Antrim et
al., 1979) and a steady increase in per capita usage is projected to reach 30-40% by the year 2000.
Significant commercial production facilities are also in operation in Canada, Argentina, Austria,
South Korea, and several European countries (see below). In 1982 HFCS accounted for 4% of the
world's production of caloric sweeteners (Food Technology, 1986b). Penetration of world market of
industrial sugars by HFCS had reached as much as 32% in 1981, and 36% in 1982 (Carasik &
Carroll, 1983). 

PER CAPITA NUTRITIVE SWEETENER CONSUMPTION TRENDS IN THE USA

Year Total, kg Sucrose, % Corn syrups HFCS, %

(other then HFCS),%

1970 55.0 84.1 15.9 -

1972 56.3 82.8 16.3 0.9
1974 55.3 79.2 18.6 2.2
1976 56.6 75.9 18.7 5.5
1978 57.8 73.1 17.3 9.7
1980 58.2 68.0 15.6 16.4

Source: Roels & de Flines, 1985.

444. The glucose isomerase market is dominated by three manufacturers: Novo Industri (Denmark),
Gist Brocades (The Netherlands), and Miles Laboratories (U.S.A.). A rough estimate of the 1983
market was 1,625 tons of glucose isomerase which in turn has created a $40 million/year market for
the immobilized enzyme. 

445. In 1982 approximately 2.15 million tons of 42% HFCS and 1.45 million tons of 55% HFCS was
produced (Poulsen, 1985). Average productivity for commercially used immobilized glucose
isomerase was approximately 2,000 kg of HFCS per kg of the enzyme. HFCS manufacturers
produced an estimated 1.2 million tons of the product in 1977 and 3.7 million tons in 1980.
Production increased to about 6.7 million tons in 1984. It is also noteworthy that the U.S. fructose
market in 1982 amounted to about $11 billion a year and was marked by high price stability
(McGraw Hill's Biotechnology Newswatch, 1982b). 

446. Today, several companies are producing fructose corn syrup in the United States with an
estimated production volume of over 3 million metric tons. The producers and brand names of their
products are listed below. A comparison of the economics of fructose syrup production versus beet
sugar production leads one to the conclusion that there is an advantage to processing corn starch
into fructose syrup in the U.S.A.

42% FRUCTOSE CORN SYRUP PRODUCERS (U.S.)

    Company Brand name

American Maize Products Company TruSweetTM

Amstar Corporation Amerose

Archer Daniels Midland Corn Sweeteners Corn SweetTM

Cargill, Inc. ISOCLEAR

Clinton Corn Processing Company ISOMEROSE®

CPC International, Inc. INVERTOSETM

The Hubinger Company HI-SWEET®

A.E. Staley Manufacturing Co. ISOSWEET®

Source: Antrim et al., 1979.

447. In 1983 in Szabadegyhaza (Hungary) a new maize processing plant was put in operation with
a processing capacity of 400 tons of native corn daily (120,000 tons/year). The yearly production in
1988 was: 50,000 tons of HFCS (42% fructose), 30,000 tons of protein feed, and 20 million liters of
ethanol. The plant's annual consumption is 95 m 3 of thermostable alpha-amylase, 90 m3 of
glucoamylase, and 35 tons of immobilized glucose isomerase (Hollo et al., 1983, 1984). 

448. As one more example in this area, in 1985 Cargill Incorporated began full operations at its new
$100 million wet-corn mill in Eddyville, Iowa. The plant, capable of producing up to half a million
tons of HFCS, employs 100 people and processes more than 20 million bushels of corn (Food
Technol., 1985a). 

