β-甘露聚糖酶末端残基变体对其酶学特性的影响

本研究用宇佐美曲霉Aspergillus usamii的5家族β-甘露聚糖酶AuMan5A为母本，借助同源建模、分子对接及分子动力学模拟等理性设计方法，将AuMan5A的N-末端和C-末端分别截去3个无规则的氨基酸残基，构建出截短的β-甘露聚糖酶 AuMan5A^N3C3.将AuMan5A和AuMan5A^N3C3的编码基因分别在毕赤酵母GS115中进行表达，对表达产物进行了初步纯化并分析比较了其酶学特性及各自的表达水平. 结果表明，reAuMan5A和reAuMan5A^N3C3的最适温度Topt均为70 ℃，reAuMan5A^N3C3在60 ℃的半衰期t_1/2⁶⁰为38 min，较reAuMan5A（t_1/2⁶⁰=40 min）略有降低；在相同表达条件下，reAuMan5A^N3C3上清液的β-甘露聚糖酶活性为73.4 U/mL，较reAuMan5A 的52.8 U/mL提高了39.0%；纯化的reAuMan5A^N3C3酶比活性为182.7 U/mg protein，较reAuMan5A的126.3 U/mg protein提高了44.7%. 与reAuMan5A相比，reAuMan5A^N3C3对角豆胶的Km值下降不明显，Vmax值有显著提高.

Effects of Terminal Variants of β-Mannanase on the Enzymatic Properties

The β-mannanase from Aspergillus usamii (AuMan5A) belongs to the glycoside hydrolase family 5. A truncated β-mannanase AuMan5A^N3C3 without 3 N-terminal and 3 C-terminal amino acid residues was designed by in silico approach using the wild type as the reference. Both AuMan5A and AuMan5A^N3C3 encoding genes were cloned and expressed in Pichia pastoris GS115. The protein products were purified for the analysis for enzymatic properties. The results showed that the temperature optima of AuMan5A and AuMan5A^N3C3 were 70 ℃. The half-life of reAuMan5A^N3C3 at 60 ℃ (t⁶⁰_1/2) was 38 min, which was shorter slightly than that of reAuMan5A(t⁶⁰_1/2=40 min). The β-mannanase activity from the expression supernatant of reAuMan5A^N3C3 was 73.4 U/mL, which was about 39.0% higher than the value (52.8 U/mL) of reAuMan5A. The specific activity of purified reAuMan5A^N3C3 was 182.7 U/mg protein, higher than that of reAuMan5A (126.3 U/mg protein). A significant increase of Vmax was detected for reAuMan5A^N3C3 with no significant change in Km as compared to the wild type.