CN106085977B - The recombination and its acquisition methods of glutamte dehydrogenase and application - Google Patents

The recombination and its acquisition methods of glutamte dehydrogenase and application Download PDF

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CN106085977B
CN106085977B CN201610400139.4A CN201610400139A CN106085977B CN 106085977 B CN106085977 B CN 106085977B CN 201610400139 A CN201610400139 A CN 201610400139A CN 106085977 B CN106085977 B CN 106085977B
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gdh
alcohol
full length
length sequence
geotrichum
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CN106085977A (en
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师俊玲
朱静
徐晓光
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Northwestern Polytechnical University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)

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Abstract

The present invention relates to the recombination of glutamte dehydrogenase and its acquisition methods and applications.The specificity of food Higher Alcohols degradation at present is poor, and limits the degradation of acid condition higher alcohol, and production of enzyme is low in thallus, time-consuming, at high cost, is not able to satisfy industrialized production.The present invention will efficient degradation higher alcohol in acid condition geotrichum candidum T3d329TF(Galactomyces geotrichum) gdh gene in bacterial strain conserved region and full length sequence cloned, expressed and recombinated enzymatic activity verifying.The present invention is that catalyst orientation reduces in food using the recombinase of can degrade in acid condition n-hexyl alcohol and isoamyl alcohol, and the advanced alcohol content especially in acid food, safety and environmental protection, controllability is strong, and production cost is low, is suitble to large-scale industrial production.

Description

The recombination and its acquisition methods of glutamte dehydrogenase and application
Technical field
The present invention relates to a kind of recombinations of enzyme, and in particular to a kind of recombination of glutamte dehydrogenase and its acquisition Method and application.
Background technique
Higher alcohol is also fusel oil, and excessive higher alcohol can generate certain toxic action to people, to damage the week of people Nervous system, central nervous system, digestive system and reproductive system are enclosed, also has injury, and cream to gravidic parent and fetus Room cancer also has relationship.Higher alcohol is more than ten times of ethyl alcohol to the anesthetic effect of nerve system of human body, its oxidation rate ratio in vivo Ethyl alcohol is slow, and residence time is also longer, therefore harmful to health.The content of Fusel Oil in Liquor is strictly limited in national standard. Currently, n-hexyl alcohol has been cited as the substance (HSDB, Hazardous Substances Data Bank) that can generate harm to human body.It is fixed It is significant to the safety for improving alcoholic beverage to advanced alcohol content is reduced.
N-hexyl alcohol and isoamyl alcohol are also the main composition for causing soybean beany flavor.Beany flavor soya-bean milk, bean powder, bean curd and It is generally existing in its reproduced goods.Since there is the habit of thousands of years human consumption soybeans in China, beany flavor can still be received reluctantly.Japan, American-European and its all states in the Southern Hemisphere people, very dislike beany flavor.Soybean and bean product are the important proteinaceous nutrients of the mankind Source, but beans raw meat problem by restrict bean product produce development and China's bean product countries in the world popularization.The type of higher alcohol, Type, mouthfeel and the quality of concentration and grape wine, beer etc. have very big relationship, as too high levels can be brought to grape wine it is bitter, Astringent taste；Bata-phenethyl alcohol too high levels can also bring offending mouthfeel to grape wine.Advanced alcohol content is more than that 100mg/L can make beer Wine taste and pouplarity are substantially reduced.Therefore, orientation reduces advanced alcohol content has very to flavour of food products and acceptance is improved Big influence.
Currently, the method that can be catalyzed higher alcohol conversion is mostly chemical conversion, but chemical conversion approach does not have because too extensive There is specific specificity, chemical contamination is more serious, and the molecular catalyst that higher alcohol is converted into corresponding ester and aldehyde etc. is difficult to obtain, And catalytic efficiency is to be improved, the disadvantages of potential insecurity factor, is not received by food service industry.Also by selecting yeast or Control forms relevant factor and film filtering controls advanced alcohol content, but ethyl alcohol and other fragrance component contents also reduce simultaneously. Production of enzyme in thallus is lower, time-consuming, at high cost, is not suitable for industrialized production.
Enzymic degradation food higher alcohols content, can be to avoid the residue problem of poisonous and harmful substance, and reaction speed is fast, raw Production process control is strong, and specificity is strong, has a good application prospect.
Summary of the invention
The object of the present invention is to provide a kind of recombination of glutamte dehydrogenase and its acquisition methods and application, Neng Gou Higher alcohol under acid condition in orientation degradation food.
