Purification and Some Properties of Chitinase B1 from<i>Bacillus circulans</i>WL-12

Watanabe, Takeshi; Yamada, Takao; Oyanagi, W; Suzuki, Kazushi; Tanaka, Hironori

doi:10.1271/bbb.56.682

Cited by 14 publications

(7 citation statements)

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“…Relatively high activity toward colloidal chitin and synthetic substrate such as 4-MU-(GlcNAc) 2 was observed (Table 1). Similar activity was also found in the chitinases from B. circulans WL-12 (Watanabe et al 1992). The recombinant enzyme completely hydrolyzed N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose and N-acetylchitohexaose to N-acetylglucosamine and N-acetylchitobiose, but did not hydrolyse N-acetylchitobiose, suggesting that the enzyme does not possess N-acetylglucosaminidase activity (Figure 2).…”

Section: Characterization Of the Purified Recombinant Chitinasesupporting

confidence: 69%

Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli

Siwayaprahm

Audtho

Ohmiya

et al. 2005

World J Microbiol Biotechnol

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A DNA fragment encoding for 598 amino acids of chitinase protein from Bacillus circulans No. 4.1 was subcloned into pQE-30 expression vector and transformed into Escherichia coli M15 (pREP4). The molecular weight of the expressed protein was approximately 66 kDa. Enzymatic activity of the recombinant protein was assayed after purification using affinity chromatography on a nickel chelating resin. The enzyme hydrolyzed N-acetylchitooligosaccharides mainly to N-acetylchitobiose, and was active toward chitin, carboxymethyl-chitin, colloidal chitin, glycol chitin and 4-methylumbelliferyl-b-D-N, N¢-diacetylchitobiose. The pH and temperature optima of the chitinase enzyme were 7.0 and 45°C, respectively. This enzyme was stable in the pH range of 5.0-9.0 and at temperatures up to 50°C. In addition, when cleaved by a proteolytic enzyme, the 20-kDa product could retain high chitinolytic activity.

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Section: Characterization Of the Purified Recombinant Chitinasesupporting

confidence: 69%

Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli

Siwayaprahm

Audtho

Ohmiya

et al. 2005

World J Microbiol Biotechnol

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“…The thermostability of chitinase II is quite remarkable; it is stable up to around 60ЊC and maintains half of its activity even at the high temperature of 100ЊC. This stability is similar to that of the thermostable chitinases of Bacillus licheniformis X-7u (34) and markedly higher than the thermal stabilities observed for chitinases of other origins (8,22,27,33,38,39,45).…”

Section: Discussionmentioning

confidence: 63%

“…Most of the bacterial chitinases have acidic pIs (22,25,45,46), and actinomycete chitinases have neutral or alkaline pIs (13,18,26,35,40). Like almost all of the other bacterial chitinases, strain K-187 chitinase has an acidic pI.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium

Wang

Chang

1997

Appl Environ Microbiol

203

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Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187. The molecular weights of FI and FII were 30,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 60,000 and 30,000, respectively, by gel filtration. The pIs for FI and FII were 5.2 and 4.8, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FI were pH 8, 50؇C, pH 6 to 9, and 50؇C; those of FII were pH 7, 40؇C, pH 5 to 10, and 60؇C. The activities of both enzymes were activated by Cu 2؉ ; strongly inhibited by Mn 2؉ , Mg 2؉ , and Zn 2؉ ; and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol. Both chitinases showed lysozyme activity. The purified enzymes had antibacterial and cell lysis activities with many kinds of bacteria. This is the first report of a bifunctional chitinase/lysozyme from a prokaryote. Chitin, a homopolymer of N-acetyl-D-glucosamine (Glc-NAc) residues linked by ␤1-4 bonds, is the most abundant renewable natural resource after cellulose (4). It is widely distributed in nature as the integuments of insects and crustaceans and as a component of fungi and algae (23). It is estimated that the worldwide annual recovery of chitin from the processing of marine invertebrates, for example, is 37,300 metric tons (32). Chitin and its derivatives are of interest because they have various biological activities, such as those of an immunoadjuvant and a flocculant of wastewater sludge, and agrochemical uses (7). Chitooligosaccharides are prepared by partial hydrolysis of chitin with hydrochloric acid or enzymatically by degradation and transglycosylation. This pretreated chitin is then mixed with a chitinolytic enzyme to hydrolyze it to the monomer or oligomer of N-acetylglucosamine. Chitinases, a group of enzymes capable of degrading chitin directly to low-molecular-weight products, have been shown to be produced by a number of microorganisms. Almost all of the reported chitinase-producing strains will use chitin or colloidal chitin as a carbon source (42). Commercial interest in the utilization of chitin and its derivatives has led to the need for inexpensive, reliable sources of active and stable chitinase preparations. The production of inexpensive chitinolytic enzymes is an important element in the utilization of shellfish wastes that not only solves environmental problems but also promotes the economic value of the marine products (42). Some animal and higher plant chitinases have lysozyme activity (chitinase/lysozyme), while bifunctional chitinases have not been isolated from microorganisms. We reported the medium composition and the character of crude chitinase from Pseudomonas aeruginosa K-187 in a previous paper (42). In the present study, we isolated two kinds of bifunctional chitinases/ lysozymes from K-187 cell-free culture broth. This paper describes the purification and characterization of these enzymes. MATERIALS AND METHODS Materials. The shrimp and crab shell powder (SCSP) ...

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“…Similar observations have been made with chitinases from Aeromonas sp. (Mitsutomi et al, 1996), Bacillus circulans WL-12 (Watanabe et al, 1992) and a 49 kDa Fig. 3.…”

Section: Discussionmentioning

confidence: 99%