CN105838729A - Novel high-activity allinase and preparation method thereof - Google Patents
Novel high-activity allinase and preparation method thereof Download PDFInfo
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- CN105838729A CN105838729A CN201610330256.8A CN201610330256A CN105838729A CN 105838729 A CN105838729 A CN 105838729A CN 201610330256 A CN201610330256 A CN 201610330256A CN 105838729 A CN105838729 A CN 105838729A
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Abstract
The invention relates to a novel high-activity allinase and a preparation method thereof. The preparation method is characterized in that a wild type allinase gene from a garlic bulb is transformed by utilizing a random mutation technology, and a high-activity allinase mutant gene, namely a novel high-activity allinase gene, is obtained; then the novel high-activity allinase gene is expressed in a bacillus subtilis expression system and pichia pastoris expression systems (including a pichia pastoris free expression system and a pichia pastoris cell surface display system) respectively, and recombination strains for producing the high-activity allinase are obtained; detections carried out after fermentation expression show that the specific enzyme activity of the novel high-activity allinase is improved by 50% in comparison with a wild type allinase.
Description
Technical field
The invention belongs to bioengineering field, relate to the ratio enzyme built by fallibility round pcr lactam enzyme by directional anagenesis in vitro
The new-type garlic propylhomoserin enzyme mutant improved alive, relates in particular to random mutation and the recombinant DNA technology of gene,
A kind of novel high vigor allinase and preparation method thereof.
Technical background
Allinase also known as S-allyl-L-cysteine sulfoxide lyase, Alkylcysteine sulfoxide lyase etc. (E.C.4.4.1.4), 1949 by
First Stoll and Seeback find.It has the forms such as dimer, trimer, the tetramer.Different plants
Albumen polymerized form is different, and the allinase such as Bulbus Allii (Alliium sativum) is dimer, and foreign
Herba Alii fistulosi (Alliium cepa) is then the tetramer.
Foreign scholar has obtained encoding the gene of allinase, but institute from Bulbus Allii Cepae, Folium Allii tuberosi, ramson and Bulbus Allii
Gene order, protein molecular quality and the biochemical property of report have many differences.The clone's such as Rabinkov is big
Bulbus Allii bulb allinase gene is up to 2200bp, for immaturity enzyme (precursor before post-translational cleavage
Alliinase) full-length gene order.This allinase exists only in garlic bulb and leaf, and is not present in
In root of Bulbus Allii, but its root has the highest enzymatic activity, thus infer the same work that Bulbus Allii root contains allinase
Enzyme.2000, Lancaster etc. is a kind of novel allinase of purification from onion root, has obtained coding ocean
The cDNA of Radix Allii Fistulosi allinase albumen, elaborates its structure and function.
1998, Weik etc. have studied allinase and recombinates in escherichia coli, saccharomyces cerevisiae and Pichia sp.
Express, it is thus achieved that restructuring allinase.2010, celebrate the clones such as duty and obtain allinase gene, and
Escherichia coli are expressed, it is thus achieved that recombiant protein inclusion body, through protein renaturation, allinase ratio of recombinating
The vigor report higher than Weik.But compared with the sample directly extracted from Bulbus Allii through phytochemistry method the most not
Preferable.2010, the allinase primers that Wu Xiaoli etc. logs according to GenBank, passes through RT-PCR
Technology is cloned first and has been logged in China's Zhejiang garlic bulb allinase gene.Zhejiang allinase ratio
Many 3 bases of Garlic propylhomoserin enzyme sequence that GenBank logs in, have 9 sites to undergo mutation.And by mesh
Gene be successfully building up on pPICZ α C yeast expression vector.Restructuring allinase has enzymatic activity,
Specific activity of enzyme is (82.09 ± 3.89) U/mg, less than the natural allinase extracted.
Bulbus Allii effect in terms of medicine and health care has obtained universal accreditation, thus the effect of allinase is the most aobvious
Obtain particularly important.The garlic tablet and the capsule that produce the most both at home and abroad reach more than 30 kinds, from Bulbus Allii respectively
Extract alliin and allinase, utilize alliin and allinase to produce complex capsule or injection.Therefore,
Carry out DNA molecular recombinant technique and build allinase expression system production allinase, and utilize random mutation
Improve the research in terms of these of enzymatic activity and stability, just become the effective way improving Bulbus Allii quasi drugs drug effect
Footpath, and by space broader with application offer for the exploitation for Bulbus Allii quasi drugs and health product.
