Nature and Science, 2009;7(2), ISSN 1545-0740, http://www.sciencepub.

net,
[email protected]

The Effect of Chemical Modification on the Thermal Stability of Protease from Local Isolate
Bacteria, Bacillus subtilis ITBCCB148

Yandri*, D. Herasari, T. Suhartati and S. Hadi

Department of Chemistry, University of Lampung, Bandar Lampung 35145 Indonesia

*E-mail: [email protected]

Abstract: This research aims to study the effect of chemical modification on the thermal stability
of protease enzyme from local bacteria isolate Bacillus subtilis ITBCCB148 which was modified
using dimethyladipimidate (DMA). To approach this aims, the production, isolation and
purification of the enzyme were done. The purified enzyme was then modified with DMA. The
number of modified lysine residue was shown by the modification degree determined using
trinitrobenzenesulfonic acid. The success in the chemical modification was done by comparing the
thermal stability of the enzyme before and after modification. The results showed that the
modified enzymes with DMA produced modified enzyme with modification degree of 69, 75 and
76%. The enzyme thermal stability of the modified enzyme with modification degree of 69, 75
and 76% at 60°C were shown with the following data: t1/2 = 96.25 min, ki = 0.0072 min-1, ΔGi
106.868 kJ mole-1; t1/2 = 119.48 min, ki = 0.0058 min-1, ΔGi 107.466 kJ mol-1; and t1/2 = 210 min;
ki = 0.0033 min-1; ΔGi 109,028 kJ mole-1, respectively, whereas the thermal stability of the
purified enzyme has data of: t1/2 = 52.1 min, ki = 0.0133 min-1, 105.168 kJ mole-1. The chemical
modification on the purified protease enzyme has been able to increase the thermal stability up to
2 – 4 times compare to that of the purified one based on the decrease of Ki value. [Nature and
Science. 2009;7(2):68-75]. (ISSN: 1545-0740).

Keywords: Protease, chemical modification, dimehyiladipimidate, Bacillus subtilis ITBCCB148

Introduction
Protease is enzyme that breaks the peptide bond into oligopeptide and amino acids. This
enzyme is widely used in industrial sector such as food, skin, detergent and pharmaceutical
industries (Rao et al., 1998). The use of this enzyme as biocatalyst in industry must fill some
criteria, which are it must have high thermal stability and is able to work in wide pH range (Vieille
and Zeikus, 1996). This condition is not own by most of the enzymes, as generally an enzyme
works at physiological condition and cannot stand against extreme condition especially
temperature and pH. In industrial processes which use the enzyme in enzymatic reaction, the use
of en enzyme in an extreme condition from its optimum condition, can cause the enzyme lose its
catalytic activity. Therefore in order to find an enzyme which can be used in industrial process, we
must be able to get enzyme which is able to work in extreme conditions. This can be achieved in a
few ways such as direct isolation from organism which lives in extreme condition (extremophilic)
or by chemical modification to the enzyme from organism which does not live in extreme
condition (mesophilic) (Wagen, 1984). The stabilization of enzyme obtained from mesophilic
microbia is preferred way to get stable enzyme (Mozhaev and Martinek, 1984). According to
Mozhaev et al. (1984), three ways to increase stability of an enzyme are amobilization, chemical
modification, and directed mutagenesis. Chemical modification is a method to increase the
stability of an enzyme which is soluble in water. The use of enzyme amobilization has a weakness,
i.e. the decrease of binding capacity or enzyme reactivity due to the mass transfer inhibition by
amobil matrix. In directed mutagenesis needs complete information about the primer structure and
the picture of three dimension structure. According to Janecek (1993) in amobil process, the work
mechanism of the enzyme in clinical sector, during interaction with receptor or other components
of cellular membrane might be altered due to the presence of long matrix. While chemical
modification, the interaction of enzyme with substrate is not hindered by the presence of insoluble
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[email protected]

