CN101173292B

CN101173292B - 1,3-propylene glycol redex enzyme isoenzyme variant gene Gln202ala and uses thereof

Info

Publication number: CN101173292B
Application number: CN2007101717596A
Authority: CN
Inventors: 李红梅; 李琳; 陈佳
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2007-12-04
Filing date: 2007-12-04
Publication date: 2011-05-04
Anticipated expiration: 2027-12-04
Also published as: CN101173292A

Abstract

The invention discloses a 1, 3-propanediol oxidation reductase isozyme variant gene Gln202Ala and the use. Mediated random mutation of fallible PCR technology is used for screening and getting 1, 3-propanediol oxidation reductase isozyme variant gene. Codon CAG of glutamine of No. 202 in the gene is mutated into a codon GCG of Alanine, thereby getting 1, 3-propanediol oxidation reductase isozyme variant gene Gln202Ala. The nucleotide sequence of the mutated gene is SEQID No.1, which can encode 1, 3-propanediol oxidation reductase isozyme mutations. The variant gene Gln202Ala is reassembled for expressing 1, 3-propanediol oxidation reductase isozyme mutations through gene engineering technology, which is used for catalyzing hydroxy-propionaldehyde and producing 1, 3-propanediol. The invention has the advantages of improving activity by 1.5 to 2 times and providing wide application prospect for chemical raw material 1, 3-propanediol of biotransformation.

Description

1, ammediol redex enzyme isoenzyme variant gene Gln 202 ala and uses thereof

Technical field

The genetic engineering bacterium that the invention belongs in the biochemical industry produces 1, and the technical field of ammediol industrial chemicals is specifically related to 1, ammediol redex enzyme isoenzyme variant gene Gln 202 ala and uses thereof.

Background technology

1, ammediol is important solvent and industrial chemicals, can be used to the polycondensates such as polyester, urethane of synthetic excellent property as monomer with it.Recently, because with 1, ammediol and terephthalic acid are that monomer synthetic polytrimethylene terephthalate (PTT) has very unique character, so many research organizations pay much of being gone abroad.At present, chemical method synthesizes 1, and the ammediol patent is had by Dutch shell (Shell) chemical company and German Degussa.And bio-transformation synthesizes 1, and the ammediol patent is then obtained by Dupont (DuPont) company and German national biotechnology center.Unite to have applied for glucose being substrate with genetic engineering bacterium direct production 1, the patent of ammediol as E.I.Du Pont Company in 1999 and second-biggest-in-the-world industrial enzyme manufacturer Genencor company.It is low that biotransformation method has power consumption, can utilize renewable resources, cleaner production, low cost and other advantages, but since in the microbial metabolism approach with synthetic 1, the catalytic efficiency of ammediol involved enzyme is low, up to now, biosynthesizing 1, the output of ammediol still can't be compared with the output of chemical synthesis.At bio-transformation glycerine synthetic 1, in the pathways metabolism of ammediol, need glycerol dehydratase and 1, the ammediol oxydo-reductase, wherein glycerol dehydratase is responsible for transformation of glycerol is become intermediate product 3-hydroxy propanal, and 1, the ammediol oxydo-reductase then is responsible for the 3-hydroxy propanal being converted into 1, ammediol.Because 1, ammediol oxydo-reductase catalytic activity in catalytic process is very poor, has seriously restricted bio-transformation and synthesized 1, the scale operation of ammediol.There is one 1 in recent findings in the e. coli k12 body, the isozyme of ammediol oxydo-reductase (its corresponding gene is yqhD), be to replace 1 in the glycerine pathways metabolism, ammediol redox enzyme catalysis intermediate product 3-hydroxy propanal generates 1, another key enzyme of ammediol, replace 1 by it, the catalytic efficiency of ammediol oxydo-reductase is apparently higher than 1, the catalytic efficiency of ammediol oxydo-reductase.But present 1, the catalytic capability of ammediol oxidoreductase isozyme is high not enough, extensive industrialization biosynthesizing 1, and ammediol also has certain difficulty.

Summary of the invention

The purpose of this invention is to provide a kind of 1, ammediol redex enzyme isoenzyme variant gene Gln 202 ala and uses thereof, overcome existing 1, ammediol oxydo-reductase poor catalytic activity in catalytic process, seriously restrict bio-transformation and synthesized 1, the shortcoming of ammediol scale operation.