Enzyme Directed Molecular Evolution in Vitro belongs to the nonideal explosives of protein, is the New Policy of protein engineering.Profit
The multiformity of molecule is createed at molecular level, in conjunction with sensitive triage techniques, rapidly with molecular biology method
Obtain preferable mutant.It is not required to understand the space structure of protein, avtive spot, catalyst mechanism etc. in advance
Factor, but create special evolution conditions artificially, simulation natural evolution mechanism, transformation enzyme gene in vitro,
Obtain the structure enzyme with some expection feature.Wherein, fallibility PCR refers to while amplifying target genes,
Utilize Taq enzyme not possess 3 ' → 5 ' proofreading functions, change Mn in reaction system simultaneously2+、Mg2+With various dNTP
Concentration, be randomly incorporated into base mispairing with certain frequency to genes of interest, cause genes of interest to occur random prominent
Become.But, the gene through once suddenling change typically is difficult to obtain satisfied result, develops the most again continuous error-prone
PCR (Sequential Error-prone PCR) strategy.The product obtained will be expanded as next by a PCR
The template of secondary PCR amplification, carries out fallibility the most repeatedly, makes the micromutation obtained each time constantly amass
Tire out and produce important beneficial mutation.Therefore, there is exclusive advantage.
Bacillus subtilis belongs to gram positive bacteria.Bacillus subtilis expression system has the advantage that 1, energy
Enough secrete various protein efficiently；2, many bacillus subtilis uses in fermentation industry are the most suitable
Long history, no pathogenicity, do not produce any endotoxin；3, bacillus micro-organism genetic background is ground
That studies carefully is fully aware of, and has growth rapidly, to nutrition without the advantage of particular/special requirement；4 codon-bias are not
Substantially；5 fermentations are simple, and bacillus subtilis is aerobic bacteria, it is not necessary to anaerobic fermentation equipment, to culture medium without spy
Different requirement, after fermentation ends, is easily separated fermentation liquid and microorganism, can enter the purification of destination protein
Recovery stage；6 have resistance, can produce multiple thermostability enzyme preparation.
Pichia sp. is a kind of unicellular lower eukaryotes, and condition of culture is common, and growth and breeding speed is rapid.
Pichia yeast expression system, when expressing gene engineering product, can effectively reduce production with large-scale production
Cost.Pichia yeast expression system has certain post translational processing ability, and the exogenous proteins of results has one
Determining the folding in degree to process and glycosylation modified, character is more stable compared with the protein of prokaryotic expression,
Pichia yeast expression system has two kinds of expression-forms, including Pichia sp. dissociate expression system and Pichia sp. thin
Cellular surface display systems.Pichia sp. dissociate expression system express exogenous gene there is certain post translational processing
Ability, the exogenous proteins of results has folding processing to a certain extent with glycosylation modified, character relatively protokaryon
The protein that expression system is expressed is more stable, and some yeast expression system has external secretion signal sequence, it is possible to
Expressed exogenous proteins is secreted into extracellular, it is easy to purification, and Pichia pastoris surface display system
In addition to the folding processing and appropriate glycosylated advantage of the post translational processing ability and albumen with exogenous gene, warp
The whole-cell catalyst that this system obtains can also reuse thus reduce production cost.Pichia anomala expression system
System has become modern molecular biology and has studied most important instrument and model, is that expression alien gene is more satisfactory
Instrument.
Summary of the invention
In place of it is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of novel high vigor allinase
Mutant gene, the present invention uses fallibility round pcr, wild type allinase is carried out random mutation, obtains
Novel high vigor allinase (Glu225Val, Glu267Phe), the specific enzyme activity of novel high vigor allinase
50% is improve than the specific enzyme activity of wild type allinase.
It is an object of the invention to complete by the following technical solutions:
A kind of novel high vigor allinase mutant gene, its gene order is SEQ ID NO:5.
The construction method of a kind of novel high vigor allinase mutant gene, by garlic bulb wild type alliin
Enzyme gene carries out random mutation, the 674th bit base A → T, the 799th bit base G → T, the 800th bit base
A → T, the 801st bit base A → C, obtain novel high vigor allinase mutant gene；Described Bulbus Allii squama
Stem wild type allinase gene order is SEQ ID NO:3.
A kind of novel high vigor allinase mutant, by the nucleotide sequence coded open country as shown in sequence 3
In raw type allinase aminoacid sequence, the aminoacid Glu of the 225th replaces with Val and the ammonia of the 267th
Base acid Glu replaces with Phe, obtains the aminoacid sequence as shown in sequence 6.
