瘤胃微生物Fosmid文库蛋白酶活性克隆的筛选与序列分析

摘要

在日粮蛋白质的降解过程中,蛋白酶是把蛋白质水解为肽或氨基酸的关键酶,由于受到纯培养技术的影响,瘤胃内产生蛋白酶活性的细菌和各种蛋白酶的遗传信息知之甚少.本实验旨在利用蛋白酶选择性培养基从瘤胃微生物Fosmid文库中筛选出含蛋白酶活性的克隆子,通过生物信息学分析获得这些克隆子的遗传信息.应用脱脂乳粉和大豆蛋白粉两种蛋白酶选择性培养基,从30 000个克隆中筛选得到14个具有蛋白酶活性的活性克隆.利用福林酚试剂法检测14个蛋白酶克隆子的酶活力,结果表明,每个克隆子分别具有不同的蛋白质分解能力.以酪蛋白为底物的克隆子酶活力介于0.59～2.74 U/mg之间,以大豆蛋白粉为底物的克隆子酶活力在0.70～7.19 U/mg之间,而且同一克隆对于不同的底物所表现的酶活力也不同.随机挑选10个活性克隆进行末端测序(GenBank登录号:JY084410～JY084429),经Blast比对后发现,45％的基因序列与已知编码基因无法匹配,pro 10F末端序列与金属肽酶匹配度为54％,属于肽酶M13家族,且该克隆蛋白酶最适pH值为7.0,为下一步研究该克隆的酶学性质和序列特征分析提供了基础资料.%During the degrading of diet protein, protease is the first key enzyme which hydrolyses protein to peptide or amino acid. However there is few genetic information of protease from rumen microbiota due to the limitation of pure-cultured method. The objective of the experiment was to screen, sequence and bioinformatics analyze the protease clone from a Fosmid library of the dairy cow rumen microbiota. Using two different protease selective medium, including 1% skim milk or 1% isolated soybean protein, respectively, fourteen protease activity clones were obtained from rumen microbiota Fosmid library containing 30 000 clones. Different protease decomposition ability of the fourteen clones was measured by Folin-phenol reagent method. The results showed that each clone had its unique ability of protease decomposition. When casein was used as the substrate, the range of enzyme activity was from 0.59 to 2.74 U/mg. While the isolated soybean protein served as substrate, the range of enzyme activity was from 0.70 to 7.19 U/mg. Furthermore, the same clone had different sizes of enzyme activity for different substrates. After end sequences often positive cloning(GenBank Accession numbers: JY084410-JY084429), the sequences were blasted by Blastn and Blastx. The results showed that 45% of the genetic sequences could not match with the known genes encoding. The end sequence of prolOF could match to metal peptidase, with the similarity of 54%, which belonged to peptidase family M13. The optimal pH of the clone was 7.0.These results provide the basicdatum for the further study on the clone's enzymatic properties and gene characteristics.