化学分子伴侣及诱导条件协同强化Thermotoga maritima α-葡聚糖磷酸化酶可溶性表达

doi:10.13560/j.cnki.biotech.bull.1985.2020-1473

摘要/Abstract

摘要：

为实现海栖热袍菌Thermotoga maritime α-葡聚糖磷酸化酶的高效制备。构建了产α-葡聚糖磷酸化酶重组菌株,并对发酵条件进行了优化。初步研究发现,重组菌株在37℃条件下进行诱导培养,摇瓶发酵24 h,酶活力仅有5.2 U/mL;SDS-PAGE蛋白电泳显示,重组酶主要以不可溶的包涵体状态存在,可溶性酶所占比例偏低。为减少包涵体的形成、提高重组酶可溶性表达水平,分别从诱导条件和分子伴侣两个方面进行了优化。首先,通过对诱导剂种类、诱导剂浓度、温度、诱导剂添加时间等诱导条件进行优化后,α-葡聚糖磷酸化酶的酶活力提高到27.0 U/mL,是优化前的5.2倍。其次,通过添加化学分子伴侣进一步提高重组酶可溶性表达水平,发现在发酵0 h添加20 mmol/L的化学分子伴侣（肌醇）后,最高酶活力为62.0 U/mL,是优化前酶活力的11.9倍;然而,共表达5种分子伴侣质粒pG-KJE8、pGro7、pKJE7、pG-Tf2和pTf16对α-葡聚糖磷酸化酶的可溶性表达均没有促进作用。

关键词: α-葡聚糖磷酸化酶, 重组表达, 诱导条件优化, 分子伴侣, 化学分子伴侣

Abstract:

In order to efficiently prepare α-glucan phosphorylase from Thermotoga maritima,a recombinant strain of producing α-glucan phosphorylase was constructed and the condition of fermentation was optimized. The preliminary study uncovered that the activity of the enzyme was only 5.2 U/mL when the recombinant strain was cultured under induction at 37℃ in shake flasks for 24 h. SDS-PAGE electrophoresis also revealed that the recombinant enzyme mostly existed as insoluble inclusion bodies,while the soluble protein in the supernatant was very low. In order to reduce the formation of inclusion body and to increase the soluble expression of recombinant α-glucan phosphorylase,both of the induction conditions and chaperones were optimized. Firstly,the induction conditions,including inducer types,inducer dosage,temperature,and induction time were optimized in shake flasks. Under optimized induction condition,the maximal enzyme activity reached 27.0 U/ml,which was 5.2 times of that before optimized. In addition,it also showed that the best chemical chaperone was inositol and the optimal dosage and additive time of inositol was 20 mmol/L and 0 h,the maximal enzyme activity was 62.0 U/mL,which was 11.9 times of that before optimized. However,the molecular chaperone plasmid（pG-KJE8,pGro7,pKJE7,pG-Tf2 and pTf16）did not enhance the soluble expression of recombinant α-glucan phosphorylase.

Key words: α-glucan phosphorylase, recombinant expression, optimization of induction conditions, molecular chaperones, chemical chaperones

段绪果, 张玉华, 黄婷婷, 丁乾, 栾舒越, 朱秋雨. 化学分子伴侣及诱导条件协同强化Thermotoga maritima α-葡聚糖磷酸化酶可溶性表达[J]. 生物技术通报, 2021, 37(8): 233-242.

DUAN Xu-guo, ZHANG Yu-hua, HUANG Ting-ting, DING Qian, LUAN Shu-yue, ZHU Qiu-yu. Synergetic Enhancing the Soluble Expression of Thermotoga maritima α-Glucan Phosphorylase by Chemical Chaperones and Induction Condition Optimization[J]. Biotechnology Bulletin, 2021, 37(8): 233-242.

图/表 7

图1 重组菌株及对照菌株发酵产α-葡聚糖磷酸化酶SDS-PAGE电泳图 M：分子量标准;1：重组菌株细胞可溶性组分;2：重组菌株细胞非可溶组分;3：对照菌株细胞非可溶组分;4：重组菌株细胞可溶性组分

Fig. 1 SDS-PAGE analysis of α-glucan phosphorylase pro-duced by the recombinant strains and the control strain M: protein standard; 1: soluble fraction of recombinant strain; 2: insoluble fraction of recombinant strain; 3: soluble fraction of the control strain; 4: insoluble fraction of the control strain

图2 乳糖浓度对重组菌生长、产酶的影响（A）和细胞破碎上清SDS-PAGE电泳图（B）

Fig. 2 Effect of lactose concentration on the recombinant strain growth and enzyme production (A) and the SDS-PAGE analysis of cell disruption supernatant of the recombinant strain (B)

图3 IPTG浓度对重组菌生长、产酶的影响（A）和细胞破碎上清SDS-PAGE电泳图（B）

Fig. 3 Effect of IPTG concentration on the recombinant strain growth and enzyme production (A) and the SDS-PAGE analysis of cell disruption supernatant of the recombinant strain (B)

图4 诱导温度对重组菌生长、产酶的影响（A）和细胞破碎上清SDS-PAGE电泳图（B）

Fig. 4 Effect of induction temperature on the recombinant strain growth and enzyme production (A) and the SDS-PAGE analysis of cell disruption supernatant of the recombinant strain (B)

图5 诱导剂添加时间对重组菌生长、产酶的影响（A）和细胞破碎上清SDS-PAGE电泳图（B）

Fig. 5 Effect of adding inducer at different times on the recombinant strain growth and enzyme production (A) and the SDS-PAGE analysis of cell disruption supernatant of the recombinant strain (B)

图6 化学分子伴侣种类（A）及肌醇浓度（B）对重组菌生长、产酶的影响空白：blank;2- 苯乙醇：2- phenyl ethanol;甘氨酸：glycine;海藻糖：trehalose;苯甲醇：phemethylol;甜菜碱：betaine;L- 脯氨酸：L- proline;氧化三甲胺：trimethyl aminoxide（TMAO）;肌醇：inositol

Fig. 6 Effect of different chemical chaperone (A) and inositol concentration (B) on the recombinant strain growth and enzyme production

图7 重组菌在最佳条件下生长曲线和产酶曲线

Fig. 7 Growth curve and enzyme production curve of recombinant strain on the optimal condition

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