链霉菌来源D-甘露糖异构酶的性质及其在制备D-甘露糖中的应用

【背景】D-甘露糖具有多种功能活性,在食品、医药、饲料等行业应用广泛。D-甘露糖异构酶可以催化D-果糖与D-甘露糖之间的相互转化,在D-甘露糖的酶法制备中具有应用潜力。【目的】克隆一个链霉菌(Streptomycessp.)来源的D-甘露糖异构酶基因(ssMIaseA)并在大肠杆菌中表达,研究其酶学性质,并用于制备D-甘露糖。【方法】从链霉菌(Streptomycessp.)中发掘一个D-甘露糖异构酶基因(ssMIaseA),构建重组表达质粒pET-28a-ssMIaseA并在大肠杆菌BL21(DE3)中表达,经Ni-NTA亲和层析纯化后测定酶学性质,利用高效液相色谱对SsMIaseA制备D-甘露糖进行研究。【结果】SsMIaseA与嗜热裂孢菌(Thermobifda fusca)来源的D-甘露糖异构酶ManI相似性最高,为60.2%。该酶比酶活为525 U/mg,分子量约为45 kD,最适pH和温度分别为7.5和45°C,在pH 6.5-10.0范围内和45°C以下保持稳定。该酶对甘露糖具有最高催化活性,其次是果糖、塔罗糖和塔格糖。利用SsMIaseA转化600 g/L D-果糖,反应8 h达到平衡,生成185 g/L D-甘露糖,转化率为31%。【结论】SsMIaseA作为新型D-甘露糖异构酶为D-甘露糖的酶法制备奠定了基础。

Characterization of a D-mannose isomerase from Streptomyces sp. and its application in the preparation of D-mannose

Background] D-mannose with many functional activities has been widely used in food, medicine, feed, etc. D-mannose isomerase can catalyze the reversible reaction between D-fructose and D-mannose, and has application potential in the enzymatic preparation of D-mannose. Objective] The D-mannose isomerase gene (ssMIaseA) from Streptomyces sp. was cloned and expressed in Escherichia coli, and its enzymatic properties were studied and used to prepare D-mannose. Methods] The D-mannose isomerase gene (ssMIaseA) from Streptomyces sp. was cloned and the recombinant expression plasmid pET-28a-ssMIaseA was constructed and expressed into E. coli BL21(DE3). After purification by Ni-NTA affinity chromatography, the enzyme properties were determined, and the preparation of D-mannose from SsMIaseA was analyzed by high performance liquid chromatography. Results] SsMIaseA shared the highest homology of 60.2% with ManI from Thermobifda fusca. The specific activity of the enzyme was 525 U/mg, and the molecular weight was about 45 kD. Its optimal pH and temperature were 7.5 and 45 °C, respectively. It was stable in the range of pH 6.5?10.0 and below 45 °C. It had the highest catalytic activity for mannose, followed by D-fructose, D-talose and D-tagatose. SsMIaseA was used to convert 600 g/L D-fructose and the reaction reached equilibrium at 8 h, producing 185 g/L D-mannose with a conversion rate of 31%. Conclusion] SsMIaseA as a new D-mannose isomerase has potential in the enzymatic preparation of D-mannose.