CN101348794B - Encoding gene of high activity glucose oxidase, preparation and use thereof - Google Patents
Encoding gene of high activity glucose oxidase, preparation and use thereof Download PDFInfo
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Abstract
The invention discloses a high-activity glucose oxidase mutant coding gene, a preparation method and application thereof. The coding gene of a high-activity glucose oxidase mutant has the nucleotide sequence as shown in SEQ ID NO.1 and coded proteins have the amino acid sequence as shown in SEQ ID NO.2. The preparation method comprises the following steps: firstly, two pairs of mutagenic primers is designed; secondly, a Escherichia coli strain DH5 alpha/ GOD-D401N/A574V is obtained; thirdly, a recombinant plasmid pPIC9kGOD-D401N/A574V is identified; fourthly, the Escherichia coli strain DH5 alpha/ GOD-D401N/A574V is cultured; fifthly, linear DNA containing a mutant gene is obtained; sixthly, a yeast Pichia strain GS115/GOD-D401N/A574V is obtained; and seventhly, the target proteins are obtained after induction and purification. The method is easy and has low cost; and the application of the glucose oxidase mutant proteins in detection and preservation of sugar foodstuff has high use value.
Description
Technical field
The present invention relates to molecular enzymology and biotechnology, be specifically related to a kind of encoding gene of high activity glucose oxidase, relate to the preparation method of this mutant strain encoding gene simultaneously.The high enzyme glucose oxidase mutant strain alive albumen of this mutator gene coding can be widely used in fields such as food, medicine and biology.
Background technology
Glucose oxidase is one of topmost toolenzyme in the biological field.The production of the glucose oxidase that uses in using at present generally all adopts aspergillus niger and Penicillium bacterial strain to do to produce bacterium, carries out the deep ventilation cultured method and produces.China and U.S. Chang Yidian mould and penicillium crysogenum are for producing bacterial classification, and Japan then filters out the rugged mould of Buddhist nun as bacterial classification commonly used.
From Updike in 1967 and Hicks GOD is fixed on Clark oxygen electrode surface, has been applied to since the blood sugar detection, GOD is widely used in many association areas such as food, feed, medicine.
In foodstuffs industry, because the existence of oxygen causes many chemical reactions that are unfavorable for quality product, and created condition for many microorganism growth.At present, many countries are widely used in GOD in various food and the food processing technology as artificial safety oxidation inhibitor.Though purposes is various, the effect of GOD mainly is to remove glucose, deoxygenation, formation hydrogen peroxide, forms four aspects of gluconic acid.Utilize its single-minded oxidasic principle, make the glucose oxidase enzyme analyser, can quick and precisely measure the glucose content in the various food simply, instruct and produce
[12]
In medicine industry, GOD is used for the external quantitative analysis of serum (slurry), urine and cerebrospinal fluid glucose as test kit, enzyme electrodes etc.; The zymin that GOD makes also can be used for removing or the formation of alleviating dental plaque, tartar and carious tooth prevents the generation of oral disease and odontopathy
[36]In addition, owing to can catalysis generate H
2O
2, also can be used for H
2O
2The treatment of responsive lymphadenomatous target goal.
GOD still is a kind of novel enzyme feed additive, can improve the animal intestinal environment, regulates diet digestion, promotes growth of animal.Contain the mixed fodder additive of glucose oxidase, lactic acid superoxide and lactoferrin, can be used for preventing livestock gastrointestinal tract infection, diarrhoea, and the effect of the growth of animal of promotion is arranged.
Because GOD has wide practical value, along with the success of GOD heterogenous expression in yeast, also the someone utilizes the means of orthogenesis to transform GOD, makes enzyme work be increased to original 1.5 times, aspect thermostability, pH stability, also obtained the mutant strain that character is improved.
Summary of the invention
The object of the present invention is to provide the proteic encoding gene GOD-D401N/A574V of a kind of high enzyme glucose oxidase mutant strain alive, have the nucleotide sequence shown in the SEQ ID NO.1 and the mutant enzyme of described sequence encoding, this mutant enzyme has the aminoacid sequence shown in the SEQ ID NO.2.Introduce sudden change by over-lap PCR in aspergillus niger GOD structure gene, be connected in expression vector, produce high enzyme GOD mutant enzyme alive, the enzyme work of this mutant enzyme is 3 times of wild-type, also has good thermostability and stability in storage simultaneously.
