耐高温耐酸稳定假密环菌(Armillariella tabescens) MAN47β-甘露聚糖酶体外分子定向进化

从已经建立的易错PCR初级突变文库中筛选得到的16个兼具耐酸、高温稳定、高酶活力的克隆出发,将其作为DNA shuffling的亲本基因,利用DNA shuffling技术,结合易化筛选和96微孔板通量筛选的方法获得耐酸、高温稳定的克隆。并且,对筛选出来的耐酸高温稳定的突变子进行测序分析和同源建模,比较分析β-甘露聚糖酶突变基因序列的生物学信息。经过两轮DNAshuffling,最终筛选得到一个耐酸高温稳定突变体1108,其在90℃时酶活力还能维持在70%;在pH 3.0时酶活力维持在70%;在高温80℃和pH 4.0的条件下,酶活力是野生型的10倍;常规条件下(pH 6.0,40℃),酶活力是野生型酶的5倍。序列比对发现耐酸、高温稳定突变体1108有三个碱基发生了改变(T289A、A535T、T1085C),导致相应的氨基酸发生了改变(Ser97Thr、Val362Ala、Ile179Leu)。根据同源建模结果和氨基酸性质研究发现,突变位点位于催化中心附近,推测第97位的突变与酶的耐酸性和活性有关,第179位和第362位的突变与酶的高温稳定性有关。实验结果为进一步了解β-甘露聚糖酶MAN47的结构与功能之间的关系提供有用参考。

Directed Evolution in vitro of Armillariella tabescens MAN47 β-Mannanase with Higher Thermalstability and Acid Tolerance

A mutant database has been built and mutants with acid tolerance or thermostability or high activity which would be taken as parent gene of DNA shuffling was also screened.Then mutants with higher thermalstablility and acid tolerance from DNA Shuffling database was searched by facilitation and 96 deep wall plate culture screen.Futhermore,some information about bioinformatics of β-mannanase by sequence comparison and homologous model was also been got.Through two cycles of DNA shuffling,a mutant database was built.Then one optimum 1108 was screened from about 104 mutants.The evoluted β-mannanase displayed both higher thermalstability and acid tolerance than wide type.The evoluted enzyme 1108 retained high activity after treatment at 90℃ for 30 min,whereas,the wild type nearly lost activity under this condition.Meanwhile,the activity of 1108 under 80℃ and acid treatment was 10 times and 5 times as wide type respectively.The sequence comparison illustrated that there were three nucleotide substitutions T289A,A535T,T1085C which carried corresponding amino acid changes Ser97Thr,Val362Ala,Ile179Leu.According to homologous modeling by SWISS-MODEL Repository and amino acid analysis,There is a foundation mutations locate near the catylyze core,So there is a speculation Ser97Thr,Val362Ala,Ile179Leu have something with acid tolerance activity and thermalstablility respectively.