Identification Of The Enzymes Related To The Chitin Degradation From Marine Metagenomics

Chitin degradation products such as chitosan and chitooligosaccharides have important application value in the fields of agriculture and food.The bioenzymatic degradation of chitin has the advantages of mild reaction,controllable conditions and environmental friendliness.The marine genome contains many genes related to chitin degradation,but at this stage it is just a collection of large amounts of genetic data,which makes it impossible for researchers to choose which genes are worthy of in-depth study.In this study,a set of bioinformatics methods to directly and efficiently mine chitin biodegradation-related enzymes from the marine metagenome was established.On this basis,two chitosanases and one chitin deacetylase were screened.This study laid an important theoretical foundation and technical support for the enzymatic preparation of chitosan and chitooligosaccharides.The chitosanase genes chis1754 and chis5 were excavated and isolated from the marine metagenome.Their functions were studied after heterologous expression in E.coli.The results showed that at the laboratory level,with 92%deacetylation chitosan as the substrate,the activity of CHIS1754 was up to 209 U mL-1.The optimal reaction temperature of CHIS1754 was 55?.After incubating at 60? for 20 minutes,the enzyme activity dropped sharply to 40%of the original enzyme activity.The optimal pH was 5.5.When incubated at 0? for 1h,the enzyme activity could still maintain 50%or more of the original enzyme activity within the range of pH 4.0-8.0.Most divalent metal ions had no significant effect on the activity of CHIS1754,while a small amount of Fe2+,Zn2+and Ca2+would inhibit its activity.The Km,kcat,and Vmax values of CHIS1754 were 5.28 mg mL-1,41.06 min-1,and 90.33 ?M min-1 mL-1,respectively,and kcat/Km was 7.78 mL mg-1 min-1.The analysis of its hydrolysates by the thin layer chromatography and high performance liquid chromatography-mass spectrometry found that the main products of CHIS1754 degrading chitosan were disaccharides,trisaccharides and tetrasaccharides,indicating that CHI S1754 was an endo-chitosanase.Subsequently,a CHIS1754 mutant with 15 single point mutations,CHIS1754T,was designed based on molecular evolution data to improve its thermal stability.The results showed that the Tm of CHIS1754T increased by 7.63?,and its residual enzyme activity at 6? for 20 minutes was four times that of the wild type.In addition,the kcat/Km of CHIS1754T was 4.8 times higher than that of wild type.The chitosanase CHIS5 had activity on chitosan with different degrees of deacetylation(DA75,DA80,DA90,DA92),and the activity on chitosan with a degree of deacetylation of 92%was the best.The optimum temperature and pH of the reaction are 60? and 5.5,respectively.It could degrade chitosan into chitosan oligosaccharide with a degree of polymerization of 2-5,which was an endo-chitosanase.In this study,a total of 22 chitin deacetylase genes were mined from the marine metagenomes of which CDA20 was soluble in E.coli.Enzyme activity detection and matrix-assisted laser analysis tandem time-of-flight mass spectrometry(MALDI-TOF-MS)analysis showed that CDA20 could hydrolyze the acetyl groups on the GlcNAc and one side of(G1cNAc)2 at the same time,while most of the published chitin deacetylases could hydrolyze chitin oligomers with polymerization degree ?2 or GlcNAc.The optimum temperature for this enzymatic reaction was 45? and the optimum pH was 8.0.When Ni2+or Co2+were added separately,the activity of CDA20 could be increased nearly twice,and the activity of Zn2+can also be increased by about 40%.When the enzyme was used in combination with chitosanase,it could effectively hydrolyze partially acetylated chitosan to chitosan oligosaccharides regardless of the mass concentration,which laied a certain foundation for the industrialization of green chitin degradation.In addition,chitin deacetylase CDA20 was mixed with chitosanases CHIS1754 and CHIS5,respectively,and it was found that the complex enzyme could effectively hydrolyze chitosan with different deacetylation degrees and generate chitosan oligosaccharides.In this study,chitosanases CHIS1754 and CHIS5 with high enzymatic activity and chitin deacetylase CDA20 with novel hydrolysis mechanism were mined from the marine metagenomic data.By constructing a multi-enzyme complex,a one-step technology platform for chitin oligosaccharide degradation was built in the laboratory,which provided a theoretical basis for the green degradation and high-value application of chitin in industry.