Heterologous Expression And Functional Study Of GH48 Family Exocellulase CelA From Bacillus Licheniformis

In recent years,the intensification of the resource crisis has led to a sharp increase in the demand for bioenergy.Lignocellulose is the most abundant biomass resource,and its biotransformation research has attracted much attention.Cellulase,especially processive enzymes,is considered to be an important component of the biotransformase system and has an important influence on the rate of transformation.In this study,the GH48 family exocellulase CelA derived from Bacillus licheniformis was cloned and heterologously expressed,and the enzymatic properties and related functional residues of the enzyme were studied.The influence of CBM domain on the catalytic properties of CelA was also studied.The specific research contents and results are as follows:?1?Cloning and heterologous expression of exocellulase CelA geneSequence analysis of exocellulase CelA derived from Bacillus licheniformis ATCC14580 showed that this enzyme contained 704 amino acids,which have no obvious signal peptide and transmembrane region,only one functional domain belonging to GH48family.The homology modeling of CelA using online software showed that CelA has a typical??/??6 barrel-shaped fold of the GH48 family enzyme,and further confirmed that CelA is a GH48 family cellulase.The celA gene was cloned from the Bacillus licheniformis ATCC14580 genome by homologous amplification and transformed into E.coli Rosetta?DE3?for heterologous expression.The recombinant protein was purified by affinity chromatography.?2?Study on the catalytic properties of CelAThe results of substrate specificity measurement showed that CelA had the highest enzyme activity when regenerated amorphous cellulose RAC was used as the substrate.The optimum pH value was 9.0,and the optimum reaction temperature was 50?,which had better temperature and pH stability.Mn²⁺and low concentrations of Na⁺,Fe²⁺and Fe³⁺promoted the enzyme activity of the recombinant enzyme;K⁺,Ca²⁺,Mg²⁺,Zn²⁺,Cu²⁺,Co²⁺and Ni²⁺inhibited the enzyme activity.Methanol,ethanol,isopropanol,?-mercaptoethanol,Tween-80,triton X-100,EDTA and high concentrations of glycerol significantly decreased enzyme activity.PMSF,urea and low concentrations of glycerol had no significant effect on enzyme activity.The smallest substrate of CelA hydrolyzed cellooligosaccharide is cellotetraose,the hydrolysate is a disaccharide molecule,and the product of the hydrolyzed pentasaccharide is a disaccharide and a trisaccharide,which is a cellobiohydrolase.?3?Study of CelA functional lociBased on the results of sequence and structure analysis,the catalytic residues,substrate binding residues and tunnel terminal loop residues which may be related to CelA function were identified.The functions of these sites were studied by site-directed mutagenesis.The results showed that E38,E49 and D228 are the catalytically active residues of CelA,in which E49 is a proton donor and D228 is a catalytic base,while E38also plays an important role in the hydrolysis of CelA.The enzymes of mutants E38A,E49A and D228N,their activity is only about 10%of the wild type.Y100,W312 and T107are residues in the tunnel entrance or tunnel that may be associated with substrate binding.The enzymatic activities of mutants Y100A,T107A and W312A are 34%,59%and 28%of wild type,respectively.These residues may be involved in guiding the sugar chain into the active site,among which aromatic residues play a greater role.After the amino acid residues E591 and E592 on the terminal loop were mutated to alanine with a small side chain,the remaining enzyme activities were 48%and 51%of the wild type,respectively,indicating that the flexibility of the terminal loop had little effect on product release,possibly they are directed to discharge the product from the cleft by generating hydrogen bonds with it.It is possible to direct the product out of the cleft by generating hydrogen bonds with it.?4?Effect of CBM domain on the catalytic properties of CelAThe fusion PCR technology was used to link the CelA catalytic domain with the carbohydrate binding domain CBM.The activity of the recombinant hybrid enzyme decreased sharply,only 9.8%of the wild type.The optimal reaction temperature and optimum pH of the enzyme did not change.But the stability is declining.