449. Fructose syrup is produced in Japan (paragraph 442) and Europe although the present
markets are somewhat limited in comparison with the United States. In Europe, where the product
is known as isoglucose, and particularly in the European Economic Community, development of
fructose syrup production has been slowed due to the strong political influence of the sugar industry
and a subsidy on exported beet sugar (Antrim et al., 1979). It is difficult to process European corn
by wet milling, and therefore a major part of the corn needed for fructose syrup production would
have to be imported without the luxury of a subsidy (Antrim et al., 1979). Even so, 1976 production
of fructose syrup in Europe was estimated to be about 100,000 metric tons (United Kingdom,
35,000 tons; Spain, 25,000 tons; West Germany, 21,000 tons; Belgium, 14,000 tons; the
Netherlands, 10,000 tons), with a projected 1980 production of 0.75-1.0 million tons. Since then
additional fructose syrup plants were constructed in France (Societe des Products du Maise and
Roquette Freres), Ireland, Italy (Liquichemica under license from Miles Laboratories and Cargill),
the Netherlands, United Kingdom (in Tilbury, under a joint venture between Schotten/Honig of the
Netherlands and Tate & Lyle of the U.K.), Yugoslavia (under a joint venture of AIPK Poljoprivreda,
Miles Laboratories, and MI-Car International, a Miles affiliate), Canada (two plants, one under joint
venture of John Labatt Ltd., Toronto and Redpath Industries Ltd., a unit of Tate & Lyle, Ltd; and the
other by Canada Starch Co., a unit of CPC International). Total world production of high-fructose
syrup by 1984 has been estimated to be about 6.7 million metric tons (Jensen & Rugh, 1987). 

C.     OPTICAL RESOLUTION OF AMINO ACIDS BY IMMOBILIZED AMINOACYLASE 

450. Utilization of L-amino acids for medicine, food, and animal feed has developed rapidly in recent
years. In 1982, the world production of amino acids was estimated as 500,000 tons, with a market
value of about US $1.3 billion (Kieslich, 1985). Of this amount 300,000 tons were used in the food
industry as monosodium glutamate, 100,000 tons of DL-methionine were used as animal feed
additives, and 40,000 tons of L-lysine were used as food and fodder constituent. 

451. These data are generally consistent with those for 1979, according to which the world
production of amino acids amounted to 424,340 tons: 270,000 tons of L-glutamic acid, 100,000 tons
of DL-methionine, and 29,000 tons of L-lysine (Hitosi, 1983). The biggest producer of methionine is
Degussa AG, the Frankfurt (West Germany) chemical and precious metals company, which
produced 75,000 tons/year of that essential amino acid, which is used in pharmaceuticals, medical
infusion solutions, and special diet foodstuffs. Japan, primarily Ajinomoto and Kyowa Hakko,
produces about two-thirds of the world volume of amino acids (Kieslich, 1985). Another significant
producer of amino acids is China with a total output of 75,000 tons annually (Han Ying-Shan, 1991).

AMOUNT OF AMINO ACIDS SOLD IN JAPAN

    Amino acid Tons

Monosodium glutamate 76,000

DL- or L-methionine 5,500

Glycine 2,700

L-lysine 2,500-3,000

DL- and L-alanine 600

L-aspartic acid 540

L-phenylalanine 90

DL- and L-threonine 40

L-tryptophan 40

L-valine 13

L-isoleucine 11

Source:Samejima & Kimura, 1980.

452. L-lysine, L-tryptophan and L- or DL-methionine are the most common amino acid animal feed
supplements, and their world-wide demand is increasing both in the health food industry and in
bioresearch. A world wide market survey of amino acid production shows that the synthetic DL-
methionine and its hydroxy analog used as feed supplements are both produced commercially from
petrochemicals, not via fermentation. L-lysine and L-tryptophan, on the other hand, are usually
produced by fermentation (Eldib et al., 1985). The Japanese companies Ajinomoto and Bio-Kyowa
were the only producers of L-lysine in the early 1970's. Since then, the South Korean firm Miwon,
the French company Eurolysine and the Mexican company Fermex have joined the ranks.
Currently, no American company produces L-lysine. In fact in the 1960's and early 1970's Du Pont,
Monsanto, and Stauffer Chemical Co. were the only large American companies producing any
amino acids. Stauffer closed its monosodium glutamate plant in 1983 because of severe
competition from foreign suppliers (Eldib et al., 1985). 

