| Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions ,the gene of PGA was cloned into two expressing vector pKKFPGA and pSMLFPGA. Both the constructed plasmids pSMLFPGA and pKKFPGA contained the PGA gene ,trc promoter and rrnB transcript terminator, differ in the replicon and antibotic marker, pKKFPGA contained multicopy replicon(COLE 1) and ampicillin marker .while pSMLFPGA contained medium-copy replicon(p15A) and tetracycline marker. As both the recombinant plasmids and the host DH5a had no lacIq gene which encode lac represser , the trc promoter was not repressed by lac represser and always active .therefore the AfPGA could be constitutively expressed without IPTG induction in the host DH5 α .In the shaking flask, the optimum pH and temperature for AfPGA expression was 7.0 and 30℃. AfPGA expressed in DH5a was repressed by fast-utilized carbon source of high concentration, while slow-utilized carbon source such as sucrose, starch and dextrin enhanced enzyme production. Corn syrup had positive effect on the AfPGA production of DH5a/pSMLFPGA.but had a little effect on that of DH5a/pKKFPGA.Contrary to corn syrup, urea increased the AfPGA production of DH5a/pKKFPGA,but decreased that of DH5a/pSMLFPGA.For DH5a/pKKFPGA, the optimized culture medium, determined by the fractional factorial experiment ,was tryptone l0g/L, yeast extract 7.5g/L, base salt 0.75V/V, MgSO4 2 g/L and dextrin 20g/L,for DH5a/pSMLFPGA , the optimized culture medium was tryptone 20g/L, yeast extract 5g/L, base salt 0.75V/V, MgSO4 1.5 g/L and dextrin 40g/L.Under optimizing condition, the activity of AfPGA reached 4000U/L(NIPAB method) for DH5a/pKKFPGA and 4800U/L for DH5a/pSMLFPGA, with a cell-density-specific activity of more than 600U/L/A600.Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effuluent was added ammonium sulphate to Imol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-lmol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6 U/mg protein.Overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of various types of PGA precursors and then formed inclusion bodies in the cytoplasm and/or periplasm. The supernatant fraction and the precipitation fractions were analyzed by Western blotFor strain DH5 a /pKKFPGA, 5-10% PGA precursors formed asinclusion bodies in the cytoplasm while no inclusion bodies formed in the periplasm, this suggested most PGA precursors were transported to the periplasm and matured to active PGA and indicated that the maturation of PGA in strain DH5 α /pKKFPGA was limited by the translocation step. For strain DH5 α /pSMLFPGA, no obvious incusion bodies were formed in the cytoplasm and the pereplasm, this indicated that the protein synthesis flux of AfPGA was modulated and explained why the PGA activity from DH5a/pSMLFPGA was higher than that from DH5a/pKKFPGA. Although some PGA precursors formed inclusion bodies in the cytoplasm of DH5a/pKKFPGA, the amount only made up 5-10 % of all the AfPGA peptides, this explained why the expression level of AfPGA in DH5 α /pKKFPGA was a little lower than that in DH5 α /pSMLFPGA. |
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