Study on Screening and Fermentation Conditions of High-yield Neutral Protease Strains

Abstract: Neutral protease is one of protease preparations earliest,found by human.Neutral protease is used widely at food industry,washing industry,leather industry and medical areas.At present,most protease applied in markets is bacteria protease.Screening of high-yield neutral protease strains is also the key point of recent studies.There are many ways to improve neutral protease yield,but simple and effective mutagenesis ways are also research problems.A neutral protease strain N19 was gained from soil and identified as Bacillus Subtilis. A mutant strain UMN-26 producing neutral protease was gained from the initial bacillus strain N19 treated with ultraviolet radiation,HNO_2 and microwave compound induction mutation.UMN-26 can produce 803U/mL neutral protease.Compared with that of the initial strain,the enzyme productivity of the strain has increased by 408%.After being propagated for ten generations,UMN-26 gets a stable heredity.It is obvious that,using physics and chemistry compound induction mutation is effective approach to increase neutral protease yield.Studying on the fermentation and production conditions of UMN-26,we gained the optimum culture medium components and the optimum culture conditions.Many factors including carbon source,nitrogen source,inorganic salt,Tween-80 were investigated in shake flask level.It was found that the concentrations of yeast-extract,corn steep liquor and sucrose had the more obvious influences.The orthogonal test of the three factors showed that the optimized culture medium was:yeast-extract 2%,corn steep liquor 1.5%, sucrose 1.5%.Through shake flask experiments,it was acquired that the best culture conditions:pH6.5,37℃,240rpm,40mL/300mL,and 2.5%(V/V)of bacterium culture for 12h.Under such conditions,the neutral protease activity reached 1378U/mL,which was 1.72 times of the initial strain.Compared the fermentation course curve of N19 with that of UMN-26,showed that biomass and pH value not occurring conspicuous change.The curve of protease activity have two peaks and the ability of protease production maintain a high level after the first peak,so we can think that UMN-26 is more suitable than N19 for manufacture.Researching the characters of the fermentation enzyme liquid,obtained results ??showed that:the optimal reaction temperature of UMN-26 was 65℃.As for the protease's thermal stability,only 50%of its activity was remained after 30 minutes incubation at 65℃and protease activity lost quickly with higher temperatures.The UMN-26 showed high activity from pH6 to pH8.0 and its optimal pH for activity was 7.0,belonged to the neutral protease.The test of pH stability indicated that the UMN-26 was more stable in pH 6.5~8.0, most stable in pH7.0.The protease activity was inhibited by EDTA(about 30%of the activity was lost),but was stimulated by Fe~(2+)(more than three times of the initial activity was achieved).This paper researched and established the fermenting and protease effecting conditions of UMN-26.The results were significant for have settled rationale for utilization of UMN-26…
Key words: neutral protease; bacillus; induction mutation; characters of enzyme

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