CN105838728B - Polygalacturonase optimization gene and its expression vector and application - Google Patents
Polygalacturonase optimization gene and its expression vector and application Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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Abstract
The invention discloses polygalacturonase optimization gene and its expression vector and applications.The present invention is not first under the premise of changing polygalacturonase amino acid sequence, comprehensively consider codon preference, the variation of G/C content, codon adaptation indexI (CAI), the deletion of unstable sequence, the factors such as mRNA secondary structure carry out the transformation of a variety of strategies to polygalacturonase gene, obtain 3 polygalacturonase optimization genes.Further above-mentioned 3 polygalacturonase genes and 1 wild type gene are transferred in Pichia pastoris and are expressed, finishing screen selects that secreting, expressing amount significantly improves, the strongest nucleotides sequence of enzyme activity is classified as polygalacturonase optimization gene shown in SEQ ID NO.3.Polygalacturonase expressed by polygalacturonase optimization gene of the present invention can effectively degrade the pectic substance in pear juice, improve crushing juice rate and light transmittance, lay a good foundation for industrialization expanding production.
Description
Technical field
The present invention relates to polygalacturonase gene more particularly to optimized polygalacturonase gene with
And recombinant expression carrier and recombinant host cell containing optimization gene, further relate to the preparation of polygalacturonase and in fruit juice
Application in processing belongs to polygalacturonase gene application field.
Background technique
Pectin is the important composition ingredient in plant cell wall, by the galacturonic acid of different esterification degrees and different side chains with
α-Isosorbide-5-Nitrae glucosides key connection forms.Contain a large amount of pectin in fruit, since it is with water imbibition and viscosity, in fruit juice production mistake
Cheng Zhong, it is sticky that the presence of pectin will cause pulp, causes juice extracting rate low and muddy, influences its yield and mouthfeel；It is dynamic
Chyme viscosity is increased in object enteron aisle, inhibits enteron aisle to the infiltration rate of nutrient to a certain extent.This can be to the battalion of livestock and poultry
It supports to absorb and utilize etc. and causes directly to influence, or even will affect the production performance of animal, animal functions of intestines and stomach is caused to be decayed.
Pectase is the general name of a kind of enzyme that can decompose pectic substance.Polygalacturonase
(polygalacturonase) be Major Members in pectin enzyme family, can hydrolytic cleavage α-Isosorbide-5-Nitrae glycosidic bond, to degrade
Pectin reduces rapidly pectin viscosity.Endo-type (endo-PG) and circumscribed-type (exo-PG) two can be divided into from the mode of action
Kind.It is widely applied to food industry, textile industry, medicine, papermaking, environmental protection, biotechnology and feed addictive etc.
Aspect.
Natural pectase is typically found in animals and plants and microorganism, but derives from the pectin production of enzyme of animals and plants
It is very low, be not suitable for being used to be mass produced, and microorganism has the speed of growth fast, nutritional requirement is low, is easy to train on a large scale
Excellent living resources of many advantages, such as the supporting as production pectase.Many microbe-derived pectases have been reported, wherein
The comparison of originated from fungus is more, such as derives from Penicillium notatum, Aspergillus.
Currently, the excellent pectase that each enterprise, China uses still depends on external import, but the price of import enzyme is high
Expensive, making enterprise, the production cost increases.With the continuous development and growth of Chinese food industry, required pectase is also more and more
It is more.Especially for the pectase preparation that yield is high, stability is good, good properties and enzymatic activity are high demand also increasingly
Greatly.With the continuous development of biotechnology, using technique for gene engineering to a variety of pectin enzyme genes from microorganism into
It has gone cloning and sequencing, has had more deep understanding to the structure, function and regulating and expressing etc. of pectin enzyme gene.With
The further investigation of Protocols in Molecular Biology is had become using some high-effective microorganism bacterial strains to produce the pectase of advantageous property
A kind of new effective way.Various countries researcher, which has filtered out from various different microorganisms, largely has advantageous property
Pectin enzyme gene, these pectases possess different property, such as height is than living, heat-resisting, low temperature resistant, acidproof.In order to improve this
The production level of a little pectases, has researcher to carry out these pectin enzyme genes in the host strains such as Escherichia coli, Pichia pastoris
Heterologous recombination expression.But the expression in these reports about polygalacturonase is not still high, and purification and recovery rate is low, limit
Make its application in the industry.Therefore, the transformation that a variety of strategies are carried out to polygalacturonase gene, obtains secreting, expressing amount
The polygalacturonase optimization gene being significantly increased, has great importance.
