Novel Strain Producing Endoglucanase And Endo-xylanase:Strain Isolation,cloning And Expression Of The Enzyme Genes,and Molecular Modification Of The Two Enzymes

As an important component of the cellulase system,Endoglucanase?endo-1,4-?-D glucanase,EC3.2.1.4?can function randomly within the long chain of cellulose,cutting it at the?-1,4-glycosidic bond and degrading it into small molecule polysaccharides.Endo-xylanase?endo-1,4-?-D-xylanohydrolase,EC3.2.1.8?is the most important component of the xylanolytic enzyme system,and it can preferentially function at the?-1,4-glycosidic bond,degrading the xylans into xylo-oligosaccharide and small amounts of xyloses,xylobioses.We isolated a batch of bacteria from alkaline lakes and silt from Alkaline environment that produce cellulase and hemicellulase.Among these bacteria,we found that alkaliphilic Streptomyces sp.H31 can simultaneously produce neutral endoglucanases and alkaline endo-xylanases that can adapt to broad changes in pH.This paper carried out the cloning and expression of the newly discovered endoglucanase gene egh31 and the newly discovered endo-xylanase gene xynh31 in this strain and performed molecular modification of these enzymes.Details are as follows.1.Screening of Streptomyces sp.H31We screened and obtained a batch of bacteria from alkaline lakes and silt that produce cellulase and hemicellulase.We found that the enzymes produced by the H31 strain had high activity,of which the endoglucanase activity was 4.12 U/mL and endo-xylanase activity was51.4 U/mL in the fermentation broth.We used 16S rDNA sequence analysis in combination with external morphology to determine that this strain belonged to the genus Streptomyces.Compared with existing industrial enzyme production strains,Streptomyces sp.H31 was an extreme alkaliphile that can be cultured in highly alkaline conditions?pH 9.0?.Such conditions can inhibit contamination by other bacteria,which is conducive to large-scale fermentation.2.Cloning and prokaryotic and eukaryotic expression of the endoglucanase gene egh31The genomic DNA of Streptomyces sp.H31 was used as a template to successfully clone the endoglucanase gene egh31.The open reading frame?ORF?of this gene was 762 bp long and encoded 253 amino acids.The protein had a theoretical isoelectric point of 5.03 and a molecular weight of 26.58 kDa.BLAST analysis showed that the encoded amino acid sequence belonged to glycoside hydrolase family 12 and had the highest similarity?83%?with the?-1,4 endoglucanase from Streptomyces sp.CC77.This gene was effectively expressed in E.coli BL21 and the fungal expression system T.reesei and enzyme activity were 13.6 U/mL and 31.46 U/mL,respectively,and the specific activity was 153.7 U/mg And 161.2 U/mg.The most suitable temperature for this enzyme was found to be 60°C and the most suitable pH to be 7.This enzyme had good stability at pH3–11.We found that metallic ions such as Ni²⁺,Ca²⁺,and Zn²⁺had significant effects in promoting enzyme activity while Fe³⁺,Mg²⁺,and Al³⁺showed inhibitory effects on this enzyme.EDTA was found to have minor effects on enzyme activity,and this enzyme exhibited good tolerance in SDS and commercial detergents.Experiments showed that this endoglucanase,Egh31,had both neutral and alkaline endoglucanase activity and was a superior industrial enzyme with a broad suitable pH range.Integrated enzymatic characteristic and gene homology analysis preliminarily determined that the egh31 gene was a new?-1,4-endoglucanase gene.3.Cloning and prokaryotic and eukaryotic expression of the endo-xylanase gene xynh31Cation ion exchange chromatography was used to isolate the endo-xylanase produced by Streptomyces sp.H31.The molecular weight of this enzyme was 42 kDa and the theoretical isoelectric point was 8.18,and the specific activity was 671.63 U/mg.The most suitable pH for xylanase activity was 7.0-9.0 and most suitable temperature was around 50°C.This endo-xylanase had high enzymatic activity and stability under neutral and alkaline conditions and had good characteristics such as heat resistance,alkaline resistance,and resistance to many types of metallic ions.This enzyme's requirements match those common in the paper-making industry,which gives it good prospects for practical application.TAIL-PCR was used to clone the full-length endo-xylanases gene from the genome of Streptomyces sp.H31.BLAST analysis showed that the amino acid sequence of this gene had the highest homology with the family 10 xylanase from Streptomyces Halstedii Jm8 but had only 82%similarity.According to sequence analysis results in combination with differences in molecular weight,isoelectric point,and enzyme characteristics of this protein,we determined that the endo-xylanase gene xynh31 in Streptomyces sp.H31 was a new gene.E.coli was used to express this gene and highest enzymatic activity was 186.3 U/mL Using E.coli to express the gene,the highest enzyme activity was up to 186.3 U/mL,and the specific activity of the recombinant endo-xylanase was 628.4 U/mg.The recombinase was basically the same as the original enzyme.The recombinant enzyme and the original enzyme were found to have the same characteristics.4.Enzyme molecular modification of the endoglucanase Egh31 and xylanase Xynh31BLAST sequence analysis and protein structure homology modeling showed that the endoglucanase Egh31 lacked a binding domain.A binding domain from the Bacillus sp.III-3 endoglucanase was added at the C-terminus of the egh31 gene.The experimental results showed that the fusion enzyme containing the addition CBD exhibited an increase in enzyme activity by 30%at 55°C.In addition,enzyme activity was 40%higher than that of the original Egh31 under 1%SDS conditions.Experimental results showed that the heat stability and SDS resistance of the endoglucanase Egh31 increased in the presence of CBD.We here used bioinformatic methods for homology modeling of the 3D protein structure of the xylanase Xynh31 and prediction of its catalytic sites.Site-directed mutagenesis was used to construct the E171D/E279D mutant and verified that the predicted 171 and 279glutamate residues were active catalytic sites.Semi-rational design was used to select the 210lysine residue in Xynh31 protein for mutation into an arginine residue.Results showed that the most suitable reaction temperature of the mutant Xynh31-K210R was higher than the recombinant Xynh31 by 5°C.In addition,after storage at 60°C for 60 min,its residual enzyme activity was still above 60%.This shows that the introduction of arginine by semi-rational design successfully increased the heat stability of the xylanase Xynh31.In this study,we obtained a high-quality endoglucanase and endo-xylanase.This study also revealed the relationship between the structure and function of endoglucanases and endo-xylanases.This study enriched the choices for industrial applications for endoglucanases and endo-xylanases and may provide a foundation and reference for the rational design of industrial enzymes.