CN104593390B - A kind of gene of coding L alanine oxidizing ferment and its application - Google Patents
A kind of gene of coding L alanine oxidizing ferment and its application Download PDFInfo
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Abstract
The invention belongs to genetic engineering and enzyme engineering field, and in particular to a kind of gene of coding L alanine oxidizing ferment and its application.The present invention provides a kind of gene of coding L alanine oxidizing ferment, and it is obtained from the saccharomycete cDNA library built, the nucleotide sequence such as SEQ ID NO of the gene:Shown in 1.The coding L alanine oxidase genes are connected structure recombinant expression plasmid after clone with expression plasmid, recombinant expression plasmid is linearized and transformation receptor bacterium after purification, screening obtains genetic engineering bacterium, and L alanine oxidizing ferment is obtained after induced expression, Purification.The L alanine oxidizing ferment that the present invention is obtained can realize the L alanine in asymmetric degradation method degraded DL alanine, so as to accumulate D alanine, it is a kind of novel method for producing D alanine, this method had not only improved its degradation efficiency, the process route for shortening production D alanine, reduction production cost but also reduced environmental pollution.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of gene of coding ALANINE oxidizing ferment and its should
With.
Background technology
Alanine is to constitute one of 20 kinds of primary amino acids of protein, is the excellent means of transport of nitrogen in blood, is also
A kind of effective glucogenic amino acid.D-alanine is mainly absorbed by internal ALANINE racemization or directly and obtained from food.
Other biologies in natural environment, such as plant, insect, vertebrate and invertebrate, also detection find D-alanine.It is right
For birds and mammal, alanine is nonessential amino acid, can be degraded to by the sugar in food after pyruvic acid and convert and obtain.
Chirality and optical activity of the alanine according to its molecular structure, can be divided into ALANINE (levorotatory substance), D-alanine (dextrogyre
Matter) and DL-Alanine (racemic material).D-alanine is widespread in nature.Have free in tissue and body fluid
D-alanine, be also found in the grey matter and white matter of human brain, also have a large amount of conjugates in all kinds of peptides, protein.
It can prevent kidney stone it has been found that D-alanine has, assist glucose metabolism, relaxing the physiological functions such as hypoglycemia.
As nutritional supplement, nutrient balance can be efficiently adjusted.D-alanine is a kind of important organic chiral source, can be used as chiral medicine
Thing, chiral additives, chiral auxiliary etc., are widely used in pharmacy and food service industry.D-alanine has optical activity as one kind
Organic acid, during some chipal compounds asymmetric syntheses effect it is irreplaceable, such as be used as production New-type wide-spectrum
Antibiotic, D- Propanolamines, the alanine protective agent of Peptide systhesis process.D-alanine also has certain immunosuppressive activity,
Also the growth of certain plants can be suppressed.
The preparation method of current D- amino acid mainly has chemical synthesis, Chiral Separation method and biological synthesis process.Change
Learn synthetic method and D-alanine is directly mainly synthesized by the method for chemistry, but this method is complicated, product purity is low, unfavorable
In large-scale production.Chiral Separation method is mainly by various methods such as chromatographic column, and the method such as ion exchange resin is to DL- third
Propylhomoserin is directly split, but this method cost is too high, yields poorly, and is not suitable for large-scale production.The existing production ammonia of D- third
The certain methods of acid, due to reasons such as yield is relatively low, resolving agent is expensive, high expensives, cause D-alanine price too high,
Using limited.
No matter which kind of method D-alanine is prepared using, there is that technological process length, step is more, accessory substance removal process is multiple
Miscellaneous, yield is low, cost it is high and easily pollution environment the shortcomings of, so as to limit the production and marketing of D-alanine.Therefore, grind
Studying carefully a kind of D-alanine production method that process route is short, production cost is low of exploitation becomes very urgent and necessary.
The content of the invention
In order to overcome the shortcomings of above-mentioned technology, the invention provides a kind of gene cloning and expression of ALANINE oxidizing ferment
Method, develop the D-alanine production method that a kind of production process route is short, cost is low.
It is of the present invention to solve the problems, such as used technical scheme：
The invention provides a kind of gene for encoding ALANINE oxidizing ferment, it is from the saccharomycete cDNA library built
It is middle to obtain, its nucleotide sequence such as SEQ ID NO:Shown in 1.
The ALANINE oxidizing ferment that the present invention is obtained is coding ALANINE oxidase gene expression product, and the enzyme is by 423
Individual amino acid composition, its molecular weight is 49523.12Da, its amino acid sequence such as SEQ IDNO:Shown in 2.
Present invention also offers a kind of recombinant expression plasmid, it is by expression plasmid and the gene of coding ALANINE oxidizing ferment
Recombinate and obtain.
