Xylanase Mutant Gly363 Saturation Engineering
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Solution Overview
Problem
Current xylanases used in industrial applications lack sufficient thermal stability and high catalytic activity, making them less effective in high-temperature environments common in industries such as feed and sugar production.
Innovation Solution
Development of xylanase mutants, specifically HwXy110A_G363R and HwXy110A_G363K, through saturation mutagenesis at the Gly363 site of the HwXy110A enzyme, which enhances thermal stability and catalytic efficiency, allowing for improved performance in high-temperature conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional xylanase is used in industrial applications, then the enzyme can perform basic hydrolysis function, but the thermal stability and catalytic activity are insufficient in high-temperature environments
Solution Approach 1:
The patent applies parameter changes by systematically varying the amino acid at position 363 (Gly363) to different residues (Ala, Val, Leu, Ile, Met, Phe, Trp, Tyr, His, Asn, Gln, Lys, Arg) to optimize both thermal stability and catalytic activity. This saturation mutagenesis approach changed the chemical parameters of the enzyme at a specific position to achieve improved performance in high-temperature industrial applications
Solution Approach 2:
The patent applies local quality by focusing modifications on a specific local region (position 363) of the xylanase molecule rather than the entire structure. By targeting this particular amino acid position for saturation mutagenesis, the invention achieved localized optimization that improved both thermal stability and catalytic activity without compromising the overall enzyme structure
2Reliability
If saturation mutagenesis is performed at Gly363 site, then thermal stability and catalytic efficiency are significantly improved, but the complexity of protein engineering process increases
Solution Approach 1:
The patent applies local quality by focusing modifications on a specific local region (position 363) of the xylanase molecule rather than the entire structure. By targeting this particular amino acid position for saturation mutagenesis, the invention achieved localized optimization that improved both thermal stability and catalytic activity without compromising the overall enzyme structure
Solution Approach 2:
The patent applies parameter changes by systematically varying the amino acid at position 363 (Gly363) to different residues (Ala, Val, Leu, Ile, Met, Phe, Trp, Tyr, His, Asn, Gln, Lys, Arg) to optimize both thermal stability and catalytic activity. This saturation mutagenesis approach changed the chemical parameters of the enzyme at a specific position to achieve improved performance in high-temperature industrial applications
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The mutants exhibit increased thermal stability and catalytic activity, with half-time at 80°C extended by 1.9 and 2.5 times and T50 values increased by 3°C and 5°C respectively, and specific activities enhanced by 24% and 53%, making them suitable for industrial applications in feed and sugar production.
Implementation Method 1
Xylanase is a key enzyme for the hydrolysis of xylan, which can cleave the β-1,4-glycosidic bond of the backbone of xylan, and thus produce small oligosaccharides or monosaccharides
Data Source
AI summary
The present invention discloses a kind of xylanase mutant and its preparation method and application, which relates to the technical field of genomic engineering and genetic engineering. Such mutant includes one or more mutants obtained by taking xylanase HwXy110A as female parent to conduct saturation mutagenesis to the site of Gly363. Specifically, relates to obtaining 19 mutants through site-directed mutagenesis, and then conducting yeast expression to them, after that, obtaining two mutants with significantly improved specific activity and thermal stability through screening of thermal stability and catalytic activity; the present invention can significantly improve the thermal stability and catalytic efficiency of xylanase through modifying the site of Gly363, and is of important guiding significance for improving the thermal stability and the catalytic efficiency of the 10th family of xylanases and other glycoside hydrolases as well as lays the foundation for its application in industrial production.