matrix, so the decrease of enzyme activity can be minimized. Based on this fact, the chemical
modification is suggested way to increase the stability of enzyme.
In order to obtain the enzyme by chemical modification with stable covalent bond can be
done by utilizing bifunctional reagent, modification with nonpolar reagent, addition of charge
group or new polar group and hydrophilization the surface of protein (Mozhaev et al., 1990).
Modification with bifunctional reagents (cross-bond reagents) has been used many times to
increase the enzyme stability. An example of bifunctional cross-bonded reagent is
dimethyladimipidate. Kazan et al. (1996) reported that penicillin G acylase has increased its pH
working range after reaction with DMA. Erarslan and Ertan (1995) have done thermostablization
of penicillin G acylase from E. coli mutant with dimethyladipimidate (DMA),
dimethylsuberimidate (DMS) and dimethyl-3,3’-dithiobispropionimidate (DTBP). The higher
thermostabilization increased was observed after cross bond with DMA at temperature above 50oC.
Based on the results of the above researches, the chemical modification using
dimethyladipimidate to increase the thermal stability of protease enzyme from local bacteria
isolate Bacillus subtilis ITBCCB148 is chosen.

Materials and Methods

Materials: Dimethyladipimidate (DMA) and other chemicals used were purchased from
Sigma Aldrich and used without further purification. Local bacteria isolate Bacillus subtilis
ITBCCB148 was obtained from Microbiolgy Laboratory, Chemical Engineering Department,
Bandung Institute of Technology, Bandung, Indonesia. Buffer pH was measured at the
temperature of use, and the pH reported is that at the temperature of the incubation.

Production of protease enzyme: the production of enzyme is done using fermentation

media containing peptone 0.5%, yeast extract 0.15%, glucose 0.036%, and NaCl 0.25% (Yandri et
al. 2007).

Purification of protease enzyme: The purification of protease enzyme is done in few steps:
the separation of enzyme liquid from the cell with cold centrifugation to get the raw enzyme
extract, precipitation with ammonium sulphate, ion exchange column chromatography and
molecule filtration column chromatography (Yandri et al., 2007, 2008).

The protease enzyme activity test and protein content determination:The protease
enzyme activity test was done based on the modified of Kunitz method (Yamaguchi et al., 1982).
Protein content determination was done based on the Lowry et al. (1951) method.

The modification of purified enzyme with DMA: Into 10 mL (0.082 mg/mL) of purified
protease enzyme in 20 mM of phosphate buffer pH 7 was added with solid DMA until the
concentration of DMA were 0.5%, 1%, and 1,5% (W/v) to reach inactivation of kinetic work at
different pH and temperature and then leave them for 1 h at room temperature (Kazan et al.,
1996)..

Determination of modification degree: Determination of modification degree was done

based on the method used by Synder and Sobocinski (1975) and as following 0.1 mL of modified
enzyme is dissolved into 0.9 mL borate buffer (pH 9.0) and then added with 25 µl 0.3 M 2,4,6-
trinitrobenzenesulfonic acid. The mixture is then shaken and left it at room temperature for 30 min.
The standard solution is made with the same composition but using the purified enzyme, while the
blank contains 1 mL borat buffer 0.1 M pH 9 and 25 µl 0.3 M 2,4,6-trinitrobenzenesulfonic acid.
The absorbance is measured at the λmax 420 nm.

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Determination of optimum pH and temperature before and after the modification: To

know the optimum pH of the enzyme before and after the modification, the phosphate buffer 0.1
M was used with pH variations of 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; and 9.0. The temperature
was kept constant at the determined optimum pH. To find the optimum temperature, the variations
of temperature used were 50; 55; 60; 65 and 70°C, and then followed by the measurement of
enzyme activity with Kunitz method.

