Screening And Mutation Breeding Of High-yield Protease Bacillus Subtilis

Bacillus subtilis is one probiotic. It is not only present in the soil, air, water, and some present in the human and animal intestinal tract. B. subtilis has a strong resistance. In the harsh environment it can save lives by endogenous spores. At the same time it also has a powerful strain of enzyme production characteristics, primarily secreted extracellular protease, amylase, cellulase enzymes. In aquaculture enzymes from Bacillus subtilis can reduce fodder residue and decompose animal excreta, in addition can also promote digestion and absorption of aquatic animals. It is widely used for the preparation of probiotics. However, in practice, due to the effect of Bacillus subtilis temperature, can not achieve the desired enzyme standard.So screening of high activity of Bacillus subtilis and improving Bacillus subtilis’ production activity becomes very meaningful.In order to obtain a strain of Bacillus subtilis that produced prolific protease.This article study the screening and identification of Bacillus subtilis protease yield and improving Bacillus subtilis produced protease activity by UV mutagenesis.The main results of this study are as follows:1. screening and identification of Bacillus subtilis protease yield under high temperature environment.We obtained 8 strains from the pond water, which were named BY1 to BY8. According to the chracteristics of morphology, cultivation, physology and 16 SrDNA squence, they were identified as Bacillus sp. We used MEGA5.1 software to build the evolutionary tree of eight Bacillus and analyze their homology. The results showed that the strains in addition BY6 are in the same branch and have 99% homology with Bacillus subtilis. The strain of BY6 is e in the same branch and has 99% homology with B. lentus. What’s more, We further determine the outcome by Biolog biochemical identification.The producing protease strains and a commercial strain were measured using Casein transparent circle and Folin-Ciocalteu method.The results shows that strain BY7 has the largest transparent circle(5.4)under the culture temperature 37 ℃, significantly higher than other bacteria; Commercial strains BS2 produced protease activity is 16.61 U/m L,and strain BY7 protease activity is 43.28 U/mL. Its production protease activity is much higher than commercial strains BS2The strain BY7 is applied to tilapia aquaculture, with streptococcus pneumoniae as the control group. The lethal rates of perfusing BY7 and NS are 0%,but according to the concentration of streptococcus pneumoniae from low to high respectively is 10% 30% 30% and30%. So we can determine that the bacillus subtilis strain BY7 is safety.2. the UV mutation of Bacillus subtilisHaving an ultraviolet mutagenesis to strain BC2 of our laboratory, and after screened by casein medium, we will get 60-100 monoclonal srains. The strains producing a transparent circle were identified, and the transparent circle diameter/colony diameter was calculated. The ratio of top ten percent of colony the transparent circle diameter/colony diameter will be stored at-80℃, and choose ratio of biggest of the colony mutagenized again. We got B38 strain after six mutation screening. We determine the protease-producing activity between B38 strain and BC2 strain. Research shows that after cultured for 96 hours, the BC2 strain protease-producing activity is 27.68U/mL after the mutafacient protease-producing activity is 86.82U/mL, increased 3.14 fold.In order to detect ultraviolet mutagenesis effects on other enzyme-producing activity of strains. We determine the cellulase-producing activity between B38 mutant strain and B32 original strain by DNS method. Research shows that after cultured for 48 hours, the BC2 strain cellulase-producing activity is 0.85U/m L after the mutafacient cellulase-producing activity is 0.97U/m L. There were no significant difference between BC2 and B38.This study analyzes the genetic stability of the mutant strain B38. The results showed that after ten times of subculture the strain producing strains of protease activity was77.01 U /m L and cellulase activity was 0.913U/mL. They maintained 88.70% and 94.6% respectively. This shows that BY7 has genetic stability.In summary, In this study a high protease activity bacillus subtilis was screened from hot spring breeding pond under cultural temperature 37℃. And by using the method of UV mutagenic treatment improved the existing strains BC2 producing protease activity under under cultural temperature 25℃. The study on provides two strains of bacillus subtilis for Microecologics.