L-Selective dipeptide synthesis using novel thermophilic enzyme from Clostridium sp.

Article abstract of DOI:10.1016/S0957-4166(01)00444-X

A novel, inexpensive, thermophilic protease-type enzyme isolated from Clostridium thermohydrosulfuricum was used for dipeptide synthesis. The enzyme showed broad substrate selectivity and enantioselectivity towards L-amino acids in peptide bond formation.

Full text of DOI:10.1016/S0957-4166(01)00444-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 2505–2508
L
-Selective dipeptide synthesis using novel thermophilic enzyme
from Clostridium sp.†
J. S. Yadav,a,* H. M. Meshram,a A. R. Prasad,a Y. S. S. Ganesh,a A. Bhaskar Rao,a G. Seenayya,b
M. V. Swamyb and M. Gopal Reddyb
aOrganic Division I, Indian Institute of Chemical Technology, Hyderabad 500 007, India
bDepartment of Microbiology, Osmania University, Hyderabad 500 007, India
Received 7 June 2001; accepted 28 July 2001
Abstract—A novel, inexpensive, thermophilic protease-type enzyme isolated from Clostridium thermohydrosulfuricum was used for
dipeptide synthesis. The enzyme showed broad substrate selectivity and enantioselectivity towards
formation. © 2001 Elsevier Science Ltd. All rights reserved.
L-amino acids in peptide bond
1. Introduction
tion.9 However, this method has the drawback of poor
availability and feasibility for industrial production.10
The design of novel biologically active polypeptides is a
new field of protein chemistry. The short chain peptides
are known for their biological importance in nutri-
tional, flavoring and pharmacological areas.2 At
present, dipeptides are synthesized by both chemical
and enzymatic routes.3 Chemical routes for peptide
synthesis, especially fragment condensation often suffer
from problems such as racemization and time consum-
ing protection/deprotection of amino acids.4 The alter-
native to chemical synthesis is the use of biocatalysts
for peptide bond formation where, the reaction often
takes place under mild, aqueous conditions with high
degrees of stereo- and regioselectivity.5 Although
enzymes, viz., proteases are becoming increasingly pop-
ular as biocatalysts in peptide formation, one of the
main drawbacks for industrial application of the
enzymes is the intrinsic instability of the enzyme in
many of the unnatural environments required for
organic reactions.6 Accordingly, much research has
focused on the enhancement of the enzyme stability at
higher temperature.7,8 Microorganisms growing in
extreme environments are reservoirs of enzymes that
could change the face of biocatalysts. Among these
enzymes, the extremozymes like thermolysin (protease)
a heat stable enzyme isolated from Bacillus thermopro-
tedyticus rokko are known to catalyze dipeptide forma-
So there is a need to explore new inexpensive
extremozymes that can be used in the field of peptide
synthesis. Herein, we report an effective method of
dipeptide formation using crude enzyme protease iso-
lated from thermophilic anaerobic Clostridium thermo-
hydrosulfuricum SV 12.1
2. Results and discussion
The crude thermophilic enzyme isolated from the
anaerobic microorganism C. therohydrosulfuricum has
shown protease-type activity and was used in catalyzing
dipeptide synthesis (Table 1). It was observed that the
isolated enzyme was stable and exhibited specific activ-
ity in biphasic media using a number of solvents such
as ethyl acetate, chloroform, t-butyl methyl ether and
hexane. The enzyme was found to be unstable in mis-
cible solvent systems.1
The isolated enzyme showed maximum stability and
selectivity in saturated ethyl acetate/buffer medium.
The optimal conditions of enzyme activity at different
temperatures and pH in the synthesis of the dipeptide
Z-L
-Asp-(b-OMe)-L-Phe-OMe (entry 1) is shown in
Fig. 1. It was observed that the maximum enzyme
activity for dipeptide bond formation is at 45°C with
the buffer medium of pH 6.5. No appreciable enzyme
activity was observed on pre-incubation of the isolated
enzyme with methanesulfonyl fluoride (Sigma chemical,
* Corresponding author. Fax: +91 40 7170512.
†
IICT communication No. 4542.
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00444-X