Another object of the present invention is to provide a kind of high enzyme glucose oxidase mutant strain alive proteic preparation method, this method is to make up Yeast expression carrier, utilize the high enzyme of anion-exchange chromatography method purifying glucose oxidase mutant strain alive albumen, this method is easy, and is with low cost.
A further object of the present invention is to provide a kind of high enzyme live glucose oxidase mutant strain albumen and the application on food and medicine thereof, and promptly a kind of high enzyme is lived glucose oxidase mutant strain albumen in wine and the application in the tea fresh keeping.。
In order to realize above-mentioned task, the present invention adopts following technical measures:
An object of the present invention is to provide a kind of nucleotide sequence, a kind of high enzyme of this sequence encoding glucose oxidase mutant strain alive albumen, have the nucleotide sequence shown in the SEQ ID NO.1, it is characterized in that comparing the point mutation that C that the G that has 1752 on the GOD structure gene sports A and 2272 sports T with wild-type.Prepare by following steps:
1. the introducing of mutant enzyme GOD-D401N/A574V catastrophe point
(1) two pairs of mutagenic primers of design, adopt the PCR fixed-point mutation method on the GOD gene wild-type GOD gene on 1752 the G C that sports A and 2272 sport T;
(2) above-mentioned PCR product is after glue reclaims purifying, carrying out enzyme with restriction enzyme SnaBI and NotI cuts, after the plasmid pPIC9k fragment of cutting through same enzyme was connected, transformed into escherichia coli DH5 α competent cell obtained coli strain DH5 α/GOD-D401N/A574V;
2. identify recombinant plasmid pPIC9kGOD-D401N/A574V: with enzyme cut, PCR method identifies recombinant plasmid, and order-checking check obtains having the nucleotide sequence shown in the SEQ ID NO.1 thus;
The present invention names the gained high enzyme proteic encoding gene GOD-D401N/A574V of glucose oxidase mutant strain that lives that encodes to have following feature:
1. the used GOD wild type gene of the present invention is from aspergillus niger, and length is 2303bp, and wherein structure gene length is 1752bp.
2. have the nucleotide sequence shown in the SEQ ID NO.1, compare with wild-type and on this structure gene, have the point mutation of two places, be the C that 1752 G sports A and 2272 and sport T.
High enzyme of the present invention glucose oxidase mutant strain alive albumen can be produced with the following method.This method comprises: following concrete operation is according to the normal experiment condition, and the condition described in " molecular cloning laboratory manual " (third edition) [J. Sa nurse Brooker etc. is write, 2003, Beijing: Science Press] is carried out.
1. the introducing of mutant enzyme GOD-D401N/A574V catastrophe point:
(1) two pairs of mutagenic primers of design, adopt the PCR fixed-point mutation method on the GOD gene wild-type GOD gene on the G C that sports A and 2272 sport T;
(2) above-mentioned PCR product is after glue reclaims purifying, carrying out enzyme with restriction enzyme SnaBI and NotI cuts, after the plasmid pPIC9k fragment of cutting through same enzyme was connected, transformed into escherichia coli DH5 α competent cell obtained coli strain DH5 α/GOD-D401N/A574V;
2. identify recombinant plasmid pPIC9kGOD-D401N/A574V: with enzyme cut, PCR method identifies recombinant plasmid, and order-checking check obtains having the nucleotide sequence shown in the SEQ ID NO.1 thus;
3. cultivate coli strain DH5 α/GOD-D401N/A574V, extract recombinant plasmid pPIC9kGOD-D401N/A574V;
4. restriction enzyme BglII enzyme is cut, and glue reclaims the big fragment of purifying, promptly obtains the required linear DNA that contains mutator gene of yeast conversion;
5. electrotransformation transforms Pichi strain GS115, obtains the production bacterial strain of mutant enzyme GOD-D401N/A574V;
6. obtained strains in the step 5 is changed in the 500mL MD substratum and be cultured to OD
600=1.2-1.5; Centrifugal collection yeast cell, and cell washed once with 20mL MM substratum; Change cell in 250mL MM substratum inducible protein generation respectively, in culture, add 1% methyl alcohol every day, cultivated 4-5 days; The centrifugal supernatant liquor of collecting;
Nearly 500mL contain target protein the culture supernatants ultrafiltration and concentration to 20-30mL, in the phosphate sodium dihydrogen buffer solution of 0.02mol/L, pH6.27, dialyse desalination 24 hours of concentrated solution.Protein purification adopts Q Sepharose
TMFast Flow anion-exchange chromatography post separates, purifying, obtains having the high enzyme glucose oxidase mutant strain alive albumen of the aminoacid sequence shown in the SEQ ID NO.2 thus;
8. make typical curve with the fresh commodity GOD of Sigma company.ABTS and 100U/mL horseradish peroxidase solution with pH5.6,0.1mol/L phosphate sodium dihydrogen buffer solution preparation 2mg/mL glucose, 0.1mg/mL.In 96 orifice plates, add ABTS and the horseradish peroxidase solution 40 μ L of 100 μ L glucose solutions, 100 μ L respectively, add 1 μ l nutrient solution or enzyme liquid, measure OD
414
Can check the activity of the claimed mutant enzyme of the present invention according to above method.Purity detecting can detect with the SDS-PAGE method.
