Development Of Proteases And Chitinases And Their Applications In Shrimp Shell Wastes Utilization

The ocean is rich in biomass such as protein and chitin,among which chitin is the secondlargest biomass except for cellulose in the nature.With the rapid development of aquaculture,the seafood processing industry produces a large amount of waste every year,especially the shells from crustaceans.Taking shrimp as an example,more than 50% of the shrimp weight is not the meat and cannot be eaten directly.In Guangdong Province,there are hundreds of thousands of tons of discarded shrimp shells produced by the shrimp processing industry each year.These wastes are often thrown into garbage dumps or the ocean,causing serious pollution and waste of resources.Shrimp shell powder made from waste contains about 50% protein,20% chitin and valuable antioxidants such as astaxanthin.However,these components are closely combined with each other in the shrimp shell and cannot be effectively utilized for animal digestion and absorption.The price of shrimp shell powder is only 450 RMB per ton,which is much lower than that of fish meal(10,000 RMB per ton)but with comparable nutritional value.Traditional physical and chemical processing techniques such as strong acid and alkali will cause a large amount of waste containing protein and other valuable fractions,and serious pollution.Protease and chitinase hydrolysis of shrimp shell is the most effective and environmentally friendly technology for its high-value utilization.At present,the lack of efficient protease and chitinase has become the bottleneck of this technology.In this study,the Pichia pastoris and Bacillus subtilis expression system was used to express exogenous proteases and chitinases genes.Five aspartic proteases and one chitinase with higher enzyme activity were obtained.They are P6281(321.8 U/m L)from Trichoderma harzianum,Candidapepsin from Candida tropicalis,SAPP1 from C.parapsilosis,Saccharopepsin and rennet M3 from Mucor circinelloides,and chitinase Chit46(31.4 U/m L)from T.harzianum.After purification by nickel column affinity chromatography,the enzymatic characteristics of P6281 and chit46 were analyzed.Their enzymatic production was at a high level compared to similar studies,laying the foundation for subsequent applications.The hydrolysis of shrimp shell waste by proteases and chitinase was studied.The results showed that aspartic protease P6281 and Saccharopepsin had the best effect for shrimp shell protein hydrolysis.The protein in shrimp shell wastes was recovered by two-step enzymolysis.Under the optimal conditions,the recovery rate of shrimp shell protein reached 91.8%.The amino acid composition of the two shrimp shell protein hydrolysates was analyzed,and the essential amino acid contents of these hydrolysates were 45.1% and 49.1%,respectively.The deproteinized shrimp shell was further treated with chitinase Chit46.Under the optimal conditions,the chitin recovery reached 88.9%.The composition of the chitin hydrolysate was analyzed and it was found to contain chitin oligomers with a polymerization degree of 2-6 and three other higher polymerization degrees.After removing the protein and chitin,the shrimp shells are extracted with ethyl acetate,and the astaxanthin can be easily extracted.The extracted product has a potent antioxidant activity.The enzymatic process avoids the use of HCl and Na OH in the traditional process,reduces environmental pollution.The mild reaction conditions avoid the destruction of the structure and activity of various components in the shrimp shell.Based on the previous step of recycling shrimp shell wastes,the chitinase was modified,and the carbohydrate-binding modules derived from B.subtilis was fused to Chit46.Three fused chitinases were successfully obtained,and their specific enzymatic activity and substratebinding activity were significantly improved.The shrimp shell wastes could be directly hydrolyzed by chimeric chitinases without the pretreatment with acid proteases,and the chimeric chitinases break the restriction that most chitinases cannot directly process shrimp shells.The hydrolysate is a good source of carbon and nitrogen,which can well support the growth of microorganisms.The in vitro simulated digestion experiments showed that the digestibility of the unhydrolyzed residue was much higher than that of shrimp shell powder.The nutritional value of the processed shrimp shell waste was improved.In summary,on the basis of high enzymatic activity aspartic proteases and chitinase,this study has focused on the recycling of shrimp shell wastes and established green processes for comprehensive utilization of shrimp shell wastes to avoid the use of strong acid and alkali reagents in traditional processes.The method achieved both great economic and practical value.