β-甘露聚糖酶AuMan5A/Af酶学性质的改善与其Asp320的相关性分析

Correlation between superior enzymatic properties of β-mannanase AuMan5A/Af and its residue Asp320

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 (³¹⁶KSPDGGN³²²) 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Asp³²⁰的相关性,定点突变构建突变体AuMan5A/Af D^320G。[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Gly³²⁰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 D^320G的酶学性质。[结果] AuMan5A/Af D^320G的最适温度Topt为70.0 ℃,变性温度T_m为71.5 ℃,介于AuMan5A (T_opt=65.0 ℃,T_m=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/K_m) 分别是AuMan5A和AuMan5A/Af的3.9和0.3倍。[结论] 将Asp³²⁰突变为Gly³²⁰显著影响了AuMan5A/Af的酶学性质,证明了Asp³²⁰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。

Abstract:

[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) of A. 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 D^320G. [Methods] Using megaprimer PCR method, we constructed a new mutant-encoding gene, Auman5A/Af ^D320G 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 GS115, and the enzymatic properties of the expressed product were analyzed. [Results] Analytical results indicated that the optimal and melting temperature of AuMan5A/Af D^320G was 70.0 ℃ and 71.5 ℃, repectively, higher than those of AuMan5A (T_opt=65.0 ℃, T_m=64.5 ℃) and lower than those of AuMan5A/Af (T_opt=75.0 ℃, T_m=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 (k_cat/K_m) was 3.9 fold and 0.3 fold that of AuMan5A and AuMan5A/Af. [Conclusion] The mutation of Asp³²⁰ into Gly³²⁰ greatly affected the temperature characteristics and catalytic activity of AuMan5A/Af, demonstrating that Asp³²⁰ plays an improtant role in temperature characteristics, specific activity and catalytic efficiency improving of AuMan5A after loop substitution.