CN105779419A - Cloning, definition and application of internally tangent polygalacturonase gene with temperature resistance and acid stability - Google Patents

Abstract

The invention discloses internally tangent polygalacturonase rEPG4 with temperature resistance and acid stability, a gene epg4 cloned by the internally tangent polygalacturonase rEPG4, a cloning vector, an expression vector, recombinant escherichia coli, a recombinant Pichia pastoris, and application of the gene epg4, the cloning vector, the expression vector, the recombinant escherichia coli and the recombinant Pichia pastoris to preparation of the internally tangent polygalacturonase rEPG4. After the internally tangent polygalacturonase gene epg4 is expressed and purified by the Pichia pastoris, the optimum pH value is 5.0, the optimum temperature is 65 DEG C, and the specific enzyme activity is as high as 12,038.1U/mg. After the rEPG4 is processed at 25 DEG C under the pH of 3.0-7.0 for 24h, 85.9% of enzyme activity is also maintained; after the rEPG4 is processed at 50 DEG C for 1h, the 100% enzyme activity is maintained; after the rEPG4 is processed at 55 DEG C for 1h, 61.2% of the enzyme activity is maintained. Therefore, enzymes have excellent acid stability and excellent heat stability.

Description

A kind of heatproof, the clone of the endo-polygalacturonase gene that acid is stable, defines and applies

Technical field

The invention belongs to genetic engineering field, be specifically related to a kind of heatproof that can decompose polygalacturonic acid or pectin, acid is stable The application of endo-polygalacturonase rEPG4.

Background technology

Pectin is complicated polysaccharide polymer, and its structure is mainly polymerized by the D-galacturonic acid of part esterification.Pectin It is the important component part of plant intermediate layer and cell wall, plays vital to facilitating organization structure of the plant and stability aspect Effect (Van Buren et al., 1991).Due to the complexity of pectin structure, so pectase is a compound enzyme system.Base Type of action in enzyme and the Preference to reaction substrate, pectase can be divided into protopectinase, pectinesterase, poly gala Alduronic acid enzyme and pectin lyase (Alkorta et al., 1998).In these compound enzymes, endo-polygalacturonase energy α-Isosorbide-5-Nitrae the glycosidic bond being made up of the galacturonic acid residues of non-esterification in enough random cutting pectin polymers, so that high poly- Right pectin generation depolymerization.This characteristic makes endo-polygalacturonase be the most important components of pectase.

Alkaline endo polygalacturonase is mainly by antibacterial output, and is widely used in papermaking, weaving, waste water process, squeezes Oil and the fermentation of coffee & tea leaf.Acidic incision polygalacturonase mainly by fungus output, its be mainly used in fruit and vegerable and The clarification of fruit wine and the digestion (Kashyap et al., 2001) of feedstuff.Current most business pectase is by Aspergillus Niger Going out, and the several endo-polygalacturonases deriving from this bacterium have been in the news, these enzymes broadly fall into acidicenzym, but In temperature higher than 50 DEG C (Guo et al., 2002.Liu et al., 2014.Singh et al., 2002), pH is unstable under the conditions of being less than 4.0 Fixed (Liu et al., 2014).Different biological treatments needs different biochemical environment, so having heatproof, and the inscribe that acid is stable Polygalacturonase, can not only improve its catalytic efficiency, and also be able to extend its range of application.With it has been reported that Endo-polygalacturonase is compared, and rEPG4 has the active and excellent absolute acid stability of efficient catalysis and heat stability, These features make it have the highest potential using value.

Summary of the invention

It is an object of the present invention to provide a kind of heatproof, the endo-polygalacturonase that acid is stable, be designated as rEPG4.

Described heatproof, the stable endo-polygalacturonase rEPG4 of acid contains 380 aminoacid, its sequence such as SEQ Shown in ID NO.1.

Wherein, 19 aminoacid of the N end of described rEPG4 are the signal peptide sequence of its prediction.Therefore, ripe rEPG4 has 361 aminoacid, sequence is as shown in SEQ ID NO.2.

The signal peptide sequence of described rEPG4 is shown in SEQ ID NO.3.

The present invention has cloned this endo-polygalacturonase gene, the endo-polygalacturonase gene that clone obtains It is designated as epg4, DNA complete sequence analysis result to show: epg4 structural gene contains 5 introns, total length 1484bp, its sequence As shown in SEQ ID NO.4.

CDNA total length 1143bp of described rEPG4, sequence is as shown in SEQ ID NO.5.