Annotate: above-mentioned yeast culture based formulas is as follows:
MD (L
-1): the basic nitrogenous source substratum of yeast (YNB) 1.7g, ammonium sulfate 5g, glucose 20g, vitamin H 400 μ g;
MM (L
-1): YNB1.7g, ammonium sulfate 5g, methyl alcohol 12mL, vitamin H 400 μ g, casein hydrolysate 10g, pH5.6.
The present invention obtains high enzyme glucose oxidase mutant strain alive albumen and has a wide range of applications in association areas such as food, medicine and biologies.
1. under the condition of pH7.0, get above-mentioned gained glucose oxidase mix with horseradish peroxidase and ABTS with phosphoric acid buffer in, add the glucose reference liquid respectively or the drinks sample reacts with it, can get glucose content in the drinks according to typical curve.The proteic advantage of high enzyme glucose oxidase mutant strain alive that the present invention protected is that its enzyme work is more than 3 times of wild-type, can effectively reduce the usage quantity of zymin.
2. the application of glucose oxidase in tea fresh keeping: glucose oxidase and its substrate glucose are mixed, be packaged in the waterproof but gas-pervious bag film, sealing is placed in the encloses container that tealeaves is housed, when seeing through film, the oxygen in the encloses container enters in the bag, under the effect of glucose oxidase, react with glucose, thereby remove oxygen in the encloses container, prevent that tealeaves is oxidized and cause quality deterioration.
Glucose oxidase is a kind of toolenzyme of widespread use, is mainly used in deoxygenation in foodstuffs industry, removes glucose; In pharmaceutical industries, be mainly used in the mensuration of blood sugar, glucose in urine; In biological field, can also make corresponding biosensor and enzyme electrodes, be used to detect glucose concn.
Described yeast expression vector pPIC9k and Pichi strain GS115 are all available from invitrogen company.Described bacillus coli DH 5 alpha is available from TaKaRa company.Can select the host cell of coupling according to used expression vector.Described recombinant plasmid pPIC9kGOD-D401N/A574V makes up for the present invention.Described coli strain DH5 α/GOD-D401N/A574V is the coli strain with plasmid pPIC9kGOD-D401N/A574V, deposit number: CCTCC NO M207085, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, preservation date: on June 19th, 2007, classification name: bacillus coli DH 5 alpha/GOD-D401N/A574V.Described encoding gene GOD-D401N/A574V is the high enzyme glucose oxidase mutant strain alive protein coding gene with nucleotide sequence of SEQUENCE NO.1.Described Protein G OD-D401N/A574V is the high enzyme glucose oxidase mutant strain alive albumen with aminoacid sequence of SEQUENCE NO.2.Advantage of the present invention and effect:
The nucleotide sequence of the GOD mutant strain that provides among the present invention and protein sequence are the gene and the protein sequences of the high enzyme of a kind of GOD mutant strain alive, do not appear in the newspapers.The work of this strain GOD mutant strain enzyme is more than 2 times of wild-type enzyme work, also has good thermostability and stability in storage, the requirement of realistic application simultaneously.And glucose oxidase has a wide range of applications in fields such as food, medicine and biologies, it is the topmost toolenzyme of field of biosensors, therefore, obtain the GOD that high enzyme is lived, has very high practical value, particularly in the analysis device of biosensor, fixedly have the enzyme of enough catalytic activitys on small area, the height that enzyme is lived seems particularly important.