453. The Japanese companies Ajinomoto, Kyowa Hakko, Tanabe Seiyaku, Mitsui Toatsu, and
Showa Denko produce L- or DL-tryptophan on an industrial scale. In Europe tryptophan is produced
by Degussa only in semi-commercial quantities. There are no U.S. companies that produce
tryptophan (Eldib et al., 1985). In 1983 two Japanese firms announced plans to produce L-lysine at
plants in the U.S.A.: Bio-Kyowa, in Cape Giradeau, Missouri (projected yearly production capacity
of 15,000 tons) and Ajinomoto, in Eddyville, Iowa, (projected yearly production capacity of 6,000
tons). The current U.S. demand for L-lysine is 24,000 tons/year. American producers sell lysine at
the current market price of $1.40/pound ($3.11/kg), and L-tryptophan at almost $9/pound ($20/kg)
(Process Biochem., 1985). 

454. The above data relate primarily to microbiological processes for producing amino acids. During
the last two decades, however, a new approach to the production of optically active amino acids
has been developed using immobilized aminoacylase, or L-amino acid acylase. The first industrial
application of immobilized enzymes occurred in 1969 when the Tanabe Seiyaku Company, Ltd.
initiated its process for the production of L-methionine. 

1.       Background 

455. Chemical synthesis of amino acids generally produces an optically inactive racemic mixture,
i.e. both the L- and D-isomers. To obtain natural L-amino acid from the chemically synthesized DL-
form, optical resolution is required. Among the many optical resolution methods, the enzymatic
method with microbial aminoacylase is one of the most advantageous procedures, yielding optically
pure L-amino acids. The enzyme is specific for the L-form and thus a chemically synthesized acyl-
DL-amino acid is hydrolyzed asymmetrically by aminoacylase to give L-amino acid and
unhydrolyzed acyl-D-amino acid. Both products are separated easily by their differing charges and
solubilities. The acyl-D-amino acids are then racemized by heat treatment into a racemic mixture of
acyl-D- and acyl-L-amino acids, and reused for the resolution procedure. Since the substrate
specificity of mold aminoacylase is broad and attacks acyl-L-amino acids with various side-chains,
the enzymatic resolution of racemates can be applied to various amino acids (Chibata, 1980). 

456. From 1954 to 1969, this enzymatic resolution method was employed by Tanabe Seiyaku Co.
Ltd. using soluble Aspergillus oryzae aminoacylase for the industrial production of several L-amino
acids. The enzyme reaction is carried out batchwise. This procedure, however, had some
disadvantages inherent to a batch process using soluble enzymes in that in order to isolate L-amino
acids from the reaction mixture, it is necessary to remove enzyme protein by pH and/or heat
treatments (Chibata, 1978a). This results in uneconomical use of enzyme, lowered yield of L-amino
acids, and increased labor. Therefore, as a result of extensive studies of the continuous optical
resolution of DL-amino acids using immobilized aminoacylase, the industrial production of L-amino
acids was switched to the immobilized enzyme process in 1969 to produce L-methionine. 

2.       Commercial preparations of immobilized aminoacylase 

457. As in the case of immobilized glucose isomerase (see paragraphs 404-417), covalently
bonded enzymes are not prevalent among preparations of immobilized aminoacylase intended for
industrial application. The best known preparations include aminoacylase immobilized by ionic
binding to DEAE-Sephadex (developed by Tanabe Seiyaku Co.), in which the enzyme is trapped by
means of fiber wet spinning (developed by Snamprogetti, see also paragraph 408) into fibers of
cellulose triacetate as microdroplets of its aqueous solution. Tanabe indicates that the preparation
of their immobilized enzyme is easy, the activity is "stable and high", and the regeneration of
deteriorated immobilized enzyme preparations is possible. This last point is particularly important
since DEAE-Sephadex is a very expensive carrier. 