Described rEPG4 removes the cDNA sequence after signal peptide sequence as shown in SEQ ID NO.6.

Wherein, the cDNA sequence of the signal peptide of described rEPG4 is as shown in SEQ ID NO.7.

Cloning vehicle, expression vector, recombination bacillus coli or recombinant yeast pichia pastoris containing above-mentioned DNA molecular is also that the present invention protects The scope protected.

Above-mentioned expression vector is the pPIC9K carrier expressed in pichia pastoris Pichia pastoris.The expression of the present invention carries Body is specially and inserts what SEQ ID NO.6 nucleotide in sequence table obtained between SnaB I and the Avr II restriction enzyme site of pPIC9K carrier Expression vector pPIC9K-repg4.

Above-mentioned recombinant yeast pichia pastoris is that described Expression vector pPIC9K-repg4 is imported the recombinant bacterium obtained in pichia pastoris phaff GS115/rEPG4。

Above-mentioned DNA molecular, cloning vehicle, expression vector, recombination bacillus coli or recombinant yeast pichia pastoris is many in preparation inscribe Application in polygalacturonase rEPG4 is also the scope of protection of the invention.

It is a further object to provide a kind of method preparing endo-polygalacturonase rEPG4, described method bag Include following steps:

1) converting pichia pastoris phaff Pichia pastoris GS115 with pPIC9K-repg4 recombinant vector, screening is recombinated Bacterial strain GS115/rEPG4；

2) recombinant bacterial strain, the expression of methanol induction recombined endo polygalactunonic acid enzyme gene epg4 are cultivated；

3) the endo-polygalacturonase rEPG4 that also purification is produced is reclaimed.

The experiment proves that, the present invention is from the genome sequence of penicillium oxalicum (Penicillium oxalicum) bacterial strain CZ1028 In be found that one coding endo-polygalacturonase gene epg4, can express in host cell this gene with produce in Cut polygalacturonase rEPG4, be used for degrade polygalacturonic acid or pectin.This enzyme is through Pichia anomala expression, purification After, optimum pH is 5.0, and optimum temperature is 65 DEG C, and enzyme-specific vigor is up to 12,038.1U/mg；Under pH 3.0-7.0 25 DEG C process the enzyme activity that 24h also keeps 85.9%, keep 100% and 61.2% at 50 DEG C and 55 DEG C after processing 1h the most respectively Enzyme activity, this illustrates that this enzyme has excellent absolute acid stability and heat stability, these features make its have the highest potential should By value.

Accompanying drawing explanation

Fig. 1: Penicillium oxalicum CZ1028 genome supposes endo-polygalacturonase total length base containing one The agarose gel electrophoresis figure of the PCR primer of cause.

The PCR primer of the cDNA of one supposition endo-polygalacturonase of Fig. 2: Penicillium oxalicum CZ1028 Agarose gel electrophoresis figure.

The volume of the mature protein of one supposition endo-polygalacturonase of Fig. 3: Penicillium oxalicum CZ1028 The agarose gel electrophoresis figure of the cDNA sequence in code district.

Fig. 4: the structure of Expression vector pPIC9K-repg4.

Fig. 5: the agarose gel electrophoresis figure of the bacterium solution PCR primer of genetically engineeredPichia pastoris bacterial strain GS115/rEPG4.

Fig. 6: preparation and purification of Recombinant suppose that the SDS-PAGE of endo-polygalacturonase rEPG4 analyzes.

The restructuring of Fig. 7: purification supposes that the TLC of the product of endo-polygalacturonase rEPG4 hydrolysis polygalacturonic acid analyzes.

Fig. 8: endo-polygalacturonase rEPG4 the suitableeest action pH and temperature and pH and temperature stability analysis.

Detailed description of the invention

1. enzyme, test kit, carrier and reagent

LA Taq enzyme, pMD^TM18-T carrier cloning test kit, Taq^TMHot Start Version test kit, restricted enzyme SnaB I and Avr II, T4DNA ligase, the RNAiso Plus and the molecular weight protein marker that extract for RNA are purchased from TaKaRa company (Liaoning, China)；Reverse Transcription box for the synthesis of cDNA Article 1 chain is purchased from TransGen Biotech Company (Beijing, China)；Bradford method determination of protein concentration test kit purchased from Generay Biotech company (Shanghai, in State)；Expression vector pPIC9K is purchased from Invitrogen company (California, USA)；Galacturonic acid, three galacturonic acids, poly- Galacturonic acid, Pericarpium Citri tangerinae pectin and Pericarpium Mali pumilae pectin are purchased from Sigma company.

