Cloning,expression And Enzymatic Properties Of A Cellulase Gene With Partial Deletion In The Binding Domain

Cellulose is a renewable resource and is abundant and extremely rich in resources.People have been exploring how to use this resource fully and efficiently.Cellulase is widely used in bio-energy,textile,food,animal husbandry and other industries.At present,the use of cellulase to catalyze cellulose degradation to produce bio-new energy is one of the hot topics of research.Cellulase,which efficiently degrades cellulose,is a core issue in this subject.In this study,from the sample of involucrata,using a screening method of substrate plate,a bacterial strain with strong cellulolytic activity was screened,and 16S r DNA and gyr B genes were used for molecular identification.The Bacillus cereus strain was identified and named.M-3.The endo-cellulase gene of strain M-3 was cloned and named egt.By analyzing the gene sequence,this study first discovered a new cellulose mutated gene egt in nature that has an 8 bp deletion at the front of the binding region,resulting in frameshift and protein translation termination.Using the Overlapping PCR technique,the non-mutated cellulase gene was named egf.The mutant cellulase gene egt and the non-mutant egf were transformed into prokaryotic expression vector and transformed into E.coli BL21（DE3）for heterogenous expression.The difference between the two cellulases was compared.The gene egt is 1146 bp in length and encodes 381 amino acids with a molecular weight of 40.06 k Da.It has a catalytic domain,a linking zone and a partial binding zone.The gene egf is 1500 bp in length and encodes 499 amino acids.It has a catalytic domain,a linking region and a complete binding region with a protein molecular weight of 56.21k Da.Cellulase EGT and EGF both have only carboxymethyl cellulose degrading activity,and no defatted cotton enzyme,microcrystalline cellulase,filter paper enzyme and glucosidase hydrolyzing activity.Effects of different conditions on the enzyme production of engineering bacteria E.coli/pET-egt and E.coli/pET-egf.The experiment found that the enzyme production reached the highest value after 6 hours of induction;the optimal concentration of the inducer IPTG was 0.4 m M;the induction temperature had little effect on the enzyme production of the engineered bacteria;the engineering strain E.coli The optimum initial p H and optimum p H of pET-egt were 7.0,while the optimum initial p H and optimum p H of E.coli pET-egf were 8.0.The difference between the enzymatic properties of cellulase EGT and EGF.The optimal reaction temperatures of cellulase EGT and EGF were 45°C and 55°C,respectively.Cellulase EGF was more stable than EGT,and it was more resistant to high temperature.The optimum p H of EGT and EGF was 7.0,In the buffer of p H 7.0has good stability and still has 90%of untreated enzyme activity in 4 hours of treatment;Mn²⁺and Fe²⁺have activation effects on both,and Fe²⁺promotes the most,Zn²⁺,Ca²⁺,K^+,NH₄²⁺and Mn²⁺has a certain inhibitory effect on it;Km of enzyme EGT and EGF are 3.09 and 8.65 mg/m L,cellulase Partial deletion of the EGT binding region resulted in a stronger affinity for the enzyme with the substrate carboxymethylcellulose;the enzyme EGT specific enzyme activity was 98.4 U/mg,and the enzyme EGF specific enzyme activity was 53.53 U/mg.The specific activity of the cellulase EGT is higher than that of the enzyme EGF,and the catalytic hydrolysis ability is stronger.In this study,heterologous expression,optimization of fermentation conditions and enzymatic properties were compared by the cellulase gene egt and cellulase gene egf.It was found that partial deletion of the binding domain increased the specific enzyme activity of the cellulase.Lay the foundation for cellulase application and binding zone research.