Preferably, described expression plasmid is eukaryon expression plasmid pPICZaA.
The present invention also provides a kind of genetic engineering bacterium, and it is to be converted Host Strains by recombinant expression plasmid and obtained.
The method that ALANINE oxidase gene prepares ALANINE oxidizing ferment is encoded present invention also offers a kind of utilize,
Comprise the steps of：
(1) gene of clones coding ALANINE oxidizing ferment；
(2) coding ALANINE oxidase gene is connected structure recombinant expression plasmid with expression plasmid；
(3) recombinant expression plasmid is linearized with purifying；
(4) by recombinant expression plasmid transformation receptor bacterium after purification, genetic engineering bacterium is obtained after screening；
(5) by genetic engineering bacterium induced expression, ALANINE oxidizing ferment is obtained after Purification.
Preferably, the recipient bacterium described in step (4) is pichia pastoris X-33.
Preferably, the genetic engineering bacterium described in step (5) carries out induced expression using methanol.
Preferably, the methanol is added to final concentration 0.6% (v/v).
Present invention also offers application of the ALANINE oxidizing ferment in D-alanine production, it is characterised in that：The L-
ALANINE in alanine oxidizing ferment energy effectively hydrolyzing DL-Alanine, its reaction conditions is：30 DEG C of temperature, lucifuge bar
32h is reacted under part.
Compared with prior art, the invention has the advantages that：
1st, the present invention is the ALANINE oxidase gene directly obtained from the cDNA of saccharomycete.
2nd, the present invention relates to the ALANINE oxidase gene clone, corresponding amino acid sequence and its by
Expression in body bacterium pichia pastoris X-33, the expression enzyme can be that bioanalysis synthesis D-alanine is carried with effectively hydrolyzing ALANINE
A kind of new method is supplied.
3rd, the method for ALANINE oxidizing ferment energy effectively hydrolyzing DL-Alanine of the invention is simply efficient, realizes asymmetric
Edman degradation Edman degraded DL-Alanine production D-alanine, had both improved its transformation efficiency, has shortened the process route of D-alanine, subtracted again
Few environmental pollution.
Brief description of the drawings
Fig. 1 is LAO genes, recombinant plasmid PmlI and PCR qualification figure, is to enter performing PCR expansion by template of recombinant cloning vector
Increase, recovery product and pPICZaA carry out double digestion respectively through PmlI and XbaI, the result is that passing through 0.8% Ago-Gel
Electrophoresis detection；Wherein, M is DNA Maker in Fig. 1-1；1 is LAO gene PCR figures；M is DNA Mark in Fig. 1-2；1 is table
Up to carrier pPICZaA；2 be recombinant vector.
Fig. 2 is recon LAO expression figures in pichia pastoris X-33 (P.pastoris X-33), is to use the pPICZaA containing sky
The recombinant bacterium of carrier is as blank control, and the fox extracting thallus protein after induced expression is analyzed through 12%SDS-PAGE；Wherein M is
DNA Mark；1st hole is that original strain expresses LAO；2-5 holes are the LAO of restructuring vector expression.
Fig. 3 is enzymatic reaction solution high-efficient liquid phase chromatogram, is after enzymatic reaction solution is pre-processed, to use RP-HPLC color
Spectrometry detects reaction result, and testing conditions are：C18 posts (5 μm, 250mm × 4.6mm), mobile phase acetonitrile-isopropanol (7: 3), post
30 DEG C of temperature, flow velocity 1.0mL/min, UV Detection wavelength 280nm；It 9.315 is ALANINE oxydasis that wherein retention time, which is,
Enzyme.
Fig. 4 is mark product D-alanine high-efficient liquid phase chromatogram, marks product D-alanine Reversed phase high performance liquid chromatography method testing result, inspection
Survey condition is：Astec chiral columns (5 μm, 150mm × 4.6mm), mobile phase：Methanol volume is that fraction is 33%, disodium hydrogen phosphate
Concentration is 0.01mol/L, PH=6.0, flow velocity 0.05ml/min, and 25 DEG C of column temperature, Detection wavelength is 200nn, sample size 5ul.
Embodiment
With reference to specific embodiment, make further details of elaboration to the present invention, but embodiments of the present invention are not
It is confined to the scope that embodiment is represented.These embodiments are merely to illustrate the present invention, not for limitation the scope of the present invention.This
Outside, after present disclosure is read, those skilled in the art can various modifications may be made to the present invention, and these equivalent variations are same
Sample falls within appended claims limited range of the present invention.
Embodiment：
1st, library source and plasmid
Saccharomycete cDNA library and pPICZaA are preserved by this laboratory.