2. bacterial strain and culture medium

1) bacterial strain

Penicillium oxalicum Penicillium oxalicum CZ1028 is the high yield poly galactose that this laboratory is obtained by mutagenesis screening Aldehydic acid enzyme bacterial strain, has been stored in China General Microbiological culture presevation administrative center, and preserving number is CGMCC 3.15505.Pasteur Pichia sp. (Pichia pastoris) bacterial strain GS115 is purchased from Invitrogen company (California, USA).Escherichia coli (Escherichia coli DH5 α) is this Laboratories Accession bacterial strain.

2) condition of culture

1. solid-state slant culture uses Potato-dextrose agar culture medium (PDA).

2. extracting the product enzyme liquid culture based component used by RNA and DNA is (g/l): Pericarpium Citri tangerinae pectin 10, (NH₄)₂SO₄ 1.4,MgSO₄·7H₂O 0.3,KH₂PO₄ 2.0,CaCl₂·2H₂O 0.4,NaNO₃5.0, Tween 80 1.0, and 1ml trace element (g/l: CoCl₂ 2.0,MnSO₄·H₂O 1.6,ZnSO₄·H₂O 1.4 and FeSO₄·7H₂O 0.5), final each composition 0.05M buffered sodium citrate Liquid is dissolved to pH 5.5.

3. yeast culture medium

YPD culture medium: 1% yeast extract, 2% tryptone, 2% glucose, solid medium the most separately needs to add 2% Agar.

YPD Geneticin (G418) solid medium: 1% yeast extract, 2% tryptone, 2% glucose, 2% fine jade Fat and different amounts of G418.

MD solid medium: 1.34% without aminoacid yeast nitrogen (YNB), 4 × 10^-5% biotin, 2% glucose and 2% Agar.

BMGY culture medium: 1% yeast extract, 2% tryptone, 100mM potassium phosphate Ph 6.0,1.34%YNB, 4×10^-5% biotin and 1% glycerol.

BMMY culture medium: 1% yeast extract, 2% tryptone, 100mM potassium phosphate Ph 6.0,1.34%YNB, 4×10^-5% biotin and 0.5% methanol.

3. polygalacturonase vigor and determination of protein concentration:

The vigor of polygalacturonase is measured, with reference to described DNS sides such as Miller with polygalacturonic acid for substrate Method (Miller.1959).

1) making of galacturonic acid standard curve

With the galacturonic acid standard solution of deionized water preparation 2g/L, with deionized water, titer is suitable in 2ml centrifuge tube It is diluted to the solution (g/L) of 8 500 μ L variable concentrations: 0.0,0.2,0.4,0.6,0.8,1.0,1.2,1.4, each Sample concentration sets three repetitions respectively.The DNS reagent of 1ml is added in all samples, mixing, boiling water boils 10 minutes, use Frozen water is cooled to room temperature, and 3420 × g is centrifuged 5 minutes, and each sample takes 200ul supernatant respectively and joins in 96 hole ELISA Plate, The light absorption value of sample is measured under wavelength 540nm.With galacturonic acid acid concentration as transverse axis (X-axis), (Y with absorbance value as the longitudinal axis Axle) make scatterplot, adding Trendline and obtaining standard curve is y=0.9591x+0.1559 (coefficient R²=0.9994).

2) enzyme activity determination and the definition of enzyme activity unit

The 450 μ L buffer (pH value sets) containing 0.5% polygalacturonic acid is added according to experiment needs in the centrifuge tube of 2mL, After being incubated 5 minutes in the water-bath of design temperature (temperature sets according to experiment needs), add 50 μ L enzyme liquid and quickly mix. Insulation reaction adds the DNS reagent of 1mL and terminates reaction boiling water bath colour developing in 10 minutes after 15 minutes.It is cooled to room temperature with frozen water, 3420 × g is centrifuged 5 minutes, takes 200 μ L reactant liquors and joins in 96 hole ELISA Plate, measures light by microplate reader and absorb at 540nm Value.Often group experiment is in triplicate, and is provided with controlled trial.Under the condition determination set, hydrolysis galacturonic acid per minute gathers Compound discharges the enzyme amount needed for 1 μm ol reducing sugar, is defined as an enzyme activity unit (U).