The preparation method of a kind of novel high vigor allinase mutant, comprises the steps:
(1) by the wild type allinase gene of fallibility PCR random mutation garlic bulb, the 674th bit base A
→ T, the 799th bit base G → T, the 800th bit base A → T, the 801st bit base A → C, obtain novel
High vigor allinase mutant code gene；
(2) by described novel high vigor allinase mutant code gene enzyme action, it is connected to express or show load
Body, obtains carrying high vigor allinase mutant code gene recombined vector；
(3) recombinant vector is converted to host cell, obtain recombinant bacterial strain；
(4) expressing described recombinant bacterial strain, purification obtains the novel high vigor Bulbus Allii ammonia as shown in SEQ ID NO:6
Acid enzyme.
A kind of expression vector containing said gene and host cell.
And, described expression vector is pBSA43, described host cell is bacillus subtilis WB600.
And, described expression vector is pPIC9K, described host cell is Pichia pastoris GS115.
And, described display carrier is pPIC9K-Flo, described host cell is Pichia pastoris GS115.
The unique distinction following points of the present invention:
1, in the present invention novel high vigor allinase gene at bacillus subtilis expression system and Pichia sp.
Expression system is expressed, respectively obtain the novel high vigor allinase recombinant bacterial strain of bacillus subtilis and
The novel high vigor allinase recombinant bacterial strain of Pichia sp. (includes that the novel high vigor allinase of Pichia sp. dissociates
Express recombinant bacterial strain and the novel high vigor allinase recombinant bacterial strain of Pichia pastoris surface display).Recombinant bacterium
After strain fermentation, novel high vigor allinase catalyst can be obtained through corresponding process.
2, the present invention uses fallibility round pcr, and wild type allinase is carried out random mutation, obtains novel
High vigor allinase (Glu225Val, Glu267Phe), the specific enzyme activity of novel high vigor allinase is than open country
The specific enzyme activity of raw type allinase improves 50%.
3, the ratio of allinase after the novel high vigor allinase recombinant bacterium of bacillus subtilis ferments in the present invention
Enzyme is lived and be can reach (105.34 ± 1.73) U/mg, and Pichia sp. is free expresses the restructuring of novel high vigor allinase
The specific enzyme activity of bacterium up to (173.72 ± 2.89) U/mg, the novel high vigor alliin of Pichia pastoris surface display
The specific enzyme activity of enzyme whole-cell catalyst is up to (196.47 ± 4.93) U/ (g stem cell).
4, the present invention uses yeast surface display system, and allinase is showed in yeast surface, yeast cells
Both as the Producer of enzyme, the carrier in immobilized enzyme can be served as again, eliminate the purification of enzyme and fixing grade is grasped
Make, simplify production link, reduce production cost.
Accompanying drawing explanation
Fig. 1 is the PCR amplification electrophoretogram of allinase mature peptide gene of the present invention, wherein: M is DNA Marker,
1 is allinase mature peptide gene；
Fig. 2 is recombiant plasmid pBSA43-alliinasem digestion verification figure of the present invention, wherein: M is DNA
Marker, 1 is that pBSA43-alliinasem is through BamHI and HindIII double digestion；
Fig. 3 is recombiant plasmid pPIC9K-alliinasem digestion verification figure of the present invention, wherein: M is DNA
Marker, 1 is that pPIC9K-alliinasem is through EcoRI and NotI double digestion；
Fig. 4 is recombiant plasmid pPIC9K-Flo-alliinasem digestion verification figure of the present invention, wherein: M is DNA
Marker, 1 is that pPIC9K-Flo-alliinasem is through SnaBI and EcoRI double digestion；
Fig. 5 is pyruvate standard curve；
Fig. 6 is with FeSO4Standard curve is drawn for standard substance；
Detailed description of the invention
Below in conjunction with embodiment, the technology contents of the present invention is described further, but the present invention is not limited solely to these
Embodiment, it is impossible to limit protection scope of the present invention with following embodiment.
The purpose technology path that the present invention realizes is as follows
Take fresh garlic bulb to grind in liquid nitrogen, carry out the extraction of total serum IgE according to Trizol reagent description
And carry out RT-PCR.With synthesis cDNA the first chain as template, design primer, expand wild type alliin
Enzyme gene order (as shown in SEQ ID NO:3).By fallibility PCR after it is connected with pET28a carrier
Carry out random mutation, it is thus achieved that novel high vigor allinase gene (as shown in SEQ ID NO:5), build
Recombinant vector and in bacillus subtilis WB600 and Pichia pastoris GS115 successful expression, obtain producing novel
The recombinant bacterial strain of high vigor allinase, obtains the new-type garlic propylhomoserin of high vigor further by fermenting extraction process
Enzyme.