Description of drawings
Fig. 1 construction of recombinant plasmid illustrates intention
(1) amplifies the GOD gene with PCR method;
(2) two pairs of mutagenic primers of design adopt the PCR fixed-point mutation method on the GOD gene Asp of 401 of wild-type GOD molecules to be sported Asn, and 574 Ala sports Val;
(3) above-mentioned PCR product is after glue reclaims purifying, carrying out enzyme with restriction enzyme SnaBI and NotI cuts, after the plasmid pPIC9k fragment of cutting through same enzyme was connected, transformed into escherichia coli DH5 α competent cell obtained coli strain DH5 α/GOD-D401N/A574V;
PCR evaluation (b) and the enzyme of Fig. 2 recombinant expression plasmid pPIC9kGOD-D401N/A574V are cut evaluation (a) result (1% agarose gel electrophoresis).
Shown in Fig. 2 (b), the stripe size that above downstream primer amplifies is about 1800bp, and is consistent with GOD structure gene size.
Intestinal bacteria-about the 9.3kb of yeast shuttle plasmid pPIC9k size contain two Bgl II restriction enzyme sites.GOD gene inside does not then have Bgl II restriction enzyme site.Plasmid pPIC9k-GOD is cut with Bgl II enzyme, and electrophoresis result is as shown in Fig. 2 (a), and Bgl II enzyme is cut pPIC9k-GOD and obtained 2.4kb and two fragments of 8.7kb, and is consistent with guess value.Above result proves that the high enzyme glucose oxidase mutant strain protein coding gene GOD-D401N/A574V that lives has been cloned into Yeast expression carrier pPIC9k.
Pichi strain pPIC9kGOD-D401N/A574V abduction delivering behind Fig. 3 purifying produces high enzyme glucose oxidase mutant strain alive albumen (10%SDS-polyacrylamide gel electrophoresis detects).
Glucose oxidase monomer molecule amount is about 80kDa, conforms to the result.
The thermostability of Fig. 4 and wild-type relatively
Water-bath was preserved in 50 minutes in 40 ℃, and wild-type and GOD-D401N/A574V enzyme are lived does not all have significantly sacrificing, and wild-type enzyme is lived and slightly descended in the time of 60 minutes, does not obviously descend and the enzyme of GOD-D401N/A574V is alive.In 50 ℃ after 1 hour, it is original about 78% that the work of mutant strain GOD-D401N/A574V enzyme is reduced to, and wild-type enzyme is lived and then reduced to originally 72%, and both are close.60 ℃ after following 10 minutes, wild-type and GOD-D401N/A574V enzyme are lived and are all declined to a great extent, and after one hour, it is original 30% that wild-type is reduced to, and GOD-D401N/A574V reduces to original about 20%.High enzyme glucose oxidase mutant strain alive Protein G OD-D401N/A574V has good thermostability, can satisfy the needs of practical application.
The stability in storage of Fig. 5 and wild-type relatively
Under 4 ℃, wild-type and GOD-D401N/A574V have stability in storage preferably, preserve the catalytic activity that still keeps after month more than 80%, but wild-type vigor lowering speed is accelerated after 30 days, wild-type has 60% vigor residue during by 40 days, and GOD-D401N/A574V still has 80% vigor residue.When room temperature (20-25 ℃) stored down for a long time, the enzyme of wild-type and GOD-D401N/A574V was lived and is all presented progressively downtrending.At preceding 25 days that preserve, the decline that the GOD-D401N/A574V enzyme is lived was a little more than wild-type enzyme.But storing the later stage, the decline that wild-type enzyme is lived is more obvious.After at room temperature storing one month, wild-type and GOD-D401N/A574V can keep the residue vigor more than 65%, and after 40 days, wild-type is 40% vigor residue only, and GOD-D401N/A574V still has 60% vigor residue.
