Chitinase from Enterobacter sp. NRG4: Its purification, characterization and reaction pattern
Ram P. Tiwari
Keywords: chemical modification, chitinase, Enterobacter sp. NRG4, purification, substrate binding.
sp. NRG4 was shown to excrete chitinase into the culture supernatant
when cultivated in medium containing chitin. A 60 kDa extracellular
chitinase was purified to homogeneity and characterized. The enzyme
hydrolyzed swollen chitin, colloidal chitin, regenerated chitin and
glycol chitin but did not hydrolyze chitosan. The chitinase exhibited
Km and Vmax values of 1.43 mg ml-1
and 83.33 µM µg-1 h-1 for swollen chitin, 1.41
mg ml-1 and 74.07 µM µg-1 h-1 for
colloidal chitin, 1.8 mg ml-1 and 40 µM µg-1
h-1 for regenerated chitin and 2.0 mg ml-1 and
33.33 µM µg-1 h-1 for glycol chitin, respectively.
The optimal temperature and pH for activity were
Chitin is composed of repeating N-acetyl D-glucosamine residues and is a component of crustacean exoskeleton, diatoms, fungal cell walls, and squid pens. Chitin is a versatile and promising biopolymer with numerous industrial, medical and commercial uses. However, it is difficult to purify and modify chemically. Hence identification of chitin modifying enzymes and elucidation of their activities could facilitate the efficient production of specific chitin products. The biodegradation of chitin requires the synergistic action of several hydrolytic enzymes for efficient and complete breakdown. The combined action of endochitinases (EC 188.8.131.52) and exochitinases [(chitobiosidases and β-N-acetyl hexosaminidase (EC 184.108.40.206)] results in the degradation of chitin polymer into the soluble N-acetyl D-glucosamine (Gkargkas et al. 2004). Chitinases are produced by different micro-organisms which generally present a wide multiplicity of enzymes that are mainly extracellular. They have received increased attention due to their wide range of biotechnological applications, especially in the production of chito-oligosaccharides and N-acetyl D-glucosamine (Pichyangkura et al. 2002), biocontrol of pathogenic fungi (Chernin et al. 1997; Mathivanan et al. 1998), preparation of sphaeroplasts and protoplasts from yeast and fungal species (Mizuno et al. 1997; Balasubramanium et al. 2003) and bioconversion of chitin waste to single cell protein (Vyas and Deshpande, 1991).
In the present investigation we report an endochitinase that was purified and characterized from a newly isolated Enterobacter sp. NRG4.
chitin was obtained from Hi-Media,
sp. NRG4 isolated from degraded stalk of mushroom was selected as
a potent chitinase producer (Dahiya et al. 2005).
The culture medium was composed of 1.0% swollen chitin, 0.5% peptone,
0.5% yeast extract, 0.1% KH2PO4 and 0.01% MgSO4.7H2O
(pH 8.0). The micro-organism was cultivated at
assay mixture contained 1 ml swollen chitin and 0.5 ml enzyme solution.
After incubation at
The protein concentration was measured using the method of Lowry et al. (1951) with bovine serum albumin as standard. For the purified enzyme, protein concentration was measured by determining the absorbance at 280 nm.
purification of chitinase was carried out in three steps. The cell
free supernatant was precipitated with 30% ammonium sulphate. The
resultant precipitate was centrifuged at 10,000 x g,
dialysed protein was subjected to ion exchanger, DEAE-Sephadex column
purified protein was loaded onto SDS-PAGE (12%) as described by Laemmli
(1970) to determine the protein profile. Native PAGE was carried
out with the aim to study the zymography pattern of chitinase. The
detailed procedure was exactly similar to the SDS-PAGE in which SDS,
mercaptoethanol and the heating step during protein sample preparation
were eliminated. The native PAGE gel was run with purified chitinase
preparation. Half of the gel was cut and stained to locate the position
of single band and the other half of the gel was placed over chitin
agar plate (1.0% swollen chitin in citrate phosphate buffer + 1.5%
agar) and incubated overnight at
The purified chitinase was characterized with respect to its optimum pH, temperature, stability at different temperatures and pH values, effect of metal ions, surfactants, organic solvents on activity and stability.
