Study On The Alkali Tolerance Mechanism Of Alkaline Xylanase Xyn466 From Cellulomonas Bogoriensis

Xylanase has important applications in the conversion of xylan compounds.However,most of the xylanases discovered so far are neutral enzymes,which are difficult to adapt to the high pH reaction conditions of the papermaking and feed industries.The use of protein engineering methods to modify and modify xylanase is considered to be an effective way to obtain alkaline enzyme sources,and the premise is to understand the alkali resistance mechanism of alkaline xylanase.In recent years,research in this area has received extensive attention.In this context,the alkaline xylanase xyn466 from the alkalophilic bacteria Cellulomonas bogoriensis was used as the research object,and its gene cloning and heterologous expression were carried out.The properties and composition of some amino acids of the recombinant enzyme were changed by site directed mutagenesis.The alkali resistance mechanism of alkaline xylanase was studied from the influence of amino acids near the catalytic residues and the composition of amino acids.The research contents and results are as follows.1.The recombinase Xyn466 was genetically cloned,and heterologous expression was realized in Escherichia coli.The specific enzyme activity of the pure enzyme component was 261.50 U/mg.The optimum pH value of the enzyme is 8.0,and it can still maintain more than 40% of the highest enzyme activity at pH 10.0;at the same time,Xyn466 has good pH stability.After 24 h of incubation in the pH range of4.0-11.0,the remaining enzyme activity It can be maintained at more than 70%,which is a good source of enzyme for the research of alkaline xylanase.2.The hydrophobicity and charge properties of the three amino acids(A167,F168 and A169)around the catalytic residue Glu166 of Xyn466 were mutated,and the activity and pH characteristics of the mutant enzyme were determined.The influence of alkali resistance,the following conclusions are drawn:(1)The changes in the hydrophobicity of amino acids at positions 167 and 168 reduce the activity of Xyn466 and at the same time have a significant impact on the pH characteristics of the enzyme.The increased hydrophobicity of amino acids reduces the activity of the enzyme under acidic conditions,increases the activity under alkaline conditions,and improves the pH stability of the enzyme.(2)The specific enzyme activity of the amino acid mutant at position 169 is not much different from that of Xyn466.The change in amino acid hydrophobicity does not have a significant impact on the pH characteristics of the enzyme,but the charge nature of the amino acid has a greater impact on the enzyme,eliminating the amino acid.The negative charge of the enzyme can shift the optimal pH of the enzyme to acidity,and the pH stability will also decrease;the introduction of positive charges can increase the optimal pH of the xylanase,reduce the activity of the enzyme under acidic conditions and increase its alkalinity Activity under conditions.3.Change the amino acid composition of the recombinase by introducing neutral hydrophilic amino acid residues or arginine residues into the xylanase Xyn466,and determine the activity and pH characteristics of the mutant enzyme to study neutral hydrophilic amino acids and The effect of arginine on the alkali resistance of xylanase,the following conclusions are drawn:(1)The change of neutral hydrophilic amino acid content does not have a significant effect on the specific enzyme activity of Xyn466,but it affects the pH characteristics of xylanase.When the content of neutral hydrophilic amino acids increases,the enzyme activity under acidic conditions is reduced,and the enzyme activity under alkaline conditions is increased,especially the activity under extremely alkaline conditions.At the same time,mutations in this direction increase Alkaline stability of xylanase.On the contrary,the reduction of neutral hydrophilic amino acids will weaken the alkali resistance of xylanase,and deamidation will shift the optimal pH of the enzyme to acidity,and also reduce the enzyme activity under alkaline conditions.(2)The change of arginine content has little effect on the enzyme activity of Xyn466,but it has a significant effect on the pH characteristics of xylanase.Although the increase of arginine content shifts the optimal pH of the enzyme to acidity,it greatly improves the activity and stability of the enzyme under extremely alkaline conditions(pH 11.0-12.0).At the same time,the results of multiple mutations showed that the more arginine content increased,the more significant the pH of the recombinase changed.(3)Based on the results of the sequence alignment,mutation of Lys in Xyn466 to Arg increased the ratio of Arg/Lys in the enzyme molecule.The results showed that the optimal pH of the mutant enzyme did not change significantly,but the enzyme was alkaline The activity under the conditions increased,while the activity under the acidic conditions decreased accordingly;at the same time,these mutations also improved the stability of the xylanase,making the enzyme stable under alkaline conditions(especially pH 12.0)improve.The research in this article provides information for the elucidation of the alkali-tolerant mechanism of alkaline xylanase,and also provides an enzyme source and possible transformation direction for obtaining alkaline xylanase suitable for industrial production.