摘要 笔者在前期研究中已从嗜热菌Dictyoglomus thermophilum中克隆并异源表达了一种β-木糖苷酶Xln-DT,通过获得高酶活、酶学性质优异的β-木糖苷酶Xln-DT突变体,进而提高其在生物燃料、食品和医药等行业中的应用价值。基于提高比酶活的目的,通过生物信息学方法确定了β-木糖苷酶Xln-DT可突变的关键氨基酸位点,在161、202和231位点处分别引入HIS/LEU、PHE/LEU和TRP,获得Xln-DT比酶活明显提高的突变型酶Xln-DT-202PHE和Xln-DT-202LEU,并分析比较突变前后的酶学性质。突变型酶Xln-DT-202PHE和Xln-DT-202LEU的酶活较Xln-DT分别提高了3.28和2.97倍,比酶活分别提高了2.86和2.54倍。Xln-DT-202PHE和Xln-DT-202LEU的最适pH下降明显,由6.0分别下降至4.5和5.0。Xln-DT-202LEU在75℃下保温2 h酶活维持不变,较Xln-DT在75℃下的温度稳定性相比有明显提高。Xln-DT-202PHE和Xln-DT-202LEU在pH 4.0~7.0的范围内保温24 h,仍能保持80%以上的剩余酶活力。突变型酶不但显著提高了酶的活力,而且热稳定性得到了极大的改善,为其在食品热加工等领域中的应用奠定了基础。 Aβ-xylosidase Xln-DT has been cloned and heterologously expressed from the thermophilic bacterium Dictyoglomus thermophilum in the previous studies.A mutant ofβ-xylosidase Xln-DT with high enzyme activity and excellent enzymatic properties was obtained,in order to improve its vital application value in bio-fuel,food and medicine industries.The key amino acid mutation sites ofβ-xylosidase Xln-DT were obtained through bioinformatics methods to improve the specific enzyme activity.Amino acids HIS/LEU,PHE/LEU and TRP were introduced at sites 161,202 and 231,respectively,and the constructed mutant enzymes Xln-DT-202PHE and Xln-DT-202LEU obtained significantly increasedβ-xylosidase activity.In addition,the enzymatic properties before and after the mutation were analyzed and compared.The expressed enzyme activities of Xln-DT-202PHE and Xln-DT-202LEU were 3.28 and 2.97 times higher than that of Xln-DT,respectively,while their specific activities were 2.86 and 2.54 times higher than that of Xln-DT,respectively.The optimal pH of mutant enzyme Xln-DT-202PHE and Xln-DT-202LEU decreased significantly from pH 6.0 to pH 4.5 and pH 5.0,respectively.The enzyme activity of Xln-DT-202LEU remained unchanged when incubated at 75℃for 2 h,which indicated significantly improved thermal stability compared with that of Xln-DT under 75℃.Xln-DT-202PHE and Xln-DT-202LEU could maintain more than 80%of the residual enzyme activity in the range of pH 4.0~7.0 after 24 h incubation.The mutant enzyme not only significantly improved the enzyme activity,but also greatly improved its thermal stability,laying a foundation for its wide application in food thermal processing and other fields.
机构地区 南方现代林业协同创新中心 南京林业大学化学工程学院
出处 《食品与生物技术学报》 CAS CSCD 北大核心 2021年第2期41-48,共8页 Journal of Food Science and Biotechnology
基金 国家“十三五”重点研发计划项目(2017YFD0601001) 南京林业大学大学生创新训练计划项目(201910298081Y)。
关键词 Β-木糖苷酶 分子生物学 定点突变 酶活 热稳定性 β-xylosidase molecular biology site-directed mutagenesis enzyme activity thermal stability
分类号 Q814 [生物学—生物工程]