activity was assayed at different pH values (pH 2.6 to 10.0) using
activity was assayed at different temperatures ranging from 35-
The effect of substrate concentration on chitinase activity was determined at different concentrations of chitin, varying between 0.25 mg ml-1 to 16 mg ml-1 (w/v). The Km and Vmax values were determined by Lineweaver-Burk's plot.
effect of metal ions on enzyme activity was studied by incorporating
these metal ions such as MgSO4. 7H2O, KCl, CaCl2,
2H2O, CuCl2, 2H2O, HgCl2,
AgNO3, CoCl2, 2H2O, ZnSO4,
FeCl3 and FeSO4 in reaction mixture at
was added to the enzyme solution in the concentration range from 1
to 100 µg ml-1 and incubated at room temperature for 1
hr. Thereafter, residual enzyme activity was determined under standard
assay conditions. The effect of sugars such as N-acetyl D-glucosamine,
glucosamine HCl, galactosamine and glucose was studied by incorporating
these sugars at
binding was determined by incubating the enzyme with 10 mg substrate
in citrate phosphate buffer (
modification of chitinase was done using several reagents such as
para chloromecuribenzoate (PCMB), N-bromosuccinimide (NBS), 5, 5'-dithiobis-(2-nitrobenzoic)
acid (DTNB), iodoacetamide and methylene blue. The effects of these
modifiers were tested by incubating the enzyme with varying concentrations
mode of action of chitinase was determined by viscometric assay (Otakara,
1961). Purified chitinase (60 µg) was added to 60 ml substrate
swollen chitin as the sole source of carbon, Enterobacter sp.
NRG4 produced chitinase in the culture medium. The chitinase was purified
using standard techniques i.e. ammonium sulphate precipitation
(30-75%), DEAE-Sephadex ion exchange chromatography and Sephadex G-200
gel filtration chromatography. When cell free supernatant was subjected
to fractional ammonium sulphate precipitation, chitinase activity
was precipitated in 30-75% salt saturation. The yield of chitinase
was 71% with a purification fold of 3.18 and specific activity of
560.5 U mg-1 protein. The dialyzed protein was loaded on
DEAE ion exchanger. After elution with 0 to
chitinase was maximally active at pH 4.5 to 8.0 thus exhibiting a
broad pH optima (Figure 2a). Determination of
pH stability of the chitinase indicated that the enzyme was stable
between pH 4.5 to 8.0 and it retained 90% of its activity in this
range (Figure 2b). The purified enzyme showed
its maximum activity at
With acid swollen chitin, colloidal chitin, regenerated chitin and glycol chitin the purified chitinase gave Km of 1.43 mg ml-1, 1.41 mg ml-1, 1.8 mg ml-1 and 2.0 mg ml-1, respectively and Vmax were 83.33 µmole µg-1 h-1, 74.07 µmole µg-1 h-1, 40.00 µmole µg-1 h-1 and 33.33 µmole µg-1 h-1, respectively (Table 2).
enzyme showed activities towards swollen chitin, colloidal chitin,
glycol chitin and regenerated chitin but exhibited no activity towards
carboxymethyl cellulose, chitosan and Micrococcus lysodeikticus
cell wall. When swollen chitin was used as substrate the activity
was taken as 100. The activities with colloidal chitin, regenerated
chitins, glycol chitin, flake chitin and crab shell chitin were 80.3,
44.7, 39.4, 5.9 and 2.3%, respectively. Enterobacter sp. NRG4
chitinase reduced the viscosity of glycol chitin significantly in
5 min due to cleavage of chitin long chains by the chitinase at
Chitinase exhibited a substrate binding capacity of 89.5, 26.2 and 15.2% for swollen chitin, flake chitin and carboxymethyl cellulose, respectively whereas no significant substrate binding was observed for pectin, starch, xylan, wheat bran and chitosan (Figure 6).
K+ and Ca2+ stimulated chitinase activity by
13, 16 and 18%, respectively whereas Cu2+, Co2+,
Ag+ and Hg2+ inhibited chitinase activity by
9.7, 15, 22 and 72.2%, respectively at 1mM concentration. At
a known specific inhibitor of chitinase inhibited Enterobacter
sp. NRG4 chitinase by 57.1 and 65.7% at a concentration of 50 and
100 µg ml-1, respectively, with an IC50 value
of 40 µg ml-1 (64 µM) (Figure 7).
