高酶活性β-甘露聚糖酶定向进化及其用于制备甘露寡糖

Directed evolution ofβ-mannanase with higher enzymatic activity and its application for mannooligosaccharide production

为了提高β-甘露聚糖酶的活性,采用易错PCR将来源于地衣芽孢杆菌(Bacillus licheniformis)KD-1的β-甘露聚糖酶基因manBl进行定向进化,并在枯草芽孢杆菌(B.subtilis)中进行表达,以筛选酶活性提高的β-甘露聚糖酶突变体。筛选得到的突变体ManBl(I91N/L211I),其比酶活性为15554.7 U/mg,是野生型ManBl的4.1倍,食品级表达的胞外酶产量达17601.3U/mL。分子动力学和分子对接结果表明,β-甘露聚糖酶在2个位点(I91N/L211I)的突变,导致蛋白催化口袋柔性增加,与底物结合能力提高,进而提高了酶的催化效率。β-甘露聚糖酶ManBl(I91N/L211I)水解魔芋胶产物主要由甘露六糖、甘露三糖和甘露二糖组成。报道的I91N/L211I 2个位点联合突变能够提高β-甘露聚糖酶活性;ManBl(I91N/L211I)食品级表达为该酶的绿色安全应用奠定了基础。

To improveβ-mannanase activity,β-mannanase gene from Bacillus licheniformis KD-1 was amplified by error-prone PCR for directed evolution and further expressed in B.subtilis to screen mutants with higherβ-mannanase activity.It was found that the specific activity of mutantβ-mannanase ManBl(I91N/L211I)was 15554.7 U/mg,which was 4.1 times of wild-type ManBl,while the extracellular yield of ManBl(I91N/L211I)expressed in B.subtilis at food-grade level reached 17601.3 U/mL.The data from molecular dynamics and molecular docking showed that the mutation at two sites(I91N and L211I)led to the enhancement of the catalytic pocket flexibility and the affinity with the substrate,thus improving the catalytic efficiency of the enzyme.Furthermore,theβ-mannanase ManBl(I91N/L211I)could hydrolyze konjac glucomannan(KGM)into mannooligosaccharides(MOS)with different degree of polymerization(DP)and main composition of mannohexaose,mannotriose,and mannobiose.The combined mutation of I91N/L211I could increaseβ-mannanase activity.And the food-grade expression of ManBl(I91N/L211I)laid the foundation for the green and safe application of the enzyme.