Oil body lipase gene from Jatropha curcas, lipase and application thereof

Mode of execution

The embodiment of combination of the following, for further description of the present invention. The following embodiment, where not indicate the specific experimental conditions, are in accordance with technical personnel in the field of conventional conditions well-known, for example, Sambrook, molecular cloning of the Russell: laboratory manual (New   York: Cold   Spring   Press   Laboratory   Harbor, 1989) in the condition of, or in accordance with the conditions recommended by the manufacturers.

Embodiment 1 the barbadosnut oil derived from lipase gene cloning and obtain

1, materials and reagents

Barbadosnut mature seed-mined salt Panzhihua county dry valley zone. The barbadosnut mature seeds soaked in water for 12 hours after the soil is buried, 28 the embryonic root [...] culture to grow about 1 cm to the shell, in -70 the storing [...].

Escherichia coli (E.coli) TOP10 strain purchased from Invitrogen Corporation. Sequencing and subclonning carrier pMD18-T purchased from the Company TaKaRa.

Ex   Taq^TM enzyme, reverse transcriptase (AMV), various restriction endonuclease and penbritin are in TaKaRa Company. VentR   DNA polymerase (high fidelity enzyme) purchased from the Company BioLabs. A small amount of glue recovery kit, PCR product recovery kit purchased from Shanghai hua Shun biotechnology Company. IPTG, agarose, agar powder, Tris, Amersham Company in SDS. Other chemical drugs to import or domestic analytical pure reagent.

2, clone method

2.1RNA extraction

Heating -70 the longer the radicle [...] about 1 cm of the germination barbadosnut seed 0.5g, the liquid nitrogen transfer grinds pulverized and 0.1g to the pre-cooling of the 1.5 ml centrifuge tube; adding 0.5 ml extract   RNAplant (purchase to Beijing day root biochemical technology Ltd), vibration and mixing, flat at room temperature 5 min; the 4 [...] , 12,000rpm centrifugal 2 min, apply the supernatant fluid diverted to the new non-RNA enzymatics a centrifuge tube, add 0.1 ml   5mol/L   NaCl solution, moderate mixing, add 0.3 ml of chloroform, and turned upside down; the 4 [...] , 12,000rpm centrifugal 10 min. Supernatant liquid phase into to the new non-RNA enzymatics a centrifuge tube, by adding the same volume of isopropanol, mixing, placed at room temperature for 10 min, 4 °C, 12,000rpm centrifugal 10 min, discard supernatant; adding 75% ethanol, the 4 [...] , 12,000rpm centrifugal 1 min, the liquid is poured out, the remaining centrifugal again after a small amount of liquid, is sucked out of the gun head for; adding 10-30ul enzyme RNA-free water, RNA after dissolving, the 4 [...] , 12,000rpm centrifugal 1 min, the enzyme RNA-free supernatant to centrifuge tube, the -70 storing [...].

The reagent is used for more than 0.1% of the processing room temperature DEPC 12 hours, and then high-pressure sterilizing 30 min; glassware for the 200 dry heat sterilization [...] 8h. For plastic products chlorofrom processing 5 min, then high-pressure sterilizing 30 min.

2.2 reverse transcription

Reverse transcription to AMV reverse transcriptase of Company TaKaRa to manual operation.

To 2.1 step the total RNA as the template, to TaKaRa with joint Company Oligo (dT)₁₈ as primer (5     -GCTGTCAACGATACGCTACGTAACGGCATGACAGTG (T)₁₈ -3     ), in table 1 anti-transcription system by adding the various components, is room temperature for 10 min, incubated 42 °C 50 min, placed on ice 2 min. The obtained reverse transcription product of the in -20 cDNA [...] storing.

Table 1 anti-transcription system

2.3 barbadosnut oil derived from lipase gene (abbrebyted JOBL) cloning

Primer design:

A pair of primers design: JLR1 : 5 the   TACT [...] -AGC   CCT   GGC   AAT the   CCTG-3 [...]

JLR2: the 5 [...] -AAT   AAG   CAG   CCC   ACC the   CAG-3 [...] ;

PCR amplification:

To 2.2 barbadosnut of steps to obtain cDNA as the template, to primer JLR1 and JLR2 barbadosnut tree oil body lipase gene full length of the cDNA PCR amplification.

Barbadosnut tree oil body PCR amplification of the lipase gene full-length cDNA of the reaction system are shown in table 2, PGR the procedure is as follows:

1.95 the [...]   4 min     (pre-denaturation)

2.95 the [...]   30s     (denaturation)

3.53 the [...]   30s     (annealing)

4.72 the [...]   50s     (extend)

5.2-4 step cycle 30 times

6.72 the [...]   5 min     (extending end)

7.4 the preservation [...].

