CN103409449A

CN103409449A - Method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of gene in coliform strain

Info

Publication number: CN103409449A
Application number: CN2012103451530A
Authority: CN
Inventors: 周哲敏; 朱龙宝; 崔文璟; 周丽; 张川
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2012-09-18
Filing date: 2012-09-18
Publication date: 2013-11-27

Abstract

The invention discloses a method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of the gene in a coliform strain, and belongs to the technical field of enzymology and enzyme engineering. Pure recombinase is obtained through gene cloning, recombinant bacterium construction, induced expression and recombinase purification, and the specific activity of the pure recombinase is 3U/mg. The method can be used for producing a great amount of phenylalanine ammonia lyase (PAL) in relatively short time, to meet industrial production demand.

Description

The gene clone of a kind of rhodotorula glutinis phenylalanine deaminase reaches the high-efficiency expression method at the large intestine bacterial classification

Technical field:

The present invention relates to the gene clone of a kind of rhodotorula glutinis phenylalanine deaminase and reach the high-efficiency expression method at the large intestine bacterial classification, belong to zymetology and technical field of enzyme engineering.

Background technology:

Rhodotorula glutinis (Rhodotorula glutinis) can be at cheap raw material such as Semen Maydis powder, syrup, molasses, dregs of beans, on the industrial wastes such as gourmet powder waste water, grow, easily realize large-scale the cultivation, and have the hyperphenylalaninemia deaminase active, it produces L-phe and sweeting agent aspartame in industrial application.But because this enzyme is inducible enzyme, after the work of growth logarithmic phase enzyme reached peak value, enzyme was lived and is descended comparatively fast, and poor stability, thalline can not be for a long time, recycling, cause production cost to remain high.For improving enzyme, live and stability, need to adopt the molecular biosciences means to carry out molecular modification to enzyme, build engineering bacteria, scale operation high purity recombinase, for suitability for industrialized production.The method of this patent is clone's rhodotorula glutinis PAL gene, builds recombination bacillus coli, realizes the high efficient expression of phenylalanine deaminase.Adopt the ammonium sulfate fractional precipitation, anionite-exchange resin, recombinase is made with extra care in gel-filtration, obtains pure enzyme.

Summary of the invention:

The purpose of this invention is to provide the gene clone of rhodotorula glutinis phenylalanine deaminase and reach the method at E. coli.

The concrete steps of present method are as follows:

(1) gene clone of PAL

(2) construction of expression vector pET-28a (+)-pal

(3) proceed to E.coliJBL21, the IPTG abduction delivering

(4) SDS-PAGE, Westernblot and enzyme activity determination

(5) recombinase is refining

Detailed step of the present invention is:

The total RNA of 1 rhodotorula glutinis extracts

(1) picking rhodotorula glutinis list bacterium colony in the seed culture medium of 5mL in 30 ℃ cultivate 24h after, get the seed liquor of 50 μ L to the triangular flask that the 50mL substratum is housed, cultivate 18-20h to the logarithmic phase (OD that grows for 30 ℃ ₆₀₀=1.0), the centrifugal 5min of 1500g collects thalline, and cold water washing thalline 2 times, abandon supernatant.

The TES of (2) 400 μ L (10mM Tris-HCl, 10mM EDTA, 0.5%SDS, pH7.5) damping fluid re-suspended cell, add 400 μ L acidic phenol, concuss 10sec, and 65 ℃ of incubation 1h, use the of short duration concussion of vortex vibrator frequently.

(3) ice is educated 5min, in 4 ℃, and the centrifugal 5min of 12000g.

(4) water is moved in the centrifuge tube of another 1.5mL, add 400 μ L acidic phenol, concuss 10sec, repeating step (3).

(5) water is moved in the centrifuge tube of another 1.5mL, add chloroform, concuss 10sec, in 4 ℃, the centrifugal 5min of 12000g.

(6) water is moved on in new 1.5mL centrifuge tube, add 3M NaAc and the ice-cold dehydrated alcohol of 1ml of 40ul, in 4 ℃, the centrifugal 5min of 12000g.

(7) add 75% ethanol of 1mL to shake fast washing RNA precipitation.

(8) be deposited on aseptic operating platform wind 15min to the alcohol volatilization fully, add the sterilized water 50 μ L that process through DEPC resuspended ,-80 ℃ of preservations are standby, adopt agarose gel electrophoresis to detect total RNA quality.

