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692 | Green Chem., 2008, 10, 692695 This journal is The Royal Society of Chemistry 2008
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Loading/mg Specic
Sample Sample preparation, protein g1 activity/g1
number additive biocatalyst biocatalyst
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Table 2 Hydrolytic properties of subtilisinchitosan gels depending on for peptide bond formation in nonaqueous media in reaction (1)
GA concentration thus possessing high activity and stability under these conditions
Specic
(Table 3).
Loading/mg Specic activity/mg1 The best biocatalytic properties were shown by the lm
Sample protein g1 activity/g1 enzyme for obtained from chitosansubtilisin mixture with addition of
number [GA] (%) biocatalyst biocatalyst hydrolysis 0.5% GA before drying. After optimization of the prepara-
5 1 22.3 0.42 0.02
tion technique we chose several samples with high activity,
4 2 35 1.1 0.03 which were capable of 2592% yields in the formation of the
7 2.5 20.2 0.7 0.06 tetrapeptide Z-Ala-Ala-Leu-Phe-pNA in dimethylformamide
acetonitrile (6/4) solution.
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It was shown that optimal GA concentration for chitosan The lms were mechanically stable after several uses and
subtilisin gels was in the range 12%, as gels obtained in prolonged storage (several weeks). Activity was essentially
these conditions possess maximum activity. Probably higher GA unchanged on reuse over a period of 34 days (99100% of
concentration leads to high crosslinking degree of chitosan, thus original), but after 3 weeks storage in dry conditions had
limiting substrate diffusion to the enzyme active centre. dropped to 82% of the original value.
Having demonstrated the basic activity of chitosansubtilisin
composites, we moved on to preparing composites immobilized Experimental
in a reactor. Simple drying of chitosan solution leads to lm
formation. The lm obtained adheres to the surface of reaction Subtilisin Carlsberg and chitosan (85% deacetylated, from
vessel (standard round bottom ask), meaning that we can crab shells) were obtained from Sigma. All other chemicals
readily make biocatalytic coatings using this technique. Films (crosslinkers, etc.) were obtained from Aldrich. All solvents
from chitosansubtilisin biocomposite, crosslinked with GA, and salts were of analytical grade or additionally puried.
were obtained by 4 methods: (1) obtaining chitosan lm, Peptides and peptide derivatives Glp-Ala-Ala-Leu-pNA (where
treatment by GA and then by subtilisin solution (sandwich), Glp- is pyroglutamil residue), Z-Ala-Ala-Leu-OCH3 and Phe-
(2) mixing subtilisin and chitosan solutions and then addition of pNA were obtained according to the procedures described by
GA before drying, (3) as (2) but addition of GA after drying and Getun et al.10 Reverse-phase HPLC analyses were performed on
(4) as (3) but the lm was lyophilised (Fig. 4). Protein loading for liquid chromatograph Altex Model 110A (United States) with
biocomposite lms appeared to be comparable to, and in some column Microsorb-MV C8 (4.6 250 mm, Rainin Instrument
cases even higher than gels. All samples were active in hydrolysis, Company, Inc., United States) with elution by linear gradients
especially sandwich. However, in low water media, where the of acetonitrile 1070% for 26.2 min in 0.1% TFA at the rate of
enzyme catalyses peptide bond formation, this biocatalyst was 1 mL min1 with detection at 220 and 280 nm.
inactive probably due to full exposure of enzyme into denaturing Amino acid analyses were performed on a Hitachi-835 amino
organic solvent. The other biocatalytic lms were active catalysts acid analyzer (Japan) after sample hydrolysis in 5.7 M HCl at
105 C in evacuated ampoules for 48 h.
Table 3 Properties of subtilisinchitosan lms, obtained using GA addition in peptide formation according to Fig. 2
694 | Green Chem., 2008, 10, 692695 This journal is The Royal Society of Chemistry 2008
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(4) addition of 0.1 mL 4 M NaOH solution and 50% absorption of the control sample, V 0 is the total volume of the
glutaraldehyde solution to 1% concentration in the mixture (gel sample (mL), t is the time of reaction (min), mbiocat is the weight
(5)); of the biocatalyst (g), and 8.9 is the molar extinction coefcient
(5) addition of 3 M Na2 SO4 solution (0.2 mL) (bres (6)). of p-nitroaniline (mM1 cm1 ).
