CN105349558A - Construction and application of ganoderma laccase pichia pastoris genetic engineering strain - Google Patents

Abstract

The invention provides construction and application of ganoderma laccase pichia pastoris genetic engineering strain. The construction comprises the following steps: cloning a laccase gene GlLac from ganoderma; carrying out EcoRI and XbaI double-enzyme cleavage; cloning the double-enzyme cleavage product into a pichia pastoris expression plasmid to construct an expression vector; finally, electrically converting the expression vector into a pichia pastoris expression host strain; carrying out cultivation, amplification and screening to obtain a pichia pastoris engineering strain. According to the construction, the yeast secreting type co-expression vector pPICZ<Alpha>A-GlLac of ganoderma laccase (Lac) is constructed, and converted into pichia pastoris X33; recombinant of high Zeocin resistance is selected out to serve as a pichia pastoris engineering strain. The extracellular expression vector pPICZ<Alpha>A of pichia pastoris is a carrier for judging whether a multi-copy gene is inserted into a pichia pastoris genome or not, and can express high-copy genes.

Description

The study on its developing of ganoderma lucidum laccase pichia pastoris gene engineering bacterial strain

Technical field

What the present invention relates to is the technology in a kind of molecular biology, genetically engineered field, specifically a kind of study on its developing of ganoderma lucidum laccase pichia pastoris gene engineering bacterial strain.

Background technology

The microbe species that nature participates in lignin degrading has fungi, actinomycetes and bacterium etc., but the most effectively, most importantly fungi (BuswellJ, OdierE, KirkT.Ligninbiodegradation.CriticalReviewinBiotechnology, 1987,6:l-60).Mainly these microorganisms can produce lignin modifying enzyme (ligninmodifyingenzymes, LMEs), mainly comprise lignin peroxidase (ligninperoxidase, LiP) (EC1.11.1.14), manganese peroxidase (manganeseperoxida, MnP) (EC1.11.1.13), laccase (laccase, Lac) oxydase of (EC1.10.3.2) and generation hydrogen peroxide, such as glyoxal oxidase (glyoxaloxidase, GLOX), aryl-alcohol oxidase (Aryl-alcoholoxidase, AAO) etc.

Laccase (EC1.10.3.2) is a kind of polyphenoloxidase of cupric, belongs to blue blue multicopper oxidase family, is extensively distributed in plant, fungi, minority bacterium and insect.The active centre of laccase generally comprises 4 cupric ions, and the catalytic oxidation of laccase is namely by the collaborative transmission electronics between cupric ion, and substrate oxidation is radical form by the electronics of abstraction reaction substrate, simultaneously by the electron transmission from substrate capture to O ₂, by O ₂be reduced to water (HakulinenN, KiiskinenLL, KruusK, SaloheimoM, PaananenA, KoivulaA, RouvinenJ.CrystalstructureofalaccasefromMelanocarpusalbo myceswithanintacttrinuclearcoppersite.NatStructMolBiol.2 002,9 (8): 601-605.).The substrate of laccase effect is quite wide in range, and typical substrate is various phenolic compound and derivative thereof, comprises simple diphenol, polyphenol, methoxy substitution phenol (as methyl catechol), amino phenol, chlorophenol etc.Under micromolecular amboceptor material exists, the oxidable substrate spectrum of laccase also will expand further.Due to the popularity of laccase substrate specificity and using water as unique by product, laccase is made to have good application prospect in the application of green bio technology, be mainly reflected in the aspect (ZhouXW such as bioreediation, association with pulp bleaching of aromatics synthesis, food and fruit juice production, soil and water, CongWR, SuKQ, ZhangYM.LigninolyticenzymesfromGanodermaspp:currentstatu sandpotentialapplications.CriticalReviewinMicrobiology, 2013,39 (4): 416-26).In the research of last century to laccase, determine the nutritional needs of whiterot fungi lignin degrading in laboratory conditions the seventies; Find the enzyme system of Degradation by White-rot Fungus xylogen the eighties, Lac, Lip and Mnp; Expand catalytic characteristics, molecular biological research the nineties, simultaneously the industrial circles such as bio-pulping, bio-bleaching, waste water from dyestuff, pulp-making waste-water applied research also mushroom development get up.

