弱酸特性D-来苏糖异构酶分子改造及D-甘露糖生产

Molecular Modification of D-lyxose Isomerase with Weak Acid Characteristic and D-mannose Production

本文旨在对来源于Thermoprotei archaeon菌株的D-LI(最适pH6.5)进行弱酸特性改造,以期该酶在pH5.5条件下生产D-甘露糖,从而抑制原有的美拉德反应,减少分离成本。基于蛋白序列比对及酸碱氨基酸置换策略,首先设计了8个单点突变,而后从中选择3个优良单点突变体E82K、P105K、E165K进一步进行两两组合双点突变。结果表明双点突变体E82K/P105K较野生酶的最适pH由6.5迁移至6.0,且在pH5.5条件下的酶活是原始酶的3.4倍。该突变体在以D-果糖和D-甘露糖为底物时的动力学参数K_(m)值分别为78.77 mmol/L和328.12 mmol/L,k_(cat)/K_(m)值分别为15.37 mmol/L^(−1)·min^(−1)和48.92 mmol/L^(−1)·min^(−1)。以80 g/L的D-果糖为底物反应10 h后,双点突变体E82K/P105K在pH5.5反应时的转化率较接近于原始酶在pH6.5时的转化率,美拉德反应程度较原始酶降低了约3~4倍,为工业上利用D-LI生产D-甘露糖提供了可行的酶制剂。

The aim of this paper was to modify the weak acid characteristics of D-LI(the optimum pH was 6.5)from Thermoprotei archaeon,so as to produce D-mannose at pH5.5,thereby inhibiting the original maillard reaction and reducing the separation cost.Based on the strategy of protein sequence alignment and acid-base amino acid replacement,eight single point mutations were designed,and then three better single point mutants E82K,P105K and E165K were selected for further double-point mutation.The results showed that the optimum pH of the double-point mutant E82K/P105K was shifted from 6.5 to 6.0,and the enzyme activity at pH5.5 was 3.4 times higher than that of the original enzyme.When D-fructose and D-mannose were used as substrates,the kinetic parameters K_(m) and k_(cat)/K_(m) of the mutant were 78.77 mmol/L and 328.12 mmol/L,15.37 mmol/L^(−1)·min^(−1) and 48.92 mmol/L^(−1)·min^(−1),respectively.After reacting with 80 g/L of D-fructose for 10 h,the conversion rate of the double point mutant E82K/P105K at pH5.5 was close to that of the original enzyme at pH6.5,and the color and degree of Maillard reaction decreased about 3~4 times compared with the original enzyme,which provided a feasible enzyme preparation for industrial production of D-mannose by D-LI.