Screening And Optimization Of Fermentation Conditions Of Bacterial Strain Producing Alkaline Protease

Alkaline protease is a group of important industrial enzyme and has been widely appliedin food industry, medical treatment, detergent industry, leather producing, brewing and otherfields. Nowadays, the enzymes for food industry are mainly produced by microorganism, andthe effect of alkaline protease is much better. Soybean peptide with good flavor and nutritioncan be obtained by hydrolyzing protein using alkaline protease. In order to screen the beststrain for soybean peptide production and industrial production, this article mainly discussedthe screening of strains producing alkaline protease, the optimization of the fermentationconditions of the strain and the experiment fermentation reactor.Fifty strains were isolated from the salt-soil from different areas and twenty of themwhich had clear zone on casein agar plate were fermented in flasks. One strain named PY41producing high yield of protease was screened. Based on morphological, physiobiochemicalcharacteristics and phylogenetic analysis of16S rDNA and gyr B gene sequences of the strain,it was identified that the strain PY41was belonged to Bacillus altitudinis. SDS-PAGEanalysis of the crude protease producing by PY41showed that the molecular weight ofprotease was26k and had a clear zone with protease activity.The effects of the medium components and the fermentation conditions on the proteaseproduction of Bacillus altitudinis PY41were studied. On the basis of single-factor test,response surface methodology of three factors and three levels was used to analyze the mainfactors affecting fermentation. Results showed that the optimal fermentation conditions wereestablished as follows: soybean cake6%, sucrose5%, glucose2%, CaCl₂0.07%, MgSO₄0.04%, KCl0.03%and Tween-800.05%with a initial pH10.0at37°C and a shaking speed of300r/min.The protease activity is up to1207U/mL with5d shaking flasks.We did the experiment of protease producing by Bacillus altitudinis PY41and Bacilluslicheniformis20203from flask-shaking fermentation to a10L fermentation reactor. The bestfermentation parameters were as follows: liquid coefficient0.6, inoculums concentration5%, temperature34°C, agitation300r/min, pH9.5-10.0and dissolved oxygen25mg/L-35mg/L.After optimizing, the protease activity of Bacillus altitudinis PY41and Bacillus licheniformis20203were1132U/mL and1876U/mL respectively,53.2%and130.5%higher than before.