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| Effect of Amino Acid H321 on the Enzymatic Properties of Hybrid β-Mannanase AuMan5Aloop |
| LI Xue-qing1, YUAN Feng-jiau1, CHENG Jian-qing2, DONG Yun-hai1, LI Jian-fang1, WU Min-chen2 |
1. School of Food Science and Technology, Jiangnan University, Wuxi 214122, China;
2. Wuxi Medical School, Jiangnan University, Wuxi 214122, China |
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Abstract Objective: AuMan5Aloop, a hybrid β-mannanase, was constructed by substituting the loop structure of wild-type AuMan5A with the corresponding one of Aspergillus fumigatus β-mannanase. To analyze the correlation between the enzymatic properties and amino acid H321 of hybrid β-mannanase, its H321 was mutated into the corresponding Gly of AuMan5A. Methods: Using the mega primer PCR method, the mutant enzyme gene, Auman5Aloop/H321G, was constructed by mutating a H321-encoding codon CAC of Auman5Aloop into a Gly-encoding GGT. Then, Auman5Aloop/H321G was expressed in Pichia pastoris GS115 mediated by expression plasmid pPICZαA, and the enzymatic properties of expressed recombinant enzyme, AuMan5Aloop/H321G, were analyzed. Results: Before and after the H321G substitution, the temperature optimum (Topt) of AuMan5Aloop or AuMan5Aloop/H321G was 75℃, higher than that (65℃) of AuMan5A. The half-life at 70℃ (t1/270) of AuMan5Aloop/H321G was 300 min, between AuMan5A (10 min) and AuMan5Aloop (480 min). Besides, its specific activity was 12.8 and 1.43 fold those of AuMan5A and AuMan5Aloop, and its catalytic efficiency (kcat/Km) was 14.1 and 1.12 fold those of the latter two, respectively. Conclusion: After H321G substitution as well as determination and comparison of temperature characteristics, specific activity and catalytic efficiency of three enzymes, the certain effect of H321 on the enzymatic properties of AuMan5Aloop was confirmed.
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Received: 15 July 2016
Published: 25 February 2017
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