Studies On Immobilization Of PGA And Expression Of PGA Gene In Eukarya

Penicillin G acylase (PGA) catalyzes the hydrolysis of penicillin G into 6-aminopenicillanic acid(6-APA). As 6-APA is the key intermediate in the production of semisynthetic penicillins, which is of great industrial interest.A pair of primers were designed based on the published PGA gene sequence of Bacillus megaterium in GenBank, the PGA gene from Bacillus megaterium 1.1741 was amplified in this study. Cloned the PGA gene into pYES2(amp⁺ ura⁺) vector controlled by T71ac promoter and expressed in Saccharomyces cerevisiae H158(his^-trp^-leu^- ura^-). 4 recombinants of Y1, Y2, Y3, Y4 were obtained, the PGA activity was detected in the culture liquid without the inducement of penylacetic acid. The PGA activity of Y4 is the highest of all, reached to 0.75 U/ml, which also has potential to enhance with ferment optimization. The PGA gene sequence was also determined, which showed high homology of 97.1ï¼…, 99.8ï¼…and 99.8ï¼…. when compared with the PGA gene sequences of three other Bacillus megaterium in GenBank.The immobilized PGA has played an important role in the manufacture of semi synthetic antibiotics. Embed method was took to make the surface functional magnetic microspheres by Fe₃O₄ particles and NaAlg. the enzyme molecules can be immobilized on the surface of the supports, so that to enhance the stability and binding efficiency. The effects of immobilized conditions were investigated. The results showed that the appropriate immobilization conditions of PGA are pH 7.5, the temperature 37℃, the reaction time 4 h, and the amount of PGA 333.3 U/g(dry weight). Under the above conditions the immobilized enzyme activity, binding efficiency and expression are 173.6 U/g(dry weight), 74.95ï¼…, 52.08ï¼…, respectively. the optimal pH and temperature of the immobilized enzyme were 7.5 and 40℃for hydrolysis of penicillin G, respectively. The immobilized enzyme had good operative stability. In addition, the Michaelis-Menten kinetic constants were evaluated for the immobilized and the freely soluble enzyme, they were 0.245 tool/l and 0.421 mol/l, respectively.