CN104419688A - Fructosyl transferase as well as gene, secreted expression vector and application thereof - Google Patents
Fructosyl transferase as well as gene, secreted expression vector and application thereof Download PDFInfo
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Abstract
The invention discloses fructosyl transferase which consists of an amino acid sequence shown in SEQ ID NO: 2. The invention further discloses polynucleotide which encodes fructosyl transferase, an expression vector containing polynucleotide, a transformant and a method of applying a fructosyl transferase gene to produce fructosyl transferase. The fermentation enzyme activity of a shake flask of constructed engineering bacteria can reach 508U/ml at the highest which is about 41 times that of an original bacterial strain. The invention further relates an application of the fructosyl transferase in preparing fructo-oligosaccharide and a method for preparing the fructo-oligosaccharide by using the fructosyl transferase.
Description
Technical field
The present invention relates to a kind of fructosyl transferase, its fructosyl transferase gene and secreted expression carrier thereof and application, belong to field of genetic engineering.
Background technology
Oligofructose (FOS), also known as oligofructose or FOS, refers under the effect of fructosyl transferase, generates kestose, GF3 and GF4 and composition thereof by the effect of transfer fructosyl.Fructooligosaccharide is a kind of nonreducing sugar, has the physiologically active that indigestible, unlikely carious tooth, bifidus bacillus proliferation function and water solutable edible fiber etc. are unique.
Fructosyl transferase (fructosyltransferase, EC2.4.1.9), belongs to Glycosylase 32 family.Fructosyl transferase is present in organic sphere widely, the fructosyl transferase different in kind in different microorganisms source.Microorganism fructosyl transferase acts on sucrose, carries out the reaction of intramolecular transfer fructosyl and generates FOS.Wherein the fructosyl transferase of Aspergillus niger origin is industrial mainly with microbe-derived enzyme source.
Along with molecular biological development, fructosyl transferase molecular biology starts from the nineties in last century, nineteen ninety-five, and the gene of the 1st fructosyl transferase is cloned.Thereafter, the fructosyl transferase gene of different plant species is studied.Wherein more to the research of filamentous fungus fructosyl transferase, and the gene of some encoding fructose based transferases is cloned.Up to the present bibliographical information is not many.
It is outer and be easy to the advantages such as follow-up modification that pichia yeast expression system has that expression amount is high, production cost is low, product can be secreted into born of the same parents, utilizes the albumen of Pichia anomala expression to have hundreds of.Pichia spp has become the exogenous protein expression system gained great popularity in recent years.
This research department clones and obtains aspergillus niger fructosyl transferase gene (fts from aspergillus niger, full-length gene), construct pET22b-cFTS, pET22b-cFTSW plasmid transformation of E. coli BL21, and build pGAPZA-cFTS, pGAPZ α A-cFTSW plasmid, after linearizing, electricity transforms Pichi strain GSll5, obtain positive colony shake flask fermentation 48h, measure nutrient solution supernatant fructosyl transferase (mainly outside born of the same parents, on a small quantity in born of the same parents) enzyme activity and be up to 508U/ml.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of novel fructosyl transferase gene, to obtain the engineering strain of high expression level fructosyl transferase, and such as Pichi strain, thus obtain a large amount of fructosyl transferases.
The present inventor is at aspergillus niger 10 (CGMCC3.316, purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC, great Tun road, Chaoyang District, BeiJing, China institute of microbiology of the Chinese Academy of Sciences, 100101)), clone obtains a kind of novel gene, and the albumen of its coding has fructosyltransferaseactivity activity.Identify that the genome nucleotide sequence of this gene is SEQ ID NO:1 through order-checking, cDNA sequence is SEQ ID NO:3, and encoding amino acid sequence is the albumen of SEQ ID NO:2, and this albumen has fructosyltransferaseactivity activity, is a kind of fructosyl transferase.The present inventor retrieves discovery further, and this gene order is not registration in GenBank, belongs to a kind of gene identification.On this basis, the present inventor expresses this gene further in E.coli and yeast, carries out functional verification, thus complete the present invention to it.