High enzyme glucose oxidase mutant strain alive Protein G OD-D401N/A574V compares with wild-type has good preservation stability, can satisfy the needs of practical application.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.Experimental technique among the embodiment, condition is carried out routinely, with reference to as Sa nurse Brooker etc., experimental technique described in the molecular cloning experiment guide (second edition,, Science Press in 1992).
The proteic acquisition of embodiment 1. high enzymes glucose oxidase mutant strain alive:
Fig. 1 has shown the structure principle and the process of recombination mutation glucose oxidase gene.Natural sophisticated glucose oxidase is a homodimer, and each monomer contains 583 amino acid, and gene length is about 1752bp.
1. the introducing of catastrophe point in the mutant enzyme GOD-D401N/A574V encoding gene:
(1) two pairs of mutagenic primers of design, adopt the PCR fixed-point mutation method on the GOD gene wild-type GOD gene on 1752 the G C that sports A and 2272 sport T;
The required primer sequence of design glucose oxidase rite-directed mutagenesis:
Upstream primer: 5 '-CCACTACGTAAGCAATGGCATTGAAG-3 '
Downstream primer: 5 '-TATGCGGCCGCTCACTGCAT-3 '
The rite-directed mutagenesis primer:
401s：5′-AGAACTACCGC
AACTGGATT-3′
401a：5′-CAATCCAGT
TGCGGTAGTTC-3′
574s：5′TTTCGGATG
TTATCTTGG3′
574a：5′CCAAGATA
ACATCCGAAAT3′
Be template with upstream primer and primer 574a with the pPIC9kGOD that is connected with the wild-type glucose oxidase gene earlier, amplify 574 long segment, the PCR cycling condition: 94 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 7min.Pcr amplification product reclaims through 1% agarose gel electrophoresis test kit.
Is template with universal primer 3 ' AOX1 and 574s with the wild-type glucose oxidase gene, amplifies 574 short-movie section, the PCR cycling condition: 94 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 20sec, 30 circulations; 72 ℃ of 5min.Pcr amplification product reclaims through 1% agarose gel electrophoresis test kit.
Two kinds of PCR products that will reclaim then mix, and add the upstream and downstream primer and carry out the PCR second time, and the PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 2min, 5 circulations; 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 7min.Pcr amplification product reclaims through 1% agarose gel electrophoresis test kit.Obtain containing the glucose oxidase gene fragment of mutational site A574V.
Be template with upstream primer and primer 574a with the glucose oxidase gene fragment that contains mutational site A574V again, amplify 401 long segment, the PCR cycling condition: 94 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min45sec, 30 circulations; 72 ℃ of 7min.Pcr amplification product reclaims through 1% agarose gel electrophoresis test kit.
Is template with downstream primer and 401s with the glucose oxidase gene fragment that contains mutational site A574V, amplifies 401 short-movie section, the PCR cycling condition: 94 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 20sec, 30 circulations; 72 ℃ of 5min.Pcr amplification product reclaims through 1% agarose gel electrophoresis test kit.
Two kinds of PCR products that will reclaim then mix, and add the upstream and downstream primer and carry out the PCR second time, and the PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 2min, 5 circulations; 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 7min.Pcr amplification product reclaims through 1% agarose gel electrophoresis test kit.Obtain containing the glucose oxidase gene fragment of mutational site D401N/A574V.
(2) above-mentioned PCR product is after glue reclaims purifying, carrying out the substep enzyme with restriction enzyme SnaBI and NotI cuts, after enzyme is cut the recovery of product usefulness PCR product recovery test kit, be connected with the plasmid pPIC9k fragment of cutting with the T4 ligase enzyme through same enzyme, after spending the night, 16 ℃ of connections connect product transformed into escherichia coli DH5 α competent cell, obtain positive bacterium colony with penbritin (Amp)-LB plate screening, through identifying (step as follows), called after coli strain DH5 α/GOD-D401N/A574V.
2. the evaluation of plasmid pPIC9kGOD-D401N/A574V:
From positive bacterium colony DH5 α/GOD-D401N/A574V culture, extract plasmid pPIC9kGOD-D401N/A574V with the plasmid extraction test kit, carrying out enzyme with restriction enzyme BglII cuts, 1% agarose gel electrophoresis inspection, the result produces 2.4Kb and 8.7Kb two bands (as Fig. 2 a), conform to the expection size.Further identifying with PCR, is template with the recombinant plasmid, uses, downstream primer carries out PCR, can obtain size and be the amplified band of 1.8kb (as Fig. 2 b), and expects that size conforms to.