Although can be separated to laccase from the plant of occurring in nature, its technique is loaded down with trivial details, and cost is high; Multiple-microorganism has the function producing laccase, as in existing instrument " a kind of method of producing laccase " (201310742301.7), utilizes the mould GZUIFR-H104.1 fungal bacterial strain of wooden fork Dai Shi to cultivate and produces laccase; " the high-efficiency cleaning production method of ganoderma lucidum laccase " (200610096638.5) utilize ganoderma lipsiense hypha fermentation to produce laccase etc., but due to the poor growth of natural product laccase bacterial strain, nutritional requirement is higher, or product forms the purifying that inclusion body (bacterial isolates) is unfavorable for target protein in born of the same parents, laccase yields poorly, production cost is high.In addition, utilize filamentous fungus to produce laccase and also there are some problems, as fermentation period is long, substratum requires high, and enzyme activity is low, and mycelia is subject to the damage etc. of high shear force in fermentor tank.Along with the development of Protocols in Molecular Biology, utilize DNA extracorporeal recombination to build all kinds of engineering strain, become possibility by the various albumen of the fermentative production of engineering strain, and create huge economic benefit.Therefore, in the production of laccase, clone's laccase gene, structure laccase gene engineering strain, realizing the heterogenous expression of laccase gene, is realize the restructuring suitability for industrialized production of laccase and the important foundation of practical application from now on.

From multiple white-rot fungi, clone laccase gene, as GenBank:AAR21094, AAR21096, ABP81837, AAG09229, BAA22153, AAM18407, CAA7701 etc. at present.Found by glossy ganoderma gene order-checking, in glossy ganoderma full-length genome, 16 laccase genes are had in Fungi Lignin oxydase family, compare with other basidiomycetes, Lucid ganoderma laccase gene copy number is only second to dust cover ghost toadstool (Coprinopsiscinerea), prompting glossy ganoderma has very strong lignin degradation ability (Chen Yuewen etc., the bioinformatic analysis of ganoderma lucidum laccase annotate genes Laccl and Cu thereof ²⁺abduction delivering, Agricultural University Of Hunan's journal (natural science edition) .2013,39 (3): 265-269), therefore, some investigators wish from glossy ganoderma, to clone laccase gene (as Genbank:AY485825; Zhang Yinbo etc., the clone of glossy ganoderma (Ganodermalucidum) laccase gene and sequential analysis thereof.Chinese biological chemistry and molecular biosciences journal, 2005,21 (5): 700-704.Zhang Guimin, Wang Yaping, Cai Litao, Ma Lixin.Exon splicing method synthesis fungi Lucid ganoderma laccase gene cDNA, Wuhan University Journal (Edition).2007,53 (6): 701-705.) utilizing engineered method to produce laccase, is our production and service for life.And in numerous expressive hosts, prokaryotic expression yields poorly, and likely form inclusion body, be unfavorable for the separation and purification of laccase, or have the shortcoming increasing separation and purification cost.Pichia spp is a kind of eukaryotic expression host developed rapidly recently, and nutritional requirement is not high, is suitable for the mass production method of high density fermentation, and its albumen secreting self is little, is conducive to the abstraction and purification of expression product.

Chinese patent literature CN1657611, open (bulletin) day 2005.08.24, disclose and a kind of there is laccase, zytase, the preparation of peroxidase activity and nucleotide sequence thereof, new laccase, zytase and peroxidase gene are cloned, construct the expression vector of laccase, zytase and peroxidase gene, expression vector is imported in yeast or filamentous fungus and express, and detect the activity of recombinase.Recombinant expressed laccase, zytase, peroxidase mixed enzyme are mixed with preparation, can apply in the industry such as papermaking, environmental protection, food and feed.But this technology does not provide by the specific implementation of pichia spp as the host of Restruction laccase or zytase or peroxidase protein; The pBARPE-Lac expression vector built only is expressed in aspergillus niger (Aspergillusniger), does not relate to the expression of pichia spp.Now there are some researches show: most of filamentous fungus all has the ability of secretion laccase, the laccase (enzyme is lived) that this technology is surveyed is that the laccase of mycelium secretion itself or the laccase of genetic engineering bacterium generation are also difficult to expect, because aspergillus niger inherently can secrete generation laccase; In addition, can secrete many kinds of enzymes in filamentous fungus while producing laccase, the multiple enzyme in the mixed solution that fermentation produces brings numerous inconvenience can to the abstraction and purification of follow-up laccase.

Summary of the invention

The present invention is directed to prior art above shortcomings, a kind of study on its developing of ganoderma lucidum laccase pichia pastoris gene engineering bacterial strain is proposed, recombinant yeast pichia pastoris positive strain is cultivated through methanol induction, can be used for preparing recombinant Ganoderma lucidum laccase (rGlLac).

