Improvement in the Enzymatic Properties of β-Mannanase（AuMan5A） by Site-Directed Mutagenesis

Abstract:

Based on comparison and analysis of primary and three-dimensional（3-D） structures of glycoside hydrolase family 5（GHF5） β-mannanases，an amino acid at the key site of Aspergillususamii GHF5 β-mannanase（AuMan5A） was subjected to site-directed mutagenesis to obtain a mutant enzyme AuMan5AG320D with superior enzymatic properties. Using the megaprimer PCR method，the AuMan5AG320D-encoding gene，Auman5AG320D，was constructed by mutating the Gly320-encoding codon GGT of Auman5A into Asp320-encoding GAC. Then，two genes，Auman5A and Auman5AG320D，were extracellularly expressed in Pichia pastoris GS115 and the enzymatic properties of expressed products were analyzed. Results indicated that the optimal temperature of AuMan5AG320D was 70 ℃ and and its half-life at 70 ℃（t1/270） was 25 min. The specific activity of AuMan5AG320D was increased from 351.2 U/mg to 1 729.1 U/mg and its catalytic efficiency（kcat/Km） was 9.3 times higher than that of AuMna5A. Through mutating Gly320 into Asp320，this work not only improved the temperature characteristics of AuMan5A，but also significantly enhanced its specific activity and catalytic efficiency.