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J. S. Yada6 et al. / Tetrahedron: Asymmetry 12 (2001) 2505–2508
Table 1. Protease type enzyme isolated from C. thermohydrosulfuricum catalyzed dipeptide synthesis
Entry no.
Substrate
Nucleophile
Producta
Yield (%)
[h]2D5
methanol
Acyl donor
1
2
3
4
5
6
7
8
Z-
Z-
L
L
-Asp-b-OMe
L
L
L
L
L
L
L
L
-Phe-OMe
-Phe-OMe
-Phe-OMe
-Phe-OMe
-Leu-OMe
-Val-OMe
-Asp-(OMe)2
-Phe-OMe
Z-
Z-
Z-
Z-
Z-
Z-
L
L
L
L
L
L
-Asp-(b-OMe)-
-Asp-
-Asp-(b-OMe)-
-Asp- -Phe-OMe
L
-Phe-OMe
53
40
−13.8
−14.7
−14.0
−14.9
+13.7
+18
-Asp
L
-Phe-OMe
Z-DL-Asp-b-OMe
Z-DL-Asp
Z-
Z-
Z-
L
-Phe-OMe
35b
39b
35
L
L
L
L
-Asp-b-OBz
-Asp-b-OBz
-Phe
-Asp-(b-OBz)-
-Asp-(b-OBz)-
L
-Leu-OMe
-Val-OMe
L
35
35
30
Z-Phe-
Boc-Gly-Gly-
L
-Asp-(OMe)2
-Phe-OMe
Boc-Gly-Gl
L
–
a To confirm the products, identical physical and spectral data (tlc, NMR, and specific rotation) were found in comparison with independently
synthesised materials.10
b Yields were with respect to
L-enantiomer.
Figure 1. Enzyme activity at different pH and temperature in the synthesis of dipeptide (Z-
L
-Asp-(b-OMe)-L-Phe-OMe).
USA), thus confirming the isolated enzyme has
protease-type activity. We have been able to separate
the dipeptide and the unreacted amino acid. We did not
isolate any acid component of the protected amino
acid. So we presume that the enzyme only catalyzed
dipeptide formation and not the hydrolyzed amino acid
due to esterase or amidase. The product formed was
precipitated out from the biphasic solvent mixture
(ethyl acetate 40%) depending upon the partition coeffi-
cient of the product in the solvent used, this seems to be
the decisive factor of the reaction towards favorable
peptide synthesis.11
Compared with the earlier reports,14 the important
feature of the isolated enzyme protease is the stereo-
selectivity and enantiomeric purity in the peptide bond
formation towards
observed that the reaction of a carboxyl donor (Z-
asp) with hydrophobic C-amidated nucleophiles ( -leu
and -val) occurred in moderate yield. The rate of
L-amino acids (entries 3, 4). It was
L
-
L
L
peptide formation was also observed by kinetic parame-
ters i.e. on Vmax and km. (Table 2). The variation of rate
of product formation Vmax correlates with the substrate
binding constant Km, which in turn depends on the
solubility of the product in biphasic media (log P).
Table 2. Kinetic parameters of the thermostable enzymea in synthesis of dipeptides
Entry no.
Dipeptide
Log Pb
tmax (h)
Km (mol min−1
)
Vmax (mol min mg−1
)
[h]2D5
methanol
1
2
3
4
Boc-
L
-Asp-
-Phe- -Asp-(OMe)2
-Asp-(OBz)- -Leu-OMe
-Asp- -Phe-OMe
L
-Phe-OMe
0.18
0.08
0.11
0.19
8
8
12
8
0.16
0.15
0.19
0.15
1.65
1.08
1.10
1.80
−14.7
−13.6
+13.7
−15.0
Z-L
Z-L
Z-L
L
L
L
a Kinetic parameters were determined from a Lineweaver Burk plot.12
b Values of (log P) were calculated as described by Laroute et al.13