The present invention is achieved by the following technical solutions:

The present invention relates to a kind of structure of ganoderma lucidum laccase pichia pastoris gene engineering bacterial strain, by cloning laccase gene GlLac from glossy ganoderma, enter Pichia anomala expression plasmid through EcoRI and XbaI double digestion rear clone and construct expression vector, finally its electricity is transformed in Pichia anomala expression Host Strains, after cultivating amplification and screening, obtains Pichia yeast engineering.

Described glossy ganoderma, adopt but be not limited to glossy ganoderma (G.lucidum51562), glossy ganoderma (G.lucidum50044), red sesame (G.lucidum00679), purple sesame (G.sinensi50817), preferred employing glossy ganoderma (Ganodermalucidum) 50817, all purchased from China General Microbiological culture presevation administrative center (ChinaGeneralMicrobiologicalCultureCollectionCenter, CGMCC).

The nucleotide sequence of described laccase gene GlLac is as shown in SeqID.No.1.

Described double digestion refers to: will intend the cDNA sequence of the Lucid ganoderma laccase gene GlLac expressed through EcoRI and XbaI double digestion.

Described Pichia anomala expression plasmid preferably past the plasmid of identical described EcoRI and XbaI double digestion, more preferably pPICZ α A.

Described structure refers to: will intend the cDNA sequence of the glossy ganoderma Lac expressed after EcoRI and XbaI double digestion, be cloned into equally through the pPICZ α A-carrier of XbaI and EcoRI double digestion, then enzyme cut back to close in product connection, transformation of E. coli DH5 α competent cell.

Described Pichia anomala expression Host Strains is preferably pichia spp X ₃₃.

Described electricity transforms and refers to: get the linearizing single copy plasmid of 5-10 μ L and add 80 μ L pichia spp X respectively ₃₃competent cell also mixes gently, proceeds to 2mm precooling electricity revolving cup, places 5min on ice, put into electroporation, electric shock, voltage: 1500V, electric shock time 5.4ms, after having shocked by electricity, add the sorbyl alcohol of 1mL, 1M precooling immediately, transfer in 1.5mL centrifuge tube after mixing, 28 DEG C, static 1 ~ 2h, every 200 μ L are coated with one piece of YPD flat board.28 DEG C, cultivate 3d, form dispersion, full mono-clonal, choose in spot inoculation 3mLYPD liquid nutrient medium.

Described cultivation amplification refers to: by the pichia spp X transformed through electricity ₃₃dull and stereotyped 30 DEG C of the YPD Agar that it is 1.0mg/mL that cell is coated containing Zeocin concentration is cultivated 3 days, and then picking transformant carries out pcr amplification detection.

Described screening refers to: the high laccase gene of having recombinated that copies of screening expresses the pichia spp recon of framework as the Pichia yeast engineering of expressing framework containing laccase gene, i.e. recombinant yeast pichia pastoris positive strain.

The present invention relates to the application of the engineering strain that aforesaid way obtains, use it for and prepare ganoderma lucidum laccase rGlLac.

Described preparation refers to: will be inoculated in BMGY pre-induction medium after the activation of described recombinant yeast pichia pastoris positive strain, transfers in 1.5LBMMY inducing culture afterwards, abduction delivering 96h; After induction, supernatant is heavily dissolved in tris-HCl damping fluid after dialysing and concentrating, and utilizes nickel ion affinity chromatograph (Ni-IDA) purification of recombinant proteins.

Described inoculation specifically refers to: by the recombinant yeast pichia pastoris X selected ₃₃positive strain is inoculated in the shaking flask containing BMGY nutrient solution, and at 28 DEG C, 250rpm cultivates after 16-18h, centrifugal and collecting cell.

Described induction refers to: induce with the resuspended postvaccinal cell of BMMY nutrient solution, it is 1% that every 24h adds methyl alcohol to final concentration.

Described dialysis is concentrated to be referred to: by the product after induction under 4000rpm, room temperature environment centrifugal 5 minutes, collect supernatant liquor, supernatant liquor loads in dialysis tubing, with the 48h that dialyses in the phosphoric acid buffer of pH6.0, every 12h changes a damping fluid, repeats until supernatant concentration is to about 40% of original volume.

Described nickel ion affinity chromatograph refers to: utilize Ni-NTA post (containing Ni-NTA agarose, the Lysisbuffer pre-equilibration column with 10 times of column volumes) purifying; Post is washed, in order to remove non-target protein with the Washbuffer of 20 times of column volumes; Target protein by Elutionbuffer wash-out out, for SDS-PAGE electrophoretic analysis.