Expression of the foreign gene in heterologous host is affected by many factors, including but not limited to listed below:
1) translation of codon preference, rare codon induction stops, the password being rarely employed in host organisms
The presence of son, corresponding nucleotide may adversely affect protein translation.
2) the polymerase sliding for repeating induction, the polymerase sliding for repeating induction will lead to archaeal dna polymerase sliding or yoke,
So as to cause frameshift mutation.In the organism of high GC content, it is understood that there may be higher degree is repeated to form by G or C nucleotide
Sequence.
3) mRNA secondary structure, secondary structure can isolate RBS sequence or initiation codon, with protein expression level
It reduces related.Loop-stem structure may also be related to transcription pausing and decaying etc..
It has been reported and shows that foreign gene can be improved in genetic engineering host by means such as gene codon optimizations
In expression.However, many practitioners not yet reach unified opinion to the general strategy of gene optimization.It is some
The preferred strategy of people is during designing heterologous gene as much as possible using the common codon in expressive host species.Separately
Some preferred strategies are to give maximum attention to the context of specific codon, to make to frequently occur in expressive host
Codon use maximize.Therefore, simple codon optimization can not always improve expression, in some instances it may even be possible to reduce
Expression.Frequently, other technological means must also be aided with, the distribution proportion such as G/C content in different genes region, password
Sub- adaptation index (codon adaptation index, CAI) etc., and large batch of verifying is carried out, it can just obtain high efficient expression
Mutated gene.
Summary of the invention
The object of the invention first is that the sequence to polygalacturonase gene optimizes, obtain one kind in yeast point
Secrete the polygalacturonase optimization gene that expression quantity is significantly increased；
The second object of the present invention is to provide containing above-mentioned polygalacturonase optimization gene recombinant expression carrier and
The recombinant host cell of the recombinant expression carrier；
The object of the invention third is that by the optimization gene of the polygalacturonase and containing the weight of the optimization gene
Group expression vector and recombinant host cell are applied to the production of polygalacturonase.
The present invention to achieve the above objectives, the technical solution taken are as follows:
The present invention is by the polygalacturonase gene pga-zj5a in the source aspergillus niger (Aspergillus niger)
(nucleotide sequence shown in SEQ ID NO.1), in the case where not changing its protein amino acid sequence, comprehensively considers password
Sub- Preference, the variation of G/C content, codon adaptation indexI (CAI), the deletion of unstable sequence, mRNA secondary structure etc. because
Element carries out the transformation of a variety of strategies to polygalacturonase gene, obtains 3 typical polygalacturonase optimization bases
Cause, pga-zj5a-m1 (nucleotide sequence is as shown in SEQ ID NO.2), pga-zj5a-m2 (nucleotide sequence such as SEQ ID
Shown in NO.3), pga-zj5a-m3 (nucleotide sequence is as shown in SEQ ID NO.4).
Invention further provides the recombinant expression carrier containing polygalacturonase optimization gene and contain this
The host cell of recombinant expression carrier；Wherein, it is preferred that the recombinant expression carrier is recombinant eukaryon expression vector, more preferably
To recombinate yeast expression vector；Preferably, the host cell is preferably yeast cells, more preferably Pichia pastoris
(Pichia pastoris) cell.