2nd, main agents
Expressive host bacterium P.pastoris X-33 (wild type), antibiotic Zeocin are purchased from invitrogen companies.Pm
Ll enzymes and XbaI enzymes are purchased from TaKaRa bio-engineering corporations.
3rd, method
3.1st, the clone of gene and sequencing
Performing PCR amplification is entered with the primer of designed, designed, using 25 μ L reaction system：10 × buffer 2.5 μ L, dTNP
0.5 μ L, forward and reverse each μ L of 0.5 μ L, RTaq enzyme of 0.5 μ L, cDNA template 0.25 of primer, moisturizing to 25 μ L.PCR conditions are：First
Step：95 DEG C of 5min, second step：94 DEG C of 50s, the 3rd step：52 DEG C of 45s, the 4th step：72 DEG C of 2min, the 5th step：72℃10min；Its
Middle second step to the 4th step is circulated 30 times.PCR primer detects through 0.8% agarose gel electrophoresis, reclaim after target gene with
PGM-T vector connections, obtain recombinant plasmid.Linked system is：The μ L of 1 μ L, PCR target gene products of pGM-T vector 5,
The μ L of 10 × T4ligase buffer, 1 μ L, T4DNA ligases 1, the μ L of distilled water 2,16 DEG C connect 12h and stay overnight.With 10 μ L connection liquid
100 μ L competence E.coliDH5 α are converted, the EP pipes of the competent cell static 30min in ice bath is will be equipped with, next moves to 42
Heat shock 90S in DEG C water-bath, is then quickly transferred to ice bath 2min by EP pipes, finally plus 900 μ L LB culture mediums mix after 37 DEG C shake
Bed concussion and cultivate 45min (150r/min).100 μ L competent cells are taken to be coated on containing Amp (100 μ g/mL), IPTG
37 DEG C of trainings on (0.5mmol/L), X-gal (0.004%) LB (0.5% yeast extract, 1% tryptone, 1% sodium chloride) flat board
Picking white colony after supporting 16 hours, is inoculated in the LB culture mediums containing Amp, and extracting plasmid enters performing PCR and digestion verification, survey
Sequence.
3.2nd, the structure of pPICZaA expression vectors
Enter performing PCR amplification, recovery product by template of recombinant cloning vector.Recovery product and pPICZaA are carried out respectively
PmlI and XbaI double digestions, double digestion system is as follows：PmlI and Xba I each 1.5 μ L, NEB Buffer2.13.0 μ L, 0.1%
BSA 3.0 μ L, pPICZaB 18.0 μ L, ddH2O 3.0 μ L, the μ L of cumulative volume 30.0.37 DEG C reaction 12h, reclaim target gene, with
Target gene has the pPICZaA fragments of identical cohesive end, and both are connected, and coupled reaction system is as follows：10×T4DNA
2.0 μ L, T4 ligase of Ligase Buffer2.0 μ L, LAO 8.0 μ L, pPICZaB endonuclease bamhi of fragment 2.0 μ L, ddH2O 6.0μ
L, Tota l20.0 μ L, 16 DEG C of reaction 12h, and Transformed E .coli DH5 α, Amp resistance screenings, extracting recombinant plasmid enter performing PCR,
Digestion is identified, the recombinant plasmid pPICZ aA-LAO after identification is sequenced, further checking.Double digestion and PCR results are shown in Fig. 1.
3.3rd, plasmid pPICZaA-maf linearisation and purifying
Linearisation：Select Sac I as restriction enzyme site, reaction system is as follows：μ L, the 10 × L Buffer15 μ of Sac I 10
L, pPICZ α A-LAO 110 μ L, ddH2The μ L of O 15, the μ L of cumulative volume 150.16 DEG C of reaction 3h.According to conventional phenol extraction, ethanol
Washing of precipitate carries out plasmid purification, is dissolved and precipitated with sterilized water.Plus isometric phenol/chloroform, 10000rpm is centrifuged after mixing
10min, carefully takes supernatant to a clean EP pipes, adds the frost absolute ethyl alcohol of two volumes, -20 DEG C of placements of refrigerator after mixing
30min, 10000rpm centrifuge 10min, abandon supernatant, and precipitation is cleaned once with 75% ethanol.
3.4th, the conversion of pichia pastoris X-33 electricity and recombination yeast screening
Drawing 10 μ L, the expression plasmid of purifying is mixed with 80 μ L X-33 competent cells, and electricity turns 5ms under 1500V,
Then 150 μ L bacterium solutions are taken to be spread evenly across the YPD flat boards containing 100 μ g/mL Zeocin.Picking has the bacterium colony of Zeocin resistances
Carry out the screening of MD/MM speeds spot.Expression plasmid expression of results in pichia pastoris X-33 is shown in Fig. 2.