458. The immobilized enzyme is prepared as follows. 1,000 liters of DEAE Sephadex A-25 and
1,100-1,700 liters of aqueous solution of aminoacylase are mixed and stirred at 35°C, pH 7.0, for 10
hr, before filtration and washing with water. The yield of activity compared with that of the initial
enzyme preparation is 50-60%. The half-life of the DEAE-Sephadex aminoacylase is equal to 65
days at 50°C, as compared with 48 days at 37°C for aminoacylase entrapped in polyacrylamide gel
(Chibata, 1978a). According to Snamprogetti's data (Marconi & Morisi, 1979), the entrapped
preparations of aminoacylase from hog kidney and microorganisms show "very good" stability under
operating conditions in the course of resolving racemic mixtures of N-acetylmethionine in that the
loss of enzyme activity is only 25-30% after 50 days of operation. 

459. Rohm GmbH (Darmstadt, FRG) used macroporous beads made of plexiglas-like material to
immobilize amino acid acylase. This carrier has a porosity of 3-4 mg/g and the enzyme is bound
covalently by oxirane groups. Because of their rigid structure, the 0.1-0.3 mm diameter beads are
pressure stable and show "good flow properties". The brand name of the immobilized aminoacylase
is Plexazym AC, and 1 g of it is as effective as 0.4 g of the original preparation under operating
conditions (Plainer & Sprossler, 1982). 
460. Recently Chinese researchers reported the immobilization of aminoacylase from A. oryzae by
adsorption on synthetic modified polyacrylamide. "High immobilized enzyme activity and high
operational stability" is mentioned regarding the immobilized preparation (loss of activity at 30 min
at 70°C is 90% for soluble enzyme, 37% for the immobilized enzyme) (Wang et al., 1992). 

3.       Technological characteristics of the processes 

461. The flow diagram for the continuous production of L-amino acids by Tanabe Seiyaku Co. is
shown in Figure 7. Substrate, i.e., N-acetyl-DL-amino acid solution, is charged continuously into an
enzyme column at a constant flow rate through a filter and heat exchanger by means of a chemical
pump. The substrate is converted to L-amino acid and N-acetyl-D-amino acid while passing through
the column. Enzyme column effluent is concentrated, and the L-amino acid is crystallized. Acetyl D-
amino acid is racemized by heating in a racemization tank and reused for optical resolution. The
system is controlled automatically and operated continuously. The reaction takes place at pH 7.0 (in
the presence of 5x10-4 M cobalt salts), 50°C with a flow velocity (in a downflow operation) of 900-
20,000 l/hr and a column volume of 1 m3. 

462. The aminoacylase column maintained approximately 70% of its initial activity after 30 days of
operation, and the half-life of the enzyme in the column is estimated to be approximately 65 days.
The ratio of height to diameter does not influence the efficiency of the process. A deteriorated
column is completely reactivated simply by addition of the amount of aminoacylase needed to
replace the deteriorated activity. The stability of the water insoluble carrier DEAE-Sephadex is very
high, and according to Tanabe Seiyaku (Chibata, 1978a) has been used for more than 8 years
without significant loss of binding capacity or physical decomposition. 

463. As an example, continuous production of L-methionine in a 1000-liter enzyme column is as

follows (Chibata & Tosa, 1976). A solution of 0.2 M acetyl-DL-methionine (pH 7.0, 5x10 -4M Co+2) is
passed through the aminoacylase column at a flow rate of 2000 l/hr at 50°C. Two thousand liters of
the effluent are evaporated, and the separated crude L-methionine is collected by centrifugation and
recrystallized from water. The yield is 27 kg (91% of the theoretical maximum yield). The residual
acetyl-D-methionine is heated at 60°C with acetic anhydride to achieve racemization. The reaction
mixture is adjusted to pH 1.8 and the separated acetyl-DL-methionine is collected and reused as
substrate. The yield is 36 kg (94% of the theoretical maximum yield). 

464. Since 1969 Tanabe Seiyaku has operated several series of enzyme reactors industrially in the
company's plants to produce L-methionine, L-valine, L-phenylalanine and other L-amino acids. 

OPTICAL RESOLUTION OF N-ACETYL-DL-AMINO ACIDS BY AMINOACYLASE IMMOBILIZED

ON DEAE-SEPHADEX IN A 1,000 LITER REACTOR OF TANABE SEIYAKU CO.
       