3 polygalacturonase optimization genes and 1 wild type gene are transferred in Pichia pastoris and are carried out by the present invention
Heterogenous expression, the results showed that wherein, turn the bacterial strain of pga-zj5a-m1, in the polygalacturonic acid of shaking flask level secretion expression
Enzyme highest vigor is 976U/mL, and the bacterial strain highest enzyme activity for turning pga-zj5a-m2 is 1778U/mL, turns the bacterium of pga-zj5a-m3
Strain highest enzyme activity is 1316U/mL.The bacterial strain highest enzymatic activity for turning wild type gene pga-zj5a is 1204U/mL.As a result table
Bright, the enzyme activity for turning the bacterial strain of pga-zj5a-m2 is apparently higher than the bacterial strain for turning pga-zj5a, pga-zj5a-m1, pga-zj5a-m3.
For further verification result, above-mentioned 4 plants of Pichia pastoris recombinant bacterial strains are induced into producing enzyme in 3 liters of fermentors respectively
Fermentation, fermentation results show: turning the yeast transformant of the wild type gene pga-zj5a polygalacturonic after methanol induction 120h
Sour enzyme activity is 10436U/mL, and turns the transformant of the pga-zj5a-m2 polygalacturonase vigor after inducing 120h and then reach
15428U/mL has been arrived, has improved 48% compared with before optimization.But turn its enzyme activity of the transformant of optimization gene pga-zj5a-m1
Power is only 8572U/ml, and 17.9% is reduced compared with wild type；Turning the transformant of pga-zj5a-m3 its enzyme activity is
10527U/ml, without significant changes compared with wild type.
The present invention will obtain after a variety of strategy transformations multiple excellent from the polygalacturonase gene of aspergillus niger
Change gene, pga-zj5a-m2 (nucleotide sequence is as shown in SEQ ID NO.3) can significantly improve the enzyme in Pichia pastoris
Secreting, expressing amount, and other optimization genes it is optimized after cannot still improve expression (pga-zj5a-m3), or even decline
(pga-zj5a-m1)。
The present invention also provides a kind of methods for preparing polygalacturonase, comprising: by SEQ ID NO.2, SEQ ID
Polygalacturonase optimization gene operability shown in NO.3 and SEQ ID NO.4 is connected to obtain with expression vector weight
Group expression vector；The recombinant expression carrier is converted into host cell, obtains recombinant bacterial strain；Cultivate recombinant bacterial strain, induction recombination
The expression of polygalacturonase, recycle and purify expressed polygalacturonase to get.
Wherein, the recombinant expression carrier is recombinant eukaryon expression vector, preferably restructured Pichia pastoris in expression carrier；Institute
The host cell stated is preferably yeast cells, preferably Pichia pastoris (Pichia pastoris) cell.
Technical solution of the present invention has the advantages that compared with prior art
Polygalacturonase optimization gene is transferred in Pichia pastoris by the present invention, and the optimization gene is in Pichia pastoris
Secreting, expressing amount significantly improve, and expressed polygalacturonase can effectively degrade the pectin class object in fruit juice
Matter is laid a good foundation for the further industrialization expanding production of the enzyme.
Term definition according to the present invention
Unless otherwise defined, otherwise all technologies used herein and scientific term all have with it is of the art
Those of ordinary skill usually understands identical meaning.
Term " recombinant host cell " or " host cell " mean include Exogenous polynucleotide cell, but regardless of using
Which kind of method is inserted into generate recombinant host cell, such as directly known in intake, transduction, f pairing or fields
Other methods.Exogenous polynucleotide can remain the non-integrated vector of such as plasmid or can be integrated into host genome.