Study of end-products and sugars on chitinase activity showed that
N-acetyl D-glucosamine, glucosamine HCl, galactosamine and glucose
inhibited enzyme activity by 10, 8, 4 and 9.1% at
inhibited chitinase activity by 17.6, 66.2 and 84.5%, respectively
An extracellular chitinase secreted by Enterobacter sp. NRG4 was purified to homogeneity by combination of ammonium sulphate precipitation, DEAE Sephadex ion exchange chromatography and Sephadex G-200 gel flitration chromatography. The chitinase showed a single band on 12% SDS-PAGE and Native PAGE indicating the complete purification of the enzyme. The molecular weight of the protein was found to be about 60 kDa by SDS-PAGE as well as by gel filtration chromatography. The chitinase from Enterobacter sp. NRG4 was active over broad pH range i.e. from pH 4.5-8.0, optimum being 5.5. Several workers have reported broad pH optima like pH 4.5-7.5 of chitinase from Bacillus cereus (Pleban et al. 1997), pH 5.0-8.0 for Aeromonas hydrophila H-2330 (Hiraga et al. 1997), pH 7.5-9.0 for Bacillus sp. BG-11 (Bhushan and Hoondal, 1998). The pH optima for other chitinases reported were pH 4.0 for Aeromonas sp. No. 10S-24 (Ueda et al. 1995), pH 5.0 for Alcaligenes xylosoxydans (Vaidya et al. 2001) and Arthrobacter sp. NHB-10 (Okazaki et al. 1999), pH 5.5 for Bacillus sp. WY22 (Woo and Park, 2003), pH 6.0 for Enterobacter sp. G-1 (Park et al. 1997), pH 5.4 and 6.6 for CHIT60 and CHIT100, respectively from Serratia plymuthica HRO-C48 (Frankowski et al. 2001), pH 6.3 for Bacillus sp. NCTU2 (Wen et al. 2002), pH 6.5 for Vibrio alginolyticus H-8 (Ohishi et al. 1996) and Vibrio sp. (Zhou et al. 1999), pH 7.0 for Monascus purpureus (Wang et al. 2002), pH 7.0-8.0 for Bacillus 13.26 (Yuli et al. 2004) and pH 10.0 for Cellulomonas flavigena NTOU1 (Chen et al. 1997).
The chitinase from the present strain was stable over wide pH range i.e. from pH 4.5 to 8.0. Other bacterial chitinase stable over broad pH range were pH 4.0 to 9.0 of Aeromonas sp. No. 10S-24 chitinase (Ueda et al. 1995), pH 6.0 to 9.0 of Pseudomonas aeruginosa K-187 (Wang and Chang, 1997), pH 5.0 to 8.0 of Aeromonas hydrophila H2330 chitinase (Hiraga et al. 1997), pH 4.0 to 9.0 for Vibrio sp. (Zhou et al. 1999), pH 6.8 to 8.0 of Bacillus sp. NCTU2 (Wen et al. 2002) chitinase and pH 4.0 to 8.5 of Bacillus cereus strain 65 (Pleban et al. 1997).
temperature activity and stability profile of Enterobacter
sp. NRG4 chitinase revealed that the enzyme was optimally active at
The Km values of the Enterobacter sp. NRG4 chitinase against different substrates were 1.43 mg ml-1, 1.41 mg ml-1, 1.8 mg ml-1 and 2.0 mg ml-1, respectively with swollen chitin, colloidal chitin, regenerated chitin and glycol chitin respectively, which are comparatively lower than the other reports in literature. The Km values of chitinase from different organisms were, 2.88 mg ml-1 for Enterobacter aerogenes (Tang et al. 2001), 1.4 mg ml-1 and 0.8 mg ml-1 for chitinase C1 and C3 from Vibrio alginolyticus H-8 against squid chitin (Ohishi et al. 1996), 3.0 mg ml-1 for Alcaligenes xylosoxydans chitinase (Vaidya et al. 2003) and Bacillus sp. WY22 chitinase (Woo and Park, 2003), 12 mg ml-1 for Bacillus sp. BG-11 chitinase (Bhushan and Hoondal, 1998).
