植物乳杆菌单宁酶的定向进化及应用

Directed Evolution and Application of the Tannase from Lactobacillus plantarum

摘要:

通过对植物乳杆菌（Lactobacillus plantarum, L. plantarum)单宁酶（Lptan1）的定向进化可提高其热稳定性和比酶活，进一步研究正向突变体（mLptan1）对绿茶茶汤中酯类儿茶素的降解效果。通过易错PCR构建Lptan1的突变文库，采用高通量筛选耐热性和比酶活提高的突变体并在大肠杆菌中表达，评价其在水解绿茶中酯类儿茶素方面的应用。筛选得到一个mLptan1，其最适反应温度（45 ℃）比Lptan1（40 ℃）提高了5 ℃。比酶活（425.6 U/mg）是Lptan1（312.3 U/mg）的1.36倍。将mLptan1应用于绿茶茶汤，在加酶量1 U/mL和最适反应温度下水解120 min，茶汤中酯类儿茶素降解率达85%，而Lptan1降解率仅为65%。结果表明，定向进化明显提高了Lptan1的最适反应温度和茶汤中酯类儿茶素的降解率，在茶类饮料中具有很好的应用前景。

Abstract:

To study the effect of positive mutant on the degradation of ester catechins in green tea, the thermal stability and specific enzyme activity of Lactobacillus plantarum tannase (Lptan1) were improved through directed evolution.. A mutant library of Lptan1 was constructed by error-prone PCR, and the mutants with enhanced thermostability and specific enzyme activity were screened and expressed in Escherichia coli. The application of these mutants in hydrolyzing ester catechins in green tea was further evaluated. A positive mutant (mLptan1) was selected with an optimal temperature (45 ℃) that was 5 ℃ higher than that of Lptan1 (40 ℃). Its specific enzyme activity (425.6 U/mg) was 1.36 times higher than that of Lptan1 (312.3 U/mg). When mLptan1 was applied to degrade green tea soup, the degradation rate of ester catechins reached 85% after hydrolysis at 1 U/mL and the optimal reaction temperature for 120 min, while the degradation rate of Lptan1 was only 65%. Directed evolution significantly improved both the optimal temperature of Lptan1 and the degradation rate of ester catechins in tea soup, which has a good application prospect in tea beverages.