The order-checking of GOD-D401N/A574V gene is undertaken by handsome (invitrogen) Bioisystech Co., Ltd on the plasmid.Sequencing primer is universal primer 5 ' AOX1 and the 3 ' AOX1 that utilizes on the pPIC9k plasmid, sequencing result shows that the GOD-D401N/A574V gene order and the desired design of acquisition is in full accord, obtain the high enzyme glucose oxidase mutant strain alive protein coding gene GOD-D401N/A574V shown in the SEQUENCE NO.1 thus, GOD-D401N/A574V full length gene 1752nt (comprising termination codon TGA), 583 the amino acid whose albumen of encoding.With the plasmid called after pPIC9kGOD-D401N/A574V that produces.
3. the extraction of recombinant plasmid pPIC9kGOD-D401N/A574V:
DH5 α/GOD-D401N/A574V is inoculated into the LB substratum, and 37 ℃ of incubated overnight are extracted plasmid pPIC9kGOD-D401N/A574V with the plasmid extraction test kit.
4. restriction enzyme BglII enzyme is cut, and glue reclaims the big fragment of purifying, promptly obtains the required linear DNA that contains mutator gene of yeast conversion.
5. electrotransformation transforms Pichi strain GS115, obtains Pichi strain GS115/GOD-D401N/A574V through screening and evaluation.
(1) preparation of GS115 competent cell:
GS115 is connected to 30 ℃ of overnight incubation in the 50mL liquid YPD substratum, is inoculated among the 500mlYPD 30 ℃ with the ratio of 2:100 again and is cultured to OD
600=1.2-1.5,4 ℃ of 1500g centrifugal collecting cells; With the ice-cold sterilized water re-suspended cell of 500mL, 4 ℃ of 1500g centrifugal collecting cells; Use the ice-cold sterilized water re-suspended cell of 250mL again, 4 ℃ of 1500g centrifugal collecting cells; Then with the ice-cold 1mol/L sorbyl alcohol re-suspended cell of 40ml, 4 ℃ of 1500g centrifugal collecting cells; Re-suspended cell is in the 1mL1mol/L sorbyl alcohol.
(2) recombinant plasmid pPIC9kGOD-D401N/A574V transforms GS115:
100 μ L protoplastiss mix with 5-10 μ g linearization plasmid DNA (BglII cuts), change ice-cold electric revolving cup over to, place 5 minutes; The mixture of electric shock cell and DNA (1.5kv, 4.2-4.9ms); Add the ice-cold 1mol/L sorbyl alcohol of 1mL, continue to place 30min; Add 0.5mLSOS (0.3 * YPD, 1mol/L sorbyl alcohol) again, placed 2 hours for 30 ℃, shake the will thalline once in a while and be difficult for precipitation; With coating solid MD substratum behind the 1mol/L sorbyl alcohol dilution thalline.Cultivate the positive bacterium colony of picking after 4-6 days for 30 ℃.Identify through embodiment 3, can produce the proteic bacterial strain called after of GOD-D401N/A574V GS115/GOD-D401N/A574V.
6. Pichi strain GS115/GOD-D401N/A574V produces GOD-D401N/A574V albumen through methanol induction:
The single bacterium colony of picking GS115/GOD-D401N/A574V overnight incubation in 20mL MD substratum from above-mentioned MD flat board; Culture changed in the 500mL MD substratum be cultured to OD
600=1.2-1.5; Centrifugal collection yeast cell, and cell washed once with 20mL MM substratum; Change cell in 250mL MM substratum inducible protein generation, in culture, add 1% methyl alcohol every day, cultivated 4-5 days; The centrifugal supernatant liquor of collecting.
7. high enzyme glucose oxidase mutant strain alive Protein G OD-D401N/A574V purifying:
Nearly 500mL contain target protein the culture supernatants ultrafiltration and concentration to 20-30mL, dialyse in the phosphate sodium dihydrogen buffer solution of 0.02mol/L, pH6.27 desalination 24 hours of concentrated solution is to guarantee the adsorption property of anion-exchange column.