Contain during described BMGY nutrient solution often rises: 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH6.0,1.34% yeast nitrogen, 4 × 10 _- ⁵% vitamin H and 1% glycerine.(being mass/volume per-cent)

Contain during described BMMY nutrient solution often rises: 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH6.0,1.34% yeast nitrogen, 4 × 10 _- ⁵% vitamin H and 0.5% methyl alcohol.(being mass/volume per-cent)

Technique effect

Compared with prior art, the present invention screens the bacterial strain of high yield laccase from white-rot fungi glossy ganoderma, and clones laccase gene, and in glossy ganoderma, to have copy number high for laccase gene, has higher activity after genetic expression; The present invention constructs the yeast secretion type co-expression carrier pPICZ α A-GlLac of ganoderma lucidum laccase (Lac), and transform pichia spp (Pichiapastoris) X33, by selecting the recon of high bleomycin (Zeocin) resistance as Pichia yeast engineering.The extracellular expression carrier pPICZ α A of pichia spp is the carrier that a qualification multi-copy gene is inserted in pichia spp genome, can express the gene of high copy.

Accompanying drawing explanation

Fig. 1 is ganoderma lucidum laccase full-length cDNA electrophorogram;

In figure: swimming lane M:DNAmarkDL2000; Swimming lane 1: the laccase gene of amplification;

Fig. 2 is the Lucid ganoderma laccase gene yeast secretion type co-expression carrier pPICZ α A-GlLac built;

Fig. 3 is the PCR qualification result of positive recombinant;

In figure: swimming lane M:DNAmarkDL12000; Swimming lane 1: Host Strains X33 genome; Swimming lane 2:1# recon genome; Swimming lane 3:34# recon genome; Swimming lane 4:36# recon genome; Swimming lane 5:37# recon genome; Swimming lane 6:pPICZ α A-GL-Laccase plasmid;

Fig. 4 is ganoderma lucidum laccase rGlLac abduction delivering SDS-PAGE result;

In figure: swimming lane M: protein Marker; Swimming lane X33 induces: contrast; Swimming lane 36# transformant: refer to 24h, 48h, 72h and 96h pichia spp X3336# transformant induction Lac protein expression; Swimming lane 36# transformant: refer to 24h, 48h, 72h and 96h pichia spp X3337# transformant induction Lac protein expression.

Embodiment

The present embodiment realizes the screening that ganoderma lucidum laccase produces enzyme superior strain first in the following manner

The Ganderma lucidum strain that the present embodiment adopts comprises: glossy ganoderma (G.lucidum51562), glossy ganoderma (G.lucidum50044), red sesame (G.lucidum00679), purple sesame (G.sinensi50817) are used to this experiment, and all bacterial strains are purchased from China General Microbiological culture presevation administrative center.

1) activation of bacterial classification: put by inclined-plane bacterial strain activate 24h to 28 DEG C of incubators after, the mycelia of picking slant tube bacterial classification is connected on PDA solid medium flat board, and 28 DEG C of constant temperature culture 5 ~ 7d are for subsequent use when mycelia covers with culture dish 2/3 area.

2) the flat board inspection of enzyme activity: the inspection of enzyme activity with PDA-Bavendamn method ( r.DemonstrationofphenoloxidasesusingBavendamm'sreactioni ntheringdishtest.ZentralblBakteriolParasitenkdInfektions krHyg.1972,127 (6): 555-563) primary dcreening operation is carried out to a few strain bacterial strains producing laccase.By methyl catechol with 0.04% concentration add PDA substratum, Bechtop inoculates with inoculation shovel the bacterium block that the diameter activated is 1cm, each bacterial strain do 3 parallel, 28 DEG C of constant temperature culture, observe each bacterial strain periphery of bacterial colonies with or without sorrel haloing generate)

By strain inoculation on the PDA substratum that with the addition of methyl catechol, cultivate and start to take pictures after 1 day, then observe the growing state of bacterial classification every day, and take pictures.The zone of oxidation method making substrate with methyl catechol carries out the primary dcreening operation of bacterial strain Laccase, and have sorrel haloing to generate, show that result is reliable, the bacterial strain namely producing zone of oxidation has laccase activity, and its zone of oxidation produces more early, illustrates that laccase generation also may be more early.Inoculate 4 ganoderma strain capables, measure colour developing loop diameter size respectively in the generation situation of 1d, 2d, 4d, 7d sorrel haloing.