1, the ammediol redex enzyme isoenzyme variant gene Gln 202 ala is characterized in that: described 1, the nucleotides sequence of ammediol redex enzyme isoenzyme variant gene Gln 202 ala is classified SEQID No.1 as.

Described 1, the ammediol redex enzyme isoenzyme variant gene Gln 202 ala is used for catalysis 3-hydroxy propanal and generates 1, ammediol.

The present invention adopts fallibility PCR orthogenesis evolution of technology 1, the ammediol reductase isoazyne, according to 1, a pair of primer of the gene design of ammediol reductase isoazyne, the concentration by reducing a kind of or two kinds of dATP dGTP dCTP dTTP among the conventional P CR and add a certain amount of MnCl ₂Adopt the TaqDNA polysaccharase of low fidelity simultaneously, make gene when amplification to a certain extent random error introduce and obtain the mutator gene library, and obtain a kind of variant gene by certain screening method, by this design philosophy, with pET28 α (+) yqhD as template DNA, adopt fallibility PCR orthogenesis technology to obtain the fallibility pcr amplification product, behind NheI-BamHI double digestion fallibility pcr amplification product, extracting size is about the gene product of 1164bp and carries out ligation with linear plasmid pET28 α that size behind the same enzyme double digestion is approximately 5300bp, after finishing, connection will connect among the product Transformed E .Coli Novablue, be applied to and contain on the 40 μ g/ml kantlex LB agar plates, 37 ℃ of incubated overnight obtain corresponding mutant library.Picking clone is inoculated into 300 μ L and contains in 96 microwell plates of LB liquid nutrient medium of 40 μ g/mL kantlex from mutant library, 37 ℃ of incubated overnight, get this incubated overnight liquid of 40 μ L and join 800 new μ L and contain in 1.2mL volumetrical 96 microwell plates of LB liquid nutrient medium of 30 μ g/mL kantlex, be cultured to OD at 37 ℃ ₆₀₀It is 0.5～0.7 o'clock, adding IPTG (final concentration 0.5mmol/L) induces and continues to cultivate 28h at 30 ℃, 4000r/min harvested cell of centrifugal half hour, pH7.5 phosphoric acid salt lysis buffer re-suspended cell with 300 μ L, behind 37 ℃ of cultivation 30min, place-80 ℃ of freezing 30min immediately, room temperature is thawed, the centrifugal 30min of 4000r/min, draw in 300 μ L supernatant liquors to, the 96 new micropore analysis plates, add 2 μ L200mmol/L 3-hydroxy propanal substrates (the 3-hydroxy propanal is dissolved among the DMSO), at room temperature cultivate 10min, the NADPH that adds 20 μ L 2mg/mL then starts reaction, is in the variation 5min of line monitoring absorption value at 340nm, filter out enzyme activity and be higher than the bacterial strain of protoenzyme and extract plasmid and carry out full-automatic dna sequencing, the discovery mutational site is Gln202Ala.

Advantage of the present invention is to have created a kind of the 3-hydroxy propanal to be had 1 of higher catalysis activity, ammediol oxidoreductase isozyme variant gene, its mutational site are Gln202Ala, with 1, ammediol reductase isoazyne gene is compared, and activity has improved 1.5-2 doubly in an example of measuring.The acquisition of this variant gene is a biosynthesizing industrial chemicals 1, and the biological production of ammediol provides wide application prospect.

Embodiment

Be embodiments of the invention below:

1. the acquisition of fallibility PCR primer design and pcr amplification product:

According to 1, the gene design of ammediol reductase isoazyne, later operation is for convenience introduced BamHI and NheI site respectively at 5 of primer ' end.

The dNTPs that in the eppendorf pipe of 500 μ l, adds 100pmol, upstream primer, each 20pmol of downstream primer, about 1ng pET28 α (+) yqhD is as template DNA, and ultimate density is 0.05mM MnCl ₂, 2.5uTaqDNA polysaccharase, the PCR damping fluid of 5 μ l, the MgCl of 12.5 μ l 25mM ₂, add aseptic distilled water then to cumulative volume 50 μ l.Reaction parameter is by 95 ℃ of sex change after 5 minutes, at 95 ℃ of 1min, and 54 ℃ of 2min, 72 ℃ of 1min, through 35 circulations, 72 ℃ are extended 2min then, obtain pcr amplification product.

