Studies On Optimize Fermentation Conditions For Producing Elastase From Pseudomonas Sp SWU-B

A strain capable of producing elatase-Pseudomonas sp preserved in our lab, was isolated and screened from an animal by-product, Pseudomonas sp was mutated by cooperation with complex mutation in the experiment for increasing elastase production. We got a high enzyme unit strain named Pseudomonas sp SWU-B. The dynamic trend of elastase secreting from Pseudomonas sp SWU-B was studied. It was also studied that the infection of diferent nutriment and growth condition on strain producing elastase. On the foundation of optimizing culture medium and researching various factors on Pseudomonas sp SWU-B producing elastase, fermentation condition for producing elastase by mutation strain was primarily ascertained. The main experimental results were summarized as followings:1. Three mutation agents possessed analogical mortal ratio trend, which positively related to the mutation agent's concentration. The curve of exist strains exhibited exponential trend. The result attested that Ultraviolet radiation (2537*10 10,20W, 30cm) irradiated 60s and doped in Ethylmethane Sulphonate (l%v/w) for 45min then inoculated into Lithium chlorate (0.3%w/w) flat. The elastase producing of mutation strain, named Pseudomonas sp SWU-B could increase 35.49% after repeated complex mutation. It illuminated that complex mutation could weaken repellence and saturation by single mutation agent.2. Complex carbohydrate promoted Pseudomonas sp SWU-B to secreting elastase. The fermentation supernatant's pH maintained 8.2+0.4 when C/N was low. Elastase was stabilization under alkaline circumstance. The optimization fermentation condition composed(g/L): glucose. 1%; sucrose, 3%; casein, 3%; NaaHPO4, 0.3%; MgSO4.7H2O,0.01%; start pH=7.2; 150rpm.3. Pseudomonas sp SWU-B could be suitable growth at 37 C, but 28 C was better for producing elastase. The stationary phase of the strain appeared at 16hr when gradually secreted more elastase. The fermentation culture medium was inoculated 4% seed strain of 12h seed age that reached the peak of producing elastase under 150r/min at 24h.4. Elastase secreting was interfered by the osmosis of strain cell wall and cell membranous. Tween80 could inspire strain to producing elastase at 0.2%(v/w) accretion. Penicillin could destroy peptidoglycan of cell wall to promote elastase secreting, but the mechanism need to be studied further.5. The elastase produced from Pseudomonas sp SWU-B was revulsive enzyme, which could be accumulated by mutagen substance. Not only elastin but also soybean protein induced elastase production. According to mutagen substance resource, it showed that 0.4%(w/w) soybean protein maximumly induced elastase secreting. Tryosine and Arginine was the better growth factor. It proved that 0.01%(w/w) Tryosine was good for elastase production.6. The strain could highly produce elastase when added nutrient and changed temperature at 12h of late-exponential phase. Defoaming substance increased dissolved oxygen that should promote the strain growth, thereby elastase accumulated than that without it.7. Pseudomonas sp SWU-B secreted 28U/mL on average after growing 24h in optimization fermentation medium. Semifinished enzyme got from stepwise salting-out of ammonium sulfate and dialyze possessed 100U / mg.pro.