Use in the present invention and be defined below:
1, aminoacid and the nomenclature of DNA nucleotide sequence
Use the generally acknowledged IUPAC nomenclature of amino acid residue, use three-letter codes form.DNA nucleotide sequence is adopted
By generally acknowledged IUPAC nomenclature.
2, the mark of new-type garlic propylhomoserin enzyme mutant
Use " aminoacid that Original amino acid position is replaced " to represent in new-type garlic propylhomoserin enzyme mutant to suddenly change
Aminoacid.Such as Glu225Val, represent that the aminoacid of position 225 is replaced to by the Glu of parent's allinase
Val, the numbering of position is corresponding to the aminoacid sequence numbering of allinase in SEQ ID NO:4.
In the present invention, alliinase represents that the original amino acid of wild type allinase is (such as SEQ ID
Shown in NO:4), alliinasem represent novel allinase amino acid sequence variants (such as SEQ ID NO:
Shown in 6).
It is bacillus subtilis WB600 for expressing the host cell of described new-type garlic propylhomoserin enzyme mutant, table
Reaching carrier is pBSA43；
It is Pichia pastoris GS115 for expressing the host cell of described new-type garlic propylhomoserin enzyme mutant, expresses and carry
Body is pPIC9K；
It is Pichia pastoris GS115 for expressing the host cell of described new-type garlic propylhomoserin enzyme mutant, shows and carry
Body is pPIC9K-Flo；
Embodiment 1
The acquisition of wild type allinase mature peptide gene
1, take fresh garlic bulb to grind in liquid nitrogen, carry out carrying of total serum IgE according to Trizol reagent description
Take and carry out RT-PCR.
2, with synthesis cDNA the first chain as template, according to Genbank serial number S73324.1 log in Bulbus Allii
Propylhomoserin enzyme sequence, its ORF frame upstream and downstream design pair of primers, respectively introduce restriction enzyme site BamH I,
HindⅢ.The amplimer of the allinase mature peptide gene of the design present invention is as follows:
Forward primer P1 (SEQ ID NO.1): 5 '-CGCGGATCCATGATCTGCCTAGTGATTTTGACATG
-3’
Downstream primer P2 (SEQ ID NO.2): 5 '-CCCAAGCTTTTAAATGAAAGGACGACGGGAG-3’
The reaction condition of its amplification is:
Amplification condition is: 95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 60 DEG C of annealing 40s, 72 DEG C of extensions
1min30s reacts 30 circulations；72 DEG C extend 10min.Pcr amplification product through 0.8% agarose gel electrophoresis,
Obtain the band (Fig. 1) of 1422bp, reclaim test kit with miniprep dna and reclaim PCR primer, obtain the present invention
Wild type allinase mature peptide gene alliinase, see sequence 3 after order-checking.
Embodiment 2
The acquisition of novel high vigor allinase gene.
1, recombiant plasmid is proceeded in bacillus coli DH 5 alpha, through BamH I, the checking of Hind III's double digestion,
Wild type allinase gene successful clone is on pET28a carrier.
2, random mutation
(1) carrying out random mutation based on fallibility round pcr, build novel high vigor allinase, design is drawn
Thing is as follows:
Forward primer P1 (SEQ ID NO.1): 5 '-CGCGGATCCATGATCTGCCTAGTGATTTTGACATG
-3’
Downstream primer P2 (SEQ ID NO.2): 5 '-CCCAAGCTTTTAAATGAAAGGACGACGGGAG-3’
In fallibility PCR reaction system, using P1 and P2 as upstream and downstream primer, with wild type allinase
Mature peptide gene is template, carries out fallibility PCR.
The reaction condition of its amplification is:
Amplification condition is: 95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 60 DEG C of annealing 40s, 72 DEG C of extensions
1min30s reacts 30 circulations；72 DEG C extend 10min.
(2) novel high vigor allinase mutant gene (alliinasem) is cloned into expression vector
In pET28a, convert e. coli bl21 (DE3), be inoculated in every hole and (contain containing 200 μ L LB fluid mediums
The Kan of 30 μ g/mL) 96 porocyte culture plates in, under the conditions of 37 DEG C 200r/min shaking table cultivate,
Work as OD600Reach 0.6, in each hole, add IPTG (final concentration 1mmol/L), at 16 DEG C, induce 16h,
Culture is carried out respectively following 3 kinds of processing modes: (1) multigelation 3 times (-80 DEG C, 15min；37 DEG C,
15min；0 DEG C, 15min)；(2) lysozyme (200 μ g/ml, 30min) is added；(3) heat treatment (100 DEG C
Boiling water bath, 20min), then under 4000r/min, centrifugal 10min removes precipitation, is transferred to by supernatant
Another block 96 orifice plate detects its enzyme activity.
