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Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells

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Abstract

Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 °C, and activity was stimulated with 25 mM Mn2+. In kinetic analysis, Chi18B showed Km values of 9.07 ± 0.65 μM and 129.27 ± 0.38 μM with the substrates 4-methylumbelliferyl-N-N′-diacetylchitobiose and α-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.

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Acknowledgments

This study was carried out with the support of "Cooperative Research Program for Agricultural Science & Technology Development (project number 20080101-030-002-001-03-00)," RDA, and by a grant number R01-2008-000-20220-0 from the Basic Research Program of Korea Science and Engineering Foundation, Republic of Korea. Shah Md. Asraful Islam is supported by scholarships from the BK21 Program, Ministry of Education and Human Resources Development, Korea.

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Authors and Affiliations

  1. Division of Applied Life Science (BK21 Program), Gyeongsang National University, Chinju, 660701, Republic of Korea

    Shah Md. Asraful Islam, Sun Joo Hong, Renukaradhya K. Math, Jong Min Kim, Myoung Geun Yun, Ji Joong Cho & Han Dae Yun

  2. Life Science, Gyeongsang National University, Chinju, 660701, Republic of Korea

    Han Dae Yun

  3. Department of Food Science, Jinju National University, Chinju, 660758, Republic of Korea

    Kye Man Cho

  4. Division of Plant Environmental Research, Gyeongsangnam-do Agricultural Research and Extension Service, Chinju, 660360, Republic of Korea

    Jae Young Heo & Young Han Lee

  5. Department of Agricultural Chemistry, Sunchon National University, Suncheon, 540742, Republic of Korea

    Hoon Kim

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  1. Shah Md. Asraful Islam
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  2. Kye Man Cho
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Correspondence to Han Dae Yun.

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Islam, S.M.A., Cho, K.M., Hong, S.J. et al. Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells. Appl Microbiol Biotechnol 86, 119–129 (2010). https://doi.org/10.1007/s00253-009-2215-1

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  • DOI: https://doi.org/10.1007/s00253-009-2215-1

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