Term " polynucleotides " or " nucleotide " mean the deoxyribonucleotide of sub-thread or bifilar form, deoxyribose core
Glycosides, ribonucleotide or ribonucleotide and its polymer.Except nonspecific limitation, otherwise the term is covered containing natural nucleotide
Known analog nucleic acid, the analog have similar to reference nucleic acid binding characteristic and be similar to it is naturally-produced
The mode of nucleotide is metabolized.Unless in addition specific limitation, otherwise the term also means oligonucleotide analogs comprising
PNA (peptide nucleic acid), the DNA analog used in antisense technology (thiophosphate, phosphamide acid esters etc.).Unless in addition referring to
Fixed, otherwise specific nucleic acid sequence also impliedly covers variant of its conservative modification (including but not limited to degenerate codon takes
Generation) and complementary series and clearly specified sequence.Particularly, can by generate one of them or more than one selected by (or
It is all) the 3rd sequence replaced through mixing base and/or deoxyinosine residue of codon realize that degenerate codon replaces
(Batzer et al., Nucleic Acid Res.19:5081 (1991)；Ohtsuka et al., J.Biol.Chem.260:2605-
2608(1985)；With Cassol et al., (1992)；Rossolini et al., Mol Cell.Probes8:91-98 (1994)).
Term " expression " refers to transcription and/or translation of the foreign gene in host cell.
Term " conversion " refers to the method introducing foreign gene in host cell.
Term " foreign gene " refers to for specific host cell, the gene order be belong to external source, or
From identical source but its original series modify or has been transformed.
Detailed description of the invention
Fig. 1 polygalacturonase wild type gene pga-zj5a codon usage frequency；
Polygalacturonase gene pga-zj5a-m1 codon usage frequency after Fig. 2 present invention optimization；
Polygalacturonase gene pga-zj5a-m2 codon usage frequency after Fig. 3 present invention optimization；
Polygalacturonase gene pga-zj5a-m3 codon usage frequency after Fig. 4 present invention optimization；
The mRNA secondary structure figure of Fig. 5 polygalacturonase wild type gene pga-zj5a；
The mRNA secondary structure figure of polygalacturonase gene pga-zj5a-m1 after Fig. 6 present invention optimization；
The mRNA secondary structure figure of polygalacturonase gene pga-zj5a-m2 after Fig. 7 present invention optimization；
The mRNA secondary structure figure of polygalacturonase gene pga-zj5a-m3 after Fig. 8 present invention optimization；
Fig. 9 yeast construction of recombinant expression plasmid schematic diagram；
Polygalacturonase vigor in 3 liters of fermentors of Figure 10 yeast strain；
Purifying (the M:Marker of Figure 11 polygalacturonase；1: unpurified polygalacturonase；2: after purification
Polygalacturonase；3: through the postdigestive polygalacturonase of Endo-H；4:Endo-H)；
Application effect of the recombination polygalacturonase of Figure 12 Different adding amount in pear juice；
Application effect of the polygalacturonase in pear juice is recombinated under Figure 13 different role time.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
Illustrate:
Used genetic recombination technology is the routine techniques in this field in following specific embodiments.In following tests
The technology that does not describe in detail in example, according in following laboratory manual or document related Sections or part carry out, comprising:
Wei Xiao,John M.Walker,Yeast Protocol Senond edition(2006)；Kriegler, Gene
Transfer and Expression:A Laboratory Manual (1990)；Ausubel et al, Current
Protocols in Molecular Biology(1994)；Sambrook et al, Molecular Cloning, A
Laboratory Manual (the 3rd edition .2001).
The optimization design and synthesis of 1 polygalacturonase gene of test example
1, test method
1.1 bacterial strains and plasmid
Aspergillus niger (Aspergillus niger) is saved by this laboratory of inventor；
Optimization gene segment is synthesized by Nanjing Jin Sirui biotechnology company.
The optimization design of 1.2 polygalacturonase genes
According to the sequence for the polygalacturonase wild type gene for cloning acquisition in aspergillus niger, gene order is carried out excellent
Change, this is in the process mainly according to following principle:
1) the encoded amino acid sequence of polygalacturonase gene pga-zj5a is not changed (such as SEQ ID NO.5 institute
Show)；
2) according to codon usage bias, as far as possible the reduction concatenated probability of rare codon, because of rare codon
It is possible that translation efficiency can be reduced；
3) according to codon preference in Pichia pastoris, change its CAI Distribution value range；
4) G/C content and distributed areas in gene order are controlled；
5) appearance of high GC and the continuous region AT are avoided；
6) loop-stem structure of mRNA (energy) is optimized to extend the half-life period of mRNA.