The technical scheme adopted by the invention is that:
The acquisition methods of glutamte dehydrogenase recombination, it is characterised in that:
It is realized by following steps:
Step 1: extraction geotrichum candidum (Galactomyces geotrichum) T3d329TF bacterial strain total serum IgE, and synthesize cDNA；
Step 2: the conserved region and full length sequence of gdh gene are cloned:
Step 3: the conserved region and full length sequence of gdh gene are expressed, including recombinant expression carrier Inducing expression in Escherichia coli of building and enzyme gene.
In step 1, geotrichum candidum (Galactomyces geotrichum) T3d329TF bacterial strain cultural method by following Step is realized:
(1) by geotrichum candidum (Galactomyces geotrichum) T3d329TF strain inoculated is in fluid nutrient medium, In 28 DEG C, cultivate 18h under conditions of 160rpm after, be transferred in fluid nutrient medium by 2.0% inoculum concentration, in 28 DEG C, the item of 160rpm Expand culture 38h under part；
The fluid nutrient medium is murphy juice dextrose broth, formula are as follows: 20% potato leaching juice 1000mL, Glucose 20g, pH are natural；
(2) gained culture is centrifuged 10min at 5000rpm, 4 DEG C, collects thallus, and suspended again with sterile water and from The heart is repeated 5 times, and 10min is finally centrifuged at 8000rpm, 4 DEG C, is collected wet thallus, is lured under 28 DEG C of MSM culture medium, 160rpm After leading processing 6h, it is centrifuged 10min at 5000rpm, 4 DEG C, collects thallus, sterile water wash 5 times, finally in 8 000 r/m, 4 It is centrifuged 10 min at DEG C, collects thallus；
The MSM culture medium is minimal medium, formula are as follows: MgSO47H2O 0.5g, KH2PO41.0g, NH4NO3 1.0 g, 6.67g/L CaCl2Solution 3mL, the FeCl of 17 g/L3Solution 3mL, FeSO4·7H2O 0.05g, NaNO31.0g steams 1 000mL of distilled water.
In step 2:
Conservative zone amplication primer are as follows:
GDH-F1 5'-GGATCCACGCCGCTCAAGGTC-3' BamHⅠ；
GDH-R1 5'-AAGCTTTACCAAGAAATCACCGTGGTC-3' HindⅢ；
Full length sequence amplimer are as follows:
GDH-F2 5'-CGGGATCCATCAAAATGGTCCAGCCTTCC-3' BamHⅠ；
GDH-R2 5'-CCCAAGCTTTTACCAGAAATCACCGTGGTCG-3' HindⅢ；
PCR amplification system is
PCR reaction condition are as follows:
The reaction condition of conserved region: 95 DEG C of initial denaturation, 5 min；94 DEG C of denaturation temperature, 30s；55.6 DEG C of annealing temperature and 58.0 DEG C, 30 s；72 DEG C of elongating temperature, 1:30 min；After 35 circulations, 72 DEG C of 10 min of extension；
The reaction condition of full length sequence: 95 DEG C of initial denaturation, 3 min；94 DEG C of denaturation temperature, 30 s；Annealing temperature 58.0 DEG C, 30 s；72 DEG C of elongating temperature, 1 min；After 33 circulations, 72 DEG C of reparations extend 7 min.
In step 3:
The building of recombinant expression carrier the following steps are included:
(conserved region pMD19-T-658-GDH() and pMD19-T-1359- are connect with pMD19-T after pcr amplification product recycling GDH(full length sequence)) and convertE.coliDH5 α competent cell, picking positive bacterium colony, after proposing plasmid PCR detection, positive gram It is grand to be sequenced；Then to recombination carrier T (conserved region pMD19-T-658-GDH() and pMD19-T-1359-GDH(full length sequence)) III double digestion of BamH I and Hind has been carried out with expression vector (pET-28as)；
Endonuclease reaction system are as follows:
Later, 37 DEG C of reactions are placed in overnight, carry out 1% agarose gel electrophoresis, gel extraction purpose band uses Omega Gel Extraction kit；Then connect expression vector, by after digestion target fragment and expression vector be attached；
Coupled reaction system are as follows:
The recombinant expression carrier built overnight, is respectively designated as the conserved region pET-28as-658-GDH(by 16 DEG C of connections) With pET-28as-1359GDH(full length sequence).Recombinant expression carrier conversionE.coliDH5 α competent cell, mention plasmid PCR and After double digestion detection, positive colony sequencing verifying；
Inducing expression of the gdh gene in Escherichia coli:
Recombinant expression carrier is convertedE.coliBL21 (DE3) competent cell, from conversion plate on pick them separately containing The conserved region recombinant expression carrier pET-28as-658-GDH(), pET-28as-1359-GDH(full length sequence) recombinant bacterium and contain The control bacterium of empty carrier is inoculated in the expression culture medium containing 50 μ g/mL Kan, 37 DEG C, 180r/min overnight incubation.Then It is transferred in the 100mL triangular flask equipped with 30mL expression culture medium (2 × YT) with 10% inoculum concentration, 37 DEG C, 180 r/m culture It is 0.6-1.0 to OD600, adds 24 μ g/mL Kan, and IPTG to final concentration 0.1mmol/L, 180r/m is added, 25 DEG C of low temperature Induce 7h；4 DEG C, 12000 × g is centrifuged 5min, collects thallus；With PBS suspension cell, ultrasonic disruption 5min in ice-water bath (200W, work 3s, and have a rest 5s, 120 times), 12000 × g is centrifuged 15min, collects supernatant measurement glutamte dehydrogenase to n-hexyl alcohol Activity, and carry out SDS-PAGE detection；Using convert recombinant bacterium BL21 (DE3)/pET28as of empty plasmid pET-28as as pair According to.
