Screening,Identification,and Genomic Analysis Of Marine Chitinase-producing Strains And Evaluation Of Enzymatic Properties Of Chitinase

Chitin is a natural polymer compound with a content second only to cellulose,which exists widely in nature and is difficult to be effectively utilized because of its high crystallinity,low solubility and stable properties.Chitinase is one of the key enzymes for the degradation of chitin.Chitinase can effectively break down insoluble chitin and degrade it into lower polymeric chitinoligosaccharides（COS）with higher solubility and physiological activity.By studying the mechanism of action and properties of chitinase,we can better understand the degradation process of chitin and help to improve the utilization of chitin resources and degradation products.Therefore,it is of great importance in production life to explore chitinases that can degrade chitin efficiently.In this study,103 strains of chitinase-producing bacteria were screened from marine source samples,and one strain was identified as an efficient chitin-degrading bacterium.Identification of the 16S r RNA gene of the strain showed that the strain belonged to the genus Microbulbifer,which was named Microbulbifer sp.J1-2.Its 16S r RNA gene had the highest similarity with Microbulbifer celer KCTC 12973（CP087715.1）,with 98.51%similarity.The strain J1-2 can produce chitinase under the induction of chitinous substrate,its optimal enzyme production temperature is 30℃,the optimal enzyme production p H is6.0.The optimal temperature of chitinase secreted by the strain is 45℃,the stability is good below 45℃.The optimal p H is 6.0,the stability of chitinase is good in p H 4.5～8.0.The genome of strain J1-2 was sequenced and bioinformatically analyzed,and the results showed that the genome size of strain J1-2 was 4929267 bp,and the GC content was 61.3%.The genome contained a total of 4259 possible protein-coding sequences,and a total of 2526 functional genes were predicted.Six chitinase genes were annotated to the RAST and CAZy databases,and the KEGG database annotation indicated that the strain has a complete pathway for chitin metabolism.Bioinformatics analysis of the annotated chitinase genes showed that all six chitinases belonged to the GH18 family of chitinases.Among them,Chi1076 and Chi3178 contain a transmembrane region,and all six chitinases have a signal peptide at the N-terminal end of the amino acid sequence.All six chitinases contain a GH18 family chitin catalytic structural domain.In addition,Chi1076,Chi3120,and Chi3121 contain two chitin-binding domains,Chi1077 contains three chitin-binding domains,and Chi3314 contains not only two chitin-binding domains but also three PKD and IG domains.3D structural simulations showed that all of them have the typical eight-strandedβho barrel structure of GH18 family catalytic domain.Clonal expression of the gene chi3120,annotated in strain J1-2,was performed for the chitinase gene.The recombinant expression strain BL21（DE3）/p ET-28a sumo-chi3120 was successfully constructed by PCR amplification of the target gene using the genome of strain J1-2 as a template.The recombinant protein was obtained after 16 h induction of recombinant bacteria by 0.1 mmol/m L IPTG.After protein purification,the enzyme activity was 27.02 U/m L and the specific enzyme activity was 1.27 U/mg.The enzymatic properties of the recombinant chitinase were determined,and the results showed that the optimum temperature of the recombinant enzyme was 50℃,and the residual enzyme activity was more than 85%after holding at 45℃for 2 h.The optimum p H was 6.5（Na₂HPO₄-Na H₂PO₄）;the residual enzyme activity was more than 90%after incubation in phosphate buffer at p H 6.5 for 1 h,and the residual enzyme activity was more than 60%after incubation at p H 4.5～9 for 1 h.The enzyme activity was slightly enhanced by 5mmol/L Ni²⁺and inhibited by Cu²⁺,Fe³⁺,Mn²⁺,DTT,EDTA,SDS,and IPA.When colloidal chitin was used as the substrate,the Michaelis constant（K_m）of Chitinase Chi3120 was calculated to be 4.32 mg/m L,the maximum reaction rate(V_max)was 0.89μmol/（m L·min）,and the catalytic constant(K_cat)was 1.69 s^-1.The enzyme also hydrolyzed powdered chitin and chitosan.The recombinant enzyme hydrolyzed colloidal chitin as monosaccharides,chitin disaccharides and trisaccharides,presumably as an endogeneous chitinase.