| Xylanase catalyzes the hydrolysis of xylan 1,4-?-D-xylosidic bond,which has important applications in many fields such as feed,food,energy conversion,etc.Among them,alkaline xylanase has been proven in the paper industry.In the process of improving pulp performance and reducing environmental pollution,the development of new enzyme resources and the study of alkali resistance mechanisms have received extensive attention in recent years.In this context,two GH10 family alkaline xylanases derived from Cellulomonas bogoriensis and Bacillus sp.NG-27 were used as research objects,and near the catalytic residues were studied by site-directed mutagenesis and sequence deletion.The nature of the amino acid and the short sequence rich in acidic amino acids may play a role in the alkali resistance of the GH10 family xylanase.The specific research results are as follows:1.Effect of amino acids near catalytic residues on enzyme alkali resistanceThe +1,+2 and +3 amino acids of the catalytic residues were studied by site-directed mutagenesis.At the +1 position of the catalytic residue,A of Xyn486 was mutated to V and N and V of BSX was mutated to A and N based on the change in hydrophobicity,and the amino acids of Xyn486 and BSX were each mutated to D and H based on the charge.The pH characteristics of the mutants were tested,and the results showed that the amino acid hydrophobicity was increased,and the optimum pH of the xylanase shifted to the alkaline and the alkali resistance was enhanced;Reduce the hydrophobicity of the amino acid,the optimum pH of the xylanase shifts to acidity,and the alkali resistance decreases;The introduction of a negative charge does not change the optimum pH and pH stability of the xylanase,but reduces the activity of the enzyme under acidic and basic conditions and the stability under alkaline conditions;The introduction of a positive charge also fails to alter the optimum pH of the enzyme,but reduces the activity of the enzyme under acidic and basic conditions and stability under acidic conditions.At the +2 position of the catalytic residue,the F of Xyn486 is mutated to I,A and N and the V of BSX is mutated to A and N based on the change in hydrophobicity,the amino acids of Xyn486 and BSX were mutated to D,H,E and D,H based on the charge,respectively.The pH characteristics of the mutants were tested.The results showed that the amino acid hydrophobicity was increased.The optimum pH of the xylanase shifted to the alkaline and the alkali resistance was enhanced.The amino acid hydrophobicity was lowered.The optimum pH of the xylanase was acidic.Shift,alkali resistance is weakened;introduction of negative charge does not change the optimum pH of xylanase and enzyme activity under acidic and alkaline conditions,but reduces the stability of the enzyme under extremely alkaline(pH 12)conditions,reducing The pH range of the enzyme activity;the introduction of positive charge fails to change the optimum pH of the enzyme,the pH range of the enzyme activity and the activity under acidic and basic conditions,but the stability of the enzyme changes to some extent,and the specific change trend is different.The enzymes show different.At the +3 position of the catalytic residue,the D of Xyn486 and E of BSX were mutated to I based on the change of hydrophobicity.Based on the charge,the amino acids of Xyn486 was mutated to Q,N,E,H and K,and the amino acids of BSX was mutated to N and H.The pH characteristics of the mutants were tested.The results showed that increasing the hydrophobicity of the amino acids had an effect on the optimum pH of the enzyme,the pH range of the enzyme activity,the activity of the enzyme under different conditions,and the pH stability of the enzyme.The stability of the enzyme under polar conditions(pH 12)is determined,while the other changes are irregular;The negative charge of the amino acid was eliminated that will reduce the alkali resistance of the enzyme;Introducing a positive charge may result Increasing the optimum pH of the enzyme reduces the stability of the enzyme under polar conditions(pH 12),but the effect of the enzyme on the activity under different conditions and the stability under acidic and neutral conditions is uncertain.2.The effect of short sequences rich in acidic amino acids on the alkali resistance of the enzymeA short sequence of three acidic amino acids that may be related to the alkali resistance of xylanase was determined by sequence alignment.The truncated mutants BSX-over-1,BSX-over-2 and BSX-over-3 of xylanase BSX,the truncation mutant Xyn486-over-2 of Xyn486,and the insertion mutation of Xyn486-insertion-1 and Xyn486-insertion-3 were constructed by fusion PCR.The properties were measured.The results showed that for the BSX truncation mutant,the optimum pH of BSX-over-1 and BSX-over-3 did not change compared with the wild type,but the enzyme activity and pH stability were reduced,especially in alkaline.The stability reduction under the conditions is most obvious;the BSX-over-2 is completely inactivated and cannot be tested for properties.The optimum pH of the insertion mutant Xyn486-insertion-1 was consistent with the wild type,but the activity under alkaline conditions and the stability in the range of pH<11 increased;The optimum pH of Xyn486-insertion-3 shifts to acidity,the enzyme activity decreases under alkaline conditions,and the pH stability decreases;Xyn486-over-2 is the same as BSX-over-2,the enzyme activity is completely lost and the nature test cannot be performed.The experimental results show that the presence of short sequences will increase the alkali resistance of the enzyme to some extent,but not every short sequence in all enzymes can improve the alkali resistance of xylanase.The results of this study provide experimental information for the clarification of the xylanase-resistant mechanism,and lay the foundation for the development of alkaline xylanase resources using protein engineering. |
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