Visual result analysis is visible, little in the difference of each bacterial strain of Initial stage of culture secretion laccase on same substratum, and after cultivating 4d, the activity of glossy ganoderma 50044 strain secretes laccase increases very fast, and after cultivation 7d, the activity of this strain secretes laccase is the highest; Glossy ganoderma 51562 and glossy ganoderma 00679 from the beginning of cultivating to the situation of cultivating the activity that terminates secretion laccase and linearly rising always, when the activity of cultivating secretion laccase during 9d reaches maximum and glossy ganoderma 50044 bacterial strain 7d, the activity of secretion laccase is close.Therefore glossy ganoderma primary dcreening operation result thinks that glossy ganoderma 50044, glossy ganoderma 51562 and glossy ganoderma 00679 are made no distinction of rank in primary dcreening operation, all can be used as the candidate strain of producing laccase and studies further.

2) screening again of ganoderma lucidum laccase superior strain: 4 strain glossy ganodermas 51562, glossy ganoderma 50044, glossy ganoderma 00679, glossy ganoderma 50817 are aseptically accessed in culture medium respectively, at 4d to 12d, the enzymic activity of laccase is measured respectively after shaking culture 4d, to determine ganoderma lucidum laccase superior strain.

The mensuration of described enzymic activity, its method and result as follows.

1. preculture: utilize the mycelia on activated solid medium, produces 1cm with inoculation in Bechtop ²the dull and stereotyped bacterial classification of left and right is connected in the 150mL triangular flask that 50mL liquid precultivation medium (the PDA liquid nutrient medium of improvement) is housed.Triangular flask is put into 28 DEG C of constant-temperature table 120rpm and cultivate 9 ~ 14d.

2. produce enzyme to cultivate: on aseptic super clean bench, 5mL pipettor is used to pipette the pre-incubated liquid containing mycelia bead of common 10mL at twice, add in the 150mL triangular flask that 40mL liquid glossy ganoderma culture medium is housed, two repetitions, be placed in after 28 DEG C of constant-temperature tables cultivate 4d and start to measure laccase activity, afterwards every day sampling and measuring 1 time.

3. Cu ²⁺produce the impact that enzyme is cultivated: on aseptic Bechtop, get the pre-incubated liquid containing mycelia bead of 10mL at twice with 5mL transfer pipet, add containing 40mLCu ²⁺in the 150mL triangular flask of culture medium, two repetitions.Start to measure laccase activity after cultivating 4d in 28 DEG C of constant-temperature tables, afterwards, every day sampling and measuring 1 time.

4. the definition of laccase activity measuring method enzyme activity and concrete operation as follows:

The enzyme amount of [laccase activity definition] definition 1min catalysis 1M substrate is a Ge Meihuo unit (U).

[operation steps] accurately pipettes 100mM propanedioic acid-sodium malonate damping fluid, each 30mL of 0.6mMABTS solution, and mixing, shakes up, 30 DEG C of water-baths.Get above-mentioned mix reagent 250 μ L and be placed in the cuvette that volume is 400uL, the zeroing of 420nm place accurately adds enzyme liquid X to be measured (10 μ L), immediate record light absorption value, 1 time is recorded every 10S, totally 7 times, get change mean value, be converted to the absorbancy change (OD of per minute ₄₂₀change).

[laccase activity calculating] enzyme contained by often liter of enzyme liquid amount alive calculation formula is as follows:

Wherein, the product molar specific absorbance that Laccase Catalyzed ABTS is formed is 36000molLcm ^-1.

It is visible that experimental result carries out intuitive analysis, the laccase activity of glossy ganoderma 00679 and glossy ganoderma 51,562 two bacterial strains is far away higher than glossy ganoderma 50044 and glossy ganoderma 50,817 two bacterial strains, and four strain glossy ganodermas are respectively 2654U/L (00679), 1175U/L (51562), 1199U/L (50044) and 782U/L (50817) at the average activity of 9d internal secretion laccase.Enzyme activity determination is thought further, and glossy ganoderma 00679 laccase production ability is the strongest, and after cultivating 4d, the activity of laccase reaches 2291U/L, cultivates 11d and reaches maximum 3267U/L.Therefore, subsequent experimental just adopts glossy ganoderma 00679 to carry out series of experiments as material.

