新型食品添加剂γ-聚谷氨酸合成酶基因的克隆及表达  

Cloning and expression of a novel food additive γ-PGA synthase gene

作　　者：王风青[1] 梁金钟[1] 毕明月[1] 肖玮[1] WANG Feng- qing LIANG Jin- zhong BI Ming- yue XIAO Wei(Key Laboratory of Food Science and Engineering, College of Heilongjiang Province, College of Food Engineering, Harbin University of Commerce, Harbin 150076, China)

机构地区：[1]哈尔滨商业大学食品工程学院,黑龙江省高等学校食品科学与工程重点实验室,黑龙江哈尔滨150076

出　　处：《食品工业科技》2017年第3期127-132,共6页Science and Technology of Food Industry

基　　金：国家支撑计划项目(2012BAD32B08);研究生创新科研资金项目(YJSCX2015-356HSD)

摘　　要：γ-聚谷氨酸(γ-poly glutamic acid,γ-PGA)特殊的分子结构,使其具有很好的乳化、增稠、抗冻等特性,可作为添加剂广泛应用于食品当中。实验以枯草芽孢杆菌Bacillus subtilis 115基因组为模板,通过PCR技术成功克隆到γ-PGA合成酶系基因pgs BCA,并进行测序,利用Ex PASy-Prot Param tool、Server 3.0 Signal P,TMHMM Server和Tmpred,PSORTB,Predict Protein,Swiss-Model Workspace软件,分别对蛋白质的生物信息进行了分析和预测,针对γ-PGA合成关键酶基因pgs B构建了表达体系,结果表明:pgs B,pgs C和pgs A基因分别含1182、450和1143个核苷酸,分别编码393、149和380个氨基酸,其对应蛋白均不存在信号肽,属于非分泌型蛋白;Pgs B、Pgs C和Pgs A分子量分别为44016.7、16302.9、42759.8 Da;Pgs B为亲水性不稳定的酸性蛋白质,二级结构以α-螺旋为主;Pgs C是疏水碱性的膜结合蛋白,存在4个强跨膜区;Pgs A为亲水稳定的碱性蛋白,在N端存在1个强跨膜区域。经聚丙烯酰胺凝胶电泳(SDS-PAGE)检测,pgs B基因被成功表达,目的蛋白相对分子质量约为45 k Da,与预测值基本相符。With special molecular structure,γ- poly glutamic acid （ γ- PGA ） has many characteristics, such as emulsification, thickening,antifreeze and so on. As an additive,T-PGA can be widely used in food.The gene of pgsBCA were cloned from Bacillus subtilis 115 genome DNA by PCR.Biological information of PgsBCA were analyzed using the online software （ExPASy - ProtParam tool, Server 3.0 SignalP, TMHMM Server and Tmpred, PSORTB, PredictProtein, Swiss- Model Workspace） based on the amino acid sequence.As the key enzyme gene of γ-PGA synthesis, expression system ofpgsB was constructed.The results showed that the pgsB,pgsC and pgsA genes contained 1182,450,1143 nucleotides and code 393,149,380 amino acids, respectively.PgsB,PgsC and PgsA did not exist signal peptide, belonged to the non secretory protein.The molecular weight of PgsB, PgsC and PgsA were 44016.7,16302.9 and 42759.8 Da, respectively. PgsB was hydrophilic, unstable acidic protein, its secondary structure was mainly alpha helical, PgsC protein containing four transmembrane domain, was highly conservative and hydrophobic alkaline membrane binding protein, PgsA was stable hydrophilic basic protein,which contained one transmembrane region in the N terminal. By SDS-PAGE test, the pgsB gene was expressed successfully and its relative molecular mass was about 45 kDa, was consistent with theoretical value.

关 键 词：Bacillus SUBTILIS 115 Γ-PGA pgsBCA基因克隆 生物信息分析 诱导表达 

分 类 号：TS202.3[轻工技术与工程—食品科学]