The sudden change library structure:

The fallibility pcr amplification product extracts the gene product that size is about 1164bp behind the NheI-BamHI double digestion, and carry out ligation with linear plasmid pET28 α (+) that size behind the same enzyme double digestion is about 5300bp, after finishing, connection will connect among the product Transformed E .Coli Novablue, be applied to and contain on the 40 μ g/ml kantlex LB agar plates, 37 ℃ of incubated overnight obtain corresponding mutant library.

The amount ratio of plasmid pET28 α (+) and plasmid pET28 α (+) yqhD and restriction enzyme when enzyme is cut:

Plasmid pET28 α (+) yqhD/ plasmid pET28 α (+) 5 μ l

NheI(10u/μl) 1μl

BamHI(10u/μl) 1μl

Buffer(10X) 5μl

Sterilized water 8 μ l

Cumulative volume 20 μ l

The proportioning of ligase enzyme and plasmid when plasmid pET28 α (+)-yqhD connects:

Plasmid DNA pET28 α (+) segment 2 μ l

Gene yqhD segment 4 μ l

T4DNA ligase enzyme 1 μ l

T4DNA ligase enzyme Buffer (10X) 2 μ l

Sterilized water 11 μ l

Cumulative volume 20 μ l

3. screening that the 3-hydroxy propanal is had the variant gene of high catalytic activity

Picking clone is inoculated into 300 μ L and contains in 96 microwell plates of LB liquid nutrient medium of 40 μ g/mL kantlex from mutant library, 37 ℃ of incubated overnight, get this incubated overnight liquid of 40 μ L and join 800 new μ L and contain in 1.2mL volumetrical 96 microwell plates of LB liquid nutrient medium of 30 μ g/mL kantlex, be cultured to OD at 37 ℃ ₆₀₀It is 0.5～0.7 o'clock, adding IPTG (final concentration 0.5mmol/L) induces and continues to cultivate 28h at 30 ℃, 4000r/min harvested cell of centrifugal half hour, pH7.5 phosphoric acid salt lysis buffer re-suspended cell with 300 μ L, behind 37 ℃ of cultivation 30min, place-80 ℃ of freezing 30min immediately, room temperature is thawed, the centrifugal 30min of 4000r/min, that draws 300 μ L contains 1, in the supernatant liquor to of the ammediol oxidoreductase isozyme 96 new micropore analysis plates, add 2 μ L200 mmol/L 3-hydroxy propanal substrates (the 3-hydroxy propanal is dissolved among the DMSO), at room temperature cultivate 10min, the NADPH that adds 20 μ L 2mg/mL then starts reaction, be in the variation 5min of line monitoring absorption value at 340nm, filter out enzyme activity and be higher than the bacterial strain of protoenzyme and extract plasmid and carry out full-automatic dna sequencing, the discovery mutational site is Gln202Ala.

By the mensuration of enzyme kinetics curve, with 1, ammediol reductase isoazyne gene is compared, and activity has improved 1.5-2 doubly in an example of measuring.

4. the recombination bacillus coli fermentative production 1, ammediol

A) picking contain this 1, the positive colony of ammediol reductase isoazyne variant gene Gln 202 ala is linked in the seed culture medium that contains 30 μ g/ml kantlex, 180r/min, 37 ℃ of shaking table incubated overnight 12h.The inoculum size of seed liquor by 2% (v/v) is linked in the fermention medium that contains 40 μ g/mL kantlex, 180r/min, 37 ℃ of shaking tables are cultured to thalline OD ₆₀₀Be about 0.7-0.9, add IPTG to final concentration 0.4mmol/L, and add 25g/L concentration substrate 3-hydroxy propanal, in 25 ℃, the 120r/min fermentation culture was cultivated 10 hours.

B) collect fermented liquid,, collect supernatant liquor at the centrifugal 15-30 of 3000-5000r/min minute. add isopyknic organic solvent chloroform, extract 1, ammediol is used for gas chromatographic analysis with rotation additional issue concentrated extracting solution then.