(3) enzyme activity detection
1. enzyme activity definition
Allinase is often catalyzed 1 molecule alliin and reacts and can produce 2 molecule acetone acid, therefore can be by surveying
Determine the concentration of acetone acid to calculate that enzyme is lived.The definition that enzyme the most of the present invention is lived is: under the conditions of 35 DEG C, Bulbus Allii ammonia
The reaction of acid enzyme catalysis alliin, generation l μ g acetone acid per minute is defined as 1 unit of activity (U).
2. concentrations of pyruvate and the drafting of absorbance value standard curve
Acetone acid can generate acetone acid-2,4-dinitrophenylhydrazone, the latter with 2,4 dinitrophenyl hydrazine effect
In cherry red in alkaline solution, at wavelength 520nm, measure light absorption value.The acetone acid of differently configured concentration is molten
Liquid, respectively takes 1mL, adds 1mL 0.1%2, and 4-dinitrophenylhydrazine fully shakes up, and adds 2mL1.5mol/LNaOH
Shake up, stand 10min colour developing, at 520nm, measure light absorption value with 752 ultraviolet spectrophotometers.Draw
Concentrations of pyruvate and the standard curve of absorbance value.
Pyruvate standard curve as it is shown in figure 5, trying to achieve regression equation is Y=0.0244X+0.0167, phase relation
Number is R2=0.9990.
The assay method of the most novel allinase enzyme work and step
Enzymatic activity is measured with acetone acid system.The alliin substrate of preparation 3mmol/L.Collect tunning, add
1.0mL substrate, reacts 5min at 30 DEG C, add 1.5mL10% trichloroacetic acid and terminate reaction.Add
0.5mL0.1%2,4-dinitro phenylhydrazine, fully mixes, and adds 7mL0.5mol/LNaOH solution and shakes up colour developing,
520nm wavelength measures light absorption value.Acetone acid total concentration and enzyme activity is calculated according to absorption value.By Folin-phenol method
Measure allinase albumen quality in solution.Obtain the Rate activity of allinase in institute's test sample product.
The mensuration that the most novel allinase enzyme is lived
The specific enzyme activity of novel high vigor allinase, allinase after sudden change after measuring original allinase and suddenling change
Specific enzyme activity than sudden change before improve 50%.
Specific enzyme activity defines: the unit of activity number of had enzyme in the protein of Unit Weight, typically uses U/mg egg
White matter represents.
(4) sequencing
Order-checking (Beijing Hua Da bio-engineering corporation) result show, now amplification obtain Glu225Val,
The novel high vigor allinase gene alliinasem of Glu267Phe, is shown in sequence 5.
Embodiment 3
The structure of the novel high vigor allinase recombinant bacterium of bacillus subtilis
1, the structure of expression vector pBSA43
PBSA43 is with bacillus coli-bacillus subtilis shuttle cloning vector pBE2 as skeleton, is cloned into one
Individual strong bacillus cereus constitutive promoter P43, and recombiant protein direct secretion can be made to fruit in culture medium
Polysaccharide saccharase signal sequence sacB and obtain.It is with AmprGene, can utilize ammonia in escherichia coli
Benzylpcnicillin resistance is as selection markers；Also there is Km simultaneouslyrGene, can be at bacillus subtilis, lichens
Bacillus cereus utilize kalamycin resistance as selection markers.
