Beta-Galactosidase Proline Substitutions for Thermal Stability
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Solution Overview
Problem
Existing β-galactosidases, particularly those derived from Bacillus circulans, face stability issues, including heat resistance, which limits their practical use in industrial oligosaccharide production, as modifications aimed at improving enzyme properties often compromise stability.
Innovation Solution
Identification of the steric structure of BgaD-D, a key β-galactosidase enzyme, followed by targeted amino acid mutations, specifically substituting proline at specific sites (K166, G307, and A833), enhances the enzyme's heat resistance through structural stabilization, leading to improved thermal stability and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If enzyme modifications are made to improve enzyme properties, then enzyme activity is improved, but heat resistance is decreased
Solution Approach 1:
The patent applies parameter changes by systematically varying amino acid residues at specific positions (166, 307, and 833) in the β-galactosidase sequence. Through site-directed mutagenesis, different amino acids were substituted at these positions to optimize both enzyme activity and heat resistance, ultimately identifying proline substitutions that simultaneously improved both properties.
Solution Approach 2:
The patent applies local quality by focusing modifications on specific local regions of the enzyme rather than global changes. The three key positions (166, 307, and 833) were identified as critical hotspots where localized amino acid substitutions could independently or synergistically affect both catalytic activity and thermal stability, allowing precise optimization of specific enzyme properties.
2Temperature
If reaction temperature is increased to improve substrate solubility and avoid contamination, then substrate solubility is improved, but enzyme stability is decreased
Solution Approach 1:
The patent changes the thermal parameter of the enzyme by modifying its amino acid composition at key positions. The proline substitutions at positions 166, 307, and/or 833 fundamentally alter the enzyme's thermal stability profile, enabling it to maintain catalytic activity at higher temperatures (40-60°C) where substrate solubility is improved and bacterial contamination is reduced.
3Productivity
If substrate concentration is increased to improve transglycosylation efficiency, then reaction efficiency is improved, but heat resistance requirements increase
Solution Approach 1:
The patent modifies the enzyme's amino acid parameters to enhance its heat resistance, thereby enabling the enzyme to function effectively at higher temperatures where substrate concentration can be increased. The proline substitutions allow the enzyme to maintain stability and activity under the high-temperature, high-substrate-concentration conditions required for efficient transglycosylation.
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 modified β-galactosidase exhibits enhanced heat resistance and activity, enabling more efficient production of oligosaccharides at higher temperatures, reducing bacterial contamination, and increasing productivity, thus advancing its practical application in industrial processes.
Implementation Method 1
β-galactosidase (EC 3.2.1.23) is an enzyme which hydrolyzes a β-D-galactoside linkage to release D-galactose
Implementation Method 2
β-galactosidase is also capable of transferring a galactoside linkage, and there are known methods in which this capability is used to produce galacto-oligosaccharides
Implementation Method 3
one or more amino acids selected from the group consisting of the following (1) to (3) is/are proline... an amino acid corresponding to lysine at position 166... an amino acid corresponding to glycine at position 307... an amino acid corresponding to alanine at position 833
Data Source
Figure 1(A)~1(B)
Figure 2
Figure 3
AI summary
The present invention has a purpose of providing a technique for increasing the heat resistance of a β-galactosidase. According to the present invention, in a reference β-galactosidase amino acid sequence which shows a 90% or more identity to the amino acid sequence of SEQ ID NO: 4, proline is substituted for one or more amino acids selected from the group consisting of the following amino acids: (1) an amino acid corresponding to lysine at position 166 of the amino acid sequence of SEQ ID NO: 4, (2) an amino acid corresponding to glycine at position 307 of the amino acid sequence of SEQ ID NO: 4, and (3) an amino acid corresponding to alanine at position 833 of the amino acid sequence of SEQ ID NO: 4.