The present embodiment comprises the following steps:

1) clone of Lucid ganoderma laccase gene (GlLac)

1.1) cultivation of Ganoderma mycelium, induction and pre-treatment: select the ganoderma strain capable 00679 of screening to be material, the liquid spawn of 5mL is drawn with pipettor, add in the triangular flask of the 250mL filling 25mL culture medium, put in constant-temperature table, 28C, 240rpm shaking culture is about 4d and collects thalline, the dH processed with DEPC (diethylpyrocarbonate, diethylpyrocarbonate) ₂the PBS buffer solution for cleaning 3 times of O preparation, is filtered dry and is placed in rapidly the frozen 10min of liquid nitrogen afterwards, standbyly takes out total serum IgE and STb gene.

1.2) extracting of STb gene, total serum IgE and the purifying of mRNA: glossy ganoderma DNA extraction method is extracted with reference to sky root (TIANGEN) DNA (or RNA) method identified in test kit and carried out.

1.3) genomic walking: adopt genomic walking test kit (TaKaRa), concrete operation step is determined by those skilled in the art's reference reagent box specification sheets (TAKARA) and practical situation.

1.4) purifying of DNA, digestion, connects and transforms: according to ordinary method well known by persons skilled in the art, as corresponding test kit operational manual and pertinent literature carry out.

1.5) DNA sequencing gained object fragment is all cloned on pMD18-T carrier, is checked order by Sangon Biotech (Shanghai) Co., Ltd..

With the STb gene of glossy ganoderma for template carries out PCR, result obtains the amplified production of about 1.5kb, with conforming to of expected results.Purifying rear clone, on pMD-18T, serves the order-checking of Hai Boya company.Sequencing result, after the BLAST comparison of NCBI is correct, designs two groups of genomic walking primers:

Primer1, as shown in SeqIDNo.2, namely 5 '-TGGCTGCGACCCGAGTTCA-3 ' and

Primer2, as shown in SeqIDNo.3, namely 5 '-TCGCCGCAGTCAACGCAGATCC-3 ',

Primer3, as shown in SeqIDNo.4, namely 5 '-CCCCGTCGTAGCGCAGGATCG-3 ' and

Primer4, as shown in SeqIDNo.5, namely 5 '-GGGCGGCGAAGATGTTGAGTGCTGT-3 ' carries out genomic walking, obtains 5 ends and 3 terminal sequences of its structure gene, then according to the DNA sequence dna design primer at structure gene two ends:

Primer5, as shown in SeqIDNo.5, namely 5 '-ATGGCCAGACTTCAATCCTTTG-3 ' and

Primer6, as shown in SeqIDNo.5, i.e. 5 '-AGCGCTCACTGGTCGTCGTTGG-3 ', carries out pcr amplification with glossy ganoderma STb gene for template and obtains its complete structure gene.

Utilize primer5, primer6 with the mRNA of glossy ganoderma for template carries out RT-PCR, amplification is to the full-length cDNA (called after GlLac) of its correspondence, and laccase full-length cDNA electrophorogram shows the fragment (as shown in Figure 1) of nearly about 1.5kb size.This fragment reclaimed and be cloned on pMD18-T, serve the order-checking of Hai Boya company, it is 1566kb that sequencing result obtains laccase full length cDNA sequence, as shown in SeqID.No.1.

2) structure of yeast expression vector and conversion

2.1) structure of expression vector and qualification: expression vector utilizes EcoRI and XbaI double digestion, purifying is reclaimed in rubber tapping, be specially: by the correct transformant containing pMD18-T-GlLac in the LB liquid nutrient medium containing penbritin 37 DEG C, 200rpm, shaking culture is spent the night, extract plasmid DNA, and with EcoRI and XbaI double digestion, reclaim the object fragment of about 1.5kb.Lac fragment and carrier pPICZ α A fragment are spent the night with T4DNA ligase enzyme (purchased from precious biotechnology (Dalian) company limited) 16 DEG C and is connected, in transformation of E. coli (E.coli) DH5 α competent cell, select the extraction that positive colony carries out plasmid, by plasmid EcoRI and XbaI double digestion, obtain about 1.5kb and 3.5kb two band, choose multiple positive colony respectively and carry out determined dna sequence.Positive co-expression plasmid called after pPICZ α A-GlLac (as Fig. 2) that sequence is correct, and for next step test.

2.2) pichia spp X ₃₃the preparation of competent cell: the instruction manual with reference to InvitrogenEasySelecteTMPichiaExpressionKit prepares yeast X ₃₃the competent cell of (purchased from Invitrogen company).

