枯草芽胞杆菌谷氨酰胺转氨酶在谷氨酸棒杆菌中的分泌表达及诱导条件的优化

Expression of the Transglutaminase from Bacillus subtilis and Optimization of Fermentation Conditions for Corynebacterium glutamicum

本文通过重叠PCR技术构建获得枯草芽胞杆菌(Bacillus subtilis)来源的谷氨酰胺转氨酶(transglutaminase,TG)(BTG)重组基因Sprodbtg,该基因依次包含SD序列、谷氨酸棒杆菌信号肽ΔS0949序列、pro D-BTG基因,并将该目的基因克隆入大肠杆菌–谷氨酸棒杆菌穿梭表达载体p XMJ19中构建重组质粒p XMJ19-Sprodbtg.随后,重组质粒电转入谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032中进行诱导表达,并对重组菌株的诱导条件进行优化.结果表明:重组菌株C.glutamicum ATCC13032/p XMJ19-Sprodbtg表达重组酶原蛋白pro D-BTG经活化后,BTG的活力达到(41.23±2.01)U/L;在重组菌培养12 h后诱导、IPTG终浓度0.8 mmol/L、诱导40 h的最优诱导条件下,BTG的活力达到最高,为(55.62±2.34)U/L,较优化前提高了约34.90%,.

Overlap PCR was used to produce Sprodbtg encode with transglutaminase（BTG） from Bacillus subtilis. The recombined gene Sprodbtg contains signal peptide AS0949 from Corynebacterium glutamicum, SD sequence and the proDBTG gene. The sprodbtg was cloned into pXMJ19 to construct secretion expression vector pXMJ19-Sprodbtg. After that, the recombined plasmid was transformed into C. glutamicum ATCC13032 to express proD-BTG. The optimized fermentation conditions were also studied. The results showed that the recombined strain C. glutamicum ATCC13032/pXMJI9-Sprodbtg accumulated proD-BTG up to a level of （41.23 ± 2.01）U/L. The optimized conditions for the induction of expression of the recombined protein proD-BTG in strain 13032/pXMJ19-Sprodbtg were as follows： growing in MMTG medium for 12 h, adding IPTG with a final concentration of 0.8 mmol/L and the subsequent incubation for 40 h. The activity of BTG reached （55.62 ±2.34） U/L, a 34.90% increase compared with the non-optimized control.