发酵乳杆菌来源4,6-α-葡萄糖基转移酶的酶学表征及产物性质鉴定  

Characterization and identification of Lactobacillus fermentum 4,6-α-glucosyltransferase and its products

作　　者：盛露菲 杨卫康 吴敬[1,2] 陈晟[1,2] Lufei Sheng;Weikang Yang;Jing Wu;Sheng Chen(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu,China 2;Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物工程学报》2021年第12期4363-4372,共10页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31972032)资助。

摘　　要：4,6-α-葡萄糖基转移酶(4,6-α-GTs)能以直链淀粉为底物合成含α(1-6)键的α-葡聚糖,在酶法合成膳食纤维中具有很大的应用潜力。根据4,6-α-GTs氨基酸序列中的保守区段设计引物,从发酵乳杆菌Lactobacillus fermentum基因组中PCR扩增得到一条假定GTFB-like 4,6-α-GTs基因(命名为gtf16),构建重组质粒pET15b-gtf16并在宿主大肠杆菌Escherichia coli BL21(DE3)成功表达,纯化后对其进行酶学表征。结果表明,该酶最适pH为5.0,最适温度为40℃。通过薄层色谱、NMR光谱、水解酶水解等检测手段对Gtf16酶转化产物进行系统的表征,发现其产物具有以下特征:与已报道的来源于罗伊氏乳杆菌Lactobacillusreuteri121菌株的4,6-α-葡萄糖基转移酶-GtfB与直链淀粉作用后的产物异麦芽寡糖结构类似;产物键型中α(1-6)键占比可达75%,产物平均分子量为23 793 Da;产物被消化酶水解后得到的抗消化成分含量可达88.22%。4,6-α-glucosyltransferases(4,6-α-GTs), which converts amylose into α(1-6) bonds-containing α-glucan, possesses great application potential in enzymatic synthesis of dietary fiber. Primers were designed according to the conserved motifs existing in the amino acid sequence of 4,6-α-GTs, and used to amplify a putative GTFB-Like 4,6-α-GTs gene(named as gtf16)from the genomic DNA of Lactobacillus. The gtf16 gene was cloned into the plasmid pET15 b, expressed in Escherichia coli BL21(DE3), followed by purification and characterization. The optimum pH and the optimum temperature of the purified enzyme were 5.0 and 40 ℃, respectively. The biotransformation product of this enzyme was systematically characterized by thin-layer chromatography, NMR spectroscopy, and hydrolysis reaction. The Gtf16-catalyzed product shows a similar structure to that of the isomalto/malto-polysaccharide(IMMP), which is the amylose-derived product catalyzed by GtfB from Lactobacillus reuteri 121. Moreover, The Gtf16-catalyzed product contains up to 75% of α(1-6) bonds and has an average molecular weight of 23 793 Da. Furthermore, the content of the anti-digestive components was 88.22% upon hydrolysis with digestive enzymes.

关 键 词：4 6-α-葡萄糖基转移酶 克隆表达 酶学性质 异麦芽寡糖 

分 类 号：TS201.2[轻工技术与工程—食品科学]