The glutamte dehydrogenase recombination that the acquisition methods obtain.
The glutamate dehydrogenase recombinase that the glutamte dehydrogenase recombination defines.
Application of the glutamate dehydrogenase recombinase in terms of orientation reduces food higher alcohols content.
The invention has the following advantages that
Operation of the present invention is simple, conversion rate is fast, the production time is short, yield is high, safety and environmental protection, production cost are low, utilizes Based on the gdh gene of geotrichum candidum, using the means of genetic engineering, being quickly obtained can be high in acid condition The glutamte dehydrogenase of effect degradation n-hexyl alcohol and isoamyl alcohol；Using enzyme process bioconversion higher alcohol, can be completed within a few hours Reaction, degradation efficiency are high；Using enzyme process bioconversion higher alcohol, Growth of Biologic Cell situation can be got rid of to the shadow of efficiency of pcr product It rings, controllability is strong；It is highly-safe, specificity is strong using enzymatic conversion method higher alcohol, it is that one kind can industrialize extensive orientation and reduce The Enzymology method of food Higher Alcohols provides new thinking and approach for the orientation reduction of industrial Higher Alcohols.
Detailed description of the invention
Fig. 1 is total serum IgE and conserved region and full length sequence amplification figure in thallus T3d329TF.
In figure, M:DNA Marker；1,2 conserved region gene amplified production of (left side) 1,2 total serum IgE (right side)；3,4 overall length bases Gene-amplification product.
Fig. 2 is the identification of recombinant protein expression-form.
Fig. 3 is the SDS-PAGE electrophoresis of recombinase after Ni column purification.
In figure, a:E. coliBL21(DE3)/pET-28as-GDH-658；B:E. coliBL21(DE3)/pET-28as- GDH-1359；M: protein standard；50 mM: the recombinase (conserved region) of purifying, as indicated with an arrow；120 mM: purifying recombinase (full-length gene), as indicated with an arrow；28as compares bacteriumE. coliBL21 (DE3)/pET-28as holoprotein.
Fig. 4 is the detection of the Western marking.
In figure, 1:28as compares bacteriumE. coliBL21 (DE3)/pET-28as holoprotein；2: recombinant bacteriumE. coliBL21 (DE3)/pET-28as-GDH-658 clasmatosis liquid supernatant；3: the recombinase of gene conserved region after purification；4: weight Group bacteriumE. coliBL21 (DE3)/pET-28as-GDH-1359 clasmatosis liquid supernatant；5: the weight of full length gene sequence after purification Group enzyme arrow meaning is target protein.
Fig. 5 is excision SUMO fusion tag.
In figure, a: recombinant bacteriumE. coliBL21 (DE3)/pET-28as-GDH-1359 clasmatosis liquid supernatant；M: albumen Standard；1,28as, compare bacteriumE. coliBL21 (DE3)/pET-28as holoprotein；2: recombinant bacteriumE. coliBL21(DE3)/ PET-28as-GDH-1359 holoprotein；3: recombinant bacteriumE. coliBL21 (DE3)/pET-28as-GDH-1359 clasmatosis liquid Supernatant；4: the SUMO label that full length sequence clasmatosis liquid supernatant is cut away by SUMO protease.B: the full length sequence of purifying Recombinase；1: the gained enzyme of purifying and the SUMO label cut away by SUMO protease.
Fig. 6 is the expression activitiy of recombinase and bacterial enzyme.
Fig. 7 is the GC-MS analysis for recombinating enzymatic n-hexyl alcohol and isoamyl alcohol converted product.
