MTHase Mutants for Trehalose Production via Amino Acid Substitution
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Solution Overview
Problem
Current methods for producing trehalose, such as using starch as a substrate with MTSase and MTHase, face challenges in yield and cost efficiency, with high-temperature enzyme systems offering high conversion rates but low protein expression, making them less suitable for industrial applications.
Innovation Solution
Development of MTHase mutants with increased enzyme activity by substituting specific amino acids, such as M140L, L202P, L218D, Y323G, F338S, and I404T, to enhance enzyme activity and substrate affinity, allowing for more efficient trehalose production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high-temperature enzyme systems are used for trehalose production, then trehalose conversion rate and thermal stability are improved, but protein expression level decreases
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of MTHase at specific positions (140, 202, 218, 323, 338, 404) to create mutants with improved properties. This molecular-level parameter change allows the enzyme to maintain high catalytic activity while achieving better protein expression levels in host organisms, thus resolving the contradiction between conversion rate and expression level.
2Ease of manufacture
If starch liquefaction liquid is used as substrate with MTSase and MTHase, then production cost is reduced, but yield and efficiency remain inferior to imported products
Solution Approach 1:
The patent improves trehalose yield by changing the kinetic parameters of MTHase through amino acid substitution. The mutants exhibit enhanced enzyme activity and altered substrate affinity, which increases the overall conversion efficiency and yield when using cost-effective starch liquefaction liquid as substrate, thus resolving the contradiction between low production cost and inferior yield.
3Productivity
If amino acid substitutions are made at positions 140, 202, 218, 323, 338, or 404 of MTHase, then enzyme activity increases, but protein structure complexity increases
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions at specific positions (140, 202, 218, 323, 338, 404) rather than throughout the entire protein structure. This localized modification approach increases enzyme activity while minimizing overall structural complexity changes, as only specific residues are altered to achieve the desired functional improvement.
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 MTHase mutants demonstrate significantly higher enzyme activity and improved substrate affinity, leading to enhanced trehalose production efficiency and reduced production costs, addressing the limitations of existing methods.
Implementation Method 1
maltooligosyl trehalose trehalohydrolase (MTHase) could be used to produce trehalose through the synergistic effect thereof by using liquefied starch as a substrate
Data Source
AI summary
The present invention discloses a maltooligosyl trehalose trehalohydrolase (MTHase) mutant and application thereof, belonging to the technical fields of gene engineering and enzyme engineering. The present invention provides a series of MTHase mutants to prepare trehalose, having a better effect. Further, the MTHase mutant is expressed in Escherichia coli BL21 (DE3) and the enzyme is optimized by fermentation, which can significantly increase the yield of enzymes.