Screening Of Alkaline Protease Mutants And Application Research On Waste Proteolysis

A large amount of hard protein waste is generated every year in the world.If this kind of protein resources can be effectively used,it can not only reduce the environmental burden,but also supplement the existing protein resources,which helps to save industrial costs and reduce waste of resources.Both traditional physical and chemical treatments have certain drawbacks.The use of microbial fermentation and enzymatic hydrolysis to solve this problem is the future development trend,but for now,this application technology is not yet mature,and there are many Limiting factors,such as a single source of protease production strains,low enzyme activity,unsuitable enzymatic properties,difficult analysis of degradation products,etc.Therefore,this topic is to screen protease mutants with excellent performance and evaluate the alkaline protease and collagen of mutants Protease activity and analysis of its application of hydrolyzed hard protein.First,the obtained alkaline protease strains N76,G97,S99 mutants derived from Bacillus clausii were compared with wild-type alkaline protease(WT),and the relative enzyme activity changes of alkaline protease and collagenase were determined,and the alkaline protease was screened.The alkaline protease activity and collagenase activity of N76A mutant increased by 14.65%and 49.26%respectivelycompared with N76.The alkaline protease activity and collagenase activity of G97N mutantincreased by 31.30%and 11.81%compared with G97,The alkaline protease activity and collagenase activity of G97I mutant increased by 26.88%and 21.26%.The alkaline protease activity and collagenase activity of S99F mutant increased by 88.62%and 90.75%compared with S99,The alkaline protease activity and collagenase activity of S99G mutant increased by 35.45%and 141.46%.In general,among the mutations at three sites,the enzyme activity at position 99 was significantly improved compared to others.Then,optimize and amplify the fermentation conditions of the S99F mutant strain obtained by mutation,using a fermentation temperature of 33-35?,a rotation speed of 150-650r/min,on a 30 L fermentor to control the solution Oxygen is maintained at above 30%.Ammonia water and 1 mol/L phosphoric acid were added automatically during fermentation to maintain the pH of the fermentation broth at 7.3-7.5.At 24 hours,the reducing sugar content is less than half of the initial content,and feeding was started.Ensure the normal growth of bacteria and enzyme production.The final fermentation vigor reached 21200U/mL.The enzyme powder was prepared by spray drying method,and the activity of the enzyme powder is 155000U/g.Use the prepared alkaline protease enzyme powder to hydrolyze chicken feathers and paw skin to optimize the hydrolysis conditions.Under the condition of 55 ?,the initial pH 10.5,the amount of alkaline protease 800 U/g substrate,the liquid-to-material ratio is 30:1,hydrolysis time 3h,the chicken feather hadthe best hydrolysis effect and the product yield was42.32%.Under the condition of 45?,the initial pH 11.5,the amount of alkaline protease 2300 U/g substrate,the liquid-to-material ratio is 30:1,hydrolysis time 6h,the chicken paw skin hadthe best hydrolysis effect and the product yield was 52.34%.The method and hydrolysis process laid the foundation for industrial production applications.