The nucleotide sequence of the above-mentioned fructosyl transferase of coding is cloned in expression vector and builds recombinant expression vector by the present inventor.Expression vector wherein used, such as, but not limited to, secreted expression carrier, includes, but not limited to pET-22b, pGAPZA or pGAPZ α A, and these carriers all can be bought from Reagent Company and obtain.
Specifically, the present inventor by from aspergillus niger 10 clone encoding fructose based transferase nucleotide sequence conveniently molecular cloning method be cloned into the pET-22b be purchased, in pGAPZA or pGAPZ α A carrier, construct recombinant expression vector pET22b-cFTS, pET22b-cFTSW, pGAPZA-cFTS and pGAPZ α A-cFTSW.Wherein pET22b-cFTS and pET22b-cFTSW is used for expressing in intestinal bacteria, pGAPZA-cFTS and pGAPZ α A-cFTSW is used for expressing in yeast.The zymoprotein with fructosyltransferaseactivity activity can be obtained after expression, be referred to as fructosyl transferase.
In preferred embodiments, provide a kind of method preparing fructosyl transferase, described method comprises the steps:
(1) nucleotide sequence of clone or the albumen shown in composite coding SEQ ID NO:2, and be cloned in suitable expression vector, build the recombinant expression vector of the nucleotide sequence containing the albumen shown in coding SEQ ID NO:2;
(2) recombinant expression vector that step (1) obtains is proceeded in suitable host cell, cultivate, express the albumen shown in SEQ ID NO:2.
It should be appreciated by those skilled in the art that aforesaid method also comprises purification step, purifying obtains the fructosyl transferase of higher degree.
Those skilled in the art should understand that, excretion vector is selected to carry out the recombinant expression vector of construction expression fructosyl transferase of the present invention, object makes target protein be secreted in culture supernatant, instead of formation inclusion body, thus the culture supernatants obtained inherently has desirable fructosyltransferaseactivity activity, and can obtain the target protein with fructosyltransferaseactivity activity through simple purification step, and not through the complex process of sex change purifying and renaturation.
Those skilled in the art should understand that, when mentioning " fructosyl transferase " of the present invention, this term except comprising the albumen that is made up of the aminoacid sequence of SEQ ID NO:2, also comprise by the aminoacid sequence of SEQ ID NO:2 through one or more aminoacid replacement, disappearance or insertion obtain there is the albumen with the same or analogous fructosyltransferaseactivity activity of aminoacid sequence of SEQ ID NO:2.
Therefore, the invention provides following:
1. a fructosyl transferase, it is:
(1) albumen be made up of the aminoacid sequence of SEQ ID NO:2; Or
(2) by the aminoacid sequence of SEQ ID NO:2 through one or more aminoacid replacement, disappearance or insertion obtain there is the albumen with the same or analogous fructosyltransferaseactivity activity of aminoacid sequence of SEQ ID NO:2.
2. the polynucleotide sequence of the fructosyl transferase of coding described in the 1st.
3. the polynucleotide sequence according to the 2nd, it is for shown in following (1) or (2):
(1) nucleotide sequence shown in SEQ ID NO:1 or 3 or 4;
(2) nucleotide sequence hybridization limited with (1) under strict conditions and the nucleotide sequence of the protein with fructosyltransferaseactivity activity of encoding.
4. containing the 2nd or the recombinant expression vector of polynucleotide sequence of encoding fructose based transferase described in 3.
5. the expression vector according to the 4th, is characterized in that, described expression vector is excretion vector.
6. the expression vector according to the 4th, expression vector wherein used is selected from pET-22b, pGAPZA or pGAPZ α A.
7. the recombinant host cell containing the polynucleotide sequence of encoding fructose based transferase described in the 2nd or the recombinant expression vector described in the 4th.
8. the recombinant host cell according to the 7th, described recombinant host cell is transgenic cell line or genetic engineering bacterium, such as, the intestinal bacteria of restructuring or pichia spp.
9. the polynucleotide sequence of the encoding fructose based transferase described in the 2nd, the recombinant expression vector described in the 4th or the 7th or the application in fructosyl transferase preparation is produced of recombinant host cell described in 8.
10. prepare a method for the fructosyl transferase described in the 1st, described method comprises the steps: cultivation the 7th or the recombinant host cell described in 8, obtains the fructosyl transferase described in the 1st.
