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Aldolase mutant as well as coding gene and application thereof

An aldolase and mutant technology, applied in the field of molecular biology, can solve the problems of low catalytic activity, poor substrate affinity, difficult to adapt to industrial production, etc. low conversion rate

Active Publication Date: 2020-11-03
浙大宁波理工学院
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  • 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

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  • Aldolase mutant as well as coding gene and application thereof
  • Aldolase mutant as well as coding gene and application thereof

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...

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Abstract

The invention discloses an aldolase mutant, a coding gene thereof and an application of the aldolase mutant in production of statins intermediates, and belongs to the technical field of molecular biology. The amino acid sequence of the aldolase mutant is as shown in SEQ ID NO. 1. According to the invention, on the basis of thermophilic bacteria Thermoga maritima wild aldolase, site-specific mutagenesis is introduced into the coding gene of the thermophilic bacteria Thermoga maritima wild aldolase, a codon (TTT) for coding phenylalanine at the 184 site is mutated into a codon (ATT) for coding isoleucine, and serine (Ser) at the 233 site is mutated into alanine (Ala). The activity of the obtained aldolase mutant in an acetal reaction by using acetaldehyde and chloroacetaldehyde as substratesis remarkably improved, catalytic efficiency is improved by 0.86 times, the mutant has a good industrial application prospect, and the problems of low aldolase catalytic conversion rate, insufficientactivity, poor substrate affinity and the like of wild thermophilic bacteria are solved.

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

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Application Information

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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
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