CN105566471A - Monellin protein mutant and preparation method thereof - Google Patents

Monellin protein mutant and preparation method thereof Download PDF

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CN105566471A
CN105566471A CN201610087481.3A CN201610087481A CN105566471A CN 105566471 A CN105566471 A CN 105566471A CN 201610087481 A CN201610087481 A CN 201610087481A CN 105566471 A CN105566471 A CN 105566471A
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monellin
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刘波
蔡成固
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Qilu University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

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Abstract

The invention discloses a monellin protein mutant and a preparation method thereof. Site-specific mutagenesis is performed on a single-chain monellin, a thermostable mutant is obtained and successfully expressed in pichia pastoris, and the technical problem that sweet protein is easy to degrade in the production and transportation process is solved; meanwhile, protein sweetness is improved, the good sweet effect can be achieved on the condition of a small amount of monellin protein mutant, and a solid foundation is laid for large-scale industrial marketization of the sweet protein.

Description

A kind of monellin protein mutant and preparation method thereof
Technical field
The present invention relates to protein mutant technical field, particularly relate to a kind of monellin protein mutant and preparation method thereof.
Background technology
Sweet protein monellin is a kind of natural sweeteners extracted in the plant Dioscoreophyllumcumminsii of West Africa, there is strong sweet taste, sugariness is about 3000 times of equal in quality sucrose under the same conditions, natural monellin albumen is by two different peptide chains by covalent bonds together, combined by A chain and B chain, research finds that natural monellin albumen at high temperature easily loses activity, two peptide chains are combined into a protein chain by gene engineering method by the people such as Kim in 1989, its thermostability is promoted greatly, keep stable under wide region PH, there is not too large change in its sugariness simultaneously.Because itself is not containing sugar, as the best sweet taste substitute food product of diabetics and cardiovascular patient, also effectively can prevent the generation of children caries, there is very large market potential.Strand monellin albumen processes and will lose activity at 65 DEG C, makes it be restricted in production and transportation.
Strand monellin albumen adopts genetic engineering means also to express successfully in other biological, but as escherichia expression system, plant expression system, subtilis expression system due to the problem of these expression system output and toxic metabolite material makes monellin albumen cannot large scale fermentation production.Adopt methanol induction of Pichia pastoris fermentation that monellin protein yield is greatly improved, but adding due to toxic substance methyl alcohol, and leavened prod safety problem becomes hidden danger.
In addition how by target protein direct secretion to outside born of the same parents, save later-period purification cost, and do not add inductor during the fermentation, reduce the microbiological contamination probability in fermenting process, also to avoid toxic metabolite material in addition, ensure that the safety of this albumen as foodstuff additive, is the problem that the present invention needs to solve.
Summary of the invention
Object of the present invention is exactly defect for above-mentioned existence and provides a kind of monellin protein mutant and preparation method thereof.The present invention has carried out rite-directed mutagenesis to strand monellin, obtains a kind of Heat Stability Mutations body, and in pichia spp successful expression, solve the technical barrier of sweet protein easily degraded in production and transport; Add albumen sugariness simultaneously, make just just can reach good sweetening effects, for this sweet protein heavy industrialization marketization is taken a firm foundation in its a small amount of situation.Employing PGAPZ α A plasmid can by target protein direct secretion to outside born of the same parents, save later-period purification cost, because this is as GADPH self-induction type promotor, do not need in fermenting process to add any inductor, decrease the microbiological contamination probability in fermenting process, simultaneously without any toxic metabolite material, ensure that the safety of this albumen as foodstuff additive.
A kind of monellin protein mutant of the present invention and preparation method thereof technical scheme is, wild-type strand monellin gene the 2nd amino acids L-glutamic acid (Glu) rite-directed mutagenesis is l-asparagine (Asn) by a kind of monellin protein mutant.
Sugariness promotes 3 times relative to wild strand monellin albumen.
Thermostability promotes 10 DEG C relative to wild strand monellin albumen.