2.3) conversion of yeast: extract recon plasmid pPICZ α A-GlLac, pPICZ α A-GlLac plasmid correct for qualification is reclaimed with rubber tapping after SacI linearizing, is specially: get the electricity of the plasmid after 5 ~ 10 μ g linearizings and transform pichia spp X ₃₃after the dull and stereotyped preliminary screening of 0.10mg/mL, transfer continuously with 96 orifice plates respectively and be cultured to concentration and reach unanimity, be then seeded to YPD (YeastPeptoneDextroseAgar) the plate screening height copy transformant respectively containing 0.5mg/mL, 1.0mg/mL, 2.0mg/mL bleomycin (Zeocin); At Zeocin final concentration be 1.0mg/mL YPD flat board on screen 10 transformants, the resistant panel of 2.0mg/mLZeocin has no transformant growth.

2.4) the PCR qualification of yeast conversion bacterium, concrete steps comprise:

1. the preparation of Yeast genome: choose in spot inoculation 3mLMD liquid nutrient medium, overnight incubation, adopt and boil-freeze-cooking method extraction pastoris genomic dna, be specially: by the centrifugal 5min of 1mL bacterium liquid 2500 × g, abandon supernatant, add 500 μ LPBS suspension precipitations in precipitation, the centrifugal 3min of 2500 × g; Abandon supernatant, precipitation is dissolved in 100 μ LTE damping fluids, and boiling water bath 10min ,-80 DEG C of freezing 30min, the centrifugal 5min of boiling water bath 10min, 1500 × g, gets supernatant as template PCR again.

2. PCR qualification, is specially: with yeast genome for template, and the PCR reaction system using carrier universal primer AOX to detect recon is 50 μ L, PCR response procedures is: 94 DEG C of 3min, (94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 2min) 35cycles; 72 DEG C of 7min.5 μ L product electrophoresis detection are got after reaction terminates.The bacterium having specific fragment to increase is regarded as positive bacteria.

The result display of PCR qualification, the YPD flat board being 1.0mg/mL from Zeocin final concentration screens the transformant grown fine, use AOX13 ' and α-factor to combine primer (primer sequence slightly) and do PCR qualification, confirm positive recombinant, PCR detected result as shown in Figure 3.Transformant produces the band of about 1.5kb, conforms to goal gene.Illustration purpose gene is integrated into pichia spp genome by homologous recombination.Preserve bacterial strain, in order to follow-up test.

3) abduction delivering of Lucid ganoderma laccase gene GlLac in pichia spp

3.1) after the recon being accredited as the positive being activated, select 36# and 37# recon (transformant) to be inoculated in respectively in BMGY (Bufferedminimalglycerol-complexmedium) nutrient solution and to increase its biomass, this substratum often rises and contains: 1% yeast extract, 2% peptone, 100mM potassiumphosphate pH6.0,1.34%YNB, 4 × 10% ~ 5% vitamin H and 1% glycerine; Then be transferred in BMMY (Bufferedmethanol-complexmedium) fermentation culture and continue to cultivate, this substratum often rises and contains: 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH6.0,1.34% yeast nitrogen, 4 × 10 _- ⁵% vitamin H and 0.5% methyl alcohol, make its OD ₆₀₀about reach 2.0.By high-density shaking flask mode at 28 DEG C, methyl alcohol final concentration is 1.0%, carries out abduction delivering.Get induction supernatant respectively at 24h, 48h, 72h, 96h, after TCA precipitation concentration, carry out SDS-PAGE detection, assessment expression.

The rGlLac Pichi strain getting PCR test positive, after methanol induction, carries out SDS-PAGE electrophoresis, and the dye protein expression of different induction time of Coomassie brilliant blue R250 the results are shown in Figure 4; Fig. 4 shows, when induction time is respectively 24h, 48h, 72h, 96h, all has molecular mass to be about the special protein band of of 57kDa, expects in the same size with rGlLac albumen.Can find out the increase of 36# transformant along with induction time, the amount of the laccase given expression to (57kD) also progressively increases, shown in arrow.Illustrate that laccase gene is at pichia spp X ₃₃in have certain expression.

3.2) laccase gene is at pichia spp X ₃₃in a large amount of abduction delivering and purifying: according to expression test result, choose 36# transformant, be inoculated in BMGY pre-induction medium after activated, transfer in 1.5LBMMY inducing culture afterwards, abduction delivering 96h.After induction, supernatant is heavily dissolved in tris-HCl damping fluid after freeze-drying is concentrated.Utilize nickel ion affinity chromatograph (Ni-IDA) purification of recombinant proteins, purifying protein measures its expression amount through Coomassie Brilliant Blue can reach more than 260mg/L.

