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Alcohol dehydrogenase and application thereof in preparing alcohol

An alcohol dehydrogenase and purpose technology, applied in the field of genetic engineering, can solve the problems of the difficulty of alcohol dehydrogenase, the high investment cost of production equipment, the slow speed of forward movement and the like

Active Publication Date: 2019-10-11
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional catalytic hydrogenation process needs to be carried out under high temperature and high pressure, which is relatively dangerous, and the investment cost of production equipment is high
[0003] Alcohol dehydrogenases are widely found in animals, plants and microorganisms. Although alcohol dehydrogenases are easy to obtain and most of them are stable, most of them are obtained through multiple screening, chromogenic methods, screening methods based on coenzyme derivatives, and screening methods based on coenzyme fluorescence. method, it is difficult to obtain alcohol dehydrogenase resistant to aldehydes based on the above method
[0004] In addition, during the reaction process of synthesizing alcohol by alcohol dehydrogenase, too high concentration of alcohol will inhibit the further progress of the reaction and slow down the reaction to move forward, resulting in a lower yield of product

Method used

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  • Alcohol dehydrogenase and application thereof in preparing alcohol
  • Alcohol dehydrogenase and application thereof in preparing alcohol
  • Alcohol dehydrogenase and application thereof in preparing alcohol

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Effect test

preparation example Construction

[0041] In the preparation method of the above recombinant alcohol dehydrogenase, the expression host cell is Escherichia coli.

[0042]The preparation method of the above-mentioned recombinant alcohol dehydrogenase enzyme, the specific process is as follows: including double digestion with BamHI and HindIII to obtain the target gene and connecting the pET-32a(+) vector, transforming it into Escherichia coli BL21(DE3), inducing with IPTG, Efficient soluble expression was obtained.

[0043] The final concentration of IPTG is 0.6-1.8mM, and the induction temperature is 18-37°C.

[0044] The recombinant alcohol dehydrogenase provided by the invention comprises the method and steps of transforming the expression host cell with the above-mentioned expression vector, culturing the transformant, and obtaining the recombinant alcohol dehydrogenase from the culture.

[0045] The last object of the present invention is achieved through the following technical scheme: the application of ...

Embodiment 1

[0054] Example 1 Establishment of metagenomic library and acquisition of positive clones, gene cloning and expression

[0055] 1. Extraction of total DNA:

[0056] Weigh 5g sample, the sample is the soil near the biomass acid hydrolysis equipment, add 13.5ml DNA extraction buffer (0.1M Tris, 0.1M EDTA-Na, 0.1M Na 3 PO 4 , 1.5M NaCl, 1% CTAB, pH 8.0), shake vigorously, add 300μl lysozyme (100mg / ml), invert 5-6 times, 37℃ water bath for 30min, add 1.5ml 20% SDS, 65℃ Water bath for 1h (during this period, turn it upside down several times every 15min), centrifuge at 8000r / min for 5min, take the supernatant, extract twice with an equal volume of chloroform, centrifuge at 8000r / min for 10min, take the supernatant, add 0.6 times the volume of isopropyl Alcohol, place at room temperature for 2h, centrifuge at 20000r / min for 20min, discard the supernatant, add 5mL of pre-cooled 70% ethanol to the pellet, centrifuge at 20000r / min for 10min, collect the DNA precipitate, air-dry, and d...

Embodiment 2

[0079] The mensuration of embodiment 2 enzyme activity

[0080] (1) Definition of alcohol dehydrogenase activity with furfural as substrate: 1 unit of alcohol dehydrogenase activity is defined as the amount of enzyme required to reduce 1 μmol of furfural per minute at pH 6.0 and 30°C.

[0081](2) Measuring principle: use furfural as a substrate to measure enzyme activity, alcohol dehydrogenase catalyzes the reduction of 1 mol of furfural to generate 1 mol of furfuryl alcohol, and at the same time oxidizes 1 mol of NADPH to generate 1 mol of NADP + , NADPH has the largest characteristic absorption peak at 340nm, within a certain range of light absorption value, the light absorption value at 340nm is proportional to the concentration of NADPH.

[0082] (3) The determination method is as follows:

[0083] Reaction system: 300 μl, including: 10 mM furfural, 0.1 mM NADPH, 50 μl diluted enzyme solution, pH 6.0 potassium phosphate buffer. In a microplate reader, detect the light ab...

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Abstract

The invention discloses alcohol dehydrogenase. The nucleotide sequence of the alcohol dehydrogenase is shown in SEQ ID NO.1. The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.2. The invention further discloses application of the alcohol dehydrogenase in a reaction for synthesizing alcohol with aldehyde as a substrate. The novel recombinant alcohol dehydrogenase has efficient soluble expression capability in an escherichia coli expression system, and the conversion rate of the recombinant alcohol dehydrogenase for synthesizing the alcohol in an aldehyde reducing reaction system is 90% or so.

Description

technical field [0001] The invention belongs to the field of genetic engineering. In particular, the present invention relates to dehydrogenases that reduce aldehydes, especially furan aldehydes, to alcohols, and to polynucleotides encoding such dehydrogenases and their use in the bioconversion of aldehydes to alcohols. Background technique [0002] Alcohol (especially furan alcohol) is an important organic chemical raw material with wide application, which can be obtained from the corresponding aldehyde by catalytic hydrogenation. Industrial hydrogenation reduction is divided into two processes: liquid phase hydrogenation and gas phase hydrogenation. The liquid-phase hydrogenation method is divided into high-pressure method and medium-pressure method. The high-pressure method is mostly operated above 9.8MPa, the temperature is controlled between 170-200°C, and chromium trioxide and copper oxide are used as catalysts, and the conversion rate is 95-100 %, the medium pressure...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C07K19/00C12N15/70C12P17/04
CPCC12N9/0006C12N15/70C12P17/04C07K2319/00
Inventor 傅尧李向杰刘孝龙汪哲仑周金龙
Owner UNIV OF SCI & TECH OF CHINA
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