Different subtilisin concentrations in the subtilisinchitosan An activity unit is dened as the amount of enzyme that
mixture were obtained using consequent dilution of 150 mg liberates 1 lmol of p-nitroaniline per 1 min under the condition
mL1 subtilisin solution which were then added to the initial described above.
chitosan solution. The biocatalyst obtained was washed 3 times
with 0.05 M Tris-HCl buffer pH 8.2 to block all remaining Synthesis of Z-Ala-Ala-Leu-Phe-pNA by immobilized subtilisin
CHO groups. The amount of immobilized protein was calculated
A portion of immobilized subtilisin (37 mg) was added to
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from the data of amino acid analysis (for samples prepared with
the solution of Z-Ala-Ala-Leu-OMe (5.1 mg, 12 lmol) and
different subtilisin concentrations) or from UV absorbance at
Phe-pNA (3.4 mg, 12 lmol) in 380 lL of the mixture of
280 nm of starting subtilisin solution subtracting absorbance of
dimethylformamideacetonitrile 6/4. The reaction suspension
washings at 280 nm.
was shaken at 20 C, with 5 lL samples being periodically taken
Different lms were formed from chitosan acetate (2%
for HPLC-analysis.
chitosan solution in 2% acetic acid was precipitated by 10-
fold volume of acetone, ltered, dried and ground). This was
then re-dissolved in acetate buffer pH 5.0 (1 mL, 1 wt%) and Conclusions
mixed with subtilisin solutions in the same buffer (250 lL, 75 mg We have demonstrated that chitosan-immobilized subtilisin
mL1 ). Then glutaraldehyde was added either before drying (5% possesses both hydrolytic (in aqueous media) and synthetic
solution, to obtain 0.5 and 1% concentration in the mixture, (9)) (in low water media) activities. In general hydrolytic activity
or after drying (0.25 or 0.5% solution, 2 mL (10)). Samples (11) of biocomposite lms containing GA was higher than for
were prepared by same way as (10) the only difference was that biocatalysts prepared without crosslinker. These lms can be
lm was lyophilized instead of drying. The obtained samples attached to the walls of a reaction vessel and used in both
were washed 3 times with 0.05 M Tris-HCl buffer pH 8.2. In media. We have shown that biocatalytic lms prepared in this
the case of sample (8) solution of acetate chitosan in acetate work are characterized by preparative simplicity, high activity
buffer pH 5.0 (1 mL, 1 wt%) was dried to obtain lm, then and stability in different media under very mild conditions.
treated with 2 mL of 0.5% glutaraldehyde solution during 2 h,
and subtilisin solution ((250 lL, 40 mg mL1 ) was added. After
incubation during 24 h at +4 C the sample was washed 3 times Notes and references
with 0.05 M Tris-HCl buffer pH 8.2. 1 B. Krajewska, Enzyme Microb. Technol., 2004, 35, 126.
2 E. Guibal, Prog. Polym. Sci., 2005, 30, 71.
Determination of immobilized subtilisin hydrolytic activity 3 D. J. Macquarrie and J. J. E. Hardy, Ind. Eng. Chem. Res., 2005, 44,
8499.
against Glp-Ala-Ala-Leu-pNA 4 P. H. Fromm, M. E. Rezac, K. Wurges and P. Czermak, AIChE J.,
2007, 53, 237.
Glp-Ala-Ala-Leu-pNA solution in DMF (50 lL, 5 mg ml1 ) was 5 L. Ferreira, M. A. Ramos, J. S. Dordick and M. H. Gil, J. Mol. Catal.
added to 2 mL of 0.05 M Tris/HCl buffer (pH 8.3) containing B: Enzym., 2003, 21, 189.
1.5 mM CaCl2 , and the mixture was incubated at 20 C for 6 A. V. Bacheva, F. M. Plieva, E. N. Lysogorskaya, I. Yu Filippova and
V. I. Lozinsky, Bioorg. Med. Chem. Lett., 2001, 11, 1005.
10 min. Then a weighed sample of chitosansubtilisin was 7 I. Yu Filippova, A. V. Bacheva, O. V. Baibak, F. M. Plieva, E. N.
added, and the mixture was incubated at 20 C, absorbance Lysogorskaya, E. S. Oksenoit and V. I. Lozinsky, RAS Bull., 2001,
was measured every minute with periodical shaking until its 50, 1896.
absorbance at 410 nm reached 0.10.4. The reaction was stopped 8 A. V. Bacheva, O. V. Baibak, A. V. Belyaeva, E. N. Lysogorskaya,
E. S. Oksenoit, V. I. Lozinsky and I. Yu Filippova, Russ. J. Bioorg.
with 1 mL of 50% acetic acid. In control samples there was Chem., 2003, 29, 502.
reverse order of addition of the biocatalyst and acetic acid. 9 A. V. Bacheva, O. V. Baibak, A. V. Belyaeva, E. S. Oksenoit, T. I.
The specic activity was calculated according to the formula: Velichko, E. N. Lysogorskaya, A. K. Gladilin, V. I. Lozinsky and
I. Yu Filippova, Biochemistry (Moscow), 2003, 68, 1896.
(A410 Ac410 ) V 0 t mbiocat 8.9 10 I. V. Getun, I. Y. Fillipova, E. N. Lysorgskaya, E. S. Oksenoit, V. V.
Anisimova, S. V. Kolobanova, A. V. Bacheva and V. M. Stepanov,
where A410 is the absorption of the mixture at 410 nm, Ac410 is the Bioorg. Med. Chem. Lett., 1997, 20, 2692.
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