2-O-Sulfation Enzyme Mutant for Heparin Production
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing non-animal-derived heparin lack efficient enzyme mutants for 2-O-sulfation and 3-O-sulfation, leading to suboptimal activity and quality control issues in heparin manufacturing.
Innovation Solution
Development of 2-OST and 3-OST enzyme mutants with specific amino acid substitutions, such as leucine to basic amino acid substitutions at position 321 for 2-OST and specific amino acid changes at positions 77, 125, 164, 167, 171, and 259 for 3-OST, enhancing trimer ratio and activity, used in microorganism-based sulfation processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type 2-OST and 3-OST enzymes are used in heparin production, then the manufacturing process can be implemented, but the enzyme activity is insufficient and quality control is problematic
Solution Approach 1:
The patent applies parameter changes by modifying amino acid sequences of 2-OST and 3-OST enzymes to create mutants with improved catalytic activity. Specific amino acid substitutions (e.g., V332M for 2-OST, E90Q for 3-OST) change the enzymatic parameters to enhance sulfation efficiency while maintaining product quality consistency
Solution Approach 2:
The patent creates copies of the original enzymes with modified sequences. By copying the wild-type enzyme structures and introducing specific mutations, the patent generates mutant versions that replicate the essential function while improving activity and reliability for industrial heparin production
2Productivity
If conventional sulfation methods are used, then the process can proceed, but the trimer ratio is low and reaction efficiency is suboptimal
Solution Approach 1:
The patent changes the physical-chemical parameters of the enzymes through amino acid mutations, specifically increasing the trimer ratio of 2-OST and optimizing 3-OST configuration. This parameter change directly improves reaction efficiency and reduces the time required for complete sulfation
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 mutant enzymes significantly improve the efficiency of 2-O- and 3-O-sulfation reactions, enabling the production of high-quality heparan sulfate with enhanced activity and quality control, suitable for producing heparin.
Implementation Method 1
a 2-O-sulfation enzyme mutant and a 3-O-sulfation enzyme mutant, and a method for using the same... an enzyme (2-OST) catalyzing the reaction of 2-O-sulfation... an enzyme (3-OST) catalyzing the reaction of 3-O-sulfation
Data Source
AI summary
The present invention provides a 2-OST mutant exhibiting a high activity. Specifically, the present invention provides a 2-O-sulfation enzyme mutant, having a substitution of a leucine residue at position 321 with a basic amino acid residue in any one amino acid sequence of: (a) the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO: 2; (c) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2; (d) the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; (e) an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; (f) an amino acid sequence having 90% or more identity to the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; and having a 2-O-sulfate transfer activity.