2, test result
By optimization, 3 polygalacturonase mutated genes pga-zj5a-m1, pga-zj5a-m2 and pga- are obtained
Zj5a-m3, nucleotide sequence are respectively shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.
G/C content variation is as shown in table 1 before and after polygalacturonase gene sequence optimisation.Polygalacturonase gene
The codon service condition for optimizing front and back is as shown in Figure 1,2,3, 4.Polygalacturonase gene optimization front and back mRNA second level knot
Structure is as shown in Fig. 5,6,7,8.
The variation of G/C content before and after 1 polygalacturonase gene sequence optimisation of table
The building of 2 polygalacturonase recombinant pichia yeast strain of test example and the preparation of zymoprotein
1, test method
1.1 bacterial strains and plasmid
Trans1-T1 competent escherichia coli cell and pEASY-BLUNT-simple carrier are purchased from the full formula gold biology in Beijing
Technology Co., Ltd.；
Expression vector pPIC9 and Pichia pastoris F-strain GS115 is Invitrogen Products；
Plasmid with wild type polygalacturonase gene
PEASY-BLUNT-simple-pga-zj5a is the building of the present inventor laboratory；
Plasmid pUC57-simple-pga-zj5a-m1, pUC57- with polygalacturonase gene after optimization
Simple-pga-zj5a-m2, pUC57-simple-pga-zj5a-m3 are designed by the present inventor, commission Nanjing Jin Sirui biology
Scientific & technical corporation's synthesis.
1.2 culture mediums and other solution
LB culture medium: 0.5% yeast extract, 1.0% peptone, 7.0,121 DEG C of sterilizing 20min of 1.0%NaCl, pH；
LB solid medium: 2% (w/v) agar powder is added in liquid LB；
YPD fluid nutrient medium: 1% yeast extract, 2% peptone, 2% glucose, 108 DEG C of sterilizing 30min；
13.4%YNB (no amino acid yeast nitrogen): determine after weighing a small amount of deionized water dissolving of 134g YNB solid
Hold 1000mL, 0.22 μm of filtration sterilization, 4 DEG C save backup；
0.02% biotin: the biotin for weighing 20mg is dissolved in 100mL deionized water, 0.22 μm of filtration sterilization, 4 DEG C of guarantors
It deposits spare；
MD solid medium: 1.34%YNB, 0.00004% biotin, 2% glucose, 1.5% agarose, 108 DEG C go out
Bacterium 30min；
1mol/L sorbierite: 182.1g D-glucitol is dissolved in 1000mL water, 0.22 μm of filtration sterilization, 4 DEG C of preservations.
BMGY culture medium: 1% yeast extract, 2% peptone, phosphate buffer (pH 6.0), 1.34%YNB,
0.00004% biotin, 1% glycerol (v/v)；
BMMY culture medium: 1% yeast extract, 2% peptone, phosphate buffer (pH 6.0), 1.34%YNB,
0.00004% biotin, 0.5% methanol；
The building of 1.3 polygalacturonase wild type genes and optimization gene yeast recombinant expression carrier
PEASY-BLUNT-simple-pga-zj5a, pUC57-simple-pga-zj5a-m1, pUC57- are extracted respectively
Simple-pga-zj5b-m2, pUC57-simple-pga-zj5a-m3 and pPIC9 plasmid, and use EcoR I (SnaB I) respectively
Double digestion processing is carried out to this 5 plasmids with Not I, respectively by polygalacturonase wild type gene pga-zj5a and 3
Optimization gene and expression vector pPIC9 digestion products are recycled and are connected, and are reflected by digestion and sequencing to positive colony
It is fixed, yeast recombinant expression carrier pPIC9-pga-zj5a and pPIC9-pga-zj5a-m1, pPIC9-pga- are thus constructed respectively
Zj5a-m2 and pPIC9-pga-zj5a-m3 (Fig. 9).