2 reverse transcriptions, obtain cDNA

(1) in the centrifuge tube of 0.5mL, add total RNA of 50ng, 1 μ L 5 '-AAGCAGTGGTATCAACGCAGAGTACGGG GG-3 ') and 5 '-(T) ₂₅VN (N=A, C, G, T; V=A, G, Ce, add water to 4.75 μ L and mix.

After (2) 72 ℃ of incubation 3min in 42 ℃ of incubation 2min, the centrifugal 10sec of 1500g.

(3) add 2 μ L 5 * buffer (250Mm Tris-HCl, 375mM KCl, 30mM MgCl ₂), 1 μ L DTT (20mM), 1 μ L dNTP (10mM), the ThermoScript II of 0.25 μ LRNase inhibitor and 1 μ L, in 70 ℃ of processing 10min, be cooled to room temperature and add 20 μ L sterilized waters after 42 ℃ of incubation 90min, obtains cDNA.

3 pcr amplifications

Design a pair of Auele Specific Primer primer F:5 '-GGAATTCCATATGATGGCCCCCTC CGTCGACTCGATC-3 '; Primer R:5 '-CGGAATTCCTAGTATGGTCTACGT CCAAAGG-3 '.0.25mL centrifuge tube add 1 μ L cDNA, each 1 μ L of primer F and R, 5 μ L 10 * buffer, 5 μ L dNTP (2mM), 0.5 μ L pfu archaeal dna polymerase, add deionized water to 50 μ L, 94 ℃ of denaturation 4min, 94 ℃ of sex change 1min, 58 ℃ of annealing 30sec, 72 ℃ are extended 2min, 25 circulations, and agarose gel electrophoresis detects.

4 build cloning vector pUCm-T-pal

(1) in the centrifuge tube of 0.25mL, add 5 μ L PCR products, 1 μ L 10 * Taq buffer, 1 μ L dATP (2mM), 1 μ LTaq enzyme (5U/ μ L) adds water to 10 μ L, the imitative extracting of phenol after 70 ℃ of reaction 30min, the ethanol precipitation, aseptic operating platform wind 15min is complete to the ethanol volatilization, resuspended with 10 μ L sterilized waters.

(2) get previous step reaction solution 5 μ L, add 1 μ L to connect buffer, 1 μ L 50%PEG, 1 μ L pUCm-T carrier, 1 μ L T ₄Ligase enzyme, add deionized water to 10 μ L, in 16 ℃ of connections of spending the night.

(5) get and connect product 10 μ L, 10 μ L 5 * KCM (0.5M KCl, 0.15M CaCl ₂, 0.25M MgCl ₂), after mixing, deionized water 30 μ L are added in the E.coli JM109 competence of 50 μ L, ice bath 30min, 42 ℃ of thermal shock 90sec, 2min on ice, the fresh LB substratum that adds 100 μ L, cultivate 30-60min for 37 ℃, get the LB flat board that 100 μ L coatings contain penbritin (50ug/mL), cultivate 10h for 37 ℃, picking list bacterium colony, access 5mL contains in the LB substratum of penbritin of 50ug/mL 37 ℃ and cultivates 10h, gets 2mL bacterium liquid, the upgrading grain, the pUCm-T-pal that the PCR checking builds, sample checks order.

5 construction of expression vector pET-28a (+)-pal

Nde1 and EcoR1 carry out double digestion to pUCm-T-pal and empty carrier pET-28a (+) respectively, glue spends the night in 16 ℃ of connections after reclaiming, Transformed E .coli JM109, at the LB flat board that contains kalamycin resistance, choose positive recombinant, extract recombinant plasmid go forward side by side performing PCR and double digestion checking, positive colony is pET-28a (+)-pal.

6 abduction deliverings

Get 1 μ L pET-28a (+)-pal, proceed to E.coli BL21, get the LB flat board that 100 μ L coatings contain kantlex (50ug/mL), cultivate 10h for 37 ℃, picking list bacterium colony in the LB substratum of 5mL 37 ℃ cultivate 10h, get the 1mL nutrient solution in the LB substratum of 100mL 37 ℃ be cultured to OD ₆₀₀Be to be added to the IPTG that final concentration is 0.4mM at 0.6 o'clock, after 26 ℃ of abduction delivering 12h, centrifugal collection thalline, carry out SDS-PAGE detection and enzyme assay.

7?Westernblot

(1) after the SDS-PAGE electrophoresis finishes, rubber tapping, transferring film damping fluid balance 3 times, each 5min.