Fig. 8 is the substrate specificity of recombinant protein.
Fig. 9 is the building of expression vector pET-28as-658-GDH.
Figure 10 is the building of expression vector pET-28as-1359-GDH.
Specific embodiment
The present invention will be described in detail With reference to embodiment.
The acquisition methods of glutamte dehydrogenase recombination of the present invention, are realized by following steps:
Step 1: extraction geotrichum candidum (Galactomyces geotrichum) T3d329TF(CCTCC NO:M 208167) The total serum IgE of bacterial strain, and synthesize cDNA；
The geotrichum candidum (Galactomyces geotrichum) T3d329TF on October 21st, 2008 is preserved in China Type Tissue Collection, address are No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road, and deposit number is CCTCC NO:M 208167.
Step 2: cloning the conserved region and full length sequence of gdh gene, obtains enzyme open reading frame GenBank accession number is KJ442577:
Step 3: the conserved region and full length sequence of gdh gene are expressed, including recombinant expression carrier Inducing expression in Escherichia coli of building and enzyme gene.
In step 1, geotrichum candidum (Galactomyces geotrichum) T3d329TF bacterial strain cultural method by following Step is realized:
(1) by geotrichum candidum (Galactomyces geotrichum) T3d329TF strain inoculated is in fluid nutrient medium, In 28 DEG C, cultivate 18h under conditions of 160rpm after, be transferred in fluid nutrient medium by 2.0% inoculum concentration, in 28 DEG C, the item of 160rpm Expand culture 38h under part；
The fluid nutrient medium is murphy juice dextrose broth, formula are as follows: 20% potato leaching juice 1000mL, Glucose 20g, pH are natural；
(2) gained culture is centrifuged 10min at 5000rpm, 4 DEG C, collects thallus, and suspended again with sterile water and from The heart is repeated 5 times, and 10min is finally centrifuged at 8000rpm, 4 DEG C, is collected wet thallus, is lured under 28 DEG C of MSM culture medium, 160rpm After leading processing 6h, it is centrifuged 10min at 5000rpm, 4 DEG C, collects thallus, sterile water wash 5 times, finally in 8 000 r/m, 4 It is centrifuged 10 min at DEG C, collects thallus；
The MSM culture medium is minimal medium, formula are as follows: MgSO47H2O 0.5g, KH2PO41.0g, NH4NO3 1.0 g, 6.67g/L CaCl2Solution 3mL, the FeCl of 17 g/L3Solution 3mL, FeSO4·7H2O 0.05g, NaNO31.0g steams 1 000mL of distilled water.
In step 1, pillar of the extracting method referring to Sangon Biotech (Shanghai) Co., Ltd. of total serum IgE is extracted Fungi total serum IgE extracting and purifying kit illustrates to carry out, and cDNA synthesis uses the Superscrip First- of Invitrogen company Strand cDNA Synthesis System for qRT-PCR kit carries out.
In step 2:
Conservative zone amplication primer are as follows:
GDH-F1 5'-GGATCCACGCCGCTCAAGGTC-3' BamHⅠ；
GDH-R1 5'-AAGCTTTACCAAGAAATCACCGTGGTC-3' HindⅢ；
Full length sequence amplimer are as follows:
GDH-F2 5'-CGGGATCCATCAAAATGGTCCAGCCTTCC-3' BamHⅠ；
GDH-R2 5'-CCCAAGCTTTTACCAGAAATCACCGTGGTCG-3' HindⅢ；
PCR amplification system is
PCR reaction condition are as follows:
The reaction condition of conserved region: 95 DEG C of initial denaturation, 5 min；94 DEG C of denaturation temperature, 30s；55.6 DEG C of annealing temperature and 58.0 DEG C, 30 s；72 DEG C of elongating temperature, 1:30 min；After 35 circulations, 72 DEG C of 10 min of extension；
The reaction condition of full length sequence: 95 DEG C of initial denaturation, 3 min；94 DEG C of denaturation temperature, 30 s；Annealing temperature 58.0 DEG C, 30 s；72 DEG C of elongating temperature, 1 min；After 33 circulations, 72 DEG C of reparations extend 7 min.