3) mensuration of protein content uses Bradford method (Bradford.1976)

4. suppose the acquisition of endo-polygalacturonase rEPG4 encoding gene epg4

1) the mycelial preparation of penicillium oxalicum CZ1028 of polygalacturonase is produced

1. the preparation of penicillium oxalicum CZ1028 spore suspension: by the spore inoculating of penicillium oxalicum CZ1028 to solid PDA inclined-plane On, 30 DEG C of quiescent culture, after 5 days, take in the triangular flask that Fresh spores is placed in the aseptic ultra-pure water of the X100 containing 0.1%triton, with three Bead in the bottle of angle fully breaks up spore.

2. penicillium oxalicum CZ1028 produces enzyme cultivation: spore liquid is inoculated in aforesaid product enzyme fluid medium (every 1L volume triangle The fluid medium Han 100ml in Ping) to final concentration 1 × 10⁵Individual/mL, be placed in 30 DEG C, 150rpm shaking table cultivate after 2 days, raw Long mycelium is used for extracting genomic DNA and RNA.

2) extraction of penicillium oxalicum CZ1028 genomic DNA: collect the mycelium produced in enzyme fluid medium, successively use nothing The EDTA of bacterium normal saline and 20mM/L washs thalline, obtains being dried mycelium, adds after liquid nitrogen is fully ground mycelium and uses SDS method extracts genomic DNA.

3) extraction of RNA and the synthesis of cDNA Article 1 chain

Using RNAiso Plus reagent to extract the total serum IgE of penicillium oxalicum CZ1028, extracting method is carried out to specifications.Use Reverse Transcription box, goes up described operational approach to specifications and is cDNA by mRNA reverse transcription therein and carries out agarose Detected through gel electrophoresis.

4) acquisition and the analysis of endo-polygalacturonase rEPG4 full length DNA are supposed

Designing a pair specific primer (primer 1), forward primer is 5 '-ATGCGGCCAGCTTTTACTTCA-3 ', downstream Primer is 5 '-TCAAGAGCACTTGGTCACACTGG-3 ', the penicillium oxalicum CZ1028 genomic DNA obtained with extraction As template, using LA Taq enzyme to carry out PCR amplification, reaction system is with reference to description, and PCR response procedures is as follows: 94 DEG C 5min→[94℃ 30sec→53℃ 30sec→72 90sec]₅→[94℃ 30sec→58℃ 30sec→72℃ 90sec]₃₀→72℃ 10min, 4 DEG C of preservations.PCR primer is carried out agarose gel electrophoresis, under gel imaging instrument, cuts about 1500bp Band, use DNA purification reclaim test kit carry out recovery go forward side by side row agarose gel electrophoresis detection, such as Fig. 1: swimming lane M is DL2000DNA Marker, containing 6 DNA fragmentations, is respectively as follows: 2.00,1.00,0.75,0.50,0.25 from big to small And 0.10kb；Swimming lane 1 is the full length DNA of the endo-polygalacturonase rEPG4 supposed.Reclaim the product of gained, Carrier T Cloning Kit is used to be connected on pMD18-T carrier and convert to escherichia coli.Primer 1 is used to carry out bacterium colony PCR, sends to order-checking by the Positive E. coli containing recombiant plasmid, and the sequence obtaining this full length gene DNA is in sequence table SEQ ID NO:4.This sequence 1484bp, with in ncbi database (http://www.ncbi.nlm.nih.gov/) Penicillium oxalicum 114-2 genome sequencing Comparative result analysis shows that this sequence contains 5 introns, and it is at SEQ ID NO:4 In position (bp) be respectively as follows: 255-319,612-673,804-873,1004-1062,1147-1231.

5) acquisition and the analysis of endo-polygalacturonase rEPG4 full-length cDNA are supposed

The Article 1 chain of the penicillium oxalicum CZ1028 cDNA to obtain is carried out as template, use primer 1 and LA Taq enzyme PCR expands, and reaction system is with reference to description, and PCR response procedures is as follows: 94 DEG C of 5min → [94 DEG C of 30sec → 53 DEG C 30sec→72℃ 60sec]₅→[94℃ 30sec→58℃ 30sec→72℃ 60sec]₃₀→ 72 DEG C of 10min, 4 DEG C of preservations.Will PCR primer carries out agarose gel electrophoresis, cuts the band of about 1100bp under gel imaging instrument, uses DNA purification Reclaim test kit carry out recovery go forward side by side row agarose gel electrophoresis detection, such as Fig. 2: swimming lane M is DL2000 DNA Marker； Swimming lane 1 is the full-length cDNA supposing endo-polygalacturonase rEPG4.Product obtained by recovery, uses T to carry Body Cloning Kit is connected on pMD18-T carrier and converts to escherichia coli.Primer 1 is used to carry out bacterium colony PCR, will Positive E. coli containing recombiant plasmid sends to order-checking, and the sequence obtaining this full length gene cDNA is the SEQ ID in sequence table NO:5.This sequence 1143bp, uses online software analysis (http://www.cbs.dtu.dk/services/SignalP/) It is shown to be: full-length cDNA (SEQ ID NO:5) is by ripe coding region (SEQ ID NO:6) and signal peptide district (SEQ ID NO:7) composition.Encoding 380 aminoacid (SEQ ID NO:1), wherein maturation zone is 361 aminoacid (SEQ ID NO:2), signal peptide district is 19 aminoacid (SEQ ID NO:3).