Ethylene glycol chitin, glycol chitin and colloidal chitin are useful substrate for enzyme assays of endo-type chitinase (Park et al. 1997). The hydrolysis pattern of purified enzyme indicated that chitinase from Enterobacter sp. NRG4 was an endochitinase. It exhibited high activity towards swollen chitin, colloidal chitin, regenerated chitin and glycol chitin as compared to flake chitin and crab shell chitin. It showed no activity towards carboxymethyl cellulose, chitosan and Micrococcus lysodeikticus cell wall. The hydrolysis products from swollen chitin were (GlcNAc)2 and GlcNAc. Enterobacter sp. G-1 was also reported to secrete an endochitinase which showed high activity towards colloidal chitin and ethylene glycol chitin more than flake chitin or soluble CMC. It could not hydrolyze flake chitosan but showed 36 to 80% activity towards deacetylated chitosan compared with colloidal chitin. The products from colloidal chitin hydrolysis were mainly (GlcNAc)2 with small amount of (GlcNAc)3 and (GlcNAc)4 (Park et al. 1997). Characteristics of purified chitinases from other reported Enterobacter spp. are summarized in Table 3. Aeromonas sp. chitinase I and II hydrolyzed colloidal chitin and ethylene glycol chitin effectively but the activity was significantly lower towards chitin and chitosan. No detectable activities towards Micrococcus lysodeikticus cell wall were observed (Ueda and Arai, 1992). Chitinase exhibited a substrate binding capacity of 89.5, 26.2 and 15.2% for swollen chitin, flake chitin and carboxymethyl cellulose, respectively. Lee et al. (2000) reported binding of Pseudomonas sp. YHS-A2 chitinase 78, 12, 0, 5 and 10% with colloidal chitin, chitin, carboxymethyl cellulose, crude chitosan and birch wood xylan, respectively.
metal ions, Mg2+, K+ and Ca2+ stimulated
chitinase activity by 13, 16 and 18%, respectively whereas Cu2+,
Co2+, Ag+ and Hg2+ and inhibited
chitinase activity by 9.7, 15, 22 and 72.2%, respectively at
plymuthica activity was stimulated by 120, 150 and 240% in presence
at 1mM and iodoacetamide at
Allosamidin inhibited chitinase activity by 57.1 and 65.7% at 50 and 100 µg ml-1, respectively. The IC50 value was 40µg ml-1 (64 µM). Other reported IC50 values were 48 µM for Bacillus sp. BG-11 chitinase (Bhushan and Hoondal, 1999) and 9.0 µM for chitinase from human serum and leucocytes (Escott and Adam, 1995).
various sugars and end products, chitinase was inhibited by 81.3%
in presence of N-acetyl D-glucosamine at 10mM concentration whereas
glucosamine HCl, galactosamine and glucose inhibited up to 19%. Chitinase
of Metarhizium anisopliae was inhibited by 28, 21 and 79% in
presence of glucose, N-acetyl D-glucosamine and D-glucosamine, respectively
In conclusion, we have purified and characterized a chitinase from newly isolated Enterobacter sp. NRG4. The capability of this chitinase to hydrolyze chitin efficiently, lower end product inhibition, broad pH activity and stability makes the enzyme industrially significant for biotechnological applications, especially in production of chitobiose and N-acetyl D-glucosamine.
BALSUBRAMANIUM, N.; Juliet, G.A.; SRIKALAIVANI, P. and LALITHAKUMARI, D. Release and regeneration of protoplasts from the fungus Trichothecium roseum. Canadian Journal of Microbiology, April 2003, vol. 49, no. 4, p. 263-268.
BHUSHAN, Bharat and HOONDAL, Gurinder Singh. Effect of fungicides, insecticides and allosamidin on a thermostable chitinase from Bacillus sp. BG-11. World Journal of Microbiology and Biotechnology, June 1999, vol. 15, no. 3, p. 403-404.
BHUSHAN, Bharat and HOONDAL, Gurinder Singh. Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp. BG-11. Biotechnology Letters, February 1998, vol. 20, no. 2, p. 157-159.
CHEN, Hsing Chen; HSU, Mei Fang and JIANG, Shann Tzong. Purification and Characterization of an exo-N,N`-diacetylchitobiohydrolase like enzyme from Cellulomonas flavigena NTOU1. Enzyme and Microbial Technology, February 1997, vol. 20, no. 3, p. 191-197.
