联合β-半乳糖苷酶和L-阿拉伯糖异构酶酶法合成D-塔格糖的研究

Enzymatic synthesis of D-tagatose from lactose with the combination ofβ-galactosidase and L-arabinose isomerase

将大肠杆菌K-12中的β-半乳糖苷酶基因lacZ和L-阿拉伯糖异构酶基因araA以串联方式克隆到载体pET-28a(+)上,并转入大肠杆菌BL21(DE3)中进行表达。通过SDS-PAGE分析发现,重组菌株能表达出大量可溶性β-半乳糖苷酶蛋白和L-阿拉伯糖异构酶蛋白。以重悬菌液为酶源,可将乳糖降解为D-半乳糖,并将D-半乳糖转化为D-塔格糖。在温度为50℃,pH 7.0的缓冲液中,经一段时间反应后,D-塔格糖的转化率可达21%以上。加入Mn^(2+)、Co^(2+)和Fe^(2+)均能够使D-塔格糖的转化率提高。

The genes encoding L-arabinose isomerase and β-galactosidase from Escherichia coli K-12 were cloned into expression vector pET-28a（ ＋ ） （ DE3 ）. L-arabinose isomerase in tandem, then the tandem recombinant plasmid was transformed into the strain E. coli BL1 and β-galactosidase could be highly expressed simultaneously in soluble form after induction with IPTG.. The resuspended cells were used as enzyme source,the lactose could be degraded into D-glucose and D- galactose, then the later was converted into D-tagatose. The conversion rate of D-tagatose could reach to 21% at 50 ℃ and pH 7.0. The conversion rate of D-tagatose increased after the addition of Mn^2＋ ,Ca^2＋ ,Co^2＋ or Fe^2＋ ions into the reaction liquid.