Fructosyl transferase described in 11. the 1st is preparing the application in fructooligosaccharide, described application comprises: take sucrose as substrate, fructosyl transferase described in the 1st or the 7th or the culture supernatants of recombinant host cell described in 8 is added in reaction system, obtain fructooligosaccharide after enzyme reaction, described fructooligosaccharide comprises kestose, GF3 and GF4 and composition thereof.
12. 1 kinds of methods preparing fructooligosaccharide, described method comprises: take sucrose as substrate, fructosyl transferase described in the 1st or the 7th or the culture supernatants of recombinant host cell described in 8 is added in reaction system, obtain fructooligosaccharide after enzyme reaction, described fructooligosaccharide comprises kestose, GF3 and GF4 and composition thereof.
Accompanying drawing explanation
Below in conjunction with in the detailed description of accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 shows the physical map of (A) recombinant expression vector pET22b-cFTS; (B) physical map of recombinant expression vector pET22b-cFTSW;
Fig. 2 shows the physical map of (A) recombinant expression vector pGAPZA-cFTS; (B) physical map of recombinant expression vector pGAPZ α A-cFTSW;
Fig. 3 display turns HPLC collection of illustrative plates (G, the glucose of glycosyl product; S, sucrose; GF2, kestose; GF3, GF3).
Sequence explanation
SEQ ID NO:1 fructosyl transferase genomic dna sequence, it is by 1941 based compositions, containing 1 intron in this sequence, is 5 ' the 1767th to 1820 of holding
The aminoacid sequence that SEQ ID NO:2 fructosyl transferase is derived, being made up of 628 amino-acid residues, is N end signal peptide sequence from nitrogen end the 1-15 amino acids residue
The cDNA base sequence of SEQ ID NO:3 fructosyl transferase, has N end signal peptide sequence, by 1887 based compositions, is called for short cFTS
SEQ ID NO:4 cFTSW sequence, namely eliminates 15 amino acid whose N end signal peptide sequences and does not comprise the nucleotide sequence of last terminator codon
The N end signal peptide sequence of SEQ ID NO:5 fructosyl transferase
Embodiment
Further describe the present invention referring to specific embodiment, but it should be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments.
Come by the following examples to set forth the present invention further, described embodiment is exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.The test method of unreceipted actual conditions in the following example, except as otherwise noted, embodiments of the invention are by the conventional art in the fields such as the molecular biology within the limit of power of employing those skilled in the art.In addition, except as otherwise noted, in this article, nucleic acid is write from left to right with the direction of 5 ' to 3 ', and aminoacid sequence is then from left to right write with the direction of aminoterminal to carboxyl terminal.Substantially all operate according to the condition described in common molecular cloning handbook.
Concrete steps are as follows:
(1) fructosyl transferase gene is cloned;
(2) expression vector containing fructosyl transferase gene is built;
(3) expression vector transformation receptor bacterium;
Described expression vector is excretion vector, is preferably pET-22b (purchased from Novagen), pGAPZA or pGAPZ α A (these two kinds of carriers are purchased from Invitrogen).
Described recipient bacterium is intestinal bacteria (E.coli BL21) or pichia spp.
(4) cultivate the recipient bacterium transformed, obtain fructosyl transferase.
Fructosyl transferase enzyme activity determination method:
Reference Wang Limei etc. (2006, food and biotechnology journal 25 (2)) sucrose solution (preparing in the 0.1M pH5.0 phosphoric acid-citrate buffer) 2ml that gets crude enzyme liquid 0.5ml and 25% suitably diluted mixes, enzymolysis 1 hour at 45 DEG C, 100 DEG C are boiled 15 minutes enzymolysis reaction.Fructosyl transferase work, by measuring the content of glucose (G) and the reducing sugar (R) discharged in enzymolysis process, calculates the quantity of fructose (F) produced in enzymolysis process and the fructose (Fc) be transferred amount according to the following formula.
F=R—G
Fc=G—F=2G-R
Under the same conditions, to add the sucrose solution of fermentoid liquid in contrast.Under these conditions, be a fructose-transferring enzyme unit of activity with the per minute enzyme amount shifted needed for 1 μm of ol fructose.
