Screening Of Marine Cellulase-producing Microorganism And The Cloning Of Cellulase Gene

Cellulase is the most abundant renewable biopolymer and the most widely distributed and abundant carbohydrate on earth.There are many materials containing cellulase in the environment,which are difficult to be degraded in traditional organic solvents and easy to cause pollution to the environment.Cellulase is a complex family of hydrolases that can hydrolyze cellulase into glucose.It can decompose cellulase into small molecules of sugars,which are more conducive to the conversion of cellulase into other substances.The abundant cellulase resources can be applied to all aspects,so the optimization of cellulase bioconversion process will alleviate the current problems of insufficient food,non-degradable waste and dependence on fossil fuels.In this paper,a cellulase producing Marine bacterium was screened from the mixed water samples of intertidal sand mud in heishi reef of Dalian by CMC medium and Congo red staining.According to morphological,physiological and biochemical characteristics,16S r DNA identification and phylogenetic evolutionary tree analysis,this strain belongs to Herbaspirillum huttiense,and named as Herbaspirillum huttiense J-1.At the same time,the fermentation conditions affecting cellulase production were also optimized,and the optimal conditions for enzyme production were obtained as follows:p H was 8.5,inoculation quantity was more than5%,temperature was 30?,rotation speed was 170 r/min,carbon source was CMC-Na,nitrogen source was peptone.Based on single factor experiment,three factors,p H,CMC-Na and peptone were selected for orthogonal experiment,the optimized fermentation conditions were as follows:p H was 8.0,CMC-Na was 1.5 g,and peptone was 1.5 g.and the cellulase activity can research as high as 10.498 U/m L after the optimization.Using Herbaspirillum huttiense J-1 genomic DNA as template and Cellulase-A/Cellulase-S as upstream and downstream primer,about 1300 bp gene fragments was amplified from J-1genomic DNA,and then it was connected with p UCm-T vetor,transferred into E.coli DH5?cells and identified by colony PCR,recombinant plasmid PCR,recombinant plasmid double enzyme digestion and sequencing.The results showed that the amplified fragment was 1235 bp with an open reading frame,which encoding a 411 amino acid composition protein.The BLAST showed that the homology of the amplified fragment with the cellulase gene sequence of WP?039788877.1 was 100%.The results of phylogenetic tree construction showed that the protein sequence similarity of the amplified fragment and Herbaspirillum Huttiense WP?039788877.1(Herbaspirillum Huttiense IAM 15032)was 100%,so it was inferred that the cloned fragment was a cellulase gene fragment.Bioinformatics analysis showed that the proposed cellulase formula was C₂₀₁₈H₃₁₃₁N₅₆₇O₅₇₄S₁₃,the molecular weight was 44936.29 Da,the isoelectric point was 8.86,the instability coefficient was 40.86,the GRAVY value was-0.188,and it was hydrophilic protein.The secondary structure is mainly composed of spiral and irregular curl.The pupative translation cellulase has a signal peptide sequence,and the shear site of the signal peptide is located between the 36th and 37th sites,which proves that the target protein is a secretory protein.Phosphorylation of proteins is mainly concentrated on the tyrosine,serine and threonine residue,and there are 29 potential phosphorylation sites on the peptide chain.its tertiary structure is mainly composed of the helical helix,and random Random coil,had a high similarity with some known cellulase.The target protein has a PRK11097 domain,which is consistent with the conserved domain of Glyco?hydro?8 family in the sequence of 19-407.It proves that the pupative protein contains the conserved domain of glycolysylase family and has the hydrolytic function of endo-1,4-d-glucanase.It also contains 48 single restriction sites so that different primers can be designed for different experiments by using different restriction sites.The expression plasmid of p ET-21a-cellulase was constructed and transferred to Escherichia coli BL21(DE3),after identification,the target fragment was correctly connected to PET-21a.The target protein expression was induced by 0.5 mmol/L IPTG under 20?and37?,respectively,and SDS-PAGE analysis was performed.The results showed that the molecular weight of the protein was about 45.0 KDa.Western Blot results showed that the target protein could be detected in the inclusion body at 20?.