CHERNIN, L.S.; DE LA FUENTE, L.; SOBOLEV, V.; HARAN, S.; VORGIAS, C.E.; OPPENHEIM, A.B. and CHET, I. Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans. Applied Environmental Microbiology, March 1997, vol. 63, no. 3, p. 834-839.
DAHIYA, Neetu; TEWARI, Rupinder; TIWARI, Ram Prakash and HOONDAL, Gurinder Singh. Chitinase production in solid state fermentation by Enterobacter sp. NRG4 using statistical experimental design. Current Microbiology, 2005. In press.
FRANKOWSKI, Jens; LORITO, Matteo; SCALA, Felice; SCHMID, Roland; BERG, Gabriele and BAHL, Hubert. Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48. Archives of Microbiology, December 2001, vol. 176, no. 6, p. 421-426.
GKARGKAS, Konstantinos; MAMMA, Diomi; NEDEV, George; TOPAKAS, Evangelos; CHRISTAKOPOULOS, Paul; KEKOS, Dimitris and MACRIS, Basil J. Studies on N-acetyl-ß-D-glucosaminidase produced by Fusarium oxysporum F3 grown in solid-state fermentation. Process Biochemistry, July 2004, vol. 39, no. 11, p. 1599-1605.
HIRAGA, Kazumi; SHOU, Lee; KITAZAWA, Mitsunori; TAKAHASHI, Saori; SHIMADA, M.; SATO, R. and ODA, K. Isolation and characterization of chitinase from a flake chitin degrading marine bacterium, Aeromonas hydrophila H-2330. Bioscience Biotechnology and Biochemistry, 1997, vol. 61, no. 1, p. 174-176.
KIM, Kyoung Ja; YANG, Yong Joon and KIM, Jong Gi. Purification and characterization of chitinase from Streptomyces sp. M-20. Journal of Biochemistry and Molecular Biology, 2003, vol. 36, no. 2, p. 185-189.
LEE, H.S.; HAN, D.S.; CHOI, S.W.; KIM, D.S.; BAI, D.H. and YU, J.H. Purification, characterization and primary structure of a chitinase from Pseudomonas sp. YHS-A2. Applied Microbiology and Biotechnology, September 2000, vol. 54, no. 3, p. 397-405.
MATHIVANAN, N.; KABILAN, V. and MURUGESAN, K. Purification, characterization and anti-fungal activity from Fusarium chlamydosporum, a mycoparasite to groundnut rust, Puccinia arachidis. Canadian Journal of Microbiology, July 1998, vol. 44, no. 7, p. 646-651.
MIZUNO, Katsushige; KIMURA, Osamu and TACHIKI, Takashi. Protoplast formation from Schizophyllum commune by a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of S. commune as a carbon source. Bioscience Biotechnology and Biochemistry, 1997, vol. 61, no. 5, p. 852-857.
OHISHI, Kazuo; YAMAGISHI, Masaoki; OHTA, Toshiya; SUZUKI, Mitsuaki; IZUMIDA, Hitoshi; SANO, Hiroshi; NISHIJIMA, Miyuki and MIWA, Tan. Purification and properties of two chitinases from Vibrio alginolyticus H-8. Journal of Fermentation and Bioengineering, 1996, vol. 82, no. 6, p. 598-600.
OTAKARA, A. Studies on the chitinolytic enzymes of black-koji mold. Part I. Viscometric determination of chitinase activity by application of glycol chitin as a new substrate. Agricultural Biological Chemistry, 1961, vol. 25, p. 50-54.
OKAZAKI, Katsuichiro; KAWABATA, Toshiyuki; NAKANO, Masahito and HAYAKA, Shigeru. Purification and properties of chitinase from Arthrobacter sp. NHB-10. Bioscience Biotechnology and Biochemistry, 1999, vol. 63, no. 9, p. 1644-1646.
PARK, Jae Kweon; MORITA, Kenji; FUKUMOTO, Ikuo; YAMASAKI, Yukikazu; NAKAGAWA, Tsuyoshi; KAWAMUKAI, Makoto and MATSUDA, Hideyuki. Purification and characterization of the Chitinase (ChiA) from Enterobacter sp. G-1. Bioscience Biotechnology and Biochemistry, 1997, vol. 61, no. 4, p. 684-689.