  Yield of amino acids (kg)
Amino acid Flow velocity 24 hr 30 days
(volume/hr)

L-alanine 1.0 214 6420

L-methionine 2.0 715 21450
L-phenylalanine 1.5 594 17820
L-tryptophan 0.9 441 13230
L-valine 1.8 505 15150

Source: Chibata & Tosa, 1976.

465. A basically similar approach has been applied by Snamprogetti (Italy) for batchwise resolution
of a racemic mixture of N-acetyl-DL-tryptophan in a small pilot plant (Marconi & Morisi, 1979; Bartoli
et al., 1978). Amino acid acylase entrapped in cellulose triacetate fibers was used to produce L-
tryptophan and N-acetyl-D-tryptophan. In the pilot process the feed tank contains 9.85 kg acetyl-DL-
tryptophan, 23.8 g CoCl2x6H2O, 1.6 kg NaOH (for pH 7.0) and 200 liters water at 45°C. This
solution is recycled for 5.5 hr through the enzyme reactor at flow velocity of 350 l/hr (after which the
hydrolysis is practically complete). The reactor is 77 by 19 cm with 4 kg of dry fiber, containing 0.28
kg protein/kg dry fiber. The hydrolyzed material is evaporated to 15 liters under vacuum and
separated, based on solubility differences, to yield 3.9 kg of L-tryptophan (95% yield) and 15 liters
of acetyl D-tryptophan solution. The latter is mixed with 3 l of acetic anhydride and held for 5 hr at
45°C to yield 4.7 kg of racemized and precipitated product (yield 96%). The racemized acetyl-D-
tryptophan is recycled to the feed tank. The small pilot plant, having a capacity of about 1 kg of L-
tryptophan/hr, has operated for several months. The activity of the aminoacylase fibers decreased
by 20% during this period, and their productivity was about 400 kg of L-tryptophan per kilogram of
fiber. The same approach was also used for the resolution of N-acetyl derivatives of DL-valine and
DL-methionine (Marconi & Morisi, 1979). 

466. In 1983 Degussa AG (West Germany) reported a plan to develop a new technology for the
production of optically and chemically pure L-amino acids, i.e., arginine, isoleucine, threonine,
proline, and tryptophan, by 1985-1988 (Harper, 1983). For the optical resolution of racemates the
company uses a membrane reactor fed with the soluble catalyst aminoacylase instead of
immobilized enzymes. A Degussa-produced N-acetyl racemic amino acid solution is cycled through
the membrane reactor where acylase, produced by Amano Pharmaceutical Co., Ltd. in Nagoya,
Japan, produces deacylated L-amino acids. The latter, of lower molecular weight as compared with
N-acetyl-D- and N-acetyl-L-amino acids, pass through the reactor's membrane. Because of its high
molecular weight, the enzyme is also retained in the vessel (McGraw Hill's Biotechnology
Newswatch, 1982c). Separated L-amino acids are collected by ion exchange or crystallization. After
racemization, the remaining solution is recycled with more of the original N-acetyl-DL-amino acid
substrate. According to a Degussa representative, the continuous-production process with the
membrane reactor is more efficient than other methods, including those with carrier-fixed enzymes
(McGraw Hill's Biotechnology Newswatch, 1982c). 

467. Another process developed by Degussa became the first industrial application of a two-
enzyme system with regeneration of a cofactor and the production of some L-amino acids from
cheap keto acids. The regeneration of cofactors like NAD or ATP, which dissociate from their
apoenzymes, is a serious problem in enzyme engineering, since dissociating coenzymes must
retain a degree of mobility in order to have access to the active centers of the two apoenzymes. The
alternative, i.e. continuous replacement of the cofactor, would be very expensive. In the Degussa
process NAD+ is coupled to polyethylene glycol and retained in the membrane reactor with keto
acid and formate dehydrogenase (Hartmeier, 1985). 

