Study On The Production Of Aminopeptidase From Bacillus Subtilis Strain Bred By The Protoplast Transformation

Aminopeptidase is a kind of protein hydrolase which catalyzes the cleavage of aminoacids from the N-terminus of the polypeptide chain. The breeding of aminopeptidasehigh-producing strain builds a good foundation for the production of aminopeptidase.In this paper, the preparation of protoplast from leucine aminopeptidase(LAP)-producing Bacillus subtilis Zj016and its regeneration conditions were studied andoptimized. Then recombinant plasmid pMA-BSAP and the prepared protoplast from B.subtilis Zj016were mixed in hypertonic buffer. Mediated by polyethylene glycol (PEG), theprotoplast-DNA mutually cohered and the corresponding genetic transformation was achieved.After regeneration of cell wall under appropriate conditions, a recombinant transformed strainB. subtilis ZH-Zj016was successfully obtained, the capacity of enzyme production is16times of the original strain. The genetic properties of the recombinant strain were proven to bestable through successive passage experiments.The fermentation medium and conditions for B. subtilis ZH-Zj016were appropriatelyoptimized, and optimal fermentation conditions for shake flask were obtained: soluble starch6g/L, soy protein powder18g/L, yeast extract14g/L, glycerol2mL/L, K2HPO43H2O4g/L,MgSO40.5g/L, CoCl20.5mmol/L, Kanamycin50μg/mL, initial pH8.0, fermentationtemperature37oC, liquid volume50mL/250mL, rotation rate220rpm, inoculum volume7‰.After fermentation for30h, the enzyme activity can be up to125U/mL. On this basis, aseries of fermentation control strategies, such as the minimum dissolved oxygen (DO) control,temperature-shift culture and cell permeability regulation, were performed and the optimalfermentation conditions for15L fermenter was confirmed as follows: medium volume of8L,initial pH8.0,7‰of inoculum volume, agitation rate of250~500r/min, fermenter pressurewithin0.06~0.08MPa,40%DO upholding (DO is coupled with agitation rate), ventilationvolume of1.2vvm, culture temperature at39oC within15h and shifts to37oC thereafter.After fermentation for27h, LAP activity produced by the strain reached138U/mL.The extraction process of LAP from B. subtilis ZH-Zj016was preliminarily established:0.15%(v/v) of flocculant was added to fermentation broth, and filtration was performed toremove all bacteria cell. Then the enzyme extract was concentrated four times byultrafiltration with a30kDa PES membrane and under the operating pressure of0.2MPa. Thefinal recovery of LAP activity was66.73%.The colony morphology, aminopeptidase production conditions, fermentation period,utilization of raw material, and protease spectrum of three aminopeptidase-producing bacteria,B. subtilis Zj016(wild strain), B. subtilis ZH-Zj016(transformation strain) and B. subtilis GC(engineering strain) were analyzed and compared. Among them, B. subtilis ZH-Zj016hasobvious advantages, such as short fermentation period, improved equipment and raw materialutilization, increased intensity of production, reduced production costs and contamination, etc.