Protein purification adopts Q Sepharose
TMFast Flow anion-exchange chromatography post, pillar 20mM SODIUM PHOSPHATE, MONOBASIC (pH6.3) balance, behind the last sample, use the 20mM SODIUM PHOSPHATE, MONOBASIC, 0-0.2MNaCl (pH6.3) gradient elution, monitor collection with nucleic acid protein detector (wavelength 280nm), glucose oxidase directly flows out (band is yellow) at first from post.Obtain the high enzyme glucose oxidase mutant strain alive Protein G OD-D401N/A574V shown in SEQUENCENO.2 of separation and purification thus.
Proteic active the detection and other zymologic properties detections of the GOD-D401N/A574V that embodiment 2 Pichi strain GS115/GOD-D401N/A574V express
Make typical curve with the fresh commodity GOD of Sigma company.ABTS and 100U/mL horseradish peroxidase solution with pH5.6,0.1mol/L phosphate sodium dihydrogen buffer solution preparation 2mg/mL glucose, 0.1mg/mL.In 96 orifice plates, add ABTS and the horseradish peroxidase solution 40 μ L of 100 μ L glucose solutions, 100 μ L respectively, add 1 μ L nutrient solution or enzyme liquid, react and measure OD after 3-5 minute
414
With the determination of protein concentration kit measurement that Bio-Rad company produces, make typical curve with BSA.
Calculate enzyme than living
As seen from the above table, high enzyme work glucose oxidase mutant strain Protein G OD-D401N/A574V enzyme activity is 3 times of wild-type, aspect enzyme work, has significantly and improves.
Thermal stability analysis:
The mutant that catalytic activity improves is followed the reduction of thermostability sometimes, and wild-type and mutant strain enzyme liquid are placed respectively under 40 ℃, 50 ℃, 60 ℃, at room temperature measures residual enzyme every sampling in ten minutes and lives, and has estimated the thermostability of enzyme.The results are shown in Figure four.Water-bath was preserved in 50 minutes in 40 ℃, and wild-type and GOD-D401N/A574V enzyme are lived does not all have significantly sacrificing, and wild-type enzyme is lived and slightly descended in the time of 60 minutes, does not obviously descend and the enzyme of GOD-D401N/A574V is alive.In 50 ℃ after 1 hour, it is original about 78% that the work of mutant strain GOD-D401N/A574V enzyme is reduced to, and wild-type enzyme is lived and then reduced to originally 72%, and both are close.60 ℃ after following 10 minutes, wild-type and GOD-D401N/A574V enzyme are lived and are all declined to a great extent, and after one hour, it is original 30% that wild-type is reduced to, and GOD-D401N/A574V reduces to original about 20%.High enzyme glucose oxidase mutant strain alive Protein G OD-D401N/A574V has good thermostability, can satisfy the needs of practical application.
Stability in storage is analyzed:
Enzyme liquid is kept under 4 ℃ and the room temperature (20-25 ℃), and regularly (every 3-4 days) sampling and measuring enzyme is lived, and has estimated the stability in storage of enzyme.The results are shown in Figure five.
Under 4 ℃, wild-type and GOD-D401N/A574V have stability in storage preferably, preserve the catalytic activity that still keeps after month more than 80%, but wild-type vigor lowering speed is accelerated after 30 days, wild-type has 60% vigor residue during by 40 days, and GOD-D401N/A574V still has 80% vigor residue.When room temperature (20-25 ℃) stored down for a long time, the enzyme of wild-type and GOD-D401N/A574V was lived and is all presented progressively downtrending.At preceding 25 days that preserve, the decline that the GOD-D401N/A574V enzyme is lived was a little more than wild-type enzyme.But storing the later stage, the decline that wild-type enzyme is lived is more obvious.After at room temperature storing one month, wild-type and GOD-D401N/A574V can keep the residue vigor more than 65%, and after 40 days, wild-type is 40% vigor residue only, and GOD-D401N/A574V still has 60% vigor residue.
High enzyme glucose oxidase mutant strain alive Protein G OD-D401N/A574V compares with wild-type has good preservation stability, can satisfy the needs of practical application.
