Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Omega-transaminase mutant with improved catalytic efficiency

A catalytic efficiency, mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low catalytic efficiency of ω-transaminase, reducing the development and application of ω-transaminase, etc.

Active Publication Date: 2019-03-19
JIANGNAN UNIV
View PDF10 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although ω-transaminase has great application and research value, the catalytic efficiency of ω-transaminase screened from wild bacteria is low, which greatly reduces the development and application of ω-transaminase.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Omega-transaminase mutant with improved catalytic efficiency
  • Omega-transaminase mutant with improved catalytic efficiency
  • Omega-transaminase mutant with improved catalytic efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Preparation and construction of ω-transaminase site-directed mutants

[0026] ω-transaminase 1 site-directed mutant I215M derived from Bacillus pumilus W3:

[0027] In the present invention, the most similar thermophilic archaea transaminase crystal structure (PDB ID: 5E25) is used as a template to construct the three-dimensional simulation structure of Bacillus pumilus W3 ω-transaminase (ω-BPAT) through the Swiss-Model online server ( figure 1 ). Through amino acid primary sequence alignment, it was found that the similarity between thermophilic archaea aminotransferase and ω-BPAT reached 51.21%, which was in line with the parameters of homology modeling. Therefore, it can be considered that ω-BPAT has a similar three-dimensional structure to thermophilic archaea aminotransferase. According to the predicted results of software analysis, mutant I215M was constructed by PCR-mediated site-directed mutagenesis.

[0028] The preparation method of site-directed ...

Embodiment 2

[0036] Example 2: Expression and purification of native ω-transaminase and site-directed mutants thereof

[0037] Pick the positive single clones transferred into the expression host Escherichia coli BL21 (DE3) and grow them in LB liquid medium (containing 30 μg / mL ampicillin) for 8-10 h, and transfer the seed fermentation broth to LB liquid medium ( containing 30 μg / mL ampicillin); Escherichia coli was cultured on a shaker at 37°C for 2 hours until OD 600=0.6 or so, add 0.05mM IPTG to induce extracellular expression of the mutant I215M recombinant strain, and continue to culture and ferment in a shaker at 15°C for 24h, then centrifuge the fermentation broth at 4°C and 8000g for 10min to remove bacteria, and collect the centrifuged fermentation supernatant. Slowly add 60% (NH 4 ) 2 SO 4 , placed at 4°C for salting out overnight. Centrifuge at 10,000 g for 20 min at 4°C to collect the precipitate. After redissolving the precipitate with 50mmol / L pH 5.3 citric acid-disodiu...

Embodiment 3

[0038] Embodiment 3: enzyme activity analysis method

[0039] The determination method of ω-transaminase activity refers to Gao, S. (Gao, S., Su, Y., Zhao, L., Li, G., Zheng, G., 2017.Characterization of a(R)-selective amine transaminase from Fusariumoxysporum. Process. Biochem. 63, 130-136.).

[0040] Take an appropriate amount of bacterial supernatant (or purified and diluted enzyme solution), add 500 μL sodium dihydrogen phosphate / disodium hydrogen phosphate buffer (100 mM, pH7.0), which contains 20 mM (R)-α-phenethylamine ( or (S)-α-phenethylamine), 20mM sodium pyruvate, 0.1mM pyridoxal 5'-phosphate (PLP), mix well, react at 45°C for 15min respectively, and then add the same amount of ethyl acetate to terminate the reaction. The absorbance of the solution at 254 nm was measured before and after the reaction.

[0041] Under the above conditions, the amount of enzyme required to catalyze 1 μmol of related ketones in 1 minute is defined as one enzyme activity unit (U / ml). ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an Omega-transaminase mutant with improved catalytic efficiency, and belongs to the technical field of the gene engineering and the enzyme engineering. Through performing site-specific mutagenesis on amino acid with a higher factor B in an Omega-transaminase crystal structure, namely mutating I at a site of Omega-transaminase 215 into M, the Omega-transaminase mutant with the improved catalytic efficiency is obtained. The catalytic efficiency Kcat / Km of the mutant I215M is improved by 17.39 times. While (R)-phenethylamine is catalyzed to generate acetophenone, an acetophenone maximum conversion rate of the mutant is improved by 6.99 times compared with native enzyme. The obtained mutant is more suitable for an application in industry compared with natural Omega-transaminase.

Description

technical field [0001] The invention relates to an omega-transaminase mutant with improved catalytic efficiency, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Aminotransferase (transaminase) belongs to the class of transferases and is usually used to catalyze the transfer of an amino group from an amino donor compound to an amino acceptor compound. According to the differences of different variable regions, the protein sequences of transaminases from different sources reported in the literature were compared and clustered, and then according to the superposition and iterative comparison of hydrophilic sites, we can divide transaminases into 4 categories. Among them, ω-transaminase belongs to the second subfamily and is often used to prepare chiral amines and unnatural amino acids, such as β-amino acids. Because ω-transaminases have a slightly larger large pocket binding region than aspartate transaminases (Asp...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1096C12N15/70C12Y206/01
Inventor 廖祥儒翟李欣赖英杰杨邵岚蔡宇杰管政兵
Owner JIANGNAN UNIV
Features
  • Generate Ideas
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com