Polypeptide Tag Enhances Nitrilase Solubility and Activity
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Solution Overview
Problem
Current methods for improving the soluble expression of nitrilase in E. coli often compromise enzyme activity and stability, making it challenging to achieve high yields of pharmaceutical intermediates like gabapentin without significant losses in catalytic performance.
Innovation Solution
A polypeptide tag comprising 5-11 amino acids with uncharged glycine at both ends and a random combination of histidine, glutamic acid, aspartic acid, lysine, and arginine in the middle, specifically GKGKG, is used to enhance the thermal stability and activity of recombinant nitrilase, allowing for efficient production of pharmaceutical chemicals by improving soluble expression and enzyme activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional fusion tags are used to improve soluble expression of nitrilase, then solubility is improved, but enzyme activity and stability are compromised
Solution Approach 1:
The patent applies parameter changes by systematically optimizing the polypeptide tag sequence, specifically using a 5-amino acid tag with uncharged glycine at both ends and basic/polar residues (K, R, H, E, D) in the middle. This precise parameter optimization achieves high solubility (87.9%) while preserving enzyme activity (2.37 times original), resolving the contradiction between solubility improvement and activity preservation.
2Stability of the object's composition
If polypeptide tags are introduced to enhance soluble expression, then solubility is improved, but thermal stability deteriorates
Solution Approach 1:
The patent optimizes tag parameters by selecting specific amino acid compositions with uncharged glycine at termini and balanced charged/polar residues in the middle. This parameter optimization achieves 87.9% solubility while simultaneously improving thermal stability, as evidenced by maintained enzyme activity at elevated temperatures, thus resolving the contradiction between solubility and thermal stability.
3Quantity of substance
If existing fusion tags are used to increase enzyme quantity, then soluble expression is improved, but catalytic performance is reduced
Solution Approach 1:
The patent applies parameter changes by designing a optimized polypeptide tag with specific sequence (5 amino acids, glycine at ends, basic/polar residues in middle) that achieves 2.37 times the original enzyme activity while maintaining high solubility. This precise parameter optimization ensures that increased enzyme quantity does not compromise catalytic performance,反而 enhances it.
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 polypeptide tag significantly enhances the soluble expression and thermal stability of nitrilase, achieving up to 3034.7 U/g dcw activity, which is 2.37 times that of the original enzyme, and allows for faster substrate hydrolysis, making it suitable for large-scale industrial applications.
Implementation Method 1
The polypeptide tag can effectively promote the whole cell catalyst activity and thermal stability of the host recombinant enzyme
Implementation Method 2
The polypeptide tag significantly enhances the soluble expression and thermal stability of nitrilase
Implementation Method 3
Nitrilases (EC 3.5.5.1) are an important type of hydrolases with a Glu-Lys-Cys catalytical triad, which can selectively and effectively catalyze the hydrolysis of cyano groups to carboxyl groups
Data Source
AI summary
The present invention provides a polypeptide tag and its application in the synthesis of pharmaceutical chemicals, the recombinant nitrilase was obtained by connecting a polypeptide tag to the N-terminus of the amino acid sequence of the nitrilase; wherein amino acids at both ends of the polypeptide tag are uncharged glycine G, and the rest are a random combination of any one or more of glycine G, histidine H, glutamic acid E, aspartic acid D, lysine K and arginine R; The activity of the recombinant nitrilase in the preparation of 1-cyanocyclohexyl acetic acid is up to 3034.7 U/g dcw, the polypeptide tag significantly improves the soluble expression of nitrilase, and the whole cell catalyst hydrolyzes 1M substrate with the same concentration 30 minutes faster than the mother enzyme. The method provided by the present invention can also be used for the biocatalytic reaction of other pharmaceutical intermediates as the substrate catalyzed by the nitrilase, improving the activity of the whole cell catalyst in reaction, and also improving the solubility of other types of nitrilases and the activity of the corresponding whole cell catalysts.