Thermal stability test and stability of the enzyme pH before and after the modification:
the enzyme thermal stability before and after the modification was done by measuring the residual
activity of the enzyme after being incubated for 0, 60, 120, 180, 240, and 300 min optimum pH
and temperature based on the method applied by Yang et al. (1996)

Determination of half life (t½), ki and ΔGi: determination of ki value (rate constant of
termal inactivation) of purified enzyme and the modified enzyme was done using the first order of
inactivation kinetics equation (Equation 1) (Kazan et al. 1997
ln(Ei / E0) = -ki t (1)
The denaturation energy change (Δ Gi) of the purified and modified enzymes was done using
the Equation (2) (Yandri, 2007):
Δ Gi = -RT ln (kih/kBT)

Results and Discussion

Determination of modification degree with DMA: Modification of the purified enzyme
with DMA was done in three concentration variations of DMA which was mixed with 10 mL of
purified enzyme in phosphate buffer pH 7.0. The concentration variations of DMA used were
0.5%, 1%, and 1.5%. The result of determination of modification degree with 2,4,6-
trinitrobenzenesulfonic acid were 69, 75, and 76%. Determination of modification degree is based
on the ratio of number of lysine residues after and before modification.

Effect of modification toward optimum pH: Based on the data obtained, the modified
enzyme with modification degree of 75% get decrease its optimum pH , while the optimum pH of
modified enzyme with modification degree of 69% and 76% are the same as the purified enzyme
(Figure 1).
110
Purified enzyme
100
90 DMA 69%
80 DMA 75%
(%)Activity

70 DMA 76%
60
50
40
30
20
10
0
5 5.5 6 6.5 7 7.5 8 8.5 9
pH
Figure 1. The Optimum pH of the Purified and Modified Enzyme (69%, 75%, 76%)

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The decrease of pH optimum might be due to the change in enzyme conformation which
causes the change on the active site of the enzyme, so the reaction ability of enzyme is also
changed in its environment. Although there is decrease in optimum pH, but the (%) activity of the
modified enzyme (DMA 75% and 76%) is higher at pH 7.5-9.0 when they are compared to
modified enzyme with DMA 69% and the purified one. The high activity value (%) of the
modified enzyme illustrates that the modified enzyme attain stability increase toward pH when
compared to that of the purified one. The purified enzyme can retain its stability at pH 6.0-7.0, but
there is a drastic decrease at pH 7.5-9.0. Whereas the modified enzymes with DMA 75% and 76%
have wider working pH range, i.e. 5.0-7.0, although there is activity decrease at pH 7.5-9.0, but
the decrease is not too high and the (%) activity is higher in that pH range compared to modified
enzyme with DMA 69% and the purified enzyme. The activities (%) of purified and modified
DMA 69% at pH 9.0 were 4% and 9 %, respectively, while those for modified DMA 75% and
76% were 20% and 26%, respectively. These results showed that the modified enzymes are more
stable toward pH especially at basic condition than the purified enzyme. This phenomenon can be
explained that at basic condition, the higher concentration of OH- ion will bind to H+ ion of
carboxyl group on enzyme molecule to form H2O. By modifying the enzyme, besides the binding
of NH2 groups of enzyme molecule by carbon group from DMA molecule, the hydrogen bond
between H atom from carboxyl group with N of amine group in DMA. Due to the binding of H+ of
carboxyl group of enzyme by OH- to form H2O is not occurred. Therefore, the modified enzyme
will be more stable at basic pH. The activity increase of modified enzyme was also occurred at
acidic condition, but not too high and less significant. Based on this discussion it has been shown
that DMA is able to increase enzyme stability toward pH.

