Cloning,Expression And Characterization Of Thermostable Chitinases From Cellvibrio Sp.79 And Cellvibrio Sp.OA-2007

Chitin is usually called chitin or chitin.It is a kind of high molecular polymer connected by multiple N-acetyl-D-glucosamine,which is connected to form a polysaccharide backbone by β-1,4 glycosidic bonds.Chitin has various sources,including bacteria,fungi,plants,insects and mammals.It is the second most abundant renewable biomass energy in nature.Chemical methods are often used in the industry to deal with them,which poses serious environmental pollution problems.The use of enzyme preparations can effectively control the damage to the environment.The chitinase that has been isolated from nature has problems such as poor thermostability,low enzyme yield and low enzyme activity.A chitinase with high thermal stability is obtained,which can realize separation,purification,packaging and transportation at room temperature,and saves expenses while omitting complicated cooling steps in production,and also reduces environmental pollution and energy waste.Moreover,in the high-temperature catalysis process,the growth of bacteria can be effectively reduced and the catalysis efficiency can be improved,which is of great significance to the industrial production of chitinase.Two chitinase sequences were obtained from the genes encoded by the thermophilic strains Cellvibrio sp.79 and Cellvibrio sp.OA-2007 that were screened from hightemperature compost in the early stage.Based on their sequence prediction analysis and homology modeling Among them,ChiWP comes from the GH18 family,and ChiPUA comes from the GH19 family.The two have no sequence homology.They are composed of signal peptide,Chitin Binding Domain(ChBD),and the link between the binding domain and the catalytic domain.The linker region and the chitinase catalytic domain are connected from the N-terminus to the C-terminus.Recombinant plasmids were constructed for its full-length sequence and catalytic region,and a total of 20 expression systems were tried.Finally,chitin was achieved in the Rosetta(DE3)-pCold expression system by fusion expression of the target protein and the Trigger Factor label.Soluble expression of ChiWPcd and ChiPUAcd,the catalytic region of the enzyme.The conditions for the induction and expression of recombinant chitinase were optimized.In the Rosetta(DE3)-pCold-TF system,the optimal induction conditions for ChiWPcd:0.6 mM IPTG at 16℃ for 22 h;the optimal induction conditions for ChiPUAcd:0.5 mM IPTG,induced at 16℃ for 20 h.The high-purity active protein sas obtained by two-step Ni affinity chromatography,and its enzymatic properties were characterized.ChiWPcd can exhibit the highest enzyme activity at pH 5.5 and 60℃,with an enzyme activity of 41.08 U/mg.After 70 min at 60℃,it can still maintain 85.4%of the remaining enzyme activity.The half-life is about 87 min.Incubating at 70℃ for 40 min can still maintain 97.2%of the remaining enzyme activity.After 40 min,the remaining enzyme activity begins to decrease drastically,and its half-life is about 55 min.When the enzyme is incubated at 80℃ for 10 min,the remaining enzyme activity It is 61.3%,and its half-life is about 18.5 min.ChiPUAcd can exhibit the highest enzyme activity at pH 7.5 and 65℃,with an enzyme activity of 29.11 U/mg.After 70 min at 65℃,it can still maintain 71.1%of the remaining enzyme activity,with a half-life of about 82 min.Incubating at 75℃ for 40 min,it can still maintain 79.5%of the remaining enzyme activity,and its half-life is about 53 min.When the enzyme is kept at 85℃ for 20 min,the remaining enzyme activity is 62.2%,and its half-life is about 28 min.Destroy the disulfide bonds at different positions in ChiWPcd and ChiPUAcd,and study their influence on the thermostability of the enzyme.Among the three pairs of disulfide bonds in ChiWPcd,the disulfide bond formed by cysteine at positions 48 and 124 causes hydrophobic interactions between adjacent Phe benzene ring residues and stabilizes the outer and outer cylinder structure of the catalytic zone.Therefore,the disulfide bond at this position Destruction has the greatest impact on the thermal stability of the enzyme,followed by the disulfide bond formed by cysteine 304 and 318,while the disulfide bond formed by cysteine 150 and 154 has little effect on the thermal stability of the enzyme.In the ChiPUAcd structure,there are disulfide bonds formed by cysteine 70 and 81 and disulfide bonds formed by cysteines 168 and 200.The disulfide bond formed by Cys168 and Cys200 is at the C-terminus of the catalytic region of the enzyme.Connecting the random coil loop at the C terminal to the sixth alpha helix of the chitinase ensures the stability of the C-terminal structure of the chitinase and plays an important role in stabilizing the main structure of the enzyme’s catalysis.