In step 3:
The building of recombinant expression carrier the following steps are included:
The building of recombinant expression carrier the following steps are included:
(conserved region pMD19-T-658-GDH() and pMD19-T-1359- are connect with pMD19-T after pcr amplification product recycling GDH(full length sequence)) and convertE.coliDH5 α competent cell, picking positive bacterium colony, after proposing plasmid PCR detection, positive gram It is grand to be sequenced；Then to recombination carrier T (conserved region pMD19-T-658-GDH() and pMD19-T-1359-GDH(full length sequence)) III double digestion of BamH I and Hind has been carried out with expression vector (pET-28as)；
Endonuclease reaction system are as follows:
Later, 37 DEG C of reactions are placed in overnight, carry out 1% agarose gel electrophoresis, gel extraction purpose band uses Omega Gel Extraction kit；Then connect expression vector, by after digestion target fragment and expression vector be attached；
Coupled reaction system are as follows:
The recombinant expression carrier built overnight, is respectively designated as the conserved region pET-28as-658-GDH(by 16 DEG C of connections) With pET-28as-1359GDH(full length sequence).Recombinant expression carrier conversionE.coliDH5 α competent cell, mention plasmid PCR and After double digestion detection, positive colony sequencing verifying.
Inducing expression of the gdh gene in Escherichia coli:
Recombinant expression carrier is convertedE.coliBL21 (DE3) competent cell, from conversion plate on pick them separately containing The conserved region recombinant expression carrier pET-28as-658-GDH(), pET-28as-1359-GDH(full length sequence) recombinant bacterium and contain The control bacterium of empty carrier is inoculated in the expression culture medium containing 50 μ g/mL Kan, 37 DEG C, 180r/min overnight incubation.Then It is transferred in the 100mL triangular flask equipped with 30mL expression culture medium (2 × YT) with 10% inoculum concentration, 37 DEG C, 180 r/m culture It is 0.6-1.0 to OD600, adds 24 μ g/mL Kan, and IPTG to final concentration 0.1mmol/L, 180r/m is added, 25 DEG C of low temperature Induce 7h；4 DEG C, 12000 × g is centrifuged 5min, collects thallus；With PBS suspension cell, ultrasonic disruption 5min in ice-water bath (200W, work 3s, and have a rest 5s, 120 times), 12000 × g is centrifuged 15min, collects supernatant measurement glutamte dehydrogenase to n-hexyl alcohol Activity, and carry out SDS-PAGE detection；Using convert recombinant bacterium BL21 (DE3)/pET28as of empty plasmid pET-28as as pair According to.
Recombinate enzymatic characterization:
(1) recombinase expression-form is identified.The recombination thallus induced in right amount and pair containing empty carrier through inducing are taken respectively According to thallus, after ultrasonic disruption, the supernatant of clasmatosis liquid is taken to precipitate (the Susana et al. after Urea treatment respectively 2006) sample carries out SDS-PAGE electrophoresis (method 1), and measures Hexanol degrading activity (method 2), determines the expression of enzyme Form.
(2) expression activitiy of recombinase and bacterial enzyme.Measure recombinase supernatant (crude enzyme liquid), recombinase after Ni column purification, The activity of pure enzyme after the thick enzyme of thallus T3d329TF and column chromatographic purifying, enzyme activity determination method is the same as method 2.
(3) substrate specificity of recombinase
Using normal propyl alcohol, n-butanol, isobutanol, n-hexyl alcohol, isoamyl alcohol, glutamic acid and α-ketoglutaric acid as substrate, enzyme activity is surveyed Surely it is carried out by method 2 and method 3.
(4) SUMO fusion tag is cut off.Take the recombinase supernatant (crude enzyme liquid) of 200 μ L and through Ni-NTA after purification pure Enzyme, is added 1 μ L of SUMO protease, and under the conditions of 4 DEG C, after 16 h of digestion, the effect of SDS-PAGE electrophoretic analysis digestion determines table Whether the albumen reached is target protein.
The molecular weight of the advanced alcohol dehydrogenase of method 1 is analyzed.With the overall molecule of its organized enzyme of Native-PAGE electrophoretic analysis Amount.Electrophoresis method is carried out referring to 2.2.6.3, measures the whole molecule amount of advanced alcohol dehydrogenase；Vertical panel SDS-PAGE electrophoresis reference Laemmli(1970 method) measures the molecular weight subunit of enzyme.The analysis condition of SDS-PAGE are as follows: 12% separation gel, 5% concentration Glue, deposition condition: 10 mA of glue electric current, 20 mA of separation gel electric current is concentrated in 200 V of voltage.Coomassie brilliant blue is used after electrophoresis R-250 dyeing, coloring agent prepare same 2.2.6.3.Destainer: 67 mL of Dan Zhengshui, 25 mL of ethyl alcohol, 8 mL of glacial acetic acid.It has decolourized The photograph of Bi Houyong Bio-Rad gel imager.