The effect of modification toward optimum pH: The modified enzymes with DMA 69%,
75%, and 76% have optimum pH the same as the enzyme before it is modified (Figure 2),
however their activity is higher than that of purified one.
Soemitro (2005) stated that the activity increase occur due to temperature increase did not
change the accuracy of the tertier structure of enzyme before and after modification which bring
close the side chain of some amino acids of stereospecific substrate side bond maker or amino
acids of catalytic side support, so the optimum temperature of the enzyme did not change. This
result agreed to those reported by Yandri et al., (2008), Kazan et al. (1997) and Francis et al.
(1992) that the chemical modification must not always cause the change of optimum temperature
of the modified enzyme. Although the optimum temperature of the modified enzyme did not
change, but the stability of the enzyme has increased at temperature range of 55 – 65oC. At 55oC
the purified enzyme has activity of 75%, while at 65oC was 24%. The modified enzymes with
DMA 69%, 75%, and 76% at 55oC have activity of 87%, 82%, and 96%, respectively, while at
65oC has activity of 42%, 60% and 40%, respectively. This result illustrated that the modification
caused the enzyme was more rigid, so the enzyme was more endure toward temperature.

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110 Purified enzyme

100
DM A 69%
90
80 DM A 75%
(%) Activity
70
60 DM A 76%
50
40
30
20
10
0
50 53 56 59 62 65 68 71 74
o
Temperature ( C)

Figure 2. Optimum Temperature of the Purified and Modified Enzyme (DMA 69%, 75%,
76%)

120
Purified enzyme

100 DMA 69%

DMA 75%
80 DMA 76%
(%) Activity

E (P ifi d
60

40

20

0
0 60 120 180 240 300
Time (min)

Figure 3. Thermal Stability Expressed as Graphic between Residual (%) activityPurified

Enzyme and Modified Enzyme (DMA 69%, DMA 75%, DMA 76%) at 60oC vs Time

The effect of modification toward thermal stability: The test of thermal stability effect to
the modified enzyme was done by calculating the percentage of residual activity of purified and
modified enzymes. This was done by incubating each enzyme at 60oC for 300 minutes. The
activity was measured every 60 minutes, and the result is presented in Figure 3.
Figure 3 illustrates the residual (%) activity of each enzyme. The purified enzyme has
residual activity of 1.47% and then after modification with DMA 69%, DMA 75% and DMA 76%
the residual activities were 10.71%, 15.87% and 36.36%, respectively. Based on graph in Figure 3,
it is clear that the modified enzymes have much higher thermal stability than that of purified one.
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In line with modification degree, the higher modification degree, the higher the thermal stability of
the enzyme, and the use of DMA as the modifying agent has a good effect on the structure
stability. The mechanism of enzyme structure stability by DMA is occurred through cross-bond of
inter and intramolecular of DMA to form small circles on enzyme, to protect the unfolding of
tertier structure of enzyme and protein oligomer dissociation into subunits. The cross bond to
lysine residue in the surface will protect the hydrophobic site of the enzyme to contact with
solvents. Besides that, there is addition of hydrogen bond between the nitrogen atom of amine
group in DMA molecule and hydrogen atom of carboxyl groups of the enzyme. Since DMA is
bifunctional reagent with two functional groups which can bind to two enzyme molecules,
consequently, the hydrogen bond form will more. Kazan et al. (1996) reported that the change of
ionization degree of organic groups on enzyme due to the formation of hydrogen bond causing the
rigidity increase of penicillin G acylase structure modified with DMA. The more hydrogen
bonding formed, the higher the modification degree used, so the enzyme rigidity is also increased.
This result supported previous result obtained that the higher the modification degree, the more
stable the enzyme will be (Kazan et al., 1996).

Table 1. The Values of Rate of Thermal Inactivation (ki), Half-life (t1/2) and Denaturation
Energy Change (ΔGi) of Purified and Modified Enzymes (DMA 69%, DMA 75%, DMA
76%)

Enzyme ki (min-1) t1/2 (min) ΔGi (kJ mole-1)