5. the structure of recombiant plasmid pPIC9K-rEPG4

Design primer 2, forward primer is 5 '-GACCTACGTATCACCCGTCGCTGAGCCG-3 ', downstream primer is 5 '- CGACCTAGGTCAAGAGCACTTGGTCACACTGG-3 ', the Article 1 chain of the penicillium oxalicum CZ1028cDNA to obtain As template, use TaKaRa Taq^TMHot Start Version test kit carries out the cDNA of the ripe coding region of PCR amplification, instead Answering system with reference to description, PCR response procedures is as follows: 95 DEG C of 5min → [30sec → 72 DEG C, 95 DEG C of 30sec → 58 DEG C 80sec]₃₀→ 72 DEG C of 10min, 4 DEG C of preservations.Use DNA purification to reclaim test kit to carry out reclaiming and carry out agarose gel electricity Swimming detection, such as Fig. 3: swimming lane M is DL2000 DNA Marker；Swimming lane 1 is for being used for building pPIC9K-rEPG4 maturation coding region Gene.Respectively the gene of Expression vector pPIC9K and supposition endo-polygalacturonase rEPG4 maturation coding region is carried out Restricted enzyme action (SnaB I+Avr II), agarose gel electrophoresis observe after, cut respectively double digestion process expression vector with become The gene of ripe coding region also uses DNA purification to reclaim test kit to reclaim.By expression vector after purification and ripe coding region base Because being attached with T4DNA ligase, reaction system is with reference to description.Connect product and convert escherichia coli, use primer 2 to enter Row bacterium colony PCR, and the recombiant plasmid of PCR Positive E. coli is carried out digestion verification, agarose gel electrophoresis detects, such as Fig. 4: Swimming lane M1 is the product that lambda bacteriophage dna restricted enzyme Hind III carries out obtaining after complete degestion, containing 7 DNA Fragment, is respectively as follows: 23.13,9.416,6.557,4.361,2.322,2.027 and 0.564kb from big to small；Swimming lane 1 is Recombiant plasmid；Swimming lane 2 is recombiant plasmid single endonuclease digestion；Swimming lane 3 is recombiant plasmid double digestion；Swimming lane 4 is not foreign gene-carrying Carrier pPIC9K double digestion；M2 is DL2000 DNA Marker.From the swimming lane 3 of Fig. 4 it can be seen that two bands, one is line Property pPIC9K carrier, another is the genes of interest being connected on carrier, it follows that conclusion: construction of recombinant plasmid success. Recombiant plasmid digestion verification crossed is taken away order-checking, shows sequencing result analysis with DNAman software, do not find gene mutation, Disappearance or frameshit situation, by named for this recombiant plasmid pPIC9K-rEPG4 (' r ' represents the meaning recombinated).

6. contain the structure of the genetically engineeredPichia pastoris of plasmid pPIC9K-rEPG4

1) recombinant plasmid transformed Pichia sp.

Respectively the carrier pPIC9K of not foreign gene-carrying and the carrier pPIC9K-rEPG4 carrying ripe encoding gene is limited Property restriction endonuclease Sac I carries out single endonuclease digestion, obtains linearization plasmid.Respectively the two linearization plasmid LiCl method is converted to Pasteur In Pichi strain GS115.Concrete grammar refers to the Pichia anomala expression test kit of Invitrogen company

2) screening of genetically engineeredPichia pastoris and qualification

1. the cell suspending liquid after being processed by LiCl is coated on MD flat board after suitably diluting, and is inverted training in 30 DEG C of constant incubators Support 3 days.

Bacterium colony on MD flat board is eluted by ultra-pure water and the bead 2. crossed by sterilization treatment, suitably takes respectively after dilution The bacterium solution of each gradient of 100ul is coated on the YPD flat board that G418 concentration is 2.0mg/mL, is inverted in 30 DEG C of constant incubators Cultivate 3-4 days.

