Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli |
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Authors: | Patcharaporn Siwayaprahm Mongkon Audtho Kunio Ohmiya Chanpen Wiwat |
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Institution: | (1) Department of Microbiology, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhya Rd., 10400 Bangkok, Thailand;(2) BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, 12120 Pathumthani, Thailand;(3) Department of Applied Microbiology, Faculty of Bioresources, Mie University, 514-8507 Tsu, Mie, Japan |
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Abstract: | Summary 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-β-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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Keywords: | Bacillus circulans No 4 1 characterization chitin chitinase purification |
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