Breeding Of Bacillus Amyloliquefaciens And Study On Mesophilic α-amylase Production

Mesophilicα-amylase is a very important industrial enzyme which is widely used in grain processing, food, brewing, textile, pharmaceutical industry, etc. But in our country, the enzyme activity of mesophilicα-amylase is very low in compare with foreign competitors. As the study of mesophilicα-amylase is very little recently, it has practical significance for the development of enzyme preparation industry to carry out breeding of Bacillus amyloliquefaciens and fermentation improvement to improve the production ofα-amylase activity.In this paper, with theα-amylase activity as the index, the original strain Bacillus amyloliquefaciens was treated with ultraviolet, microwave and LiCl, and the mutant strains was screened by resistant plate with 2-DOG and tunicamycin. The Compound mutant condition: ultraviolet radiation for 6min, microwave radiation for 20s, LiCl 1.2%, screening condition: 2-DOG 5%, tunicamycin 50μg/mL. A stable genetic mutant named YL 1-5, whoseα-amylase activity is 19.3% higher than the original strain, was selected on screening-plates contain 1.2% LiCl.As the production of Bacillus amyloliquefaciens YL 1-5 mesophilicα-amylase(BAA) began at the logarithmic late phase of the biomass raising, it showed continuous producing feature-enzyme producing part-coupling with cell growth. The addition of 1.5% zeolite(40~60mesh), or 2% zeolite(20~40mesh), or 2% bentonite can effectively absorb NH4+ produced during fermentation, and the addition of 1.5% zeolite(40~60mesh) can improve enzyme activity by 11.88%. The optimized medium in shaking flask contains: corn meal 16%, cotton seed meal 8%, corn steep liquor powder 1%, zeolite(40~60mesh)1.0%, CaCO3 0.8%; The optimized culture conditions: using of baffled conical flask(250mL), volume of medium 25mL/250mL, initial pH7.0, seed age 16h, the inoculum size 5%(V/V), the culture temperature 34℃, rotation speed 190rpm. After optimized, the enzyme activity was 533.2U/mL, 27.19% higher than before. Because of the lower biomass yield, results of batch fermentation on fermenter was not as good as in shaking flask. After fed-batch from 30h till the end, the cell can grew better, which resulted in a higher enzyme activity-521.6U/mL.The characters of mesophilicα-amylase produced by Bacillus amyloliquefaciens YL1-5 include: the optimum temperature is 60℃, the optimum pH is 6.0. Under 90℃, the addition of 0.05mol/L Ca2+ can improve enzyme activity by 20% than without Ca2+. In the range of pH6.5~9.5, BAA can maintain high activity, stayed in pH8.0 buffer for 24h, only 3% of enzyme activity was lost, which means BAA can be stored in alkaline condition. BAA was definite activated with metal ion Na+,Li+,Mg2+, strong inhibited with Hg2+, little inhibited with Sr2+,Zn2+,Mn2+,Ba2+.