Purification and characterization of a chitinase from Serratia proteamaculans |
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Authors: | Muhammad Aamer Mehmood Xiang Xiao Fauzia Yusuf Hafeez Yingbao Gai Fengping Wang |
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Institution: | 1. National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan 2. Key Laboratory of Marine Biogenetic Resources, State Oceanic Administration, Xiamen, People’s Republic of China 3. Third Institute of Oceanography, State Oceanic Administration, Xiamen, People’s Republic of China 4. COMSATS Institute of Information Technology, Islamabad, Pakistan
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Abstract: | A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S?1) and crystalline β-chitin (17.20 ± 0.83 S?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger. |
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