Material and reagent:
Restriction enzyme used, PCR reagent is purchased from NEB company; RNA extraction and Reverse Transcription box are purchased from Promega company; T4DNA ligase enzyme, DNA Marker etc. is purchased from precious biotech firm; PEASY-B carrier, competent escherichia coli cell DH5 α, BL21, PCR purification kit is purchased from Quan Shijin, and primer is purchased from Hua Da gene, and DNA and plasmid extraction kit are purchased from a day root biology; Electrotransfer is purchased from Bio-Rad company; YPD and YPD-Zeocin is all according to the operational manual preparation of Invitrogen company; Other reagent are the analytical reagent bought both at home and abroad.
Embodiment 1: the clone of aspergillus niger fructosyl transferase gene
Extract aspergillus niger 10 (CGMCC3.316, purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC, great Tun road, Chaoyang District, BeiJing, China institute of microbiology of the Chinese Academy of Sciences, 100101)) genomic dna.Design primer carries out PCR reaction, and obtain aspergillus niger fructosyl transferase full genome encoding sequence (FTS), primer sequence wherein used is as follows:
sense primer：
5′-TAGGCGGATCCCATGAAGCTTCAAACGGCTTC-3′
anti-sense primer：
5′-AGGGCGGA ATTCTTA AGACTGACGATCCGGC-3′
Extract aspergillus niger 10 total serum IgE, and reverse transcription becomes cDNA.Design primer carries out PCR reaction, and obtain aspergillus niger fructosyl transferase cDNA encoding sequence (cFTS is SEQ ID NO:3), primer sequence used is as follows with fructosyl transferase DNA primer sequence.
The structure of pET22b-cFTS carrier
Primer sequence used is with fructosyl transferase DNA sequence dna primer.
The fructosyl transferase cDNA sequence (SEQ IDNO:3) increased with BamH I and EcoR I double digestion and pET22b (purchased from Novagen).The object fragment of cutting through enzyme and carrier pET22b are connected with T4DNA ligase enzyme and spend the night, and heat shock method transforms large intestine competent cell, select positive strain and extract plasmid and namely obtain recombinant plasmid pET22b-cFTS.
The structure of embodiment 2:pET22b-cFTSW carrier
Primer sequence is as follows:
cffs-p11：
5’-CTCGGAATTCAGCCTCTCCTTCCATGCAGAC-3’
cffs-p2w：
5’-TAGCGGCCGCAGACTGACGATCCGGCCAAG-3’
By the fructosyl transferase nucleotide sequence called after cFTSW utilizing above-mentioned primer amplification to obtain, its sequence is shown in SEQ ID NO:4.
CFTSW (SEQ ID NO:4) is as follows with the difference of cFTS (SEQ ID NO:3):
CFTS is the cDNA sequence comprising own signal peptide (front ten five amino acids of protein amino acid sequence), by 1887 based compositions; CFTSW is the cDNA sequence not comprising native signal peptide, is made up of 1839 bases (not comprising last terminator).
The cFTSW sequence (SEQ ID NO:4) increased with EcoR I and Not I double digestion and carrier pET22b (purchased from Novagen).The object fragment of cutting through enzyme and carrier pET22b are connected with T4DNA ligase enzyme and spend the night, and heat shock method transforms large intestine competent cell, select positive strain and extract plasmid and namely obtain recombinant plasmid pET22b-cFTSW.
The structure of embodiment 3:pGAPZA-cFTS carrier
Primer sequence is as follows:
ffs-p1：
5′-CTCGGAATTCATGAAGCTTCAAACGGCTTC-3′
cffs-p2：
5′-TAGCGGCCGCTTAAGACTGACGATCCGGC-3′
High-fidelity Tag enzymatic amplification fructosyl transferase cDNA sequence (SEQ ID NO:3), spreads cultivation containing pGAPZA plasmid (purchased from Invitrogen) coli strain simultaneously and extracts pGAPZA plasmid.The fructosyl transferase cDNA sequence (SEQ ID NO:3) of EcoR I and the amplification of Not I double digestion and carrier pGAPZA, T4DNA ligase enzyme spends the night the object fragment and carrier that connect and cut through enzyme, transformation of E. coli competent cell, selects positive strain extraction plasmid and namely obtains recombinant plasmid pGAPZA-cFTS.