Using 1% agarose gel electrophoresis detection PCR product. The operation of the detected product recovery kit recovery (recovery method refer to Shanghai hua Shun biotechnology Company glue recovery kit specification to be), then the recovered product is connected to the pMD18-T carrier (method according to Takara provided by the Company pMD18-T carrier of a specification),   TOP10 E.coli transformed into the competent cell, is coated evenly on the lb plate culture medium, 37 the culture [...] 12 hours. Random picking single colony switching to a new flat plate lb, and the template the fungus is , to JLR1 and JLR2 as primer, by referring to the table 2 amplification system according to the above-mentioned reaction conditions after verification the colony PCR, choose positive colony swings the fungus , the Beijing three thibeault biotechnology Company sequencing, the nucleotide sequence of the oil body lipase gene, see SEQ   NO   ID:1.

Table 2PCR amplification barbadosnut tree oil body lipase gene full-length cDNA system

Embodiment 2 the barbadosnut oil derived from lipase gene in the Escherichia coli expression of

1, experiment material

1.1 material, strain and plasmid vector

Barbadosnut seed: mined dry panzhihua salt-side-areas of the valley.

Amylose   resin: purchased from NEB Company

Strain: Escherichia coli (E.coli) BL21 and Top10 purchased from Invitrogen Corporation.

Plasmid vector: pMAL-c2E, Amp^R   B N   E purchased from the Company.

1.2 kit, enzyme

Kit:   RNA   PCR   Step One   Kit (AMV) (Company TaKaRa);   Kit   Gel E.Z.N.A   extraction (50) D2501-01 (  Bio-tek Omiga, USA);   Kit   Cycle-Pure E.Z.N.A (  Bio-tek Omiga, USA);   Ver2.SolutionI   Kit Ligation (Company TaKaRa);   KitI   Plasmid E.Z.N.A   Miniprep (Omiga Bio-tek, USA).

Enzymatics: Ex-Taq   polymerase (Company TaKaRa);   I EcoR (15U/the  l, Company TaKaRa);   I Sal (12U/the  l, Company TaKaRa); Xho   I (15U/ul, TaKaRa Company); T4 ligase (350U/the  l, TaKaRa Company).

2, method

2.1 barbadosnut oil derived from lipase gene (abbrebyted JOBL) expression fragment preparation

The embodiment 1 preparation of the oil body lipase gene from jatropha curcas JOBL by the SignalP 3.0Server-length cDNA sequence were found to predict, before JOBL of 68 amino acid may be a section of guide sequence, as organelles protein containing the information required for positioning, and the section 68 amino acid there is a shearing site: ILAL-I. The software structure of the membrane-spanning protein DNAMAN and TMHMM-2.0 analysis found, the protein with four transmembrane region, position are respectively: a 1st infers: 54-82, infers a 2nd: 323-351, infers a 3rd: 436-456 infers and a 4th: 468-494. Conservative domain analysis software for analysis shows that, its 220 to 400 Lipase_ 3 position amino acid conservative the structure area of the family. According to the above-mentioned bio-informatics of the results of the analysis and the pMAL-c2E design restriction enzyme cutting sites of the plasmid vector expressing primer MFP1 upstream and downstream expression primer MFP2.

Upstream expression primer:

MFP1: the 5 [...] >   ATT ggg   TTT GAT     CTC     GAA   TTC   AAC   TCT   CTT   C   ATT (EcoR   I)

Downstream expression primer:

MFP2: the 5 [...] >   CTT   GAC gag   ATG     GTC   TCC   CAA   GAA TTA     G (Sal   I)

Note: in the above-mentioned primer, the enzyme restriction site is underlined text

Lipase gene barbadosnut clones coding for the signal peptide removed JOBL N end and (MATPPVNFLIVNPQKGRKRDLFKYLVTKNKKSGMSFLDSSEESIKGGVANDHRWIL LVSIIIRRILALINTPLKYLGYVVDFILNLISQNGGISGILSNSLHGKLIIPRRGS) a 1st C end of the transmembrane structure two transmemebrane structure precursor protein (IIPMRVNVLWEIFRSFLISHIHGPEYKESWFCTLFRVLGLVLPGISAHSPVDYVNSVRL GRERATPLLSLKSFARKL) DNA fragment.  Step TaKaRa One to   RNA   PCR Kit (AMV) of the Company using that configuration of the reaction system, to after mixing barbadosnut total RNA as the template to take RT-PCR reaction, PGR the procedure is as follows:

1.95 the [...]   4 min     (pre-denaturation)

2.95 the [...]   30s     (denaturation)

3.53 the [...]   30s     (annealing)

4.72 the [...]   50s     (extend)

5.2-4 step cycle 30 times

6.72 the [...]   5 min (extending end)

7.4 the preservation [...].