3. with toothpick, the relatively macrocolony on the YPD flat board containing 2.0mg/mL G418 is inoculated in YPD fluid medium, 30 DEG C, cultivating 24h in the shaking table of 250rpm, the fermentation liquid of generation is used for being thalline PCR, identifies recon.Use forward primer 5 ' AOX:5 '-GACTGGTTCCAATTGACAAGC-3 ' and reverse primer 3 ' AOX:5 '-GCAAATGGCATTCTGACATCC-3 ' Primer carries out thalline PCR.Thalline PCR reaction condition is as follows: 95 DEG C of 5min → [30sec → 72 DEG C, 92 DEG C of 30sec → 52 DEG C 2min]₃₀→ 72 DEG C of 10min, 4 DEG C of preservations.PCR primer after agarose gel electrophoresis as shown in Figure 5: swimming lane M1 is 1kb DNA ladder, containing 10 DNA fragmentations, the most respectively 10.00,9.00,8.00,7.00,6.00,5.00, 4.00,3.00,2.00 and 1.00kb；The PCR primer of swimming lane 1 is to convert single bacterium colony that pPIC9K-rEPG4 obtains as template Amplification；The PCR primer of swimming lane 2 is the amplification as template of the single bacterium colony that obtains of the carrier pPIC9K to convert not foreign gene-carrying； Swimming lane M2 is DL2000 DNA Marker.It can be seen that PCR has the band of a 1600bp, length from the swimming lane 1 of Fig. 5 For the original sequence length (referring to the catalogue of the carrier pPIC9K of Invitrogen company) on carrier and foreign DNA length Sum, swimming lane 2 it can be seen that PCR has the band of a 500bp, the original sequence length on a length of carrier.

4. the positive colony toothpick after being verified by PCR is inoculated in the finger-type bottle containing 2ml BMGY culture medium respectively, 30 DEG C, The shaking table of 250rpm is cultivated to overnight, and the most at room temperature 3000 × g is centrifuged 5 minutes, finally overhangs precipitation containing 4ml In the finger-type bottle of BMMY culture medium, condition of culture is still 30 DEG C, and 250rpm adds the methanol of 0.5% volume therebetween every 24h. After cultivating 72h, collection fermented supernatant fluid is at 50 DEG C, surveys the polygalacturonase vigor of each positive colony under the conditions of pH 5.0, Finally determining that the bacterial strain of the highest polygalacturonase vigor is expressed in a strain, named GS115/rEPG4, outside not carrying The named GS115/CK of control strain of source genophore pPIC9K.

7. the expression of epg4 gene and purification in genetically engineeredPichia pastoris

1) induction genetically engineeredPichia pastoris bacterial strain GS115/rEPG4 expresses recombinant protein rEPG4

The GS115/rEPG4 bacterial strain toothpick being grown on solid YPD plate is inoculated in respectively containing 50ml BMGY culture medium In 250ml triangular flask, 30 DEG C, the shaking table of 250rpm is cultivated and is reached 2-4 to OD600, then takes this fermentation liquid of 25ml in room temperature Lower 3000 × g is centrifuged 5 minutes, finally precipitation is overhang in the 250ml triangular flask containing 50ml BMMY culture medium, under similarity condition Cultivate, add the methanol of 0.5% volume therebetween every 24h after and sample reservation with for SDS-PAGE detect (Laemmli.1976), cultivation 5 days is amounted to.

2) purification of recombinant protein rEPG4

1. at 4 DEG C, within the conditions of 1,1325 × g centrifugal 10 minutes, the induction GS115/rEPG4 fermented supernatant fluid of 5 days is collected.

2. at 4 DEG C, with 15ml super filter tube (Millipore) that molecular cut off is 30.0kDa by collection under the conditions of 5000 × g Fermented supernatant fluid concentrates, with the K of 0.02mM in concentration process₃PO₄The fermentation liquid in concentrated solution changed by buffer (pH 6.0).

3. the concentrated solution 4 DEG C preservation will collected, takes the molecular sieve (HiLoad 16/600 of wherein part GE company Supredex 75 column) it is purified (flow velocity: 1.0ml/min, buffer: 0.02mM K₃PO₄, collect: 1ml/ manage)

4. all of collecting pipe is saved in 4 DEG C of refrigerators.Detecting often pipe with SDS-PAGE and collect liquid purity, final acquisition only has one The albumen of band, result as shown in Figure 6: swimming lane M is molecular weight protein marker, comprises 6 bands, be followed successively by from top to bottom from Top to bottm it is followed successively by 97.2,66.4,44.3,29.0,20.1 and 14.3kDa；Swimming lane 1 is purified product.Can from Fig. 6 Arriving, only have a protein band in swimming lane 1, the molecular weight that this band is corresponding is about 38.0kDa.Described on end, according to above-mentioned Method successful purification has obtained recombinant protein rEPG4.

