青春双歧杆菌蔗糖磷酸化酶诱导表达条件的优化及其酶学性质  

Induced Expression Condition Optimization and Enzymatic Property Characterization of Sucrose Phosphorylase from Bifidobacterium adolescentis

作　　者：朱乐婷 宋文军[1] 娄婷婷 吴子健[1] 张宏宇[1] 王丹芸 张得光 王亦心 董淑元 ZHU Leting;SONG Wenjun;LOU Tingting;WU Zijian;ZHANG Hongyu;WANG Danyun;ZHANG Deguang;WANG Yixin;DONG Shuyuan(Tianjin Key Laboratory of Food Biotechnology,School of Biotechnology and Food Science,Tianjin University of Commerce,Tianjin 300134,China;Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin Key Laboratory of Industrial Microbiology,School of Biological Engineering,Tianjin University of Science&Technology,Tianjin 300457,China)

机构地区：[1]天津商业大学生物技术与食品科学学院天津市食品生物技术重点实验室,天津300134 [2]天津科技大学生物工程学院工业发酵微生物教育部重点实验室,天津市工业微生物重点实验室,天津300457

出　　处：《食品研究与开发》2024年第23期194-202,共9页Food Research and Development

基　　金：大学生创新创业训练计划项目(202410069212、202310069097);天津市科技计划项目(20YFZCSN00630)。

摘　　要：蔗糖磷酸化酶(sucrose phosphorylase,SPase)不仅能够水解蔗糖产生α-葡萄糖-1-磷酸,还能对多种底物进行糖基化修饰。来源于青春双歧杆菌的蔗糖磷酸化酶(Bifidobacterium adolescentis sucrose phosphorylase,Ba SP)的双突变体L341I_Q345S(Ba SP-IS)对曲二糖的合成具有高选择性。为进一步挖掘Ba SP-IS合成曲二糖的潜力,该文将Ba SP-IS在大肠杆菌中异源表达,以水解活性为指标,通过单因素试验和响应面法对其诱导表达条件进行优化,并对其转糖基化活性及酶学性质进行研究。结果表明,Ba SP-IS的最佳表达条件为异丙基-β-D-硫代半乳糖苷(isopropylthio-β-D-galactoside,IPTG)浓度0.4 mmol/L、诱导时间17 h、诱导温度25℃,在此条件下,粗酶液的水解活性达到11.88 U/mL。酶学性质研究结果表明,Ba SP-IS在合成曲二糖时的最适反应温度为55℃、最适反应pH值为7.0,动力学参数K_(m)值为(145.749 78±7.897 30)mmol/L,最大反应速率V_(max)值为(0.091 57±0.001 72)mmol/(L·min)。Sucrose phosphorylase(SPase)can hydrolyze sucrose to produce α-glucose-1-phosphoric acid and perform glycosylation on various substrates.The mutant L341I_Q345S of sucrose phosphorylase derived from Bifidobacterium adolescent L341I_Q345S(Ba SP-IS)had high selectivity for the synthesis of kojibiose.The potential of Ba SP-IS in the synthesis of kojibiose was explored.The Ba SP-IS gene was expressed in Escherichia coli,and the induced expression conditions were optimized by single factor experiments and response surface methodology with the hydrolytic activity as an indicator.Furthermore,the transglycosylation activity and enzymatic properties were studied.The results showed that the optimal expression conditions for Ba SP-IS were isopropylthio-β-D-galactoside(IPTG)concentration of 0.4 mmol/L,induction time of 17 h and induction temperature of 25 ℃.Under these conditions,the hydrolytic activity of the crude enzyme reached 11.88 U/mL.Enzymatic property studies showed that the optimal reaction conditions for Ba SP-IS was 55 ℃ and pH7.0.The kinetic parameters K_(m) and V_(max) values of Ba SP-IS were(145.749 78±7.897 30)mmol/L and(0.091 57±0.001 72)mmol/(L·min),respectively.

关 键 词：蔗糖磷酸化酶 曲二糖 大肠杆菌 异源表达 酶学性质 

分 类 号：TS201.25[轻工技术与工程—食品科学]