C) 1, ammediol output adopts gas chromatography determination.GC conditions is: fid detector, and 2m * Φ 3m stainless steel packed column, filler is homemade polymer microsphere GDX2401 (110 order), carrier gas is N ₂, 250 ℃ of injector temperatures, 220 ℃ of column temperatures, 250 ℃ of detector temperatures.Use product in the quantitative fermented liquid of external standard method, sample size is 2 μ l.Experimental result shows, when the starting point concentration that adds substrate 3-hydroxy propanal was 25g/L, with containing 1, ammediol reductase isoazyne variant gene Gln 202 ala recombination bacillus coli carried out fermentative production 1, ammediol, 1, ammediol output can reach 15.7 g/L, proved with contain this 1, ammediol reductase isoazyne variant gene Gln 202 ala recombination bacillus coli can efficiently express 1 really, the ammediol oxydo-reductase is with worker's variant enzyme, and is used for fermentative production 1, ammediol.

SEQUENCE?LISTING

＜110〉Shanghai University of Science and Technology

＜120〉1, ammediol redex enzyme isoenzyme variant gene Gln 202 ala and uses thereof

<160>1

<170>PatentIn?version?3.3

<210>1

<211>1164

<212>DNA

＜213〉e. coli k12

<220>

＜223〉the codon CAG of glutamine of the 202nd of coding sports the codon GCG of L-Ala

<400>1

1 atgaacaact?ttaatctgca?caccccaacc?cgcattctgt?ttggtaaagg?cgcaatcgct

61 ggtttacgcg?aacaaattcc?tcacgatgct?cgcgtattga?ttacctacgg?cggcggcagc

121?gtgaaaaaaa?ccggcgttct?cgatcaagtt?ctggatgccc?tgaaaggcat?ggacgtgctg

181?gaatttggcg?gtattgaacc?aaacccggct?tatgaaacgc?tgatgaacgc?cgtgaaactg

241?gttcgcgaac?aaaaagtgac?ttttctgctg?gcggttggcg?gcggttctgt?actggacggc

301?accaaattta?tcgctgcagc?ggctaactat?ccggaaaata?tcgatccgtg?gcacattctg

361?caaacgggcg?gcaaagagat?taaaagcgcc?atcccgatgg?gctgtgtgct?gacgctgcca

421?gcaaccggtt?cagaatccaa?cgcaggcgcg?gtgatctccc?gtaaaaccac?aggcgacaag

481?caggcgttcc?attctgccca?tgttcagccc?gtatttgccg?tgctcgatcc?ggtttatacc

541?tacaccctgc?cgtcgcgtca?ggtggctaac?ggcgtagtgg?acgcctttgt?acacaccgtg

601?gaagcgtatg?ttaccaaacc?ggttgatgcc?aaaattcagg?accgtttcgc?agaaggcatt

661?ttgctgacgc?tgatcgaaga?tggtccgaaa?gccctgaaag?agccagaaaa?ctacgatgtg

721?cgcgccaacg?tcatgtgggc?ggcgactcag?gcgctgaacg?gtttgatcgg?cgctggcgta

781?ccgcaggact?gggcaacgca?tatgctgggc?cacgaactga?ctgcgatgca?cggtctggat

841?cacgcgcaaa?cactggctat?cgtcctgcct?gcactgtgga?atgaaaaacg?cgataccaag

901?cgcgctaagc?tgctgcaata?tgctgaacgc?gtctggaaca?tcactgaagg?ttcagacgat

961 gagcgtattg?acgccgcgat?tgccgcaacc?cgcaatttct?ttgagcaatt?aggcgtgccg

1021?acccacctct?ccgactacgg?tctggacggc?agctccatcc?cggctttgct?gaaaaaactg

1081?gaagagcacg?gcatgaccca?actgggcgaa?aatcatgaca?ttacgctgga?tgtcagccgc

1141?cgtatatacg?aagccgcccg?ctaa

Claims

1.3-propylene glycol redex enzyme isoenzyme variant gene Gln 202 ala is characterized in that: described 1, the nucleotides sequence of ammediol redex enzyme isoenzyme variant gene Gln 202 ala is classified SEQID No.1 as.

2. claim 1 is described 1, and the ammediol redex enzyme isoenzyme variant gene Gln 202 ala is used for catalysis 3-hydroxy propanal and generates 1, ammediol.