The structure of embodiment 4:pGAPZ α A-cFTSW carrier
Primer used is as follows:
cffs-pl：
5′-CTCGGAATTCGCCTCTCCTTCCATGCAGAC-3′
cffs-p2w：
5′-TAGCGGCCGCAGACTGACGATCCGGCCAAG-3′
High-fidelity Tag enzymatic amplification is containing the fructosyl transferase cDNA sequence (cFTSW, SEQ ID NO:4) of its own signal peptide sequence, and spread cultivation coli strain containing pGAPZ α A plasmid (purchased from Invitrogen) upgrading grain simultaneously.CFTSW sequence and the pGAPZ α A of EcoR I and the amplification of Not I double digestion, T4DNA ligase enzyme spends the night the object fragment and carrier that connect and cut through enzyme, and transformation of E. coli competent cell, selects positive strain extraction plasmid and namely obtain recombinant plasmid pGAPZA-cFTSW.
Embodiment 5:pET22b-cFTS, pET22b-cFTSW recon transform
Plasmid is extracted, heat-shock transformed e. coli bl21 expression strain after the positive strain of recombinating spreads cultivation.
Embodiment 6:pGAPZA-cFTS, pGAPZ α A-cFTSW recon transform
Plasmid is extracted after the positive strain of recombinating spreads cultivation, be linear plasmid with BspH I single endonuclease digestion, total amount is that 5-10ug carries out electricity turn, proceeds to pichia spp GSll5 competent cell (pichia spp GSll5 purchased from Invitrogen, conventionally prepared by its competent cell).Electroporation is set to voltage 1500v, electric capacity 25uF, resistance 200 Ω.
Embodiment 7: the screening of recon and Molecular
Add 400ul LB substratum after heat-shock transformed e. coli bl21, after 37 DEG C of recoveries, coating ammonia benzyl concentration is the LB flat board of 100ug/ml.With the single bacterium colony grown for template, carry out PCR reaction with gene specific primer, obtain corresponding size strip and be namely defined as positive strain.
Electricity adds 1ml Sorbitol Solution USP after transforming Pichia pastoris GS115, after 30 DEG C of recoveries, coating Zeocin concentration is the YPD flat board of 100ug/ml, the single colony inoculation YPD liquid nutrient medium grown is cultivated, get supernatant liquor and measure fructosyltransferaseactivity activity, the culture supernatant of the positive pichia spp bacterium colony that microbiotic Zeocin screens all detects fructosyltransferaseactivity activity, the ability expressing fructosyl transferase due to each bacterial strain has difference, can choose the high engineering strain of fructosyltransferaseactivity activity for further research.
Embodiment 8:pET22b-cFTS, pET22b-cFTSW recon positive strain shake flask fermentation
Picking positive strain list bacterium colony, 5ml Tube propagation spends the night.Get in 1ml to 50ml LB triangular flask, 37 DEG C, 250rpm, shakes bacterium 2.5-3h, is placed in 5 minutes on ice, and (thalline solubility is OD6000.6-0.8) adds 10mM IPTG50-100ul and induce, 25 DEG C, and concussion is cultivated.Sampling is at set intervals surveyed enzyme and is lived (that is, getting culture supernatant survey enzyme to live), and after 24 hours, enzyme activity reaches 1.68U/ml.
Embodiment 9:pGAPZA-cFTS, pGAPZ α A-cFTSW recon positive strain shake flask fermentation
With reference to Invitrogen company operational manual, picking positive strain list colony inoculation in 5ml YPD substratum, 28 DEG C, 250rpm shaking table overnight incubation, be transferred in 50ml YPD substratum triangular flask in the ratio of 1-4%, the same terms is cultivated, and every 12 hours, sampling was surveyed enzyme and lived (namely, get culture supernatant survey enzyme to live), cultivate after 48 hours, enzymatic activities reaches the highest, is 508U/ml.