1% agarose electrophoresis detection, determining whether there is any mixed belt PCR product. Of the amplified DNA fragment by Shanghai hua Shun biotechnology Company glue recovery kit specification to be glue recovery.

2.2 preparation of expression vector

Will carry pMAL-c2E E.coli of expression plasmid   Top10 strain in 50 ml   lb liquid culture medium (containing 50 the   Amp g/mL), 37 the shaking cultivation [...] 12h rear, 4000rpm centrifugal 5 min collect the thalli.  KitI E.Z.N.A   Plasmid Miniprep according to (  Bio-tek Omiga, USA) kit for extracting plasmid specification. Using 1% agarose gel electrophoresis detection. -20 the storage [...].

2.3 enzyme expression fragment, and cloning

The embodiment 2.1 expression JOBL step preparation of fragments and 2.2 preparation of the expression vector Sal EcoR and   I respectively through double-enzyme   I (enzyme restriction conditions refer to takara Company EcoR   I and Sal specification to the use of   I), in the 37 after complete digestion [...] ,   Kit E.Z.N.A   Cycle-Pure according to (  Bio-tek Omiga) enzyme reagent kit for purification of the product to the specification. Product with 1% agarose gel electrophoresis detection enzyme and estimates the effect of the amount of DNA, the expression vector expressing JOBL DNA fragment with the mol ratio of the 5 [...] 1 is the 5 mixed   l, to the 5   I Solution connection fluid   l (Company TaKaRa), fully mixed, for the 16 is [...] 12 hours. The connecting-piece conversion TOP10 competent cell, lb/Amp the screening to the seed. Picking white colony of the bacterial colony PCR, enlarged culture extract recombinant plasmid, by identifying and sequencing enzyme. The verification correct recombinant plasmid pMAL-c2E-JOBL into   BL21 E.coli in, and the flat plate are cultured on lb/Amp (specific methods refer to molecular cloning manual).

2.4 barbadosnut oil derived from lipase gene fragment analysis SDS-PAGE the induced expression and

Inoculated in a positive single colony containing Amp (50 subsidence g/mL) in lb liquid culture medium, 37 the shaking cultivation [...] 12 hours, morrows according to 5% pH7.0 switching to the proportion of fresh lb culture medium (containing 50 the   Amp g/mL) in, the 32 to shaking cultivation [...] OD₆₀₀ value is 0.6, taking 1 ml of bacterial expression as compared with before. Adding isopropyl thio-β-D galactopyranoside (IPTG) to a final concentration of 1 mm induced, for the 32 [...] , 220rpm shaking cultivation under the condition of 4h the sampling after 1 ml to 1.5 ml in EP tube clean. 4000rpm centrifugal 10 min collect the thalli, with sample buffer liquid 1 ×SDS 50ul mixing, 100 the heat shock [...] 5 min, ultrasonic crushing 5 min then 10000rpm centrifugal 1 min precipitation cell debris, the supernatant 20ul in 12% polyacrylamide gel electrophoresis. Electrophoresis using constant voltage mode, concentrated in the voltage constant 90V, when the bromo phenol blue relocated to the separating glue, voltage the accent is 160 V. Until the bromo phenol blue close to the edge of the gel electrophoresis is stopped. Gel with coomassie brilliant blue R250 containing methanol-glacial acetic acid solution of fixed dyeing 4h above, de-colorant which is for (45% methanol, 45% H₂ O, 10% glacial acetic acid) decolourizations 4-8h.

Collecting the thalli SDS-PAGE analysis of total protein. The results showed that, after the IPTG induction, recombinant plasmid pMAL-c2E-JOBL Bl21 the Escherichia coli expression of a new belt, its molecular weight is about 80kD, consistent with the prediction (see Figure 1). PMAL-c2E-JOBL by the recombinant plasmid expression in Escherichia coli.

2.5 barbadosnut oil derived from lipase purification of recombinant protein

Amylose-resin for (purchase to Company NEB) affinity column of the recombinant plasmid pMAL-c2E-JOBL Bl21 the Escherichia coli expression of the product of the purification. -20 the preservation [...] thalline according to the 1 [...] 25 heavy suspended the proportion of buffer crosses the column (20mmol/L   Tris-HCl, pH7 . 4,   NaCl 200mmol/L, 1mmol/L   EDTA) in, ultrasonic crushing strains in the ice water bath (300W, ultrasonic 5s, interval 15s) 3 min, centrifugal 12000rpm 10 min, supernatant fluid is collected for purification of the target protein. 15 times column volume of buffer crosses the column balance column (1.0 cm × 10 cm, 20mV, 1A) the rear, in order to 1 ml/min flow rate of the supernatant fluid crosses the column , after baseline restoration to 3 times column size eluant (column buffer solution + 10mmol/L maltosaccharides) elution, branch collection, collecting the eluant of identifying SDS-PAGE. Merger containing target protein component, vacuum freeze-drying, electrophoresis can be obtained pure oil body lipase recombinant protein.

