Study On Fermentation Technology Of GL-7-ACA Acylase Production By Genetically Engineered Bacterium And Enzyme Engineering

Glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) is one of important industrial enzymes for the production of 7-aminocephalosporanic acid (7-ACA) by two-step enzymatic process. Its yield and expression are precondition to improve the production of 7-ACA. The GL-7-ACA acylase gene is constitutively expressed in recombinant Escherichia coli BL21(DE3)pYW-GA, so the strategy of feeding mode plays a key role in the fermentation process.In this paper, it was researched the process control of recombinant Escherichia coli BL21(DE3)pYW-GA in fed-batch culture. The fed-batch fermentation for producing GL-7-ACA acylase was carried out in 5L autocontrol fermentor from the process of flask shaking culture, to reduce the concentration of acetate acid and highly expression, different feeding models were adopted in fermentation process, simultaneously controlled rotate speed and dissolved oxygen. Effect of constant rate, pH control and exponential feeding models on the cell yield and GL-7-ACA acylase activity were investigated. Under the pH8.0 control feeding condition, the maximal OD₆₀₀ reached 35, and the maximal activity was 3463.8U/L in pH7.0 control feeding.Immobilized Metal Ion Affinity Chromatography(IMAC) as a selective separation process are now gaining wide application in recombinant protein purification. A new metal chelating carrier (EIP-ARG-IDA-Co²⁺) was prepared to immobilize GL-7-ACA acylase. This support can effecifically purify and immobilize GL-7-ACA acylase with His-tag from evaluated proteins in one-step. On the process of immobilization, the optimum immobilization conditions of enzyme were as follows:enzyme load was 100U/g carriers time was 16h, pH was 7.0. The optimum temperature and pH of immobilized enzyme was 50℃and 8.0 respectively, it showed that immobilized enzyme was superior to free enzyme at pH and temperature, In addition, Michaelis constant, stabilities to heat, operational stability and carrier reuse were compared and studied.This study estabilished the optimal feeding mode and method of immobilizing GL-7-ACA acylase, it provided a basic work for large production of GL-7-ACA acylase and cephalosporins in industrial fermentation.