中文摘要：

目的：将loop置换杂合β-甘露聚糖酶AuMan5A^loop的H321突变回野生型酶AuMan5A对应的Gly,以分析杂合酶的酶学性质与H321的相关性。方法：采用大引物PCR技术将AuMan5A^loop基因（Auman5Aloop）编码H321的密码子CAC突变为Gly的GGT,构建突变酶基因Auman5A^loop/H321G;借助表达质粒pPICZαA将该突变酶基因在Pichia pastoris GS115中实施表达,并分析重组表达产物AuMan5A^loop H321G的酶学性质。结果：AuMan5A^loop/H321G置换前后的最适温度Topt均为75℃,高于AuMan5A的65℃;AuMan5A^loop/H321G在70℃的半衰期t1/2^70为300 min,介于AuMan5A（10 min）和AuMan5A^loop（480 min）之间;AuMan5A^loop/H321G比活性分别为AuMan5A和AuMan5A^loop的12.8和1.43倍;催化效率kcat/Km为后两者的14.1和1.12倍。结论：通过H321G置换及对3种酶的温度特性、比活性和催化效率的测定及比较,证实了H321对AuMan5A^loop的酶学性质有一定的影响。

英文摘要：

Objective： AuMan5A^loop,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,Auman5A^loop/H321G G,was constructed by mutating a H321-encoding codon CAC of Auman5 Aloopinto a Glyencoding GGT. Then,Auman5A^loop/H321G Gwas expressed in Pichia pastoris GS115 mediated by expression plasmid pPICZαA,and the enzymatic properties of expressed recombinant enzyme,AuMan5A^loop / H321 G,were analyzed.Results： Before and after the H321 G substitution, the temperature optimum（Topt） of AuMan5A^loop or AuMan5A^loop/H321G was 75℃, higher than that（65℃） of AuMan5A. The half-life at 70℃（ t1/2^70） of AuMan5A^loop/H321G was 300 min,between AuMan5A（10 min） and AuMan5A^loop（480 min）. Besides,its specific activity was 12. 8 and 1. 43 fold those of AuMan5A and AuMan5A^loop,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 AuMan5A^loop was confirmed.