新型食品添加剂γ-聚谷氨酸合成酶基因的克隆及表达

发布时间：2018-08-23 11:22

【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。
[Abstract]:纬 -poly glutamic acid (纬 -PGA) has good emulsification, thickening and anti-freezing properties, and it can be widely used as additive in food. Using the genome of Bacillus subtilis Bacillus subtilis 115 as template, the 纬 -PGA synthase gene pgs BCA was cloned by PCR technique and sequenced. The ex PASy-Prot Param tool Server 3.0 Signal PTMHMM Server and TmpredPSORTB Predict protein Swiss-Model Workspace software were used. The biological information of protein was analyzed and predicted, and the expression system of 纬 -PGA biosynthesis key enzyme gene pgs B was constructed. The results showed that the pgs C and pgs A genes contained 1182450 and 1143 nucleotides respectively, encoding 393149 and 380 amino acids, respectively. Their corresponding proteins have no signal peptide. The molecular weights of PGS BGS C and Pgs A are 44016.7C and 16302.9U 42759.8, respectively. The secondary structure of PGS C is hydrophobic basic membrane binding protein, and its secondary structure is 伪 -helix. There were four strong transmembrane regions (PGS A) as hydrophilic and stable basic proteins, and one strong transmembrane region in N terminal. The PGS B gene was successfully expressed by polyacrylamide gel electrophoresis (SDS-PAGE). The relative molecular weight of the target protein was about 45 kDa, which was consistent with the predicted value.
【作者单位】：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;
【基金】：国家支撑计划项目(2012BAD32B08) 研究生创新科研资金项目(YJSCX2015-356HSD)
【分类号】：Q78;TS202.3

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