2, the structure of novel high vigor allinase expression vector pBSA43-alliinasem
The novel high vigor allinase gene obtained will be built through the double enzyme of BamHI and HindIII through fallibility PCR
After cutting with as the bacillus subtilis expression vector pBSA43 connection of double digestion, construction recombination plasmid
pBSA43-alliinasem.PBSA43-alliinasem is converted bacillus coli DH 5 alpha competent cell,
Select positive transformant, extract plasmid and carry out digestion verification and check order, determine that structure obtains recombinant bacterial strain
JM109/pBSA43-alliinasem。
3, expression vector pBSA43-alliinasem converts bacillus subtilis WB600
In 60 μ L competent cells, add 1 μ L (50ng/ μ L) pBSA43-alliinasem mix and turn
Move on to ice-cold electricity and convert in cup (1mm), after ice bath 1-1.5min, shock by electricity once (25 μ F, 200 Ω,
4.5-5.0ms).Shock by electricity complete after, immediately add 1mL recovery medium (LB+0.5mol/L sorbitol
+ 0.38mol/L mannitol).After 3h is cultivated in 37 DEG C of shaking table concussions, recovery thing is coated on LB flat board,
Cultivate 24-36h, picking positive transformant, it is thus achieved that bacillus subtilis recombinant bacterial strain for 37 DEG C
WB600/pBSA43-alliinasem。
Embodiment 4
The novel high vigor allinase of Pichia sp. dissociates and expresses the structure of recombinant bacterium
1, the structure of novel high vigor allinase Expression vector pPIC9K-alliinasem
The novel high vigor allinase gene obtained will be built through EcoRI and NotI double digestion through fallibility PCR
Afterwards with as the yeast expression vector pPIC9K ligase of double digestion be attached, connection product is turned
Change in e. coli jm109 (DH5 α) competent cell, through Amp resistance screening, select positive transformant；
Extract positive transformant plasmid, shake through 37 DEG C and after pipe is cultivated, extract plasmid, and carry out single, double enzyme action just step
Card, and by the named pPIC9K-alliinasem of recombiant plasmid correct for digestion verification；Digestion verification is correct
Positive colony deliver to Beijing Hua Da Gene science limited company order-checking, to further ensure that genes of interest
Correctness, finally determines and builds the recombinant bacterial strain JM109/pPIC9K-alliinasem that acquisition is correct.
2, the structure of novel high vigor allinase recombinant bacterial strain and novel high vigor allinase high expressed bacterial strain
Screening
(1) preparation of linearisation recombinant expression plasmid pPIC9K-alliinasem
Recombiant plasmid converts before Pichia pastoris GS115 at electricity, the recombinant expression plasmid that first will build
PPIC9K-alliinasem carries out linearisation to improve recombiant plasmid integration effect on Pichia chromosome
Rate.Converting every time and need linearization plasmid DNA 5-20 μ g, and plasmid is the purest, transformation efficiency is the highest.Permissible
Linearisation enzyme action is carried out respectively with these 2 kinds of restricted enzyme of Sac I and Sal I.After enzyme action is complete, with little
Amount DNA reclaims test kit and reclaims linearisation digestion products.
(2) linearization plasmid pPIC9K-alliinasem electricity converts Pichia pastoris GS115, positive transformant
Qualification and the screening of novel high vigor allinase superior strain
1. DNA linearized to 80 μ L competent cells and 5-20 μ g is joined 1.5mL pre-cooling
In centrifuge tube, mixing, reactant liquor is transferred in the conversion cup of ice bath in advance；
2. ice bath is equipped with the conversion cup 5min of conversional solution, the parameter recommended according to electricity rotary device, carries out finishing red ferment
Female electricity converts:
3. after pulse, immediately to converting the sorbitol solution of the 1mol/L of addition 1mL pre-cooling in cup, turning
Change liquid to transfer in a new 1.5mL centrifuge tube；
4. 30 DEG C of quiescent culture l-2h, absorption Pichia pastoris GS115 electricity turns liquid 200 μ L and is coated on MD training
Support on base；
5. cultivate until transformant occurs for 30 DEG C；
6. during picking transformant list bacterium colony is dissolved in 10 μ L deionized waters, take 2 μ L bacterium solution, add
Lyticase wall breaking enzyme, 30 DEG C of reaction l0min, reactant liquor is immediately placed in-80 DEG C of refrigerators freezing l0min,
Making yeast cell wall crack, the genome of release carries out PCR as template.To proceed to empty plasmid pPIC9K's
Pichia pastoris GS115/pPIC9K, as comparison, determines positive transformant.
7., on the basis of determining positive transformant, first height is screened by the resistant panel containing variable concentrations Geneticin
The transformant of geneticin resistant, measures the new-type garlic ammonia of the transformant of these high geneticin resistant the most respectively
The enzyme of acid enzyme is lived, to obtain the superior strain GS115/pPIC9K-alliinasem of novel high vigor allinase.
Embodiment 5
The structure of the novel high vigor allinase recombinant bacterium of Pichia pastoris surface display
1, the structure of recombiant plasmid pPIC9K-Flo-alliinasem
By fallibility PCR purified product and carrier pPIC9K-Flo after SnaBI and EcoRI double digestion, will be new
Type height vigor allinase gene is connected in carrier pPIC9K-Flo, and connection product is transformed into escherichia coli
In DH5 α competence, screening and culturing in the LB solid medium containing Amp, picking positive transformant, training
Supporting rear upgrading grain, double digestion is identified and checks order, named pPIC9K-Flo-alliinasem.