GOD-D401N/A574V still has good thermostability and stability in storage when enzyme is lived raising, can satisfy requirement of actual application.
The application in the glucose content in enzymatic assays wine of embodiment 3 glucose oxidases
Under the condition of pH7.0, get above-mentioned gained glucose oxidase mix with horseradish peroxidase and ABTS with sodium phosphate buffer (0.1mol/L) in, wherein the glucose oxidase final concentration is 10U/mL, and the horseradish peroxidase final concentration is 10U/mL, and the ABTS final concentration is 0.1mg/mL.
Get above-mentioned mixed solution 3mL, add glucose reference liquid or drinks sample 0.5mL 37 ℃ of water-bath 10min with it respectively, measure its absorption at the 414nm place.Can get glucose content in the drinks (liquor, red wine, beer) according to typical curve.The present invention is containing in the glucose 150mg/L, and absorbancy and sample glucose content have good positive correlation.So measurement range is at 0-150mg/L.
The application of embodiment 4 glucose oxidases in tea fresh keeping
With glucose oxidase and its substrate glucose together with the mixed of mol ratio 1:100000, be packaged in the waterproof but gas-pervious bag film, sealing is placed in the encloses container that tealeaves is housed, add-on is every liter of 6-8g mixture, when seeing through film, the oxygen in the encloses container enters in the bag, under the effect of glucose oxidase, react with glucose, thereby the oxygen in the removal encloses container, prevent that tealeaves is oxidized and cause quality deterioration, the quality guaranteed period of tealeaves to extend to 18-24 month.。
Glucose oxidase is a kind of toolenzyme of widespread use, is mainly used in deoxygenation in foodstuffs industry, removes glucose; In pharmaceutical industries, be mainly used in the mensuration of blood sugar, glucose in urine; In biological field, can also make corresponding biosensor and enzyme electrodes, be used to detect glucose concn.
SEQUENCELISTING
＜110〉Wuhan Virology Institute,Chinan academy of Sciences
＜120〉a kind of encoding gene of high activity glucose oxidase and preparation method and application
＜130〉a kind of encoding gene of high activity glucose oxidase and preparation method and application
<160>2
<170>PatentIn?version3.1
<210>1
<211>2303
<212>DNA
<213>Aspergillus?niger
<400>1
<210>2
<211>583
<212>PRT
<213>Aspergillus?niger
<400>2
Claims (3)
1. isolating high enzyme glucose oxidase mutant strain protein coding gene of living, its sequence is the nucleotide sequence shown in the SEQ IDNO.1.
2. isolating protein, its sequence is the aminoacid sequence shown in the SEQ ID NO.2.
3. intestinal bacteria is characterized in that: intestinal bacteria (Escherichia coli) DH5 α/GOD-D401N/A574V, CCTCC NO.M207085.
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| CN101955953A (en) * | 2010-09-09 | 2011-01-26 | 中国农业科学院生物技术研究所 | Glucose oxidase mutant gene, expression and application thereof |
| EP2796547B1 (en) * | 2013-04-24 | 2016-09-14 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Novel glucose oxidase variants |
| CN103397005B (en) * | 2013-08-07 | 2014-12-17 | 苏州昆蓝生物科技有限公司 | Production method of glucose oxidase |
| CN103525778B (en) * | 2013-10-18 | 2015-07-01 | 江南大学 | Glucose oxidase mutant with high catalytic activity |
| CN103555682B (en) * | 2013-11-15 | 2015-08-05 | 西南大学 | The separation purification method of glucose oxidase |
| CN105002147B (en) * | 2015-05-05 | 2018-06-15 | 广东溢多利生物科技股份有限公司 | The mutation glucose oxidase and its encoding gene and application that expression quantity improves |
| CN105039362B (en) * | 2015-07-21 | 2019-01-08 | 湖北大学 | A kind of gene mutation improves antioxidative glucose oxidase and its method |
| CN110577939B (en) * | 2018-06-07 | 2021-01-26 | 青岛红樱桃生物技术有限公司 | Glucose oxidase mutant with improved heat resistance as well as coding gene and application thereof |
| CN111004786B (en) * | 2019-12-25 | 2021-12-07 | 广东溢多利生物科技股份有限公司 | Glucose oxidase and carrier and application thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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