Embodiment 10: Yeast engineering bacteria produces the enzymolysis product analysis of fructosyl transferase
With 25% sucrose for substrate, fructosyl transferase content is 8 unit every gram of sucrose, pH5.0, reacts at 45 DEG C, (such as, reacts latter 20 minutes, 2 hours, 12 hours, 24 hours) at set intervals and gets appropriate reaction mixture and carry out product analysis.HPLC analyzes and shows, the kestose (GF2) first occurred in product, GF3 (GF3) is finally GF4.In reaction solution, Nutriflora P is the highest accounts for 58% of total mass.Fig. 3 display turns the HPLC collection of illustrative plates of glycosyl product, and spectrum data and Nutriflora P in reaction solution be the highest accounts for the 58% consistent of total mass, and do not show GF4 in this HPLC collection of illustrative plates, reacting prolongation can occur GF4.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention shown particularly and describe, but will be understood by those skilled in the art that, do not deviating from by under the condition of the spirit and scope of the present invention as defined in the claims, the change of various forms and details can be carried out wherein, the arbitrary combination of various embodiment can be carried out.
Claims (10)
1. a fructosyl transferase, it is:
(1) albumen be made up of the aminoacid sequence of SEQ ID NO:2; Or
(2) by the aminoacid sequence of SEQ ID NO:2 through one or more aminoacid replacement, disappearance or insertion obtain there is the albumen with the same or analogous fructosyltransferaseactivity activity of aminoacid sequence of SEQ ID NO:2.
2. the polynucleotide sequence of coding fructosyl transferase according to claim 1.
3. polynucleotide sequence according to claim 2, it is for shown in following (1) or (2):
(1) nucleotide sequence shown in SEQ ID NO:1 or 3 or 4;
(2) nucleotide sequence hybridization limited with (1) under strict conditions and the nucleotide sequence of the protein with fructosyltransferaseactivity activity of encoding.
4. the recombinant expression vector of the polynucleotide sequence containing the encoding fructose based transferase described in Claims 2 or 3.
5. expression vector according to claim 4, is characterized in that, described expression vector is excretion vector.
6. expression vector according to claim 4, expression vector wherein used is selected from pET-22b, pGAPZA or pGAPZ α A.
7. containing the polynucleotide sequence of encoding fructose based transferase according to claim 2 or the recombinant host cell of recombinant expression vector according to claim 4.
8. recombinant host cell according to claim 7, described recombinant host cell is transgenic cell line or genetic engineering bacterium, such as, the intestinal bacteria of restructuring or pichia spp.
9. the polynucleotide sequence of encoding fructose based transferase according to claim 2, recombinant expression vector according to claim 4 or the application of the recombinant host cell described in claim 7 or 8 in fructosyl transferase preparation is produced.
10. prepare a method for fructosyl transferase according to claim 1, described method comprises the steps: to cultivate the recombinant host cell described in claim 7 or 8, obtains fructosyl transferase according to claim 1.
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| CN106467899A (en) * | 2015-08-17 | 2017-03-01 | 中国科学院天津工业生物技术研究所 | A kind of Aspergillus niger strain of high yield fructose-transferring enzyme and its application |
| CN106467899B (en) * | 2015-08-17 | 2020-05-05 | 中国科学院天津工业生物技术研究所 | Aspergillus niger strain capable of producing fructose transferase in high yield and application thereof |
| CN105219660A (en) * | 2015-09-18 | 2016-01-06 | 上海交通大学 | The special strain therefore of synthesis of oligonucleotides fructose and the method for the synthesis of oligofructose thereof |
| CN105441512A (en) * | 2016-01-20 | 2016-03-30 | 天津科技大学 | Method for efficiently preparing fructo-oligosaccharide and enzymic preparation thereof |
| CN105441512B (en) * | 2016-01-20 | 2019-02-19 | 天津科技大学 | A kind of method and its enzyme preparation preparing oligofructose |
| CN109415747A (en) * | 2018-05-25 | 2019-03-01 | 邦泰生物工程(深圳)有限公司 | A kind of preparation method of enzyme modification stevioside and alternation enzyme processed and application |
| CN109415747B (en) * | 2018-05-25 | 2022-05-03 | 邦泰生物工程(深圳)有限公司 | Preparation method of enzyme modified stevioside, enzyme for preparation and application |
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| CN104419688B (en) | 2017-12-22 |
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