8. the enzymatic property of the recombinant protein rEPG4 of purification

1) substrate specificity and product analysis

As it was previously stated, after the gene epg4 of recombinant protein rEPG4 compares with ncbi database, find that it is that the inscribe supposed is many Polygalacturonase, so must detect its substrate specificity and product analysis, with the acetate buffer solution of 0.1M respectively Preparation is containing the reaction substrate of the pH5.0 of 0.5% polygalacturonic acid, Pericarpium Citri tangerinae pectin and Pericarpium Mali pumilae pectin, check weighing group egg at 65 DEG C White matter rEPG4 is for the enzyme activity of each substrate, to hydrolyze enzyme activity that polygalacturonic acid produces for 100%, under other substrate Enzyme activity conversion for enzyme activity.It was found that recombinant protein rEPG4 for polygalacturonic acid, Pericarpium Citri tangerinae pectin and The enzyme activity of apple pectin is respectively 100%, 33.8% and 19.0%.This description of test recombinant protein rEPG4 is poly gala Alduronic acid enzyme.Containing pH5.0, the substrate of 1% polygalacturonic acid adds a certain amount of recombinant protein rEPG4,40 DEG C of guarantors Temperature 48 hours, sampling at set intervals is boiled the rear 4 DEG C of preservations of inactivation, taken sample is carried out thin layer chromatography (TLC) experiment (Esquivel et al.,2004).Result is as shown in Figure 7: during reaction 0 minute (min) was to 30 minutes, and poly-half Lactobionic acid is degraded rapidly, has part three galacturonic acid (G3) to generate, and has no that galacturonic acid (G1) generates；Reaction 1 Hour to 24 hours, product is mainly galacturonic acid polymer and three galacturonic acids；Find anti-after reacting 48 hours Answer product to be mainly three galacturonic acids, and have no that galacturonic acid substantially generates.Substrate specificity and TLC result show egg of recombinating White matter rEPG4 is typical endo-polygalacturonase.

2) Optimun pH and temperature (Temperature)

The reaction substrate of the pH2.2-7.0 of 0.5% polygalacturonic acid is contained with the citrate-phosphate disodium hydrogen buffer of 0.1M, At 50 DEG C, measure the different pH value impact on the endo-polygalacturonase vigor of the rEPG4 of purification by preceding method. If the high enzymatic activity measured is 100%, the enzyme activity of other pH value is enzyme activity with the ratio conversion of high enzymatic activity (Relative activity)；Result is shown in that Fig. 8-A: the Optimun pH of endo-polygalacturonase rEPG4 is pH 5.0.

The polygalacturonic acid solution of 0.5% is configured, according to aforementioned mensuration poly gala with the acetate buffer solution of the 0.1M of pH 5.0 The method of alduronic acid enzyme activity (25-80 DEG C) the most at different temperatures measures the endo-polygalacturonase of purification The enzyme activity of rEPG4.If the enzyme activity under optimum temperature is 100%, the enzyme activity conversion at a temperature of other is enzyme activity. Result is shown in that Fig. 8-B: the optimum temperature of endo-polygalacturonase rEPG4 is 65 DEG C.So can set pH5.0, 65 DEG C is the optimum reaction conditions of rEPG4, surveys rEPG4 protein concentration after purification by Bradford method, obtains its specificity ratio Enzyme activity is up to 12,038.1U/mg.

3) pH stability and temperature stability

The endo-polygalacturonase rEPG4 of purification is carried out pH Detection of Stability, is respectively adopted 0.1M citrate-phosphate Disodium hydrogen buffer (pH 2.2-7.0), 0.1M Tris-HCl buffer (pH 7.5-9.0).Enzyme liquid is stored in different pH value Buffer in, place respectively after 24 hours in 25 DEG C, under pH5.0,65 DEG C (optimal condition), measure different pH buffer In residual enzyme activity (Residual activity).Found that the endo-polygalacturonase rEPG4 of purification exists After being incubated 24 hours in pH3.0-7.0 buffer, still the enzyme of remaining 85.9% is lived (see Fig. 8-C), and result above shows the inscribe of purification Polygalacturonase rEPG4 has excellent absolute acid stability.

