Screening Of Alkaline Protease-producing Marine Bacteria Strain And Study On Fermentation Conditions

Protease is a kind of enzyme which can catalyze protein hydrolysis into small peptides and amino acids in a certain temperature and pH conditions. Alkaline protease can hydrolyze protein peptide bonds at the condition of alkaline pH. High molecular protein will degrade into low molecular peptides and all kinds of amino acids while protease adds in aquatic feed. Nutrients are easy to be digested and absorbed, improving the aquatic feed utilization rate. The environment of aquatic animal digestive tract is neutral or alkaline. So neutral or alkaline proteases are usually used as additives in aquatic feed.Marine microorganisms have their own unique advantages because of the diversities and uniqueness of the marine environment. The alkaline protease with specific activity of marine microorganisms is more compatibility as aquatic feed additive. And screening for marine bacteria provides good resources for probiotics in the aquaculture.In this project, a marine bacterial strain with higher alkaline protease-producing was isolated from73different marine bacterial strains which were screened from marine samples in the sea of DaLian, China. Based on the morphological and16S rDNA sequence analysis, the strain named as HS-A156was identified as Bacillus subtilis.The crude enzyme was extracted from the fermentation broth of the strain HS-A156. The results showed that the optimal temperature for the alkaline protease activity was40℃. Enzyme activity was quite stable at temperature from25℃to45℃. And the optimal pH was9.0. Enzyme activity was quite stable at pH from8.0to11.0.With single factor trial, the optimal carbon source was glucose and the optimal nitrogen source was the mixture of beef extract and yeast powder. The enzyme activity was enhanced in the presence of Ca+. With orthogonal experiment, the optimal protease-producing liquid medium was determined as glucose1.5%、beef extract1.0%、yeast powder2.0%and CaCl20.05%. The optimal culture conditions were obtained as the initial pH8.0, fermentation temperature30℃and inoculation quantity3%. Under the optimal conditions, the enzyme activity of the fermented broth of the strain HS-A156reached538U/mL, which is3.7times higher than that under original conditions. The study could provide a basis on application and development of the probiotics and aquatic feed additive in the future.