3.5th, positive transformant Fiber differentiation
Picking positive transformant is inoculated in 25mL BMGY, and in 30 DEG C, 180r/min shaking tables concussion and cultivate to OD660 is 3
When, thalline is collected, cell is resuspended to OD660 to 1.0 with BMMY, 100% methanol is added per 24h to final concentration of 0.5%, induction
Culture.
3.6th, recombinant yeast crude enzyme liquid is extracted
Recombinant yeast X33-pPICZ α A-LAO are centrifuged off thalline through methanol induction, take 20 μ L of supernatant liquid to be concentrated by ultrafiltration
Obtain 10 μ L enzyme liquids.
3.7th, converted by substrate of cholesterol
Prepare 1%DL- alanine and add the mixing of 10ml enzyme liquids, 30 DEG C of lucifuges react 12h.Amberlite is used after reaction
Fat extracts D-alanine, and supernatant is equipped with into LewatitSP-112 (H by one+Type) then pillar be washed with water, D-alanine
Use 2mol/L HS2O4, collect reaction solution and reaction result detected using reversed-phased high performace liquid chromatographic, testing conditions are：Astec hands
Property post (5 μm, 150mm × 4.6mm), mobile phase：Methanol volume is that fraction is 33%, and disodium hydrogen phosphate concentration is 0.01mol/L,
PH=6.0, flow velocity 0.05ml/min, 25 DEG C of column temperature, Detection wavelength is 200nn, sample size 5ul, as a result sees accompanying drawing 3 and accompanying drawing 4.
It is foregoing to the present invention specific illustrative embodiment description be in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to above-mentioned teaching, can be much changed
And change.The purpose of selecting and describing the exemplary embodiment is that explaining that the certain principles and its reality of the present invention should
With so that those skilled in the art can realize and using the present invention a variety of exemplaries and
A variety of selections and change.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (4)
1. a kind of application of ALANINE oxidizing ferment in D-alanine production, it is characterised in that：The ALANINE oxidizing ferment
ALANINE that can be in effectively hydrolyzing DL-Alanine, its reaction conditions is：30 DEG C of temperature, reacts 32h under the conditions of lucifuge；
Encode the nucleotide sequence such as SEQ ID NO of ALANINE oxidase gene:Shown in 1；The amino acid sequence of ALANINE oxidizing ferment
Row such as SEQ ID NO:Shown in 2.
2. application according to claim 1, it is characterised in that the ALANINE oxidase gene prepares ALANINE oxygen
Change the method for enzyme, comprise the steps of：
(1) gene of clones coding ALANINE oxidizing ferment；
(2) coding ALANINE oxidase gene is connected structure recombinant expression plasmid with expression plasmid；
(3) recombinant expression plasmid is linearized with purifying；
(4) by recombinant expression plasmid transformation receptor bacterium after purification, genetic engineering bacterium is obtained after screening；
(5) by genetic engineering bacterium induced expression, ALANINE oxidizing ferment is obtained after Purification.
3. application according to claim 2, it is characterised in that：Recipient bacterium described in step (4) is pichia pastoris X-33.
4. application according to claim 2, it is characterised in that：Genetic engineering bacterium is entered using methanol described in step (5)
Row induced expression.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884565A (en) * | 2006-05-29 | 2006-12-27 | 安徽华恒生物工程有限公司 | Process for producing D-alanine using microbe |
| CN101649344A (en) * | 2009-07-23 | 2010-02-17 | 嘉兴学院 | Biological preparation technology of D-lactamine |
| CN103555642A (en) * | 2013-10-12 | 2014-02-05 | 江南大学 | Construction method and application of L-amino acid oxidase production recombinant strain |
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2015
- 2015-01-19 CN CN201510025185.6A patent/CN104593390B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884565A (en) * | 2006-05-29 | 2006-12-27 | 安徽华恒生物工程有限公司 | Process for producing D-alanine using microbe |
| CN101649344A (en) * | 2009-07-23 | 2010-02-17 | 嘉兴学院 | Biological preparation technology of D-lactamine |
| CN103555642A (en) * | 2013-10-12 | 2014-02-05 | 江南大学 | Construction method and application of L-amino acid oxidase production recombinant strain |
Non-Patent Citations (2)
| Title |
|---|
| "L-氨基酸氧化酶的研究进展";余志良 等;《中国生物工程杂志》;20121231;第32卷(第3期);第125-135页 * |
| "Molecular Cloning of the L-Amino-acid Oxidase Gene from Neurospora crassa";Denise M.等;《The Journal of Biological Chemistry》;19901005;第265卷(第28期);第17246-17251页 * |
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