Embodiment 3 the barbadosnut oil derived from lipase activity detection of recombinant protein

1, the pH conditions derived from different oil body lipase barbadosnut the effects of recombinant proteinase activity

 H.P.Chan & Samuel   Nixon with the method of Michael, extraction fatty acid, NaOH titration method for determining enzyme activity. Specific steps are as follows:

1.1 preparation of the crude enzyme

With reference to embodiment 2 of the method, will carry the recombinant plasmid pMAL-c2E-JOBL Escherichia coli BL21 of the IPTG induced expression 4 hours later, to the 5 two 1.5 ml of bacterial in EP the 1.2 ml, 5000g centrifugal 10 min, to supernatants, collect the thalli, and then respectively to 7 a 1.5 ml containing BL21 bacteria to EP tube pH4.0, pH5 . 0, pH6.0, pH7.0, pH8 . 0 the disodium hydrogen phosphate citric acid buffer solution and pH9.0 the 0.05mol/L glycine sodium hydroxide buffer solution (buffer solution preparation method: song ping editor-in-chief, "biochemistry and molecular biology experiment tutorial-", 2003, higher education press, P229 and P233) 0.6 ml, the BL21 bacterial re-suspension mixing, ultrasonic crushing bacterial cells 5 min, 5000g centrifugal 3 min, the supernatant into a clean 1.5 ml tube in the EP, that is, in order to spare the crude enzyme.

1.2 substrate processing

To the 2 support 1.5 ml in the tube of the EP 200ul concentration is 0.1M butyrin of (butyrin is three acid radical glycerine a, are commonly used as the substrate of the lipase activity detection) and 400ul mass percentage 5% of the arabic gum, ultrasonic emulsifying 5 min spare.

1.3 enzymatic reaction

To apply the above-mentioned 1.1 in different pH of the buffer solution of the sample to the crude enzyme 200ul are respectively transferred to the clean 1.5 ml in the EP tube, of the substrate has been emulsified good 100ul, mixing, for the 30 [...] , 150rpm oscillating reaction 40 min then, stirred 5 min terminate the enzyme activity. At the same time, in order to distill water instead of the crude enzyme to compare.

1.4 lixiviating of free fatty acid

To 7 in the tube a by adding chloroform EP : = 4 the n-heptane is [...] 3 (volume ratio) solution of 0.8 ml, oscillation 3 min later, by adding pH2.4 the 0.01mol/L disodium hydrogen phosphate citric acid buffer solution 200ul and 0.1M phosphoric acid 100ul, oscillation 1 min after 3500g centrifugal 3 min, to the upper layer solution, then adding 0.01M of HCl   200ul, oscillation 1 min, centrifugal 3500g 3 min, to the upper layer solution, the lower organic phase is dissolved in an organic solution of the free fatty acid.

1.5NaOH titration computation enzyme vitality

Draw an organic solution of the above-mentioned free fatty acid 200ul in the tube to clean EP, adds by drops two drop of phenol to select indicator, mixing, with 25 mm   NaOH titration solution, until the solution so far appear pink, recording titration of the amount of NaOH, computing lipase activity (enzyme activity definition: per mg protein per minute to catalytic butyrin generated for butyrate lnmol 1U), its result see Figure 2. From Figure 2 it can be seen in, the oil derived from lipase barbadosnut of the recombinant protein can be catalytic three butyric acid glycerides hydrolyzed to release free fatty acids, the lipase activity, the pH value to 4.0, , enzyme activity is not detected, the pH value is 5-8 time, as the pH value rise, the enzyme activity is increased, the pH value of 8.0 time, the enzyme activity of the largest detected, to 29.06U, when the pH value of 9.0, enzyme activity only pH value of 8.0 of the 60%. Can be determined from this oil body lipase from jatropha curcas of the recombinant protein for optimal reaction buffer pH=8.0.

2, the different temperatures of the oil derived from lipase barbadosnut the effects of recombinant proteinase activity

To 7 a 1.5 ml are added respectively in the EP tube 200ul using the most suitable reaction buffer solution (pH=8.0) processing and crude 100ul has been emulsified good substrate (concentration is 0.1M butyrin of), mixing, the 7 supporting EP pipe are respectively placed in the 15 [...] , the 20 [...] , the 25 [...] , the 30 [...] , the 35 [...] , the 40 [...] , the 45 [...] different temperature environment, such as the enzyme activity measured in the influence of temperature, determination results see Figure 3. In Figure 3 can be seen, the 40 an enzyme vitality [...] highest, to 43.6U, is the optimal enzyme action temperature. In the 40 [...] the following, with the rise of the temperature, enzyme activity is enhanced; higher than the 40 [...] , enzyme activity with the increase and decrease of the temperature.