2 reaction system of method: (pH4.0,0.1 mol/L) containing 10 mL citric acid solution of higher alcohol, higher alcohol used For normal propyl alcohol, n-butanol, isobutanol, n-hexyl alcohol and isoamyl alcohol, initial concentration is 20 mmol/L.1 mL of enzyme solution dosage.React item Part: 28 DEG C, 160 r/m isothermal reaction, 1 h.After reaction, it is remained with the higher alcohol in gas chromatographic detection reaction system Amount.
Test method: when measuring the higher alcohol degrading activity of enzyme solution, 1 mL enzyme solution is directly added in above-mentioned reaction system It is reacted；Control is that the reaction system of enzyme solution is replaced with 1mL citric acid solution (pH4.0,0.1 mol/L).
Method 3(1) spectrophotometric determination.The measurement of glutamic acid and α-ketoglutaric acid carries out at 30 DEG C.Reference Choudhury and Punekar(2009) method be measured and data analysis, glutamate dehydrogenase enzymatic activity is expressed as A340 The variation of NADP (H) light absorption value under the conditions of nm.Unit degrading activity (U) is defined as every milligram of albumen reducing/oxidizing per minute 1 μmolNADP+/NADPH。
(2) experimental condition: chromatographic column: Shimadzu Wondasil type C18 chromatographic column (250 × 4.6 mm)；Detector: purple External detector；Detection wavelength: 215 nm；Column temperature: 30 DEG C；Sample volume: 20 μ L；Mobile phase: containing the water-soluble of 1% chromatographic grade acetic acid Liquid: methanol=95:5.Mobile phase pH:2.5；Flow velocity: 1 mL/min.
Embodiment 1:
Step 1: thallus preparation:
By 208167 strain inoculated of geotrichum candidum CCTCC NO:M in murphy juice dextrose broth (20% potato Juice 1000mL is soaked, glucose 20 g, pH are natural) in, 18h is cultivated under conditions of 28 DEG C, 160 rpm, by 2.0% inoculum concentration It accesses and expands culture in above-mentioned culture medium, condition is same as above, and incubation time 38h, gained culture is centrifuged at 5000rpm, 4 DEG C 10min collects thallus, and is suspended and be centrifuged again with sterile water, is repeated 5 times, is finally centrifuged 10min at 8000rpm, 4 DEG C, Wet thallus is collected, with 28 DEG C of MSM culture medium, under 160rpm after induction processing 6h, 10min is centrifuged at 5000rpm, 4 DEG C, is collected Thallus sterile water wash 5 times, is finally centrifuged 10 min at 8 000 r/m, 4 DEG C, collects thallus and extracts total serum IgE.
Step 2: thallus Total RNAs extraction:
In thallus method for extracting total RNA according to Sangon Biotech (Shanghai) Co., Ltd. pillar fungi total serum IgE Extracting and purifying kit illustrates to carry out, and detects integrality using 1% ethidium bromide gel (180V, 10min), Q5000 is micro ultraviolet Spectrophotometric determination A260nm/280nmValue and rna content.
Experimental result: total serum IgE quality is preferable, and the band of 28S, 18S and 5S are than more complete, and the ratio of 28S and 18S are in 1-2: Between 1；Its A260nm/A280nm value is 1.97, and between 1.8-2.0,926 μ g/mL, RNA purity of content is higher.
Embodiment 2:
Step 1: thallus preparation:
With embodiment 1.
Step 2: the clone of conserved region and full length sequence:
1. conservative zone amplication primer:
GDH-F1 5'-GGATCCACGCCGCTCAAGGTC-3' BamHⅠ
GDH-R1 5'-AAGCTTTACCAAGAAATCACCGTGGTC-3' HindⅢ
Full length sequence amplimer:
GDH-F2 5'-CGGGATCCATCAAAATGGTCCAGCCTTCC-3' BamHⅠ
GDH-R2 5'-CCCAAGCTTTTACCAGAAATCACCGTGGTCG-3' HindⅢ
2. the conserved region of gdh gene and the PCR amplification system of full length sequence
3. PCR reaction condition
The reaction condition of conserved region: 95 DEG C of initial denaturation, 5 min；94 DEG C of denaturation temperature, 30 s；55.6 DEG C of annealing temperature With 58.0 DEG C, 30 s；72 DEG C of elongating temperature, 1:30 min；After 35 circulations, 72 DEG C of 10 min of extension.
The reaction condition of full length sequence: 95 DEG C of initial denaturation, 3 min；94 DEG C of denaturation temperature, 30 s；Annealing temperature 58.0 DEG C, 30 s；72 DEG C of elongating temperature, 1 min；After 33 circulations, 72 DEG C of reparations extend 7 min.
