一株产谷氨酰胺转氨酶菌株的分离、鉴定及其tgl的克隆表达  

Isolation and identification of a transglutaminase producing strain and its tgl cloning and expression

作　　者：张莹莹[1] 石楠[1,2] 杜萍萍[1] 申培立 李志辉[1] 于宏伟[1] 卢海强[1] 苏旭东[1] 张伟[1] 檀建新[1] 

机构地区：[1]河北农业大学食品科技学院,河北省农产品加工工程技术中心,河北保定071001 [2]河北大学生命科学学院,河北保定071002

出　　处：《食品工业科技》2015年第11期141-146,共6页Science and Technology of Food Industry

基　　金：多谷物主食冷冻面团的关键技术研究(13227105D);植物多酚类功能性物质对A(形成的影响(冀人社字[2010]195号)

摘　　要：用稀释平板法从土壤样品中分离到166株细菌菌株,通过凝胶法初筛和Folk比色法复筛,得到一株产谷氨酰胺转氨酶(transglutaminase,TGase)的菌株,通过形态学、生理生化特征和16S rDNA序列比对证明该菌株是枯草芽孢杆菌(Bacillus subtilis),命名为TGase1318。克隆了该菌株TGase的编码基因tgl,序列长738bp,编码由245个氨基酸组成的蛋白质;TGase的氨基酸序列与NCBI公布的B.subtilis的TGase相似性达94%-100%。用B.subtilis表达载体p TZ和E.coli表达载体p ET21b分别构建了含tgl的重组质粒,转化B.subtilis WB800和E.coli BL21,tgl基因表达产物在70℃下可催化BSA交联,表明tgl在B.subtilis和E.coli中获得了表达且表现出TGase活性,这为其在食品工业上的开发利用打下基础。By using the dilution plate method, 166 bacterial strains isolated from soil samples and one strain, TGasel3]8,which could produce transglutaminase, was obtained after screening with gel formation method and Folk' s colorimetry method.The strain was identified as Bacillus subtilis according to the morphology, physiological and biochemical characters as well as 16S rDNA sequence homological analysis.The tgl gene,which was a 738 bp long nucleotide,was cloned from the strain and encoded a 245 residue long TGase.Homological analysis of TGase peptide sequence revealed that it shared 94%-100% conservation with TGase of other B.subti/is strains released by NCBI.The expression recombinant plasmids pTZ-tgl and pET21b-tgl were constructed and transformed into B.subtilis WB800 and E. coli BL21, respectively.The results of BSA crosslink at 70℃ indicated the tgl gene was expressed in both B.subtilis and Ecofi with TGase activity.This study laid a foundation for the application of TGase from B.subtffis TGase1318 to food industry.

关 键 词：谷氨酰胺转氨酶 枯草芽孢杆菌 克隆和表达 16S RDNA 

分 类 号：TS201.3[轻工技术与工程—食品科学]