Glutamine Metabolism: Nutritional and Clinical Significance

Industrial Production of L-Glutamine1

Isao Kusumoto2
Ajinomoto Co., Inc., Kawasaki, Japan

ABSTRACT The industrial production of L-glutamine (L-Gln) started with its fermentation in the late 1960s.
Currently, it is manufactured for use as pharmaceuticals and health foods at the worldwide annual production of

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⬃2000 metric tons. To manufacture high quality L-Gln at a low cost, it is of prime importance to obtain a strain of
microorganism with good production efficiency and minimum by-products. Furthermore, to obtain the final
crystalline powder product, the efficient removal of impurities contained in the fermentation broth becomes
paramount. Therefore, the industrial process is designed to take into account characteristics of the fermentation
broth as well as chemical, physical and biological properties of L-Gln. Points that should be considered in the
process design and typical industrial production of L-Gln are described in this report. J. Nutr. 131: 2552S–2555S,
2001.

KEY WORDS: ● glutamine ● amino acid ● ion exchange resin ● resin chromatography ● crystallization
● polymorphism

The industrial production of amino acids is briefly re- acids used in pharmaceutical products mainly for intrave-
viewed before discussing the production technology of L- nous and enteral nutrition is ⬃15,000 tons. The annual
glutamine (L-Gln).3 Historically, the industrial production demand for L-Gln as a pharmaceutical ingredient, such as
of amino acids started with the availability of monosodium for the treatment of gastric ulcer, and as a health food
glutamate (MSG) in 1909. MSG was discovered in 1908 by ingredient is estimated to be ⬃2000 tons.
Dr. Kikunae Ikeda as a basic taste substance of kelp that is
a traditional seasoning in Japan. Currently, MSG is used General manufacturing process for amino acids
worldwide as a flavor enhancer. Originally, MSG was man-
ufactured by extraction from acid hydrolysate of plant pro- The manufacturing methods of amino acids are catego-
tein. Small-scale production of various amino acids fol- rized as: 1) extraction from hydrolysates of animal or plant
lowed but they were produced by the same extraction protein, 2) chemical synthesis, 3) fermentation, and 4)
method used in MSG production. In the late 1950s, fer- enzymic. Although DL-methionine for feed additives use
mentation technology was established and used for the and glycine, without an asymmetric carbon, are manufac-
commercial production of MSG. This was the beginning of tured in a large scale by chemical synthesis, most amino
modern amino acid production. Since then, fermentation
technologies for various amino acids have been established.
Production of L-Gln by fermentation started in the late
1960s.
Current total annual worldwide consumption of amino
acids is estimated to be over 2 million tons (Ajinomoto,
estimate). The annual demand for amino acids as MSG-
based flavor enhancers, and as feed additives comprised
mainly of L-lysine hydrochloride, DL-methionine and L-
threonine, is estimated to be 1 million tons for each amino
acid (Ajinomoto, estimate). The annual demand for amino

1
Presented at the International Symposium on Glutamine, October 2–3,
2000, Sonesta Beach, Bermuda. The symposium was sponsored by Ajinomoto
USA, Inc. The proceedings are published as a supplement to The Journal of
Nutrition. Editors for the symposium publication were Douglas W. Wilmore, the
Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School
and John L. Rombeau, the Department of Surgery, the University of Pennsylvania
School of Medicine.
2
To whom correspondence should be addressed.
E-mail: [email protected]
3
Abbreviations used: L-Gln, L-glutamine; MSG, monosodium glutamate. FIGURE 1 Schematic of the fermentation process.

0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences.

2552S
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FIGURE 4 Flow diagram of the isolation process.

FIGURE 2 L-Gln-producing bacterium. As stated, most impurities are removed by the crude isola-
tion process. The purification process is relatively simple and
performed frequently. Careful performance of the fermenta-
acids are manufactured by fermentation and enzymic meth- tion and isolation processes, as well as the combination of
ods. Several amino acids including L-leucine, hydroxy L- these two processes, is critical to manufacture a high quality
proline, L-tyrosine and L-cystine are still being manufac- product with high productivity.
tured by extraction in addition to fermentation and
chemical synthesis. L-Gln is manufactured by several man- Manufacturing method of L-Gln
ufacturers throughout the world, all using fermentation.
The manufacturing process of an amino acid by fermen- The following describes production of L-Gln by the fermen-
tation comprises fermentation, crude isolation and purifi- tation process. It is essential to the outcome of the fermenta-
cation processes. In the fermentation process, the desired tion process to maintain a clean and sterile fermentation tank.
amino acid is specifically produced by the fermentation Compared with wild-type strains, L-Gln-producing strains are
microorganism. In the crude isolation process, most impu- weak and are compromised in a contaminated environment.
rities contained in the fermentation broth are removed by Furthermore, it is important to maintain the tank under pos-
combining various technologies. Final purification is per- itive pressure by aeration during fermentation to prevent con-
formed to ensure the required quality for the intended use. tamination by other microorganisms and external materials.
The final product is obtained as a crystalline powder. The The fermentation medium consists of glucose as a carbon
product is released only after quality tests have verified that source, ammonia as a nitrogen source, a small amount of
the product meets specific requirements, and the normal minerals and vitamins as growth factors. Control factors dur-
functioning of each process step has been verified. All ing fermentation are pH, temperature and dissolved oxygen
manufacturing processes for the production of amino acids (Fig. 1).
for medical use must comply with current good manufac- An L-Gln-producing bacterium is shown in Figure 2.
turing practice requirements. This strain is derived from an improved strain of L-glutamic

