重组枯草芽孢杆菌表达4-木糖醇脱氢酶的发酵和反应条件优化

Optimized fermentation and reaction conditions to express xylitol-4-dehydrogenase in recombinant Bacillus subtilis

L-木酮糖(L-苏式-戊酮糖)是一种昂贵的稀有戊糖,而4-木糖醇脱氢酶(xylitol-4-dehydrogenase,XDH)是生物合成L-木酮糖(L-xylulose)的关键酶。为了促进L-木酮糖的生产,对枯草芽孢杆菌外源表达4-木糖醇脱氢酶的发酵条件进行优化,并对以木糖醇为底物静息细胞催化反应合成L-木酮糖的反应条件进行研究。通过单因素实验确定了最佳培养基的成分(g/L)为:蔗糖25,尿素6,MgSO_40. 05,MnSO_40. 01,FeSO_40. 01,初始pH值8. 0。最佳发酵条件为:装液量40 mL(250 mL锥形瓶),接种量1. 67%,发酵培养温度28℃。在优化条件下4-木糖醇脱氢酶酶活为5. 183 U/L,与初始酶活相比提高了231. 2%。最优反应条件:底物质量浓度20 g/L,50mmol/L甘氨酸-氢氧化钠缓冲溶液(pH 10. 0),反应温度45℃,优化反应条件下转化率达到17. 74%。通过对菌株发酵和反应工艺条件的优化,为后续L-木酮糖工业化生产奠定了基础。

L-xylulose ( L-threo-pentulose) is one of the rare sugars, and xylitol-4-dehydrogenase (XDH) is a key enzyme to biosynthesize L-xylulose. To accelerate the production of L-xylulose, conditions to express XDH in Bacillus subtilis and to catalyze L-xylulose formation from xylitol with resting cell were optimized. The optimal medium had an initial pH of 8 and contained 25g/L sucrose, 6g/L urea, 0.05g/L MgSO4,0.01g/L MnSO 4, and 0.01g/L FeSO4. The optimal fermentation condition was as follows: 40 mL liquid volume in a 250 mL conical flask, 1.67% inoculum size and cultured at 28 ℃. Under the optimized condition, the XDH activity improved by 231.2% compared against its initial activity and reached 5.183 U/L. Moreover, the optimal reaction condition was: 20g/L substrate, 50 mmol/L glycine-NaOH buffer solution (pH=10.0) and reacted at 45 ℃, which resulted in the yield of 17.74% L-xylulose. In conclusion, this study lays a foundation for industrial production of L-xylulose by optimizing the fermentation and reaction conditions of a strain.