PICHYANGKURA, Rath; KUDAN, Sanya; KULTIYAWONG, Kamontip; SUKWATTANASINITT, Mongkol and AIBA, Sei Ichi. Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase. Carbohydrate Research, March 2002, vol. 337, no. 6, p. 557-559.
PINTO, A. de S., BARRETO, C.C., SCHRANK, A., ULHAO, C.J. and VAINSTEIN, M.H. Purification and characterization of an extracellular chitinase from the entomopathogen Metarhizium anisopliae. Canadian Journal of Microbiology, 1997, vol. 43, no. 4, p. 322-327.
SUTRISNO, A.; UEDA, M.; ABE, Y.; WAKAZAWA, M and MIYATAKE, K. A chitinase with high activity towards partially N-acetylated chitosan from a new, moderately thermophilic chitin degrading bacterium, Ralstonia sp. A-471. Applied Microbiology and Biotechnology, January 2004, vol. 63, no. 4, p. 398-406.
TAKAHASHI, Mamoru; TSUKIYAMA, Tadashi and SUZUKI, Tomoo. Purification and some properties of chitinase produced by Vibrio sp. Journal of Fermentation and Bioengineering, 1993, vol. 75, no. 6, p. 457-559.
TANG, Y.; ZHAO, J.; DING, S.; LIU, S. and YANG, Z. Purification and properties of chitinase from Enterobacter aerogenes. Wei Sheng Wu Xue Bao (Acta Microbiologica Sinica), February 2001, vol. 41, no. 1, p. 82-86.
UEDA, M.; FUJIWARA, A.; KAWAGUCHI, T. and ARAI, M. Purification and some properties of six chitinases from Aeromonas sp. No. 10S-24. Bioscience Biotechnology and Biochemistry, 1995, vol. 59, p. 2162-2164.
VAIDYA, R.J.; SHAH, I.M.; VYAS, P.R. and CHHATPAR, H.S. Production of chitinase and its optimization from a novel isolate Alcaligenes xylosoxydans: potential antifungal biocontrol. World Journal of Microbiology and Biotechnology, October 2001, vol. 17, no. 7, p. 62-69.
VAIDYA, Rajiv; ROY, Satarupa; MACMIL, Simone; GANDHI, Sapan; VYAS, Pranav and CHHATPAR, H.S. Purification and characterization of chitinase from Alcaligenes xylosoxydans. Biotechnology Letters, May 2003, vol. 25, no. 9, p. 715-717.
VYAS, P.R. and DESHPANDE, M.V. Enzymatic hydrolysis of chitin by Myrothecium verrucaria chitinase complex and its utilization to produce SCP. Journal of General and Applied Microbiology, 1991, vol. 37, no. 3, p. 267-275.
WANG, S.L. and CHANG, W.T. Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in shrimp and crab shell powder medium. Applied Environmental Microbiology, February 1997, vol. 63, no. 2, p. 380-386.
WANG, San Lang; YEN, Yue Horng; TSIAO, Wei Jen; CHANG, Wen Teish and WANG, Chuan Lu. Production of antimicrobial compounds by Monascus purpureus CCRC31499 using shrimp and crab shell powder as a carbon source. Enzyme and Microbial Technology, August 2002, vol. 31, no. 3, p. 337-344.
WEN, Chih Min; TSENG, Chien Sheng; CHENG, Chih Yu and LI, Yaw Kuen. Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2. Biotechnology and Applied Biochemistry, June 2002, vol. 35, no. 3, p. 213-219.
WOO, Cheol Joo and PARK, Heui Dong. An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product. Biotechnology Letters, March 2003, vol. 25, no. 5, p. 409-412.
YULI, Purwani E.; SUHARTONO, Maggy Thenawidjaja; RUKAYADI, Yaya; HWANG, Jae Kwan and PYUN, Yu Ryang. Characteristics of thermostable chitinase enymes from the Indonesian Bacillus sp. 13.26. Enzyme and Microbial Technology, 2004, vol. 35, no. 2-3, p. 147-153.
ZHOU, S.N.; YANG, C.Y.; LU, Y.J.; HUANG, L.; CAI, C.U. and LIN, Y. C. Isolation and characterization of chitinase from marine bacterium Vibrio sp. World Journal of Microbiology and Biotechnology, December 1999, vol. 15, no. 6, p. 745-746.
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