(2) cut out and the equal-sized filter paper of adhesive tape and pvdf membrane, filter paper and film are put into to transfering buffering liquid 10min.

(3) by carbon anode plate, filter paper, pvdf membrane, gel, filter paper, negative electrode carbon plate order are from the bottom up put well, and alignment, use the glass stick roll extrusion, until centre does not have bubble, switch on power, and 200mA transferase 12 .5h.

(4) after transferring film finishes, wash film 3 times with TBS, each 5min.

(5) add coating buffer steadily to shake 2h.

(6) by film from coating buffer, taking out, add the primary antibodie of coating buffer dilution, place 12h for 4 ℃.

(7) abandon primary antibodie, wash film 4 times with TTBS, each 5min.

(8) add two of coating buffer dilution and resist, steadily shake 2h.

(9) abandon two and resist, wash film 4 times with TTBS, each 10min; TBS washes 2 times, each 10min.

(10) add demonstration liquid, lucifuge develops the color and when band occurs, puts into the distilled water cleaning, in imager, watches result.

8 recombinases are refining

(1) ammonium sulfate precipitation

The recombinant bacterium fermented liquid is centrifugal remove supernatant after, thalline washing 2 times, thalline adopts ultrasonic disruption, the centrifugal 5min of 10000g, get supernatant, in supernatant liquor, adds the ammonium sulfate powder, to saturation ratio be 35%, slowly stir it dissolved fully.Regulate the pH value, make the pH of enzyme liquid remain on 8.7,10000 * g low-temperature centrifugation 10min after standing half an hour, after the collection supernatant liquor continued to add ammonium sulfate to saturation ratio to be 55%, 4 ℃ of standing a few hours, low-temperature centrifugation was collected albumen precipitation.By resolution of precipitate in the damping fluid of 25mM Tris-HCl pH 8.7 and 4 ℃ the dialysis a few hours;

(2) ion exchange chromatography

Enzyme liquid after dialysis is joined to the good anion-exchange column Resource Q of damping fluid balance that uses in advance 25mM Tris-HCl pH 8.7.By the same buffer that contains 0-0.5M sodium-chlor, carry out linear gradient elution, flow velocity is 1mL/min, and the detection wavelength is 280nm, collects activated part.

(3) gel permeation chromatography

With ammonium sulfate (saturation ratio 55%), concentrate and use a small amount of Tris-HCl pH 8.7 damping fluids to dissolve whole active parts of collecting, enzyme liquid after concentrated is joined and uses in advance in the Superdex 75 10/300GL chromatography columns that 25mmol/L Tris-HCl pH 8.5 damping fluid balances are good, by the same buffer that contains 0.5M sodium-chlor, carry out wash-out, flow velocity is 0.4mL/min, the detection wavelength is 280nm, collect activated part, carry out simultaneously SDS-PAGE.

The accompanying drawing explanation:

The schema that Fig. 1 recombinant bacterium builds

Fig. 2 pcr amplification result

Fig. 3 expression vector pET-28a (+)-pal gel electrophoresis figure

Fig. 4 pET-28a (+)-pal double digestion result

Fig. 5 recombinant bacterium abduction delivering SDS-PAGE result: 1 BL21; 2 do not add inductor; 3 induce

The purge process of Fig. 6 PAL: 1 full cell; 2,3 ammonium sulfate precipitations; 4,5Resource Q; 5 gel-filtrations

The Westernblot result of Fig. 7 PAL

Embodiment:

Material and detection method

Rhodotorula glutinis (Rhodotorula glutinis), the preservation of key lab of the industrial biotechnology the Ministry of Education of Southern Yangtze University.

Enzyme activity determination method: adopt HPLC to measure product styracin, chromatographic column C18,5 μ m, 250mm * 4.6mm; Moving phase is 5% methyl alcohol; Flow velocity 1mL/min; Detect wavelength 290nm; Column temperature is room temperature.Enzyme is lived and defined: 40 ℃, it is 1U that per minute transforms the required enzyme amount of L-Phe generation 1umol trans-cinnamic acid.

Embodiment 1:

Be equipped with in the triangular flask of 250mL of 50mL substratum and access rhodotorula glutinis, cultivate 18-20h to the logarithmic phase (OD that grows for 30 ℃ ₆₀₀=1.0), adopt the total RNA of acid phenol extraction, employing Auele Specific Primer 5 '-AAGCAGTG GTATCAACGCAGAGTACGGGGG-3 ' and 5 '-(T) ₂₅VN (N=A, C, G, T; V=A, G, C)-3 ' and carry out reverse transcription, obtain cDNA.