The expression of 1.4 polygalacturonase wild type genes and its optimization gene in Pichia pastoris
Correct recombinant plasmid will be verified to be linearized with restriction enzyme Sal I, it is linearized after plasmid piece
Section is converted with electric shock conversion instrument to P.pastoris GS115 competent cell after ethanol precipitation, is applied on MD plate,
It is placed in 28 DEG C of incubators and cultivates 2 days.
1.5 produce polygalacturonase recombinant yeast pichia pastoris in the screening of shaking table level
From picking monoclonal on MD plate, put onto the MD plate of corresponding label；MD plate is placed in 28 DEG C of incubators and is trained
Educate 48h.Selection normal growth transformant be inoculated in the porous plate containing 600 μ L BMGY culture mediums, be placed on 28 DEG C,
48h is cultivated on 200rpm shaking table；Bacterium solution after shaking table being cultivated 48h is placed on 5000r/min centrifugation 5min, removes supernatant, respectively
600 μ L BMMY culture mediums are added in hole, are placed on 28 DEG C, 200rpm induction culturing.A methanol (additional amount is added after 12h
To reach methanol final concentration in system 0.5%), after induction culturing 48h, obtained bacterium solution is placed on 5000r/min centrifugation
10min takes supernatant to measure enzymatic activity, is screened out from it with the active transformant of polygalacturonase.
Initial screening there is the active transformant of polygalacturonase, carries out secondary screening again.Above-mentioned transformant is connect
Kind is in containing in 10mL BMGY culture medium, and 28 DEG C, 200rpm shaking table cultivates 48h, and 5000r/min is centrifuged 5min, removes supernatant,
After addition 5mL BMMY methanol induction culture medium suspends again, 28 DEG C, 200rpm shaking table culture adds a methanol every 12h
(additional amount is that methanol final concentration in system is made to reach 0.5%), after inducing 48h, 5000r/min is centrifuged 10min, and supernatant is taken to examine
Survey polygalacturonase vigor.
The fermentation of 1.6 polygalacturonase recombinant yeast pichia pastoris
The highest transformant of enzyme activity in secondary screening is chosen, the expression water of its polygalacturonase in installation for fermenting is studied
It is flat.The single bacterium colony for choosing each transformant is seeded in 50mL YPD fluid nutrient medium, cultivates 12h on 28 DEG C of shaking tables.By its
It is all transferred to containing in 200mL YPD fluid nutrient medium, 28 DEG C, 200rpm is cultivated for 24 hours, it is all then accessed 3L fermentor
In, initial medium liquid amount is 2000ml, and at 28 DEG C, 1000r/min fermented and cultured, fermentor parameter is set as pH5.0, temperature
30 DEG C of degree, ventilating ratio 2:1.It after culture 16 hours, adds carbon source (25% glucose of 400ml), after 6h, starts to carry out mixed feeding
Stage (the 25% glucose+12.5ml methanol of 100ml) after mixed feeding 4h, starts stream plus methanol, into the induction producing enzyme stage.It opens
Begin to take fermented sample to measure polygalacturonase vigor every 12h, enzyme activity increases with the extension of induction time after induction
Add, when enzyme activity is begun to decline, stop fermentation, fermentation liquid is centrifuged (10000r/min, 10min, 4 DEG C), clear enzyme solution in collection.
The measuring method of 1.7 polygalacturonase vigor
Polygalacturonase can decompose low-esterified pectin or polygalacturonic acid is oligogalacturonans, product phase
The reproducibility of substrate is increased, therefore measures the vigor of this enzyme using DNS (3,5- dinitrosalicylic acid) method.One enzyme activity
Unit (1U) is defined as under determination condition, discharges enzyme amount required for 1 μm of ol D- (+) galacturonic acid per minute.
