Isolation And Identification Of Endophytic Bacteria Producing Amylase From Corn Seeds,Their Fermentation Conditions And Enzyme Activity Analysis

Corn is an important feed material.In order to improve the feed digestibility of animals,corn seeds endophytic bacteria had been isolated and identified in this study.A strain of Bacillus velezensis was selected and the optimized fermentation conditions for producing amylase and analyzed the enzyme activity.The research results were as follows:A strain D1 was isolated from corn seeds.The strain was identified by morphological observation 16S rDNA sequenced and analyze of phylogenetic tree analysis.The sequence was 99.9%similar to B.velezensis of,and named B.velezensis D1.In order to improve the enzyme production performance of the strain,the composition of the fermentation medium was optimized.The final medium composition was determined by adding 1.0 g of corn flour,1.5 g of soybean meal powder,0.04 g of CaCl2 and 1.4 g of K2HPO4 to 100 mL of medium.Then,the enzyme production conditions(culture temperature,culture time,initial pH.loading volume,inoculation amount,shaker rotation speed,and carbon to nitrogen ratio)of the strain were tested by single factor.The main factors affecting the fermentation conditions(culture time,culture temperature,initial pH,loading volume)were selected,and the optimal fermentation conditions were optimized by L9(34)orthogonal test.The result showed that the enzyme activity was the highest when the culture conditioned were 44?,60 h,pH 7.5 and 100 mL/250 mL.And the influence of four factors on enzyme production was:culture temperature>loading volume>culture time>initial pH.The enzyme activity of amylase was studied,it was found that the optimal reaction temperature of the enzyme was 60?,and the optimal pH was 8.The enzyme still had strong stability at 50?.It was stable between pH 5.0 and 6.0,and the enzyme has strong acid resistance.When metal ions are added to make the final concentration of the substrate solution 1 mmol/L,Ca2+and Cu2+ had obvious promotion effect on enzyme activity,Mg2+,K+,Zn2+,Al3+,Fe3+,Mn2+,Na+and Fe2+had different degrees of inhibition on enzyme activity,and Fe2+has the most obvious inhibitory effect on enzyme activity.The enzyme has a broad ability to break down cereal starch.