2, the structure of Pichia sp. recombinant bacterium
By recombiant plasmid correct for order-checking after SalI linearisation, convert Pichia pastoris GS115 with electrotransformation,
MD plate screening recon, it is thus achieved that the novel high vigor allinase recombinant bacterium of Pichia pastoris surface display
GS115/pPIC9K-Flo-alliinasem。
Embodiment 6
The expression in bacillus subtilis recombinant bacterium of the novel high vigor allinase and preparation
Bacillus subtilis recombinant bacterial strain WB600/pBSA43-alliinasem is inoculated in LB fluid medium
In (receiving mycin containing card, 30 μ g/mL), 37 DEG C, 200r/min overnight incubation, by 1% inoculum concentration transfer in
In 50mL fresh culture, 200r/min cultivates 48h, can prepare novel high vigor allinase thick
Enzyme liquid, then uses salt fractionation method to precipitate novel high vigor allinase, collects protein precipitation, after dissolving,
Dialysis desalination, then after ion-exchange chromatography, gel chromatography, lyophilization prepares novel high vigor allinase
Pure enzyme enzyme powder.
Embodiment 7
Novel high vigor allinase dissociates the expression and preparation expressed in recombinant bacterium at Pichia sp.
Novel high vigor allinase recombinant bacterium is expressed by free for the Pichia sp. being incubated on YPD solid plate
Be seeded in YPD fluid medium, 30 DEG C, 250r/min cultivate 24h.It is transferred to newly with the inoculum concentration of 1%
In fresh BMGY culture medium, cultivating 24h for 30 DEG C, then 6000r/min is centrifuged 5min and obtains thalline, proceeds to
In BMMY culture medium.30 DEG C, 250r/min, mend methanol every 24h so that it is final concentration is maintained at 0.5%V/V,
The crude enzyme liquid of available new-type garlic propylhomoserin enzyme mutant after cultivating 120h, then uses salt fractionation method precipitation new
Type height vigor allinase, collect protein precipitation, after dissolving, dialyse desalination, then through ion-exchange chromatography,
After gel chromatography, lyophilization prepares novel high vigor allinase pure enzyme enzyme powder.
Embodiment 8
The preparation of the novel high vigor allinase whole-cell catalyst of Pichia pastoris surface display
The novel high vigor allinase weight of Pichia pastoris surface display that will be incubated on YPD solid plate
Group bacterium be seeded in YPD fluid medium, 30 DEG C, 250r/min cultivate 24h, with 1% inoculum concentration switching
In fresh BMGY culture medium, cultivating 24h for 30 DEG C, then 6000r/min is centrifuged 5min and obtains thalline,
Proceed in BMMY culture medium, 30 DEG C, 250r/min cultivate 120h, mend methanol every 24h so that it is eventually the denseest
Degree is maintained at 0.5%V/V, is then centrifuged for collection and takes thalline, washes 1-2 time with distilled water, adds protective agent,
The novel high vigor allinase whole-cell catalytic of Pichia pastoris surface display is prepared by vacuum lyophilization
Agent.
Embodiment 9
Recombinant bacterium enzyme activity determination
Measure enzyme after the three strain recombinant bacterium fermentations that will obtain to live, the wherein novel high vigor alliin of bacillus subtilis
In enzyme recombinant bacterium fermentation after fermentation liquid, the specific enzyme activity of novel allinase can reach (105.34 ± 1.73) U/mg,
Pichia sp. dissociate express novel high vigor allinase recombinant bacterium fermentation after fermentation liquid in specific enzyme activity up to
(173.72 ± 2.89) U/mg, the novel high vigor allinase whole-cell catalyst of Pichia pastoris surface display
Specific enzyme activity up to (196.47 ± 4.93) U/ (g stem cell).And bacillus subtilis wild type allinase
In recombinant bacterium fermentation after fermentation liquid, the specific enzyme activity of allinase can reach (70.23 ± 2.13) U/mg, finishes red ferment
Female free specific enzyme activity expressed in wild type allinase recombinant bacterium fermentation after fermentation liquid up to (115.82 ±
2.46) U/mg, the specific enzyme activity of Pichia pastoris surface display wild type allinase whole-cell catalyst up to
(130.98 ± 3.93) U/ (g stem cell).
