新型碱性低分子量β-葡萄糖苷酶基因的分离与酶学性质研究

Gene isolation and characterization of a novel alkaline β-glucosidase with low molecular mass

目的:获得新型β-葡萄糖苷酶编码基因,并对其表达产物进行酶学性质研究。方法:采用宏基因组学方法提取兔盲肠内微生物总DNA,构建DNA文库,通过筛选培养基获得产β-葡萄糖苷酶的克隆,测序获得其插入基因序列,氨基酸多重序列比对分析其序列特征,并用DNS显色法测定表达产物在不同条件下的酶活力。结果:在文库中获得一个基因片段nglu07,能编码一个低分子量的β-葡萄糖苷酶。nglu07基因片段长180bp,编码60个氨基酸残基。与已提交的糖苷水解酶Ⅰ家族成员进行氨基酸多重序列比对表明nglu07具有预测的活性位点Glu4与底物结合位点Tyr47,其最适反应温度为50℃,最适pH为10.0,粗酶活为8.12U/mL。结论:成功获得一新型低分子量的碱性β-葡萄糖苷酶编码基因,其蛋白温度稳定性高,在pH4.0～11.0的环境中均能保持较高的酶活力,具有作为食品工业用酶以及碱性酶的潜力,尤其在食品饮料加工、棉织品生物整理、洗涤及废纸脱墨等工业方面具有一定的应用价值。

Objective：Intended to obtain a gene encoding β-glucosidase applied for constructing engineering bacterium,and analyzed the enzymic properties of the β-glucosidase.Method：Using the metagenomic library and plate screening method for screening clones generating β-glucosidase,and the clones were validated by sequencing.Furthermore,multiple amino acid sequence alignment was checked for the inserted sequence.The characteristices of the enzyme product by E.coli expression system were measured under different conditions,such as different temperature,substrates.Result：A β-glucosidase-encoding gene fragment from the microbes in rabbit cecum was cloned,named nglu 07.The putative protein with molecular mass of 6.68ku was encoded by the gene（180bp）.An incomplete metallo-dependent_hydrolases superfamily domain was identified through the on line NCBI with BLASTP program and SMART online prediction.The amino acid multiple alignment showed that nglu 07 was similar to the glycoside hydrolase familyⅠ β-glucosidases and demonstrated that the Glu4,Tyr47 may be the predicted active site and binding site,respectively.The low sequence similarily with other β-glucosidases denoted that nglu 07 was likely to a novel member of glycoside hydrolase family.Analysis of the β-glucosidases activity showed that its optimum temperature and pH were 50℃ and pH 10.0,respectively.The crude enzyme activity of fermentingliquor was about 8.12U/mL.Conclusion：We successfully isolated a β-glucosidases-encoding gene.The enzyme showed the high-alkaline-tolerance characteriaztion and temperature stability.Taking these advantages,the novel alkaline β-glucosidase could be implied in the food,juice,detergent and recycled paper industries.