Enzyme activity determination method: 450 μ l, 0.2% substrate (polygalacturonic acid) and 50 μ l enzyme solutions (appropriate dilution) are at 40 DEG C
10min is reacted, 750 μ L DNS solution are added and terminate reaction；Control group first adds 450 μ l, 0.2% substrate and 750 μ l DNS to terminate
Liquid reacts 10min, finally plus 50 μ l enzyme solutions (appropriate dilution).Reaction mixture is set 5min in boiling water bath to develop the color, is cooled to room
Temperature surveys its light absorption value at 540nm.Polygalacturonase vigor is calculated by standard curve.
2, experimental result
Screened respectively turn polygalacturonase wild type gene (pga-zj5a) and optimization gene (pga-zj5a-m1,
Pga-zj5a-m2, pga-zj5a-m3) each 120 plants of yeast transformant.Wherein, pick out turn pga-zj5a enzyme activity it is highest
Bacterial strain, in the case where shaking flask is horizontal, polygalacturonase vigor is 1204U/mL；Turn the bacterial strain highest enzyme activity of pga-zj5a-m1
For 976U/mL, the bacterial strain highest enzyme activity for turning pga-zj5a-m2 is 1778U/mL, turns the bacterial strain highest enzyme activity of pga-zj5a-m3
Power is 1316U/mL.The enzyme activity for turning the bacterial strain of pga-zj5a-m2, which is significantly higher than, turns pga-zj5a, pga-zj5a-m1, pga-
The bacterial strain of zj5a-m3.
It can be seen that (as shown in Figure 10) from the expression in 3L fermentor, turn the yeast transformant of wild type gene
Pga-zj5a vigor of polygalacturonase after methanol induction 120h reaches up to 10436U/mL, and turns pga-zj5a-
15428U/mL is then reached after the Induction 120h of m2,48% is improved compared with wild type.But optimization gene pga-
Zj5a-m1 fermentation level is only 8572U/ml, and 17.9% is reduced compared with wild type；The fermentation level of pga-zj5a-m3 is
10527U/ml, without significant changes compared with wild type.
The purifying and application of the recombination polygalacturonase of test example 3
1, experimental method
The purifying of 1.1 recombination polygalacturonases
By high fermentation liquid centrifugation (10000r/min, 10min, 4 DEG C) the removal cell fragment of collection and insoluble impurity.
0.1 μm of microfiltration membranes of supernatant suspension remove remaining thallus, then carry out ultrafiltration with the ultrafiltration membrane of 10-kDa, removing salinity with
Pigment just obtains crude enzyme liquid.It is pre- flat using NTA0 buffer (20mM Tris-HCl, pH 6.5,0.5M NaCl, 10% glycerol)
Weigh His-tag nickel ion affinity chromatograph column, and crude enzyme liquid is loaded into the chromatographic column of this pre-equilibration, uses NTA500 buffer later
(20mM Tris-HCl, pH 6.5,0.5M NaCl, 10% glycerol, 0.5M imidazoles) is linearly washed with the elution rate of 4mL/min
It is de-, collect the eluent at different peaks；Enzyme activity test is carried out to all eluents, the eluate concentration for there are a large amount of enzyme activity is set
For the standard for eluting destination protein；Purified with the eluent of this concentration to enzyme solution, enzyme activity determination is carried out after collection, it is all
Purification step all operates at 4 DEG C, detects protein purification effect by SDS-PAGE.
Application of the 1.2 recombination polygalacturonases in pear juice
Stripping and slicing after fresh pyrus nivalis is enucleated, is put into juice extractor the Vitamin C squeezed the juice, and add 3.5g after weighing 700g
Acid solution.Pulp removes pulp residue with 8 layers of filtered through gauze and obtains experiment fruit juice after juicing.For the enzyme for detecting Different adding amount
Application effect of the liquid in pear juice takes the pear juice of 50mL, and addition enzyme solution makes its final concentration of 0,1,2,5,10U/mL fruit juice, 40
DEG C processing 1h.Not add the pear juice of enzyme as blank control.After having handled, the pear juice of enzymatic treatment is filtered with filter paper, and before measurement
The filtrate volume of gained pear juice in 2min.After having filtered, measuring it in wavelength with spectrophotometric is the light transmittance at 660nm.