Embodiment 10
Novel allinase Antioxidative Activity Determination
1, the mensuration of DPPH radical scavenging activity
Method with reference to Brand-WilliamsW..Hereinafter operate and enter under oxygen free condition and aerobic conditions respectively
OK: with ultra-pure water mixing alliin and restructuring allinase crude enzyme liquid, make alliin/allinase mixture
Solution (final concentration of 10mmol/Lalliin, allinase crude enzyme liquid total protein content is 0.275mg/mL),
Enzymic catalytic reaction different time in 37 DEG C of water-baths, takes l mL sample and 4mL concentration is 0.12mmol/L
DPPH ethanol solution (volume fraction is 95%), mixing, under room temperature lucifuge reaction 30min, if any precipitation
Centrifugal 5min under the conditions of 5500r/min.It is that 95% ethanol solution makees reference, in 517nm by volume fraction
Measure light absorption value.According to following formula calculating each sample liquid clearance rate to DPPH free radical:
Clearance rate/%=[1-(Ai-Aj)/Ac] × 100%
In formula: Ai: for adding the light absorption value of DPPH solution after sample liquid；
Aj: for the light absorption value of sample liquid；
Ac: the light absorption value of DPPH solution during for not adding sample liquid.
2, the mensuration (FRAP pH-value determination pH) of total antioxidant capacity
Taking each need testing solution, suitably after dilution, pipettor takes 100 μ L, adds FRAP working solution 3mL, mixed
Even reaction 20min, reads absorbance at 593nm.With FeSO4Standard curve is drawn (such as figure for standard substance
Shown in 6), trying to achieve regression equation is:
Y=0.3584x-0.0043, coefficient R2=0.9986
The total antioxidant capacity of sample represents with FRAP value: 1FRAP unit is equivalent to 1mmol/L FeSO4, i.e.
The total antioxidant capacity of sample is equivalent to FeSO4Mmol/L number.
Claims (8)
1. a novel high vigor allinase mutant gene, it is characterised in that: its gene order is SEQ ID
NO:5.
2. a construction method for novel high vigor allinase mutant gene as claimed in claim 1,
It is characterized in that: garlic bulb wild type allinase gene is carried out random mutation, the 674th bit base A → T,
799th bit base G → T, the 800th bit base A → T, the 801st bit base A → C, obtain novel high vigor
Allinase mutant gene；Described garlic bulb wild type allinase gene order is SEQ ID NO:3.
3. a novel high vigor allinase mutant, it is characterised in that: by the core as shown in sequence 3
In the wild type allinase aminoacid sequence of nucleotide sequence coding, the aminoacid Glu of the 225th replaces with
The aminoacid Glu of Val and the 267th replaces with Phe, obtains the aminoacid sequence as shown in sequence 6.
4. a preparation method for novel high vigor allinase mutant as claimed in claim 3, it is special
Levy and be: comprise the steps:
(1) by the wild type allinase gene of fallibility PCR random mutation garlic bulb, the 674th bit base A
→ T, the 799th bit base G → T, the 800th bit base A → T, the 801st bit base A → C, obtain novel
High vigor allinase mutant code gene；
(2) by described novel high vigor allinase mutant code gene enzyme action, it is connected to express or show load
Body, obtains carrying high vigor allinase mutant code gene recombined vector；
(3) recombinant vector is converted to host cell, obtain recombinant bacterial strain；
(4) expressing described recombinant bacterial strain, purification obtains the novel high vigor Bulbus Allii ammonia as shown in SEQ ID NO:6
Acid enzyme.
5. the expression vector of the gene comprised described in claim 1 and host cell.
Expression vector and the host of novel high vigor allinase gene the most according to claim 5 are thin
Born of the same parents, it is characterised in that: described expression vector is pBSA43, described host cell is bacillus subtilis
WB600。
Expression vector and the host of novel high vigor allinase gene the most according to claim 5 are thin
Born of the same parents, it is characterised in that: described expression vector is pPIC9K, described host cell is Pichia pastoris GS115.
Expression vector and the host of novel high vigor allinase gene the most according to claim 5 are thin
Born of the same parents, it is characterised in that: described display carrier is pPIC9K-Flo, described host cell is Pichia sp.
GS115。
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| CN113637714A (en) * | 2021-09-28 | 2021-11-12 | 湖北工业大学 | Method for preparing alliin by fermenting garlic planting and processing wastes |
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| CN114916561A (en) * | 2022-05-27 | 2022-08-19 | 山东大学 | Biopesticide for preventing and treating crop pathogenic bacteria |
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