The preparation method of described a kind of monellin protein mutant, comprises the following steps:
(1) wild-type strand monellin protein expression vector is built;
(2) mutator gene is obtained by carrying out rite-directed mutagenesis in step (1), picking positive transformant sequence verification;
(3) the correct Plastid transformation of order-checking in step (2) is entered in pichia spp, filter out the pichia spp of successful conversion;
(4) induction expression protein in YPD substratum, the time is 3 days;
(5) protein purification of will express in step (4).
Step (1) is, builds the expression plasmid PGAPZ α A containing wild-type strand monellin protein gene.
Step (2) specific primer on the mutated site: upstream P1:[5 '-CC cTCGAGaAAAGAGAGGCTGAAGCTGGT aACtGGGAG-3 ']; Downstream P2:[5 '-TT gCGGCCGCtTAGGGAGGAGGCACAGGTCCGTTG-3 '].
In step (2), PGAPZ α A empty carrier by Xhol with NotI endonuclease digestion PCR primer and after cutting with enzyme is connected, be transformed in bacillus coli DH 5 alpha competent cell, coat on the LLB solid medium containing 25 μ g/mlZeocin, the positive single bacterium colony of incubated overnight picking also extracts plasmid, sequence verification sudden change result, builds mutant plasmid PGAPZ α A-E2N and is saved backup by correct mutant plasmid.
Pichia spp described in step (3) is Pichia pastoris GS115.
A kind of monellin protein mutant of the present invention and preparation method thereof beneficial effect is: the present invention has carried out rite-directed mutagenesis to strand monellin, obtain a kind of Heat Stability Mutations body, and in pichia spp successful expression, solve the technical barrier of sweet protein easily degraded in production and transport; Add albumen sugariness simultaneously, make just just can reach good sweetening effects, for this sweet protein heavy industrialization marketization is taken a firm foundation in its a small amount of situation.Employing PGAPZ α A plasmid can by target protein direct secretion to outside born of the same parents, save later-period purification cost, because this is as GADPH self-induction type promotor, do not need in fermenting process to add any inductor, decrease the microbiological contamination probability in fermenting process, simultaneously without any toxic metabolite material, ensure that the safety of this albumen as foodstuff additive.
Accompanying drawing explanation
Fig. 1 is thermal treatment protein electrophoresis figure under differing temps different time.
In figure, the nonheat-treated albumen of M.-Marker, 1-, 2-65 DEG C of thermal treatment 4h, thermal treatment 6h, 4-75 DEG C thermal treatment 6h, 5-70 DEG C thermal treatment 8h, 6-70 DEG C thermal treatment 6h, 7-80 DEG C of thermal treatment 2h at 3-65 DEG C.
Embodiment:
In order to understand the present invention better, describe technical scheme of the present invention in detail with specific examples below.
Embodiment 1
The preparation of strand monellin mutator gene albumen
(1) structure of wild-type strand monellin protein carrier
By Sino-U.S.'s calm and peaceful Bioisystech Co., Ltd synthesis wild-type strand monellin full genome and at two ends difference primer XhoI and NotI two restriction endonuclease sites, goal gene fragment and PGAPZ α A empty plasmid is cut through enzyme, connect with T4DNA ligase enzyme 37 DEG C and spend the night, be transformed in DH5 α competent escherichia coli cell on the LLB solid medium coated containing 25 μ g/mlZeocin, incubated overnight picking positive transformant, extract plasmid sequence verification with plasmid extraction kit, successfully build wild-type monellin protein carrier PGAPZ α A-SCM.
(2) to wild-type strand monellin albumen rite-directed mutagenesis
Goal gene fragment cut with XhoI and NotI two kinds of restriction enzymes, reflection system is as follows:
System of being cut by enzyme cuts 6h at 37 DEG C of enzymes, and enzyme is cut rear use 2% agarose gel electrophoresis and is separated goal gene and plasmid vector, adopts glue to reclaim kits target DNA fragment; A pair Auele Specific Primer is designed: upstream P1:[5 '-CC for mutational site cTCGAGaAAAGAGAGGCTGAAGCTGGT aACtGGGAG-3 ']; Downstream P2:[5 '-TTGCGGCCGCTTAGGGAGGAGGCACAGGTCCGTTG-3 ']; XhoI site is introduced in upstream, and NotI site is introduced in downstream, carries out PCR reaction according to following system:
Above-mentioned PCR reaction is carried out according to following program:
95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 53 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C of ends extend 10min.PCR terminates rear use 1% agarose gel electrophoresis and detects.PCR primer spent the night with being connected at the open circular plasmid carrier T4DNA ligase enzyme 37 DEG C reclaimed with glue after XhoI with NotI two kinds of restriction enzyme ferment treatment 6h, concrete reaction system is as follows:
