基因工程菌发酵表达核苷磷酸化酶条件的优化

Optimization of Gene Engineering Bacteria Fermentation Conditions for Expression of Nucleoside Phosphorylase

目的 优化苷磷酸化酶（包括嘌呤和嘧啶核苷磷酸化酶）基因工程菌的发酵表达条件。方法通过工程菌摇瓶培养，测定吸光度D值，考马斯亮蓝（Bradford）法测定蛋白，SDS—PAGE电泳和凝胶成像扫描分析表达量，优化表达条件；通过正交试验优化50L发酵罐发酵条件。结果摇瓶培养起始pH为7．0～7．2，于30℃培养4h，加入终浓度为0．4mmol／L的异丙基硫代半乳糖苷（IPTG）诱导8h后收获菌体，可得到较高的生物量和重组酶蛋白表达量。50L发酵罐的最佳条件为起始pH为7．0～7．2，于32oC培养4h，加入终浓度为0．4mm01／L的IPTG诱导9h后收获菌体，每升发酵液可得2g以上的酶蛋白。结论基因工程菌发酵表达核苷磷酸化酶产量较高，可工业化生产．为酶法合成核苷酸类似物的研究奠定了基础。

Objective To optimize the fermentation conditions of gene engineering bacteria for expression of nucleoside phosphorylase （including PNPase and PyNPase）. Methods By adopting the engineering bacteria shaking culture, the absorbance D value was deter- mined, the protein was measured by the Bradford method, the expression quantity was detected by the SDS- PAGE electrophoresis and the gel- imaging scanning, so the expression conditions were optimized; the orthogonal experiment was adopted to optimize the fermenta- tion condition of the 50 L fermentor. Results The initial pH was 7.0- 7.2 in the shaking culture and the incubation was performed for 4 h under 30 ℃, then isopropyl thiogalaetoside （IPTG） with a concentration of 0.4 mmol/L was added for 8 h induction and the bacteria were finally harvested, which could obtain higher biomass and recombinant enzyme protein expression quantity. The experimental results revealed that the optimal conditions of the 50 L fermentor were as follows： with the initial pH value of 7.0- 7.2, the incubation lasted for 4 h under 32℃, the bacteria were harvested after adding 0. 4 mmol/L IPTG for 9 h induction. Each liter of fermentation broth could obtain at least 2 g apoenzyme. Conclusion The expression of gene engineering bacteria can obtain the higher output of nu- cleoside phosphorylase, which can be used for the industrial production and establishes the basis of synthesizing the nucleotide analogues by the enzymatic method.