Embodiment 2:

Adopt primer primer F:5 '-GGAATTCCATATGATGGCCCCCTCCGTCGACTC GATC-3 '; Primer R:5 '-CGGAATTCCTAGTATGGTCTACGTCCAAAGG-3 '), the PCR condition: 94 ℃ of denaturation 4min, 94 ℃ of sex change 1min, 58 ℃ of annealing 30sec, 72 ℃ are extended 2min, 25 circulations.The PCR product is cut glue and is reclaimed, and is connected with the pUCm-T carrier, proceeds to E.coli JM109, picking list bacterium colony, and the upgrading grain obtains cloning vector pUCm-T-pal.Nde 1 and EcoR 1 carry out double digestion to pUCm-T-pal and empty carrier pET-28a (+) respectively, glue spends the night in 16 ℃ of connections after reclaiming, proceed to E.coli JM109, at the LB flat board that contains kalamycin resistance, choose positive recombinant, extract the recombinant plasmid expression vector pET-28a (+) that performing PCR and the double digestion checking build-pal that goes forward side by side, pET-28a (+)-pal is proceeded to E.coli JMBL21, choose positive single bacterium colony, 37 ℃ are cultured to OD ₆₀₀Be to be added to the IPTG that final concentration is 0.4mM at 0.6 o'clock, after 26 ℃ of abduction delivering 12h, centrifugal collection thalline, carry out SDS-PAGE detection and enzyme assay.

Embodiment 4:

The centrifugal collection thalline of recombinant bacterium fermented liquid, thalline adopt ultrasonic disruption, and the centrifugal supernatant that goes obtains crude enzyme liquid, and crude enzyme liquid is with ammonium sulfate precipitation (35-55%), and resolution of precipitate is in the damping fluid of 25mmol/L Tris-HCl pH 8.7 and 4 ℃ of dialysed overnight; Enzyme liquid after dialysis is joined to the good anion-exchange column Resource Q of damping fluid balance that uses in advance 25mmol/L Tris-HCl pH 8.7.By the same buffer that contains 0-0.5mol/L sodium-chlor, carry out linear gradient elution, flow velocity is 1mL/min, and the detection wavelength is 280nm, collects activated part.With ammonium sulfate, concentrate and use Tris-HCl pH 8.7 damping fluids to dissolve whole active parts of collecting, enzyme liquid after concentrated is joined and uses in advance in the Superdex 75 10/300GL gel columns that 25mmol/L Tris-HCl pH8.5 damping fluid balance is good, by the same buffer that contains 0.5mol/L sodium-chlor, carry out wash-out, flow velocity is 0.4mL/min, the detection wavelength is 280nm, collect activated part, carry out simultaneously SDS-PAGE.

Claims

1. a rhodotorula glutinis phenylalanine deaminase (PAL) gene clone reaches at E. coli.

2. phenylalanine deaminase gene claimed in claim 1 is from rhodotorula glutinis (Rhodotorula glutinis JN-1).

3. rhodotorula glutinis claimed in claim 2 obtains (culture presevation numbering CCTCC NO:M2011490) from soil, separating.

4. claim 1 is described, it is characterized in that, builds cloning vector pUCm-T-pal and expression vector pET-28a (+)-pal.

5. claim 1 is described, it is characterized in that, gene clone Host Strains and expressive host bacterium are respectively E.coliJM109 and E.coliBL21.

6. claim 1 is described, it is characterized in that, adopts IPTG to induce recombinant bacterium to express phenylalanine deaminase.

7. claim 1 is described, it is characterized in that, adopts western blot to identify the successful expression of restructuring PAL.

8. claim 1 is described, it is characterized in that, the enzyme that adopts HPLC to measure restructuring PAL is lived.

9. claim 1 is described, it is characterized in that, adopts ammonium sulfate precipitation, and resin anion(R.A) and gel-filtration three one-step refinings, obtain pure restructuring PAL.

10. the seed culture medium of the described cultivation rhodotorula glutinis of claim 2 is: 1% yeast extract, 1% peptone, 0.5% sodium-chlor, pH6.0; Induce and produce phenylalanine deaminase agar medium in seed culture, adding 0.1% L-phe.

11. method claimed in claim 5, the substratum of E.coliJM109 and BL21 are (LB): 1% Tryptones, 0.5% yeast extract, 1% sodium-chlor, pH 7.0.