两种UDP-葡萄糖脱氢酶对透明质酸生物转化的影响  

Effects of two UDP-glucose dehydrogenases on hyaluronic acid biotransformation

作　　者：郭东会[1] 韩剑[2] 刘伟丰[2] 傅震洲[2] 朱启忠[1] 陶勇[2] 

机构地区：[1]山东大学海洋学院,山东威海264209 [2]中国科学院微生物研究所,北京100101

出　　处：《生物工程学报》2014年第11期1691-1700,共10页Chinese Journal of Biotechnology

基　　金：中国科学院微生物研究所工业微生物组学改造及应用创新培育基金"第二批"资助~~

摘　　要：分别从大肠杆菌和化脓链球菌中扩增出编码UDP-葡萄糖脱氢酶基因ecohas B和spyhas B,并将其插入T7表达载体p RX2构建重组质粒p RXEB和p RXSB。在大肠杆菌BL21(DE3)中重组表达,并对经镍柱纯化后的UDP-葡萄糖脱氢酶的酶学性质进行分析。酶学性质研究表明:spy Has B的最适反应温度是30℃,最适p H 10,最适条件下的比活力是12.2 U/mg;eco Has B的最适反应温度是30℃,最适p H 9,最适条件下的比活力是5.55 U/mg。从多杀巴氏杆菌扩增出的透明质酸合成酶基因pmuhas A分别与ecohas B和spyhas B构建共表达载体p BPAEB和p BPASB。将其转化到大肠杆菌BW25113中,经生物转化生产透明质酸(HA),并对转化条件进行了优化。结果表明:重组菌株进行透明质酸转化时,UDP-葡萄糖脱氢酶酶活力越高,稳定性越好,HA产量越高;转化条件优化后,p BPAEB/BW25113和p BPASB/BW25113在摇瓶中的产量分别是1.52和1.70 g/L,比之前报道的提高了2-3倍。We amplified genes encoding UDP-glucose dehydrogenase, ecohas B from Escherichia coli and spyhas B fromStreptococcus pyogenes. Both ecohasB and spyhasB were inserted into T7 expression vector pRX2 to construct recombinant plasmids pRXEB and pRXSB, and to express in E. coli BL21（DE3）. After nickel column purification of UDP-glucose dehydrogenases, the enzymes were characterized. The optimum reaction condition of spyHasB was at 30 ℃ and pH 10. The specific activity reached 12.2 U/mg under optimum condition. The optimum reaction condition of ecoHasB was at 30 ℃ and pH 9. Its specific activity reached 5.55 U/mg under optimum condition. The pmuhasA gene encoding hyaluronic acid synthase was amplified from Pasteurella multocida and ligated with ecohasB and spyhasB to construct the coexpression vectors pBPAEB and pBPASB, respectively. The co-expression vectors were transformed into E. coli BW25113. Hyaluronic acid（HA） was produced by biotransformation and the conditions were optimized. When recombinant strains were used to produce hyaluronic acid, the higher the activity of UDP-glucose dehydrogenase was, the better its stability was, and the higher the HA production could reach. Under the optimal conditions, the yields of HA produced by pBPAEB/BW25113 and pBPASB/BW25113 in shake flasks were 1.52 and 1.70 g/L, respectively, and the production increased more than 2-3 folds as previously reported.

关 键 词：UDP-葡萄糖脱氢酶 酶学性质 透明质酸 生物转化 

分 类 号：Q55[生物学—生物化学]