Engineering Of Bacillus Tequilensis 1,3-1,4-β-glucanase High Stability In Acidic Condition

1,3-1,4-β-glucanases can strictly hydrolyzingβ-1,4-glycosidic bonds to degrade high molecular weightβ-glucans into oligo-oligosaccharides.It was widely used in industries such as beer brewing,feed making,etc.However,industrial environment which have high temperature and low p H,severely limited their application.Although the specific activity and thermostability ofβ-glucanase have been fully improved,the acid resistance had not satisfied industrial applications.The mechanism of acid resistance is still a fuzzy picture.Therefore,the improvement of acid resistance ofβ-glucanases was significant for its industrial application.In this thesis,a Bacillusβ-glucanase with high acidic resistance and catalytic efficiency was successfully constructed by semi-rational strategies including sequence alignment,surface charge engineering and molecular dynamics simulations.Theβ-glucanase was highly expressed in Escherichia coli BL21（DE3）.Moreover,the mechanism ofβ-glucanases acidic resistance was revealed.The main results were as follows:（1）Twenty-six potential sites ofβ-glucanase acid-resistant were identified.Revelation of potential sites related to acidic resistance ofβ-glucanase.Twenty-six potential residue sites were selected to be mutated by three strategies.Nonconserved residues were identified as potential sites based on amino acid sequence alignment and secondary structure comparison.Glutamine and asparagine residuals on the surface were identified as potential sites by surface charge engineering.Flexible regions were identified as potential sites by molecular dynamics simulations.（2）Three single mutants with improved enzymatic properties was obtained.Potential residue sites were substitute for specific residuals by site-directed mutagenesis.Thirty single-point mutants were highly expressed in E.coli BL21（DE3）by plasmid p ET-28a（+）as vectors.Mutant I133L,V134A and Q1E had improved stability and catalytic efficiency at acidic condition after comparing enzymatic properties and kinetic parameters.（3）A double mutant Q1E/I133L for industrial application was obtained.Combined mutant Q1E/I133L,Q1E/V134A,I133L/V134A and Q1E/I133L/V134A were constructed by site-directed mutagenesis.Q1E/I133L was a good potential candidate for industrial application by comparing enzymatic properties.It could enhance 86.9%and 100.05%relative activity after incubation at p H5.0 and 6.0.The catalytic activity and k_cat/K_m were 1.17 and 1.77 times higher than wild type,respectively.（4）The acid tolerance mechanism ofβ-glucanase was preliminarily revealed.The exposed structure was responsible for decreased acidic resistance by fluorescence spectroscopy analysis.Increased hydrophilcity around catalytic pocket and improved negative charge of N-terminal was responsible for improvement of catalytic efficiency at acidic contion.The more stable catalytic pocket and N/C-terminal might be main reason for improved acidic resistance based on molecular dynamic simulations.