In order to verify recombination polygalacturonase under different time to the application effect of pear juice, it is with 5U/ml fruit juice
Recombinase additive amount, measurement differential responses time (15min, 30min, 60min, 90min, 120min) under pear juice crushing juice rate and
Light transmittance.Its method for handling pear juice is as described above, using the pear juice for not adding enzyme as blank control.
2, test result
Fermentation liquid takes supernatant after being centrifuged, and after ultrafiltration concentration and affinitive layer purification, obtains electrophoretically pure recombination
Polygalacturonase.(Figure 11) is analyzed through SDS-PAGE, the enzyme of purifying has the similar band of 2 molecular size ranges, and (41kDa is left
It is right), it is greater than theoretic molecular weight of albumen 37kDa.With glycoside hydrolase (Endo-H) to recombination polygalacturonase desugar
After base, a protein band is only shown, this is electrophoretically pure recombination polygalacturonase.
By the fresh pear juice squeezed in 40 DEG C of recombination polygalacturonase (0,1,2,5,10U/mL fruits with various concentration
Juice) processing 1h after, the crushing juice rate maximum of pear juice can be improved 41.8%, and light transmittance improves about 3 times (Figure 12).In addition, with
5U/mL fruit juice has detected under the differential responses time polygalacturonase to pear juice crushing juice rate and clarification as enzyme solution additive amount
Spend the influence (Figure 13) of (light transmittance).The result shows that adding penetrating for the pear juice of polygalacturonase when reaction 120min
Rate has been increased to 94.3% compared to 57.4% in reaction 15min；The crushing juice rate of pear juice is increased to from 6.06mL (15min)
10.37mL (120min), improves 71.12%.
It can be seen that the polygalacturonase of Aspergillus niger origin has important application potential in fields such as fruit juice productions,
Its gene is after the sequence optimisation of a variety of strategies, in multiple optimization genes, optimization gene pga-zj5a-m2 (SEQ ID NO.3)
Secreting, expressing level in Pichia pastoris significantly improves, and other optimization genes cannot still improve expression (pga-zj5a-
M3), or even decline (pga-zj5a-m1).Compared with domestic and international research, the present invention recombinates the final expression of polygalacturonase
Amount reaches higher expression, lays a good foundation for further industrialization expanding production.
Claims (12)
1. polygalacturonase optimization gene, which is characterized in that its nucleotides sequence is classified as shown in SEQ ID NO.3.
2. the recombinant expression carrier containing polygalacturonase optimization gene described in claim 1.
3. recombinant expression carrier according to claim 2, it is characterised in that: the recombinant expression carrier is recombination eukaryon table
Up to carrier.
4. recombinant expression carrier described in accordance with the claim 3, it is characterised in that: the recombinant eukaryon expression vector is recombination ferment
Female expression vector.
5. recombinant expression carrier according to claim 4, it is characterised in that: the recombinant yeast expression vector is that recombination is finished
Red Yeast expression carrier.
6. the host cell containing any one of the claim 3-5 recombinant expression carrier.
7. host cell according to claim 6, it is characterised in that: the host cell is yeast cells.
8. host cell according to claim 7, it is characterised in that: the yeast cells is Pichia pastoris.
9. polygalacturonase optimization gene described in claim 1 is preparing the application in polygalacturonase.
10. applying according to claim 9 characterized by comprising by polygalacturonic acid described in claim 1
Being connected with expression vector of enzyme optimization gene operability obtain recombinant expression carrier；The recombinant expression carrier is converted into place
Chief cell obtains recombinant bacterial strain；Recombinant bacterial strain is cultivated, the expression of induction recombination polygalacturonase recycles and purifies institute's table
The polygalacturonase reached.
11. applying according to claim 10, it is characterised in that: the recombinant expression carrier is recombinant eukaryon expression load
Body；The host cell is yeast cells.
12. applying according to claim 11, it is characterised in that: the recombinant eukaryon expression vector is Pichia pastoris table
Up to carrier；The yeast cells is Pichia pastoris.
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