468. In a Rohm GmbH pilot process N-acetyl-DL-amino acids are converted to L-amino acids in a
packed bed reactor at 37°C by Plexazym AC (see paragraph 459). N-acetyl-DL-methionine, 0.7 M
at pH 8, is hydrolyzed 80% at a space velocity of 6 hr-1. The rate of hydrolysis decreases to about
30% when the space velocity is increased to 25 hr-1. A yield of 500 kg L-methionine/kg Plexazym
AC is reached in a 90 day reactor run (Plainer & Sprossler, 1982). 
469. Recently, Tanabe Seiyaku announced a new approach for the continuous production of L-
alanine from L-aspartic acid developed by the company (Chibata, 1982). That approach used
immobilized Pseudomonas dacunhae cells with high L-aspartate beta-decarboxylase activity. The
decarboxylase enzyme shows high enantiomer selectivity reacting only with L-aspartic acid. Thus,
L-alanine and D-aspartic acid can be produced simultaneously from D,L-aspartic acid. D-Aspartic
acid is used as an important intermediate for semisynthetic penicillin. However, this continuous
decarboxylase system generates problems associated with evolution of CO 2 gas during the
reaction. It was difficult both to maintain the plug flow of the substrate solution under normal
pressure and to keep a constant pH of the reaction mixture in the reactor because of
CO2 effervescence. The company therefore designed a closed column reactor that performs the
enzyme reaction at an elevated pressure of 10 kg/cm2. Since liberated CO2 gas is mixed into the
reaction mixture, the complete plug-flow of the substrate solution is maintained and the pH of the
reaction mixture is not changed appreciably. Moreover, the efficiency of immobilized cells to
produce L-alanine in the closed column system at high pressure increases by 1.5 times (from 250 to
360 mmole/l/hr) as compared with the efficiency at the normal pressure conditions; the stability of
the immobilized cells is not affected by pressure elevation (Chibata, 1982). Tanabe Seiyaku
established a continuous production process for L-alanine in 1982 and succeeded in making it a
functioning commercial enterprise. 

4.       Economic estimations 

470. A comparison of the cost for production of L-amino acids by soluble and immobilized amino
acid acylase in shown in Figure 8. With the immobilized enzyme, the purification procedure for
product become simpler and the yield is higher than in the case of the soluble enzyme. Therefore,
less substrate is required for the production of a unit amount of L-amino acid. As the immobilized
aminoacylase is stable, the cost of enzyme is reduced markedly compared with that of the soluble
enzyme. In the case of immobilized enzyme, the process is controlled automatically, and the labor
cost is also reduced substantially. As a result the overall production cost of the immobilized enzyme
process is about 60% of that of a conventional batch process using soluble enzyme (Chibata,
1978a). The cost of the enzyme in this process is only approximately 1-2 per cent of the overall
operating budget, as estimated in 1984 (Chaplin, 1984). 

5.       Scale of the processes 

471. By 1971 the reported capacity of the Tanabe Seiyaku process (using immobilized
aminoacylase) was greater than 700 kg of L-amino acids per day (Suckling, 1977). Immobilized
aminoacylase columns usually produce up to 750 kg of product per day (Chaplin, 1984). Based on
1984 data, this enzyme technology can be used to produce over 50,000 tons of L-amino acids
annually (Chaplin, 1984). However, it was reported in 1984 that presumably less than 250 tons of L-
amino acids is produced by this technology per year, and the estimated immobilized enzyme
amount is less than 1 ton/year (Poulsen, 1985). Amano (Japan) also uses immobilized
aminoacylase in industry (Poulsen, 1985). 

472. In 1981 Degussa installed an experimental 5 ton/year plant in Konstanz, West Germany,
producing L-alanine, -methionine, -valine, -phenylalanine, and -tryptophan. In 1982 the production
volume of the plant increased to 60 tons/year as a result of a new method of separating amino acids
from protein hydrolysates by means of ion exchangers. The company planned to install a new 6,000
tons/year plant for L-lysine production by fermentation method at Valencia de Don Juan, Spain in
1984 (Harper, 1983; McGraw Hill's Biotechnology Newswatch, 1982c), and produced 10-15 tons
per month of L-methionine, L-valine and L-phenylalanine by means of the two-enzyme system with
cofactor regeneration (Hartmeier, 1985) (see also par. 467). 

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