以甘油为唯一碳源的氧化葡糖杆菌适应性驯化及催化合成米格列醇中间体6NSL

氧化葡糖杆菌(Gluconobacter oxydans)来源的山梨醇脱氢酶可催化N-羟乙基葡萄糖胺合成6-脱氧-6-氨基(N-羟乙基)-α-L-呋喃山梨糖,即合成降血糖药物米格列醇的关键中间体。本文采用适应性驯化策略,以甘油为唯一碳源,通过40 g/L、60 g/L、80 g/L和100 g/L甘油梯度连续传代培养,筛选获得了一株以甘油为碳源的高活力菌株G.oxydans A-3-D,扫描电镜结果表明该细胞表面褶皱较原始菌株有显著增加。在80 g/L甘油培养基摇瓶培养24 h后,菌体浓度为4.58 g DCW/L,山梨醇脱氢酶的发酵体积酶活与比酶活分别为原始菌株G.oxydans ZJB-605的1.3倍及1.5倍。此外,在摇瓶培养条件下对影响催化反应进程的关键因素进行了考查,结果表明在摇瓶体系中,G.oxydans A-3-D的最适催化反应条件为80.0 g/L底物、2.0 g DCW/L菌体细胞、20 mmol/L Mg~(2+)浓度,15℃反应48 h后底物转化率达到90.8%,6NSL累积浓度为72.6 g/L,较G.oxydans ZJB-605有显著提升。

Use of glycerol as sole carbon source for G.oxydans adaptation domestication and bioconversion of miglitol intermediate 6NSL

Sorbitol dehydrogenase (SLDH),originated from Gluconobacter oxydans,is capable to catalyze N-hydroxyethyl glucamine into 6-deoxy-6-amino (N-hydroxyethyl)-α-L-furansorbose (6NSL),which is a key intermediate for synthesis of the hypoglycemic drug miglitol.A high-activity strain G.oxydans A-3-D was obtained by glycerol gradient adaptation with 40 g/L,60 g/L,80 g/L,100 g/L as sole carbon source.SEM observation demonstrated that the plasma membrane folds in cell surface area was significantly increased in G.oxydans A-3-D if compared with that of the original strain G.oxydans ZJB-605.After 24 h cultivation in 80 g/L glycerol medium,4.58 g DCW/L cells were obtained in G.oxydans A-3-D and the catalytic activity of SLDH was significantly increased by 1.3-folds for total enzymatic activity and 1.5-folds for specific enzymatic activity,respectively,compared with that of G.oxydans ZJB-605.In addition,key parameters involved in bioconversion processes were investigated.Based on the data obtained under the optimized catalytic conditions in shake-flask (including 80 g/L substrate,2.0 gDCW/L cell and 20 mmol/L Mg2 +),the conversion rate reached to 90.8％ and 72.6 g/L 6NSL was accumulated within 48 h at 15 ℃2,which was significantly enhanced if compared with that of G.oxydans ZJB-605 strain.