The Recombinant E.COLI Fermentation And Producing (+)Gamma-Lactamase And The Enzyme Immobilization Research

(-)γ-lactam product can be used for the preparation of carbon ring nucleoside analogues, such as abacavir and peramivir.In this paper, we mainly studied the (+)γ-lactamase fermentation conditions optimization as well as the (+)γ-lactamase enzyme immobilization methods.In the fermentation tank (15L), we successfully developed a method suitable for growth of E. coli after trying different culture conditions. Firstly, we cultivated the E. coli until the beginning of logarithmic phase at 37℃,then we raised the cultivated temperature to 42℃ until the end of fermentation, in the meantime, we added lactose to induce producing enzyme at the end of logarithmic phase and once more we added the same amount of lactose after 6h, finally we obtained the max enzyme activity. The experiment of whole cell demonstrated that e.e value can reach more than 95% under the condition of 50℃,1h, and the reaction system consist of 0.16g wet bacteria,2ml substrate (5g/L), the results can meet the demand of industrial production.Then we studied ion exchange resin and HA resin as the immobilized carrier. We found that ion exchange resin can not be used in high concentration(200g/L) of substrate, because the enzyme molecule released easily from the carrier in high concentration of substrate. HA resin demonstrated better effects in immobilization experiment, it is 1200U/g of the enzyme activity capacity. The best crosslinking temperature, crosslinking time and glutaraldehyde concentration were 30℃,6h and 4%, respectively. Immobilized enzyme kinetic experiments showed that the Km value increased from 3.93g/L to 14.54g/L, and the Vmax decreased from 0.89g/L to 0.63g/L The optimum temperature (80℃)and pH value (pH 8.0) of both the free and immobilized enzyme were the same. However, the pH tolerance range increased after immobilization from pH 7.0-9.0 to pH 4.0-10.0. Subsequently, the values of the apparent activation energy (Ea) of the free and immobilized (+)Y-lactamase were 40.03kJ/mol and 33.61 kJ/mol, respectively. Experimental studies demonstrated that the best operating temperature is 50℃ and the resin and substrate consumption ratio is about 2:1. A reusability assay demonstrated that 50% of the enzyme activity remained after 60 repeated batch experiments. Experimental studies showed that the industrial production prospects is considerable of the immobilized enzyme.In addition, we also studied the new type of enzyme carrier. We modified the carboxyl of graphene oxide with epoxy chloropropane in order to obtain a new enzyme immobilization material.The infrared spectrum scanning results showed that epoxy chloropropane was reacted successfully on graphene oxide. But it is difficult to put the new carrier into application, because the machining process of graphene oxide is complex and lead to a high cost.