Linked system is transformed in bacillus coli DH 5 alpha competent cell, coat on the LLB solid medium containing 25 μ g/mlZeocin, the positive single bacterium colony of incubated overnight picking also extracts plasmid, sequence verification sudden change result, builds mutant plasmid PGAPZ α A-E2N and is saved backup by correct mutant plasmid.
(3) mutator gene Plastid transformation enters Pichia pastoris GS115
By PGAPZ α A-E2N plasmid through AvrII linearizing, adopt ethanol precipitated DNA methods to concentrate linearizing DNA, and detect with agarose gel electrophoresis.
Prepared by Pichia pastoris GS115 competent cell: first inoculate the mono-bacterium colony of pichia spp GS11 in 5mlYPD substratum, and in 30 DEG C, 250rpm/min cultivates 24h, get in the 500ml shaking flask that 1ml nutrient solution is inoculated into containing 100mlYPD substratum and cultivate 12-16h, until bacterium liquid is proceeded to during OD600=0.8-1.2 in the centrifuge tube of 50ml precooling, at 4 DEG C, centrifugal 5min under 5000g, abandon supernatant; With the resuspended thalline of the sterilized water of 25ml precooling, at 4 DEG C, centrifugal 5min under 5000g, supernatant discarded, repeats this step 2-3 time; The resuspended thalline of 1mol/L sorbyl alcohol of every effective 5ml precooling, 4 DEG C, centrifugal 5min under 5000g, abandon supernatant, repeat this step 2-3 time; Finally use 200 μ L sorbyl alcohols resuspended, be distributed in the centrifuge tube of 1.5ml, often pipe 80 μ L.Get a pipe competent cell add 10 μ L linearizings and concentrated after DNA, mix gently, and the specification proceeding to precooling is ice bath 5min in 0.2cm pole cup; Be 1.5Kv in shock parameters, 25 μ F, shock by electricity under 200 Ω, and shocked by electricity the rear 1mol/L sorbyl alcohol adding rapidly 1ml precooling, then proceeded to by mixed solution in 1.5ml centrifuge tube, at 30 DEG C of recovery 1h; Draw 200 μ L be coated with and contain on the YPDS solid medium of 100 μ g/mlZeocin, flat board is cultivated 3 days at 30 DEG C, observes positive transformant, adopt PCR to identify positive single bacterium colony.
(4) checking of target protein and purifying
PCR is verified in the 250ml shaking flask that correct positive transformant is inoculated into containing 50ml, in 30 DEG C, 250rpm/min cultivates 3 days.4 DEG C, the centrifugal fermented liquid of 12000rpm get 900 μ L supernatant liquors add 100 μ L trichoroacetic acid(TCA)s mixing, spend the night at 4 DEG C, under 10000rpm/min, centrifugal mixed solution, abandons supernatant, and with acetone wash precipitation 2-3 time, by SDS-PAGE testing goal protein expression result.
Choose in the 1L shaking flask that the brighter positive transformant of target protein band is inoculated in containing 200mlYPD substratum, at 30 DEG C, inducing culture 3 days under 250rpm.Low-temperature centrifugation collects fermented supernatant fluid, and slowly adding solid ammonium sulfate to massfraction is the 12h that saltouts at 70%, 4 DEG C, at 4 DEG C, collecting precipitation under 10000rpm/min, precipitation distilled water dissolves, and put into the dialysis tubing 4 DEG C dialysis 24h that molecular weight cut-off is 8000, period changes water 3 times.Coomassie Brilliant Blue is adopted to measure the concentration of target protein and pass through the sweet active that the solution after tasting dialysis tentatively determines albumen.
Embodiment 2
The mensuration of strand monellin mutain thermostability and sweet taste threshold value
(1) qualification of albumen thermostability
The albumen of same Batch purification is processed 2h, 4h, 6h, 8h, 10h respectively at 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, sample after process detects through SDS-PAGE, observe the band brightness of gel, tentatively determine the denaturation temperature of albumen and the highest tolerable temperature.Mutain E2N processes 6h at 75 DEG C and still has albumen to there is (Figure of description Fig. 1) process 2h at 80 DEG C under after testing, and comparatively wild-type strand monellin albumen thermostability improves 10 DEG C.
(2) mensuration of albumen sweet taste threshold value
Control group adopts the sucrose of 10000 μ g/ml, albumen is diluted to gradient from 0.25 to 150 μ g/ml.Successively sample is allowed and to be tasted by the healthy grownup of 5 male 5 female 10, taste from concentration is minimum, each concentration increases by 10 μ g/ml, till experiencing sugariness, concentration is benchmark according to this, then reduce by 1 μ g/ml concentration below at every turn, until impression is less than sweet taste, Using such method reduces concentration gradient gradually thus determines the sweet taste threshold value of sample, different sample centre pure water is gargled, each albumen net result gets trial test result mean value (table 1) of 10 people, E2N mutain sweet taste threshold value is 0.25 μ about g after measured, comparatively wild-type monellin albumen enhances about 3 times.
Table 1
Sweeting agent Sweet taste threshold value (μ g/ml)
Sucrose 10000±150
Wild-type strand monellin albumen 0.80±0.05
E2N sudden change strand monellin albumen 0.25±0.05
。

