Characterization and optimization of heterologous expression in Escherichia coli of the chitinase encoded by the chiA gene of Bacillus halodurans C-125 |
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| Affiliation: | 1. Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280, Coahuila, Mexico;2. Department of Biotechnology, Universidad Autónoma Metropolitana Iztapalapa, 09340 México, D.F., Mexico;3. Animal Nutrition Department, Universidad Autónoma Agraria Antonio Narro, Saltillo 25315, Coahuila, Mexico;1. Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland;2. Department of Special Analyses, Research and Teaching Institute for Brewing (VLB – Berlin e.V.), Seestr. 13, D-13353 Berlin, Germany;1. College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China;2. Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Busan 609-735, South Korea;3. Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, PR China;4. Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710, South Korea;1. Núcleo de Flebotomíneos, Fundação Estadual de Produção e Pesquisa em Saúde, Secretaria de Saúde do Rio Grande do Sul, Porto Alegre, RS, Brazil;2. Núcleo de Vigilância de Roedores e Vetores, Secretaria Municipal de Saúde de Porto Alegre, Porto Alegre, RS, Brazil;3. Centro de Referência Nacional e Internacional para Flebotomíneos, Centro de Pesquisas René Rachou, Belo Horizonte, MG, Brazil;1. Department of Animal Science and Biotechnology, Tunghai University, 1727, Section 4, Taiwan Boulevard, Taichung 40704, Taiwan, ROC;2. Department of Food Science, Tunghai University, 1727, Section 4, Taiwan Boulevard, Taichung 40704, Taiwan, ROC;3. Best Center for Cellular Nutrition, Han-Sient Trading Co., Ltd., 3F., 57, Section 2, Zhonghua Road, Sinzhuang District, New Taipei City 24246, Taiwan, ROC |
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| Abstract: | The diversity of the biotechnological applications of chitinolytic enzymes requires different enzyme-producing strains with different properties suitable for each process. In this work the chitinase encoded by the chiA gene of Bacillus halodurans has been studied. The protein shows a modular structure characterized by the catalytic domain of glycosyl hydrolases family 18 (GH18), fibronectin type III domain (FnIIID) and a carbohydrate-binding module family 5 (CBM5). The expression of the gene in Escherichia coli has made it possible to demonstrate the functionality of the protein which is active in the temperature range of 5–55 °C and pH values of 5.5–8.5 while maintaining a high stability under suboptimal conditions. The enzyme hydrolyzes colloidal chitin and different p-NP(GlcNAc)n (n = 1–3) by an “-exo” type mechanism according to the information deduced from its sequence. The production of the protein was optimized by constructing recombinant strains, and the effect of the expression vector used, the cell density of the culture, the concentration of inducer and the induction time were studied. Based on its spectrum of activity, stability and mechanism of action, it arises as an enzyme of potential interest for production of N-acetyglucosamine or conversion of chitin into biologically active chito-oligosaccharides. |
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| Keywords: | Chitinase Expression optimization |
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