Above-mentioned concrete enforcement can carry out local directed complete set to it by those skilled in the art in a different manner under the prerequisite not deviating from the principle of the invention and aim; protection scope of the present invention is as the criterion with claims and can't help above-mentioned concrete enforcement and limit, and each implementation within the scope of it is all by the constraint of the present invention.

Claims (11)

1. the structure of a Lucid ganoderma laccase gene GlLac Yeast engineering bacterium strain, it is characterized in that, by cloning laccase gene GlLac from glossy ganoderma, enter Pichia anomala expression plasmid through EcoRI and XbaI double digestion rear clone and construct expression vector, finally its electricity is transformed in Pichia anomala expression Host Strains, after cultivating amplification and screening, obtains Pichia yeast engineering;

The nucleotide sequence of described Lucid ganoderma laccase gene GlLac is as shown in SeqID.No.1.

2. method according to claim 1, it is characterized in that, described glossy ganoderma, adopts glossy ganoderma (G.lucidum51562), glossy ganoderma (G.lucidum50044), red sesame (G.lucidum00679), purple sesame (G.sinensi50817).

3. method according to claim 1, is characterized in that, described double digestion refers to: will intend the cDNA sequence of the Lucid ganoderma laccase gene GlLac expressed through EcoRI and XbaI double digestion.

4. method according to claim 1, it is characterized in that, described structure refers to: will intend the cDNA sequence of the glossy ganoderma Lac expressed after EcoRI and XbaI double digestion, be cloned into equally through the pPICZ α A-carrier of XbaI and EcoRI double digestion, then enzyme cut back to close in product connection, transformation of E. coli DH5 α competent cell.

5. method according to claim 1, is characterized in that, described electricity transforms and refers to: get the linearizing single copy plasmid of 5-10 μ L and add 80 μ L pichia spp X respectively ₃₃competent cell also mixes gently, proceeds to 2mm precooling electricity revolving cup, places 5min on ice, put into electroporation, electric shock, voltage: 1500V, electric shock time 5.4ms, after having shocked by electricity, add the sorbyl alcohol of 1mL, 1M precooling immediately, transfer in 1.5mL centrifuge tube after mixing, 28 DEG C, static 1 ~ 2h, every 200 μ L are coated with one piece of YPD flat board.28 DEG C, cultivate 3d, form dispersion, full mono-clonal, choose in spot inoculation 3mLYPD liquid nutrient medium.

6., according to an application for the recombinant yeast pichia pastoris positive strain described in above-mentioned arbitrary claim, it is characterized in that, use it for and prepare ganoderma lucidum laccase rGlLac.

7. application according to claim 6, it is characterized in that, described preparation refers to: after described recombinant yeast pichia pastoris positive strain inoculation, in BMGY nutrient solution, add methyl alcohol induce, then collect supernatant liquor and carry out heavily being dissolved in tris-HCl damping fluid after dialysis concentrates, finally utilizing nickel ion affinity chromatograph purification of recombinant proteins.

8. application according to claim 7, is characterized in that, described inoculation specifically refers to: by the recombinant yeast pichia pastoris X selected ₃₃positive strain is inoculated in the shaking flask containing BMMY nutrient solution, and at 28 DEG C, 250rpm cultivates after 16-18h, centrifugal and collecting cell; Contain during described BMMY nutrient solution often rises: 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH6.0,1.34% yeast nitrogen, 4 × 10 ^-5% vitamin H and 0.5% methyl alcohol.

9. application according to claim 7, is characterized in that, described induction refers to: induce with the resuspended postvaccinal cell of BMGY nutrient solution, it is 1% that every 24h adds methyl alcohol to final concentration; Contain during described BMGY nutrient solution often rises: 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH6.0,1.34% yeast nitrogen, 4 × 10 ^-5% vitamin H and 1% glycerine.

10. application according to claim 8, it is characterized in that, described dialysis is concentrated to be referred to: by the product after induction under 4000rpm, room temperature environment centrifugal 5 minutes, collect supernatant liquor, supernatant liquor loads in dialysis tubing, change a damping fluid with the 48h that dialyses in the phosphoric acid buffer of pH6.0, every 12h, repeat until supernatant concentration is to 40% of original volume.

11. application according to claim 8, is characterized in that, described nickel ion affinity chromatograph refers to: utilize Ni-NTA column purification; Post is washed, in order to remove non-target protein with the Washbuffer of 20 times of column volumes; Target protein by Elutionbuffer wash-out out, for SDS-PAGE electrophoretic analysis.