Claims (8)

1. a monellin protein mutant, is characterized in that, is l-asparagine (Asn) by wild-type strand monellin gene the 2nd amino acids L-glutamic acid (Glu) rite-directed mutagenesis.
2. a kind of monellin protein mutant according to claim 1, is characterized in that, sugariness promotes 3 times relative to wild strand monellin albumen.
3. the mutain utilizing directed mutagenesis method to obtain as claimed in claim 1 is characterized in that: thermostability promotes 10 DEG C relative to wild strand monellin albumen.
4. the preparation method of a kind of monellin protein mutant as claimed in claim 1, is characterized in that, comprise the following steps:
(1) wild-type strand monellin protein expression vector is built;
(2) mutator gene is obtained by carrying out rite-directed mutagenesis in step (1), picking positive transformant sequence verification;
(3) the correct Plastid transformation of order-checking in step (2) is entered in pichia spp, filter out the pichia spp of successful conversion;
(4) induction expression protein in YPD substratum, the time is 3 days;
(5) protein purification of will express in step (4).
5. the preparation method of a kind of monellin protein mutant according to claim 4, is characterized in that, step (1) is, builds the expression plasmid PGAPZ α A containing wild-type strand monellin protein gene.
6. the preparation method of a kind of monellin protein mutant according to claim 5, is characterized in that, step (2) specific primer on the mutated site: upstream P1:[5 '-CC cTCGAGaAAAGAGAGGCTGAAGCTGGT aACtGGGAG-3 ']; Downstream P2:[5 '-TT gCGGCCGCtTAGGGAGGAGGCACAGGTCCGTTG-3 '].
7. the preparation method of a kind of monellin protein mutant according to claim 6, it is characterized in that, in step (2), be connected by XhoI with NotI endonuclease digestion PCR primer and with PGAPZ α A empty carrier by after be transformed in bacillus coli DH 5 alpha competent cell, coat on the LLB solid medium containing 25 μ g/mlZeocin, the positive single bacterium colony of incubated overnight picking also extracts plasmid, sequence verification sudden change result, builds mutant plasmid PGAPZ α A-E2N and is saved backup by correct mutant plasmid.
8. the preparation method of a kind of monellin protein mutant according to claim 7, is characterized in that, the pichia spp described in step (3) is Pichia pastoris GS115.
CN201610087481.3A 2016-02-16 2016-02-16 Monellin protein mutant and preparation method thereof Pending CN105566471A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109627307A (en) * 2019-01-28 2019-04-16 中国药科大学 Heat-resisting sweet protein hairpin-monellin and preparation method thereof
WO2023222111A1 (en) * 2022-05-20 2023-11-23 南京百斯杰生物工程有限公司 Sweet protein monellin mutant having high sweetness and method for preparing same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1202495A (en) * 1998-06-12 1998-12-23 中国农业科学院生物技术研究中心 Gene of coded high-sweetness monellin and use of its product albumen

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1202495A (en) * 1998-06-12 1998-12-23 中国农业科学院生物技术研究中心 Gene of coded high-sweetness monellin and use of its product albumen

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIU等: "Modification of the Sweetness and Stability of Sweet-Tasting Protein Monellin by Gene Mutation and Protein Engineering", 《BIOMED RESEARCH INTERNATIONAL》 *
XUE等: "Role of protein surface charge in monellin sweetness", 《BIOCHIMICA ET BIOPHYSICA ACTA》 *
吴刚等: "甜蛋白Monellin在毕赤酵母中的分泌表达", 《高技术通讯》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109627307A (en) * 2019-01-28 2019-04-16 中国药科大学 Heat-resisting sweet protein hairpin-monellin and preparation method thereof
CN109627307B (en) * 2019-01-28 2020-11-17 中国药科大学 Heat-resistant sweet protein hairpin-monellin and preparation method thereof
WO2023222111A1 (en) * 2022-05-20 2023-11-23 南京百斯杰生物工程有限公司 Sweet protein monellin mutant having high sweetness and method for preparing same

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