(120V, 30-60min) is detected using 1% agarose electrophoresis, Sangon Biotech (Shanghai) Co., Ltd. surveys Sequence.
Experimental result: PCR amplification, obtaining target fragment is respectively 658 bp and 1359 bp, and sequence verification is correct.
Embodiment 3:
Step 1: thallus preparation:
With embodiment 1.
Step 2: the expression of enzyme gene conserved region and full length sequence:
(1) (conserved region pMD19-T-658-GDH() and pMD19-T- are connect with pMD19-T after pcr amplification product recycling 1359-GDH(full length sequence)) and convertE.coliDH5 α competent cell, picking positive bacterium colony, after proposing plasmid PCR detection, Positive colony has been sequenced.Then to recombination carrier T (conserved region pMD19-T-658-GDH() and pMD19-T-1359-GDH(overall length Sequence)) and expression vector (pET-28as) carried out III double digestion of BamH I and Hind.
(2) 37 DEG C of reactions are placed in overnight, carry out 1% agarose gel electrophoresis, gel extraction purpose band is solidifying with Omega Plastic recovery kit purifying.Then connect expression vector, by after digestion target fragment and expression vector be attached.
16 DEG C of connections are overnight.The recombinant expression carrier built is respectively designated as the conserved region pET-28as-658-GDH() With pET-28as-1359GDH(full length sequence).Recombinant expression carrier conversionE.coliDH5 α competent cell, mention plasmid PCR and After double digestion detection, positive colony sequencing verifying.
(3) recombinant expression carrier is convertedE.coliBL21 (DE3) competent cell.It picks them separately and contains from conversion plate Have the conserved region recombinant expression carrier pET-28as-658-GDH(), pET-28as-1359-GDH(full length sequence) recombinant bacterium and Control bacterium containing empty carrier is inoculated in the expression culture medium containing 50 μ g/mL Kan, and 37 DEG C, 180 r/min were cultivated Night.Then it is transferred in the 100 mL triangular flasks equipped with 30 mL expression culture medium (2 × YT) with 10% inoculum concentration, 37 DEG C, It is 0.6-1.0 that 180 r/m, which are cultivated to OD600, adds 24 μ g/mL Kan, and IPTG to 0.1 mmol/L of final concentration is added, 180 R/m, 25 DEG C of low temperature 7 h of induction.4 DEG C, 12 000 × g is centrifuged 5 min, collects thallus.With PBS suspension cell, in ice-water bath 5 min(200 W of ultrasonic disruption, work 3 s, and have a rest 5 s, and 120 times), 12 000 × g is centrifuged 15 min, and it is standby to collect supernatant With.To convert recombinant bacterium BL21 (DE3)/pET28as of empty plasmid pET-28as as control.
Using 1% agarose electrophoresis detect connection effect, SDS-PAGE analyze expression product, chromatographic just oneself Alcohol degradation amount: with embodiment 2.
Experimental result:
Gene conserved region and full length sequence are successfully transferred to expression bacteriumE.coliBL21 (DE3), and efficient soluble-expression.
Embodiment 5:
Step 1: thallus preparation:
With embodiment 1.
Step 2: recombination enzymatic characterization:
(1) the recombination thallus induced in right amount and the control thallus containing empty carrier through inducing are taken respectively, through ultrasonic disruption Afterwards, it takes the supernatant of clasmatosis liquid to precipitate the sample of after Urea treatment (Susana et al. 2006) respectively, carries out SDS-PAGE electrophoresis (method 1), and Hexanol degrading activity (method 2) is measured, determine the expression-form of enzyme.
(2) expression activitiy of recombinase and bacterial enzyme.Measure recombinase supernatant (crude enzyme liquid), recombinase after Ni column purification, The activity of pure enzyme, enzyme activity determination method 2 after the thick enzyme of thallus T3d329TF and column chromatographic purifying.
(3) substrate specificity of recombinase.With normal propyl alcohol, n-butanol, isobutanol, n-hexyl alcohol, isoamyl alcohol, glutamic acid and α- Ketoglutaric acid is substrate, enzyme activity determination method 2 and 3.
(4) SUMO fusion tag is cut off.Take the recombinase supernatant (crude enzyme liquid) of 200 μ L and through Ni-NTA after purification pure Enzyme, is added 1 μ L of SUMO protease, and under the conditions of 4 DEG C, after 16 h of digestion, the effect of SDS-PAGE electrophoretic analysis digestion determines table Whether the albumen reached is target protein.
Using SDS-PAGE and the distribution of chromatographic albumen and higher alcohol degradation amount: with embodiment 4.