FIGURE 3 Time course of L-Gln fermentation. FIGURE 5 Stability of L-Gln in aqueous solution.
2554S SUPPLEMENT

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FIGURE 6 Solubility of several amino acids, including L-Gln. FIGURE 8 Decomposition of L-Gln in aqueous solution at 35°C.

acid producing bacteria capable of high quality and high change resin treatment, chromatographic treatment and
productivity L-Gln, while minimizing the formation of by- crystallization.
products. The main points to be considered in designing processes for
Figure 3 illustrates the fermentation profile of L-Gln. amino acid production are characteristics of the desired amino
The amount of fed glucose is decreased with proliferation acid and impurity levels in the fermentation broth. The chem-
of the fermentation bacteria. L-Gln begins to accumulate ical, physical and biological properties of the amino acid are all
when the growth of the bacteria reaches a certain level. important.
L-Gln is stable around the isoelectric point (pH 5.65), but
When the initial glucose concentration decreases, more
glucose is added to the batch to improve productivity and to if the pH shifts from the isoelectric point to either acid or
increase the accumulation of L-Gln. Each step of fermenta- alkaline conditions, L-Gln is easily hydrolyzed to L-glutamic
tion is controlled by optimizing pH, temperature and dis- acid and ammonia. Figure 5 shows stability of L-Gln in aque-
solved oxygen. ous solution.
The solubility of several amino acids, including L-Gln, is
shown in Figure 6. The solubility of L-glutamic acid and
Crude isolation process L-histidine HCl increases with increasing temperature.
In contrast, the solubility of L-Gln is barely affected by
Figure 4 illustrates a typical process for the isolation of temperature as shown by the flat solubility curve. Conse-
crude L-Gln. The purpose of the isolation process is to quently, cooling crystallization is not applicable for harvest-
obtain crude L-Gln with adequate purity from the fermen- ing L-Gln.
tation broth. The broth is centrifuged or filtered through a Purification of amino acids by crystallization is an effective
membrane filter to separate cells and debris. It is desirable means to produce polymorphism. For example, as shown in
that crude crystals are harvested through the direct crystal- Figure 7, two crystal forms can be used. After crystallization of
lization of the supernatant or filtrate. If it is difficult to L-glutamic acid in the ␣-form, the crystals are dissolved, and
harvest crude crystals with adequate purity, then prepara-
tory steps are required usually involving repeated ion ex-

FIGURE 9 Simplified production flow chart of the L-Gln manufac-

FIGURE 7 Amino acids crystal forms. turing process.
 L-GLUTAMINE INDUSTRIAL PRODUCTION 2555S

then recrystallized in the ␤-form. In this manner, it is possible other amino acids, L-Gln is an amino acid that is difficult to
to remove impurities based on their different affinities for the design. Therefore, the purity of the fermentation broth is of vital
two crystal forms. importance to obtain high purity L-Gln with high productivity.
Unfortunately, L-Gln occurs only as one crystal form. The flow diagram depicted in Figure 9 briefly illustrates the
Therefore, to use crystallization for purification, there is no entire process of L-Gln production.
way other than the inefficient simple repetitive crystallization
of the one crystal form of L-Gln. CONCLUSION
Figure 8 shows the biodegradability of L-Gln in aqueous The industrial production of L-glutamine has been reviewed
solution at 35°C. Because L-Gln is easily degraded by micro- with a historical background of the industrial production of
organisms, proper control of the process as well as aspects of amino acids. From the standpoint of the manufacturing indus-
process design is imperative. try of glutamine, it is hoped that basic and clinical research on
The intrinsic properties of L-Gln need to be considered when glutamine will continue and further contribute to the im-
designing its isolation and purification processes. Compared with provement of human health.

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