基因工程菌发酵表达核苷磷酸化酶条件的优化

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。

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

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.