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Carboxylic acid reductase recombinant plasmid as well as construction method and application thereof

A technology of recombinant plasmid and reductase, applied in the fields of genetic engineering and microorganisms

Active Publication Date: 2021-05-14
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are few reports on the preparation and development of carboxylic acid reductase suitable for the synthesis of 1,2-propanediol from D-lactic acid. At present, there is only one study on the application of carboxylic acid reductase MavCAR to reduce lactic acid to synthesize 1,2-propanediol, and the carboxylic acid reductase MavCAR Acid reductase only synthesized less than 1mM R-1, 2-propanediol 4 hours after being introduced into the engineered Escherichia coli strain, so it is urgent to find a carboxylic acid reductase that can efficiently reduce lactic acid to synthesize 1,2-propanediol

Method used

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  • Carboxylic acid reductase recombinant plasmid as well as construction method and application thereof
  • Carboxylic acid reductase recombinant plasmid as well as construction method and application thereof
  • Carboxylic acid reductase recombinant plasmid as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of recombinant bacteria containing carboxylic acid reductase SrCAR gene sequence.

[0026] Synthesize the fragment of the gene (shown in SEQ NO.3 in the sequence listing) encoding carboxylic acid reductase SrCAR in the present invention by chemical synthesis, and combine it with the phosphopantetheinyl group derived from Bacillus subtilis (Bacillus subtilis) The transferase gene fragment (shown in SEQ NO.4) was connected to the pRSFDuet-1 vector, transformed into Escherichia coli competent cells, the positive cloned plasmid was extracted and analyzed by restriction enzyme map, and the recombinant plasmid pRSFDuet-SrCAR carrying the SrCAR gene was verified to be obtained -Bssfp, recombinant expression vector pRSFDuet-SrCAR-Bssfp as shown in the figure figure 1 shown. The thiolation domain of carboxylic acid reductase requires post-transcriptional modification, which covalently attaches the phosphopantetheinyl group to the conserved serine catalyz...

Embodiment 2

[0030] The correct clone identified in Example 1 was cultured overnight, then transferred to LB medium containing 1 μg / mL kanamycin for cultivation, until the bacterial solution OD 600When the concentration is 0.6-0.8, add IPTG with a final concentration of 1.5mM to induce expression at 30°C. After induction for 8 hours, collect the bacterial liquid, 8000rpm, 5min, discard the supernatant medium, wash twice with PBS equal volume, add Appropriate amount of PBS to make the resuspended bacteria OD 600 =40, add 15% (v / v) glycerin, 1mM phenylmethylsulfonyl fluoride (PMSF), and mix on a vortex machine; break with an ultrasonic breaker, ultrasonic break conditions: the working time is set to 20min, every Work for 5 seconds, rest for 4 seconds, set the power to 25%, and obtain crude enzyme solution of carboxylic acid reductase after crushing. The obtained carboxylic acid reductase crude enzyme solution was filtered with a 0.22 μm membrane to remove impurities, and 2 mL of the crude e...

Embodiment 3

[0031] Example 3: Research on enzymatic properties of carboxylic acid reductase SrCAR.

[0032] The pure enzyme solution obtained in Example 2 was subjected to research on enzymatic properties, including enzyme activity, specific activity, optimum temperature and optimum pH, etc. Enzyme activity was measured by UV spectrophotometer.

[0033] Carboxylate reductase enzyme activity assay uses 1mL reaction system, which contains 400mM Tris-HCl (pH=7.5), 5mM D-lactic acid, 10mM MgCl 2 , 150mM NaCl, 1mM ATP, 0.15mM NADPH, 1mM DTT and 20μL of enzyme, set the wavelength at 340nm, measured once every 2s, measured for 3min, blank control without enzyme. One activity unit is defined as the amount of enzyme used to oxidize 1 μmol of NADPH per minute as one enzyme activity unit. The specific enzyme activity was calculated according to the following formula:

[0034]

[0035] Where: V T is the total reaction volume, ml; V S is the sample volume, ml; ΔA is the change value of absorba...

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Abstract

The invention discloses a carboxylic acid reductase recombinant plasmid. The recombinant plasmid is obtained by connecting a carboxylic acid reductase gene and aphosphopantetheinyl transferase gene to a pRSFDuet-1 carrier. The invention further provides recombinant bacteria containing the recombinant plasmid, and carboxylic acid reductase SrCAR is further produced by using the recombinant bacteria and is applied to production of 1,2-propylene glycol. According to the present invention, the efficient synthesis approach of the biological preparation of 1,2-propylene glycol is achieved, the application prospect is high, the 1,2-propylene glycol is produced by using the carboxylic acid reductase SrCAR to catalyze glucose, and the yield of 1,2-propylene glycol within 4 h under the condition that the gene of the escherichia coli strain is not knocked out is 4-5 times of the yield of 1,2-propylene glycol synthesized by using a gene knockout escherichia coli strain containing a carboxylic acid reductase MavCAR in literature reports.

Description

technical field [0001] The invention relates to the fields of genetic engineering and microorganism technology, in particular to a carboxylic acid reductase recombinant plasmid and its construction method and application. Background technique [0002] Traditional chemical methods to catalyze the reduction of carboxylic acids to aldehydes not only require noble metals as catalysts, but also have disadvantages such as extreme reaction conditions, poor tolerance to adjacent functional groups, and side reactions. Carboxylic acid reductase (CAR, EC 1.2.1.30 and EC 1.2.1.31) is a multifunctional enzyme widely distributed in bacteria, fungi and some plants, which can efficiently catalyze the chemical reaction from carboxylic acid to aldehyde. Using carboxylic acid reductase to biologically reduce carboxylic acids to corresponding aldehydes not only has mild reaction conditions, broad substrate spectrum, and strong pertinence, but also can specifically identify carboxylic acid group...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/53C12N15/54C12N1/21C12N9/02C12P7/18C12R1/19
CPCC12N15/70C12N9/0008C12N9/1205C12P7/18C12Y102/0103C12Y102/01031C12Y207/01034
Inventor 欧阳嘉陶渊明邹丽花郑兆娟
Owner NANJING FORESTRY UNIV
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