Purified enzyme 0.0133 52.1 105.168

DMA 69% 0.0072 96.25 106.868
DMA 75% 0.0058 119.48 107.466
DMA 76% 0.0033 210 109.028

Half-life (t½) and rate of thermal inactivation (ki): The half lifes of the modified enzymes
have increased 2-4 times compared to the purified enzyme. Stahl (1999) stated that half-life will
determine the stability of the enzyme. The results obtained in this research, the half-lives of
modified enzyme with DMA 69%, 75% and 76% have all increased from 52.1 minutes to 96.25,
119.48 and 210 minutes, respectively. These data showed us that the higher the modification
degree used, the longer the half-life will be. The decrease of ki value (Table 1) showed that the
decrease of enzyme denaturation has occurred on the modified enzymes. The increases of enzyme
stability based on the decrease of ki value are 2-4 times compared to that of purified enzyme. The
lower of value ki illustrates that the enzymes is less flexible in water due to the bond formation
between DMA and NH2 group of side chain lysine residue on the surface. This causes the decrease
in protein unfolding so the structure of enzyme will be more rigid and more stable. The same
result was also obtained by Kazan et al. (1996) where the rate of thermal inactivation of DMA
cross-bonded with penicillin G acylase (PGA) was always lower than the pure PGA.

The energy change due to denaturation (ΔGi): Table 1 shows the ΔGi increase of
modified enzyme compared to that of the purified enzyme. The higher ΔGi value of the modified
enzyme demonstrates that the enzyme is more rigid and less flexible in water. The tertier structure
of the denaturated enzyme has changed and the enzyme structure that is more rigid has stronger
bond, so the enzyme conformation is not easily opened that the tertier enzyme structured can be
maintained. To denature this sort of enzyme will require more energy, so the ΔGi value obtained
will also be higher. Therefore the higher ΔGi value indicates that the enzyme is more rigid, less
flexible and is hard to denature and vice versa. Kazan et al. (1996) reported that the higher ΔGi of
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[email protected]

penicillin G Acylase which crossed-bond to DMA indicated the stabilization of PGA by cross
bond at extreme pH. In this research, the ΔGi value of purified enzyme obtained was 105.168 kJ
mole-1 while for the modified enzyme with DMA 69%, 75%, 76% were 106.868; 107.466;
109.028 kJ mole-1, respectively. The increase of enzyme stability is higher with the increase of
modification degree, although the ΔGi value is not too high, but the thermal stability is increased
2-4 times.

Conclusion
Based on the results obtained and the discussion above, it can be concluded that: the
optimum temperature of the modified enzyme is the same as the purified enzyme, but the stability
is increased at 55 – 65oC. The thermal stability test at storage temperature of 60oC for 300 min
shows the modified enzyme with DMA (69%) has residual activity of 10.71%, t1/2 = 96.25 min; ki
= 0.0072 min-1; ΔGi = 106.868 kJ mole-1.
DMA (75%) has residual activity of 15.87%; t1/2 = 119.48 min; ki = 0.0058 min-1; ΔGi = 107.466
kJ mole-1. DMA (76%) has residual activity of 36.36%; t1/2 = 210 min; ki = 0.0033 min-1; ΔGi =
109.028 kJ mole-1. While the purified enzymes has residual activity of 1.47%; t1/2 = 52.1 int, ki =
0.0133 min-1, 105.168 kJ mole-1. The modification of enzyme with DMA has been able to increase
the thermal stability of the enzyme up to 2-4 time based on the decrease of ki value.

Future works and suggestion

Based on the results obtained, we suggest to do further research to find out the three
dimension structure of protease enzyme B. subtilis ITBCCB148 to explain the stability of the
enzyme. The modification of this enzyme with other bifunctional reagent is interesting to be done
to compare the stability might be obtained.

Acknowledgements
The authors would like to thank to The Directorate of Research and Community Services,
Directorate General of Higher Education, The Ministry of National Education of Republic of
Indonesia that provide fund for this project to be undertaken through Hibah Bersaing XV/2
Research Grant Scheme 2008 with contract number of 010/SP2H/PP/DP2M/III/2008, 6 March
2008

Correspondence to:
Dr. Yandri
Department of Chemistry, University of Lampung, Gedongmeneng Bandar Lampung 35145
Indonesia
Tel: +62-721-701609 ext. 706; Fax.: +62-721-704625; E-mail: [email protected]

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