Characterization of Chitinase C from a Marine Bacterium,
            <i>Alteromonas</i>
            sp. Strain O-7, and Its Corresponding Gene and Domain Structure

Tsujibo, Hiroshi; Orikoshi, Hideyuki; Shiotani, Kayoko; Hayashi, Miyuki; Umeda, Junko; Miyamoto, Katsushiro; Imada, Chiaki; Okami, Yoshiro; Inamori, Yoshihiko

doi:10.1128/aem.64.2.472-478.1998

Cited by 70 publications

(27 citation statements)

References 44 publications

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“…With the deletion of the A region, the enzyme lost 30 -40% of its activity toward colloidal and soluble chitin; its activity toward pNP-(GlcNAc) 2 and pNP-(GlcNAc) 3 dropped 50 -60% (Table II). In addition, we proved that the digesting ability of Chi1⌬A⌬ChBD toward chitooligomers (Glc-NAc) [3][4][5][6] also dropped compared to Chi1⌬ChBD, by analyzing the digesting processes by TLC [ Fig. 4(A,B)].…”

Section: Discussionmentioning

confidence: 99%

“…4(A)] demonstrated that it was not a good substrate for the enzymes. After prolonged incubation time (2 h), Chi1 and Chi1⌬ChBD could digest all (GlcNAc) [3][4][5][6] substrates completely, with (GlcNAc) 2 as the main product and traces of GlcNAc monomer [(GlcNAc) 2 was the only product from (GlcNAc) 4 ] [ Fig. 4(B)].…”

Section: Digesting Pattern and Ability Analysis Of Chi1 And Its Truncmentioning

confidence: 99%

“…So far, plenty of chitinase genes have been cloned; some have been characterized. [2][3][4][5][6] The chitinases (EC 3.2.1.14) form two distinct families (families 18 and 19) in the glycoside hydrolase classification system. 7 Bacterial chitinases, except a few from streptomycetes, 8,9 belong to family 18, usually with a modular organization.…”

Section: Introductionmentioning

confidence: 99%

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The C‐terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis

Wang

Zhou

et al. 2003

Proteins

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The chitinase gene chi1 of Aeromonas caviae CB101 encodes an 865-amino-acid protein (with signal peptide) composed of four domains named from the N-terminal as an all-beta-sheet domain ChiN, a triosephosphate isomerase (TIM) catalytic domain, a function-unknown A region, and a putative chitin-binding domain (ChBD) composed of two repeated sequences. The N-terminal 563-amino-acid segment of Chi1 (Chi1DeltaADeltaChBD) shares 74% identity with ChiA of Serratia marcescens. By the homology modeling method, the three-dimensional (3D) structure of Chi1DeltaADeltaChBD was constructed. It fit the structure of ChiA very well. To understand fully the function of the C-terminal module of Chi1 (from 564 to 865 amino acids), two different C-terminal truncates, Chi1DeltaChBD and Chi1DeltaADeltaChBD, were constructed, based on polymerase chain reaction (PCR). Comparison studies of the substrate binding, hydrolysis capacity, and specificity among Chi1 and its two truncates showed that the C-terminal putative ChBD contributed to the insoluble substrate-protein binding and hydrolysis; the A region did not have any function in the insoluble substrate-protein binding, but it did have a role in the chitin hydrolysis: Deletion of the A region caused the enzyme to lose 30-40% of its activity toward amorphous colloidal chitin and soluble chitin, and around 50% toward p-nitrophenyl (pNP)-chitobiose pNP-chitotriose, and its activity toward low-molecular-weight chitooligomers (GlcNAc)3-6 also dropped, as shown by analysis of its digestion processes. This is the first clear demonstration that a domain or segment without a function in insoluble substrate-chitinase binding has a role in the digestion of a broad range of chitin substrates, including low-molecular-weight chitin oligomers. The reaction mode of Chi1 is also described and discussed.

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Section: Discussionmentioning

confidence: 99%

Section: Digesting Pattern and Ability Analysis Of Chi1 And Its Truncmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The C‐terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis

Wang

Zhou

et al. 2003

Proteins

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“…Being a polymer of N-acetylglucosamine, it can be a potential source of biomass. The seafood industry acts as a major source of chitinous wastes, the recycling of which is extremely important to retain the carbon-nitrogen balance in the ecosystem (Tsujibo et al 1998). The insolubility of chitin and its inertness to most chemical agents necessitates the use of more feasible biological processes.…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a thermophilic and acidophilic chitinase from Microbispora sp. V2

et al. 2002

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Aim: Purification and characterization of a chitinase from Microbispora sp. V2. Methods and Results: The chitinase from Microbispora sp. V2 was purified to homogeneity by gel filtration chromatography with 4AE6% recovery. It had a molecular weight of 35 kDa and showed maximum activity towards p-nitrophenyl-b-D D-N,N¢-diacetylchitobiose, indicating a chitobiosidase activity. The enzyme had a pH optimum of 3AE0 and temperature optimum of 60°C. It was stable in a wide pH range from 3AE0 to 11AE0, retaining 61% activity at pH 3AE0 and 52% activity at pH 11AE0. It retained 71% activity at 30°C and 45% activity at 50°C, up to 24 h. The enzyme activity was not inhibited by any of the metal ions tested except Hg 2+ , in the presence of which only 10% activity was retained. Conclusions: The 35 kDa chitinase from Microbispora sp. V2 has an acidic pH optimum and a high temperature optimum. It is fairly stable and active, and degrades chitin efficiently, although the growth of the culture and enzyme production is slow. Significance and Impact of the Study: This report is the first detailed study of a chitinase from Microbispora sp. V2, isolated from hot springs. The chitinase from Microbispora sp. V2 may have potential applications in the recycling of chitinous wastes, particularly due to its thermophilic and acidophilic character. Studies at molecular level may provide further insight on the chitinolytic system of Microbispora spp. with respect to the number and types of chitinases and their regulation.

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“…In the case of the marine bacterium Alteromonas sp. strain O-7, at least four di¡erent chitinases (ChiA, ChiB, ChiC, and ChiD) and three di¡erent N-acetylglucosaminidases (GluNAcaseA, Glu-NAcaseB, and GluNAcaseC) are produced in the presence of chitin [15]. Vibrio harveyi produces about 10 chitinases when grown on chitin, six of which have been cloned and identi¢ed.…”

Section: Introductionmentioning

confidence: 99%

Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay

Ramaiah¹

2000

FEMS Microbiology Ecology

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PCR primers specific for the chiA gene were designed by alignment and selection of highly conserved regions of chiA sequences from Serratia marcescens, Alteromonas sp., Bacillus circulans and Aeromonas caviae. These primers were used to amplify a 225 bp fragment of the chiA gene from Vibrio harveyi to produce a chiA gene probe. The chiA PCR primers and probe were used to detect the presence of the chiA gene in an assemblage of 53 reference strains and gave consistent results. Selected chiA fragments amplified by PCR were cloned and sequenced from nine known strains and from Chesapeake Bay isolates 6d and 11d. This confirmed the specificity and utility of the primers for detection of chiA-positive environmental strains. Over 1000 bacterial isolates from Chesapeake Bay water samples were tested for the presence of the chiA gene which was found to be present in 5^41% (average 21%) of the culturable bacterial community. The approach developed in this study was valuable for isolation and enumeration of chiA-positive bacteria in environmental samples. ß

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Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure

Cited by 70 publications

References 44 publications

The C‐terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis

The C‐terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis

Purification and characterization of a thermophilic and acidophilic chitinase from Microbispora sp. V2

Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay

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