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

A kind of aldolase mutant and its coding gene and application

A mutant and aldolase technology, applied in the field of molecular biology, can solve problems such as difficulty in adapting to industrial production, poor substrate affinity, and low catalytic activity, and achieve improved catalytic efficiency, improved aldolase reaction activity, and solved catalytic low conversion rate

Active Publication Date: 2021-12-21
浙大宁波理工学院
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The aldolase expressed by the thermophile Thermotoga maritima has good thermal stability, but its catalytic activity is still low, and its substrate affinity is poor, so it is difficult to adapt to large-scale industrial production

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
  • A kind of aldolase mutant and its coding gene and application
  • A kind of aldolase mutant and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Synthesis of wild-type aldolase coding gene of thermophile Thermotoga maritima

[0034] Thermotoga maritima wild-type aldolase gene DERA used in the present invention Thm It is artificially synthesized, and its gene sequence comes from NCBI. The purpose of using the artificially synthesized gene is to obtain the mutant aldolase gene, and there is no specific requirement for the carrier.

[0035] 2. Preparation of single mutants

[0036] According to the nucleotide sequence of the mutated aldolase gene, the primers for mutating at the 233rd position were designed:

[0037] 233 site upstream primer: 5'-GAATAGGAACGGCATCGGGAGTTAAGATC-3';

[0038] Downstream primer at position 233: 5'-CTTAACTCCCGATGCCGTTCCTATTCTATCAG-3'.

[0039] The PCR reaction system was: 29.5 μL of purified water; 10 μL of KD plus buffer; 2.5 μL of DMSO; 1 μL of upstream primer; 1 μL of downstream primer; 4 μL of dNTPs; 1 μL of template; 1 μL of KD plus; the total volume was 50 μL.

[0040] The P...

Embodiment 2

[0059] Synthesis of Side Chain Precursors of Statins

[0060] In a 100ml reaction system, add 20ml of the crude enzyme solution prepared in Example 1, add acetaldehyde and chloroacetaldehyde in batches or in batches to a final concentration of 200mM and 100mM, and catalyze the reaction at 25°C and 700rpm for 12h. To prepare Escherichia coli DERA, thermophile Pyrobaculum arophilum DERA, and wild-type DERA under the same conditions Thm Crude enzyme solution was used as a control.

[0061] Add twice the volume of acetone to the reaction solution, centrifuge at 10,000rpm for 10 minutes, take the supernatant, evaporate the acetone by rotary evaporation, extract 3 times with ethyl acetate, combine ethyl acetate, and rotary evaporate to obtain a yellow oily liquid, which is a statin drug Crude product of side chain precursors. The product can be detected by gas chromatography, and the condition parameters are: injection at 280°C; detector at 280°C; column temperature: 100°C for 2mi...

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 aldolase mutant, its encoding gene and its application in the production of statin intermediates, and belongs to the technical field of molecular biology. The amino acid sequence of the aldolase mutant is shown in SEQ ID NO.3. Based on the wild-type aldolase of Thermotoga maritima, the present invention introduces site-directed mutation into its encoding gene, and mutates the codon (TTT) encoding phenylalanine at position 184 to a codon encoding isoleucine ( ATT), by mutating serine (Ser) at position 233 to alanine (Ala), the obtained aldolase mutant has significantly improved aldolase activity in the aldolase reaction with acetaldehyde and chloroacetaldehyde as substrates, and the catalytic efficiency 0.86 times higher, the mutant has good industrial application prospects, and solves the problems of low aldolase catalytic conversion rate, insufficient activity and poor substrate affinity of wild-type thermophilic bacteria.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an aldolase mutant, its coding gene and its application in the production of statin drug intermediates. Background technique [0002] Statins are the most classic and effective lipid-lowering drugs. The synthesis of the bichiral side chain in its structure is a key step in the entire synthesis process. The bichiral center greatly increases the difficulty of synthesizing the statin side chain. Complexity, poor stereoselectivity, large environmental pollution and other defects, and biocatalytic methods have significant advantages such as mild reaction conditions, high stereoselectivity, and environmental friendliness, and are an important way to synthesize statin side chain intermediates in recent years. [0003] At present, some biological enzymes have been found to catalyze the aldol condensation reaction. Among them, the aldolase DERA of Escherichia coli has been widel...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P7/24C12R1/19
CPCC12N9/88C12N15/70C12P7/24C12Y401/02004
Inventor 吴志革金志华金庆超杨郁陈琦邱志龙
Owner 浙大宁波理工学院
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com