中文摘要：

【目的】为改善宇佐美曲霉5家族β-甘露聚糖酶（AuMan5A）的酶学性质,本实验室前期将AuMan5A底物结合凹槽内一个7肽（~（316）KSPDGGN322）组成的loop替换为烟曲霉5家族β-甘露聚糖酶对应的氨基酸片段（PSPNDHF）,得到loop替换突变酶AuMan5A/Af。为揭示AuMan5A/Af酶学性质显著改善与其Asp（320）的相关性,定点突变构建突变体AuMan5A/Af（D320G）。【方法】采用大引物PCR技术将AuMan5A/Af基因（Auman5A/Af）中编码Asp320的密码子GAC突变为Gly（320）的GGT,构建出突变体基因Auman5A/Af（D320G）,并在毕赤酵母GS115中进行表达,分析表达产物AuMan5A/Af（D320G）的酶学性质。【结果】AuMan5A/Af（D320G）的最适温度T_（opt）为70.0℃,变性温度T_m为71.5℃,介于AuMan5A（T_（opt）=65.0℃,Tm=64.5℃）和AuMan5A/Af（T_（opt）=75.0℃,T_m=76.6℃）之间;在70.0℃的半衰期为40 min,高于AuMan5A的10 min,但较AuMan5A/Af的480 min显著缩短;比活性分别是AuMan5A和AuMan5A/Af的2.7和0.3倍;催化效率（k（cat）/Km）分别是AuMan5A和AuMan5A/Af的3.9和0.3倍。【结论】将Asp（320）突变为Gly（320）显著影响了AuMan5A/Af的酶学性质,证明了Asp~（320）对AuMan5A/Af温度特性改善、比活性和催化效率显著提高的重要作用。

英文摘要：

[Objective] AuMan5A is a glycoside hydrolase （GH） family 5 β-mannanase from Aspergillus usamii. To improve its enzymatic properties, we have previously constructed a mutant with loop substitution, AuMan5A/Af, by substituting a loop of seven residues （316KSPDGGN322） in its substrate binding groove with the corresponding region （PSPNDHF） ofA. fumigatus GH family 5 β-mannanase. To reveal the correlation between the superior enzymatic properties of AuMan5A/Af and its residue Asp320, site-directed mutagenesis was used to obtain a new mutant enzyme AuMan5A/Af D320G. [Methods] Using megaprimer PCR method, we constructed a new mutant-encoding gene, Auman5A/AfD320G by mutating an Asp320-encoding codon GAC of Auman5A/Af into a Gly320-encoding GGT. Then, Auman5A/Af D320G was extracellularly expressed in Pichia pastoris GS 1 15, and the enzymatic properties of the expressed product were analyzed. [Results] Analytical results indicated that the optimal and melting temperature of AuMan5A/Af D320C was 70.0 ℃ and 71.5 ℃, repectively, higher than those of AuMan5A （Topt=65.0 ℃, Tm=64.5 ℃） and lower than those of AuMan5A/Af （Topt=75.0 ℃, Tm=76.6 ℃）; its half-life at 70.0 ℃ was 40 min, 10 min longer than that of AuMan5A but greatly shorter than 480 min of AuMan5A/Af. Besides, its specific activity was 2.7 fold and 0.3 fold that of AuMan5A and AuMan5A/Af, respectively, and its catalytic efficiency （kcat/Km） was 3.9 fold and 0.3 fold that of AuMan5A and AuMan5A/Af. [Conclusion] The mutation of Asp322 into Gly322 greatly affected the temperature characteristics and catalytic activity of AuMan5A/Af, demonstrating that Asp322 plays an improtant role in temperature characteristics, specific activity and catalytic efficiency improving of AuMan5A after loop substitution.