Study On The Process Of Producing D-p-Hydroxyphenylglycine Catalyzed By Recombinant Hydantoinase And Carbamoyl Hydrolase Engineering Bacteria

As a big country in the use of antibiotics,China had a considerable demand for antibiotics and a shortage of antibiotics production.Among them,the important β-lactam antibiotics,due to their good antibacterial activity,had become essential and important antibiotics in life.D-p-hydroxyphenylglycine,an important pharmaceutical intermediate used to synthesize or semi-synthesize β-lactam antibiotics,had excellent pharmacological properties,such as low adverse effects,not easy to allergic,rapid absorption,blood with high drug concentration,long duration of action,excellent oral effect,and broad antibacterial spectrum,it was one of the most sought-after pharmaceutical intermediates in China.D-p-Hydroxyphenylglycine did not have natural products,so it can only be obtained by chemical synthesis.However,due to the industrial pollution caused by chemical synthesis,as well as environmentally friendly,green chemistry policy calls,it had high selectivity and strong catalytic efficiency.The green and environmentally friendly biological enzyme method had become the darling of today.In this project,Escherichia coli was selected as the host cell,and D-hydantoinase and D-carbamyl hydrolase engineering bacteria suitable for industrial production were constructed,and the fermentation and culture conditions were optimized to increase the expression of foreign proteins.Finally,the conditions for the catalytic production of the two enzymes were optimized,and a simple,efficient and environmentally friendly enzymatic synthesis process of D-p-hydroxyphenylglycin was obtained.This topic is based on single factor experiments to investigate the influence of inducer concentration,temperature,p H,carbon source and nitrogen source on the enzyme activity of DHD and DCB recombinant bacteria.Further,the response surface method is used to optimize the composition of the rate-limiting enzyme DCB medium.The optimal medium formula after optimization is: starch and glucose as the carbon source of DHD and DCB recombinant bacteria,soy peptone as the nitrogen source,and arabinose as the inducer.The result of fermentation in the 5 L fermentor was 53.4 g/L of DHD cells and 46.7 g/L of DCB cells.Finally,the conditions of D-HPG catalyzed by the two enzymes were optimized,and the molar yield could reach 96.19 %,which provided theoretical and technical support for the industrial production of D-hydroxyphenylglycine.