J. S. Yada6 et al. / Tetrahedron: Asymmetry 12 (2001) 2505–2508
2507
The peptide bond formation of Z-
L
-asp with various
The protein precipitate was dissolved in 0.1 M phos-
phate buffer pH 6.5 and enzyme present was partially
purified on passing the protein solution through Sep-
hadex G-50. The fraction having protease-type activity
was freeze-dried and was used as the source of enzyme.
The specific activity of the protease enzyme isolated
was found to be 28 IU/mg protein (Bradford method)
using casein as substrate.15 All the organic solvents used
were of analytical grade. Standard protease enzyme
thermolysin (B. thermoprotedyticus) obtained from
Sigma Chemicals Co, USA.
hydrophobic nucleophiles was carried out using differ-
ent thermostable enzymes thermolysin and protease
type extremozyme isolated from C. thermohydrosulfu-
ricum (Table 3). Peptide bond formation with protease
isolated from C. thermohydrosulfuricum proceeded
stereoselectively with broad substrate specificity when
compared to thermolysin obtained from B. thermopro-
tedyticus rokko.
3. Conclusion
The biocatalytic peptide bond condensations were per-
formed as follows: the acid component (10 mmol) and
the amine component (20 mmol) were taken in phos-
phate buffer solution (0.1 M, pH 6.5, 10 ml), which was
pre-saturated with ethyl acetate (40%). To this reaction
medium isolated protease enzyme/thermolysin (40 mg,
crude) was mixed and then incubated at 40°C in an
orbital shaker at 200 rpm for different time intervals.
The product formation was followed by TLC and
HPLC using an analytical reversed phase (C18)
column.16 the reaction was terminated by adding 50%
aqueous ethanol containing 8% trifluoroacetic acid, the
protein precipitate was filtered off and the product
formed was dissolved in a minimum amount of
methanol for crystallization. The dipeptides obtained
were conformed by spectral data.
In summary, the extremoprotease enzyme isolated from
anaerobic Clostridium species is a novel biocatalyst in
synthesis of dipeptides. In comparison with ther-
molysin, the protease enzyme isolated from anaerobic
bacterium is stable at higher temperature with broad
substrate specificity and high stereoselectivity towards
-amino acids. Further work on application of the
enzyme in synthesis of tri- and tetrapeptides is currently
in progress.
L
4. Experimental
An anaerobic extremophilic bacterium strain C. ther-
mohydrosulfuricum SV 12 was isolated from local soil
samples (deposited in microbial culture center OU).
The bacterium was identified on the basis of cell mor-
phology growth characteristics and DNA/DNA
hybridization. The strains were grown under anaerobic
conditions (0.002 in inert (nitrogen) atmosphere in
serum vials containing bacterium strain grew in pre-
reduced PYE (peptone yeast extracts) medium and
incubated 0.1 M phosphate buffer for 8 days, at opti-
mal conditions of pH 6.8 at 45°C. The thermostable
protease type enzyme was produced as extracellular
enzyme in the medium of the extremophilic organism
C. thermohydrosulfuricum. The fermented medium con-
taining protease-type enzyme activity was partially
purified. Briefly, the medium was centrifuged to remove
cellular debris and protein in the culture medium was
precipitated by adding 30% ammonium sulfate solution.
For the compounds in entries 5 and 6 (Table 3), the
substrates used are unprotected. The low rate of synthe-
sis may be due to the higher hydrophilicity of the
substrates/products. The isolation of the products
requires the separation of the biphasic system,
lyophilization of the aqueous layer and crystallization
in methanol to give dipeptides.
Acknowledgements
One of the authors Y. S. S. Ganesh is thankful to CSIR
for the award of a research fellowship.
Table 3. Comparison of peptide bond formation using thermostable protease
Entry
Product
Rate of synthesisa (peptide bond formation)
Thermolysinc
Proteaseb
1
2
3
4
5
6
Z-
Z-
Z-
Z-
L
-Asp-(b-OMe)-
-Asp-(b-OMe)-
-Phe- -Asp(OMe)2
-Asp(b-Obz)-
L
-Phe-OMe
110
\5
40
55
30
80
35
20
20
16
12
D
L-Phe-OMe
L
L
L
L
-Leu-OMe
L
-Asp-(b-OMe)-
-Phe-
L
-Phe-OMed
d
L
L
-Asp(OMe)2
15
a Rate of synthesis of peptide were expressed in mM of product formed per minute per mg of protein (quantitated by r.p. HPLC).
b Isolated from C. thermohydrosulfuricum SV12.
c Isolated from B. thermoproteolitions.
d See experimental procedure.

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