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