Experimental result:
Efficient soluble-expression is realized to gained gene, active component is mainly in supernatant fraction；Gene conserved region and overall length sequence The expression product of column is respectively 24.3 and 50.3 kDa；Two expression products all have higher alcohol and glutamic acid catalytic activity, and The expression product activity of gene conserved region is higher than the expression product activity of full length gene sequence.
The contents of the present invention are not limited to cited by embodiment, and those of ordinary skill in the art are by reading description of the invention And to any equivalent transformation that technical solution of the present invention is taken, all are covered by the claims of the invention.

Claims (3)

1. a kind of application of glutamte dehydrogenase in terms of orientation reduces food higher alcohols content, it is characterised in that:
The higher alcohol is n-hexyl alcohol and isoamyl alcohol, and the glutamte dehydrogenase is defined by glutamte dehydrogenase recombination, institute Glutamte dehydrogenase recombination is stated to be obtained by following methods:
Step 1: extraction geotrichum candidum (Galactomyces geotrichum) T3d329TF bacterial strain total serum IgE, and synthesize cDNA；
The geotrichum candidum (Galactomyces geotrichum) T3d329TF on October 21st, 2008 is preserved in Chinese allusion quotation Type culture collection, deposit number are CCTCC NO:M 208167；
Step 2: the conserved region and full length sequence of gdh gene are expanded and are cloned:
Conservative zone amplication primer are as follows:
GDH-F1 5'-GGATCCACGCCGCTCAAGGTC-3' BamHⅠ；
GDH-R1 5'-AAGCTTTACCAAGAAATCACCGTGGTC-3' HindⅢ；
Full length sequence amplimer are as follows:
GDH-F2 5'-CGGGATCCATCAAAATGGTCCAGCCTTCC-3' BamHⅠ；
GDH-R2 5'-CCCAAGCTTTTACCAGAAATCACCGTGGTCG-3' HindⅢ；
Step 3: the conserved region and full length sequence of gdh gene are expressed, the structure including recombinant expression carrier Build the inducing expression with enzyme gene in Escherichia coli.
2. a kind of glutamte dehydrogenase according to claim 1 answering in terms of orientation reduces food higher alcohols content With, it is characterised in that:
In step 1, geotrichum candidum (Galactomyces geotrichum) T3d329TF bacterial strain cultural method by following steps It realizes:
(1) by geotrichum candidum (Galactomyces geotrichum) T3d329TF strain inoculated is in fluid nutrient medium, 28 DEG C, cultivate 18h under conditions of 160rpm after, be transferred in fluid nutrient medium by 2.0% inoculum concentration, in 28 DEG C, the condition of 160rpm 38h is cultivated in lower expansion；
The fluid nutrient medium is murphy juice dextrose broth, formula are as follows: 20% potato leaching juice 1000mL, grape Sugared 20g, pH are natural；
(2) gained culture is centrifuged 10min at 5000rpm, 4 DEG C, collects thallus, and suspended and be centrifuged again with sterile water, It is repeated 5 times, 10min is finally centrifuged at 8000rpm, 4 DEG C, collect wet thallus, induced under 28 DEG C of MSM culture medium, 160rpm After handling 6h, it is centrifuged 10min at 5000rpm, 4 DEG C, collects thallus, sterile water wash 5 times, finally in 8 000 r/m, 4 DEG C 10 min of lower centrifugation collect thallus；
The MSM culture medium is minimal medium, formula are as follows: MgSO47H2O 0.5g, KH2PO41.0g, NH4NO3 1.0 G, 6.67g/L CaCl2Solution 3mL, the FeCl of 17 g/L3Solution 3mL, FeSO4·7H2O 0.05g, NaNO31.0g, distilled water 1 000mL。
3. a kind of glutamte dehydrogenase according to claim 1 answering in terms of orientation reduces food higher alcohols content With, it is characterised in that:
In step 2:
PCR amplification system is
PCR reaction condition are as follows:
The reaction condition of conserved region: 95 DEG C of initial denaturation, 5 min；94 DEG C of denaturation temperature, 30s；55.6 DEG C of annealing temperature or 58.0 DEG C, 30 s；72 DEG C of elongating temperature, 1.5min；After 35 circulations, 72 DEG C of 10 min of extension；
The reaction condition of full length sequence: 95 DEG C of initial denaturation, 3 min；94 DEG C of denaturation temperature, 30 s；58.0 DEG C of annealing temperature, 30 s；72 DEG C of elongating temperature, 1 min；After 33 circulations, 72 DEG C of 7 min of extension.
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