基于易错PCR的假密环菌Armillariella tabescens MAN47 b-甘露聚糖酶耐高温定向进化

Directed evolution by error-prone PCR of Armillariella tabescens MAN47 β-mannanase gene toward enhanced thermal resistance

摘要:

本研究利用易错PCR技术突变假密环菌Armillariella tabescens MAN47 β-甘露聚糖酶野生型基因, PCR产物与大肠杆菌-酿酒酵母穿梭表达载体pYCα上连接, 在大肠杆菌DH5α中扩增后电转入酿酒酵母Saccharomyces cerevisiae, 构建了库容为104的初级突变体库, 筛选得到耐高温最佳突变株M262。DNS法测得80oC处理30 min后最大酶活力为25 U/mL, 较之野生型最适条件酶活力提高了4.3 倍。序列分析表明, 突变体有3 个碱基发生了突变: T343A/ C827T/ T1139C,相应的氨基酸改变为Ser115Thr/Thr276Met/Val380Ala, 利用SWISS-MODEL数据库同源建模显示, 这3 个突变氨基酸分别位于第4 个β折叠的第6 个氨基酸、第6 个α螺旋的第1 个氨基酸、第10 个α螺旋和第11 个β折叠之间的转角。

Abstract:

Firstly, We used error-prone PCR to induce mutations on Armillariella tabescens MAN47 β-mannanase gene, Secondly, we cloned the mutated fragments into secreted expression vector pYCα, Then the recombinant plasmids were transformed into Saccharomyces cerevisiae BJ5465 after amplified and extracted in DH5α cells. Through three cycles of error-prone PCR we built a mutant database, Then we screened one optimum (named M262) from about 104 mutants. The evoluted MAN47 β-mannanase displayed both higher thermal stability and activity than wide type. The evoluted enzyme M262 retained high activity after treatment at 80oC for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of M262 can reach to 25?U/mL, which is 4.3 times as wide type under optimum temperature. In addition, pH stability and pH range of evoluted enzyme M262 were both improved compared with wild-type enzyme. The optimum pH was estimated to be similar to that of wild-type enzyme. The sequence comparison illustrated that there were three nucleotide substitutions (T343A/C827T/T1139C) which carried corresponding amino acid changes (Ser115Thr/Thr276Met/Val380Ala). According to homologous modeling by SWISS-MODEL Repository, three mutated amino acids located at the sixth amino acid of the fourth β-sheet, the first amino acid of the sixth a-helix, the turn between the tenth and eleventh β-sheet, respectively.