The endo-polygalacturonase rEPG4 of purification is carried out temperature tolerance detection, is respectively adopted different storage temperatures (50 DEG C, 55 DEG C and 60 DEG C), dilute enzyme pH 5.0 buffer containing 0.5% polygalacturonic acid, then dilution enzyme liquid In the water-bath of said temperature be incubated 1 hour, every 15 minutes sampling and in optimal condition under measure enzyme activity.Not do The enzyme activity of enzyme liquid processed as above is 100%, and the residual enzyme activity conversion after different temperatures insulation is enzyme activity.Result See that the endo-polygalacturonase rEPG4 of Fig. 8-D: purification keeps after processing 1h at 50 DEG C and 55 DEG C the most respectively The enzyme activity of 100% and 61.2%, illustrates that this enzyme has preferable temperature stability.

4) mensuration of enzyme kinetics constant Km, Vmax of rEPG4

Prepare the polygalacturonic acid solution (mg/ml) of variable concentrations respectively, according to this research measures the many polygalacturonics of inscribe The reaction system of acid enzyme, measures the enzyme activity of above-mentioned variable concentrations substrate under optimal condition, and protein concentration detection is used Bradford method.Concentration of substrate (S) and the double reciprocal plot of response speed (V), count according to the equation that double-reciprocal plot draws Calculation obtains the Km value of rEPG4: 1.8mg/ml, Vmax are 32,747.1U/mg.

5) metal ion, the Organic substance impact on rEPG4 enzyme activity

1. the 0.5% polygalacturonic acid solution of pH5.0 is configured to the metal ion chlorination containing variable concentrations (1mM or 2mM) The reaction substrate of thing, measures the enzyme activity of rEPG4 under optimal condition.The results are shown in Table 1: find Mn²⁺And Cu²⁺Right The enzyme activity of rEPG4 has relatively high inhibition effect, wherein Cu²⁺The most obvious.Ca²⁺、Co²⁺、Zn²⁺, and Fe²⁺Enzyme is lived and also has Certain inhibitory action, other Action of Metal Ions are inconspicuous.

2. the 0.5% polygalacturonic acid solution of pH5.0 is configured to (10mM or 20mM) containing variable concentrations organic instead Answer substrate, under optimal condition, measure the enzyme activity of rEPG4.The results are shown in Table 2: find the SDS enzyme activity to rEPG4 Having strong inhibition effect, the enzyme activity of rEPG4 is not affected by other Organic substance.

Claims (8)

1. a heatproof, the endo-polygalacturonase rEPG4 that acid is stable, it is characterised in that its aminoacid sequence is such as Shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

2. the gene epg4 of the endo-polygalacturonase rEPG4 that a kind has been cloned described in claim 1, it is characterised in that Its nucleotide sequence is as shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

3. comprise the cloning vehicle of DNA molecular described in claim 2, expression vector, recombination bacillus coli or recombinant yeast pichia pastoris.

Expression vector the most according to claim 3, it is characterised in that described expression vector is at pichia pastoris The pPIC9K carrier expressed in Pichia pastoris, is specially and inserts between SnaB I and the Avr II restriction enzyme site of pPIC9K carrier Expression vector pPIC9K-the repg4 that in sequence table, SEQ ID NO.6 nucleotide obtains.

Recombinant yeast pichia pastoris the most according to claim 3, it is characterised in that described recombinant yeast pichia pastoris is by described table Reach carrier pPIC9K-repg4 and import the recombinant bacterium GS115/rEPG4 obtained in pichia pastoris phaff.

6. the DNA molecular described in claim 2 or the cloning vehicle described in claim 3, expression vector, recombination bacillus coli, And the application that recombinant yeast pichia pastoris is in preparing endo-polygalacturonase rEPG4.

7. the preparation method of the endo-polygalacturonase rEPG4 described in claim 1, it is characterised in that include following step Rapid:

1) converting pichia pastoris phaff Pichia pastoris GS115 with pPIC9K-repg4 recombinant vector, screening is recombinated Bacterial strain GS115/rEPG4；

2) recombinant bacterial strain, the expression of methanol induction recombined endo polygalactunonic acid enzyme gene epg4 are cultivated；

3) the endo-polygalacturonase rEPG4 that also purification is produced is reclaimed.

8. the application in degraded polygalacturonic acid or pectin of the endo-polygalacturonase rEPG4 described in claim 1.