CN104419688B - A kind of transfructosylase and its gene, its secreted expression carrier and application - Google Patents
A kind of transfructosylase and its gene, its secreted expression carrier and application Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01009—Inulosucrase (2.4.1.9)
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Abstract
The invention discloses a kind of transfructosylase, the transfructosylase is by SEQ ID NO：2 amino acid sequence composition；Also disclose the polynucleotides for encoding the transfructosylase, expression vector containing the polynucleotides, transformant, and the method for application transfructosylase gene production transfructosylase, the engineering bacteria shake-flask fermentation enzyme activity of structure reaches as high as 508U/ml, is about 41 times of original strain enzyme activity.The method that fructooligosaccharide is prepared the invention further relates to application of the transfructosylase in fructooligosaccharide is prepared and using the transfructosylase.
Description
Technical field
The present invention relates to a kind of transfructosylase, its transfructosylase gene and its secreted expression carrier and should
With belonging to field of genetic engineering.
Background technology
FOS (FOS) is also known as Fructooligosaccharides or FOS, refers in the presence of transfructosylase,
By shifting fructosyl effect generation ketose, Nystose and GF4 and its mixture.Fructooligosaccharide is a kind of
Nonreducing sugar, has indigestible, will not unique physiology such as carious tooth, Bifidobacterium Bifidum proliferation function and water solutable edible fiber
Activity.
Transfructosylase (fructosyltransferase, EC2.4.1.9), belong to the family of glycosidase 32.Fructosyl
Transferase widely exists in living nature, and the transfructosylase property in different microorganisms source is different.Microorganism fructosyl turns
Enzyme effect is moved in sucrose, carries out intramolecular transfer fructosyl reaction generation FOS.The transfructosylase of wherein Aspergillus niger origin is
Industrially mainly with microbe-derived enzyme source.
With the development of molecular biology, transfructosylase molecular biology starts from last century the nineties, nineteen ninety-five,
The gene of 1st transfructosylase is cloned.Thereafter, the transfructosylase gene of different plant species is studied.Wherein
Research to filamentous fungi transfructosylase is more, and the gene of some encoding fructose based transferases is cloned.Arrive
Document report is not many so far.
Pichia yeast expression system has that expression quantity is high, production cost is low, product can be secreted into extracellular and be easy to subsequently repair
The advantages that decorations, has hundreds of using the albumen of Pichia anomala expression.Pichia pastoris turns into the external source to gain great popularity in recent years
Protein expression system.
This research department is cloned from aspergillus niger obtains aspergillus niger transfructosylase gene (fts, full-length gene), constructs
PET22b-cFTS, pET22b-cFTSW plasmid simultaneously convert e. coli bl21, and build pGAPZA-cFTS, pGAPZ α A-
CFTSW plasmids, electricity conversion Pichi strain GSll5, obtains positive colony shake flask fermentation 48h after linearisation, determines nutrient solution
Supernatant transfructosylase (mainly extracellular, on a small quantity in intracellular) enzyme activity is up to 508U/ml.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of new transfructosylase gene, to obtain high expression
The engineered strain of transfructosylase, such as Pichi strain, so as to obtain substantial amounts of transfructosylase.
The present inventor aspergillus niger 10 (CGMCC3.316, it is commonly micro- purchased from China Committee for Culture Collection of Microorganisms
Bio-Centers (CGMCC, BeiJing, China Chaoyang District great Tun roads institute of microbiology of the Chinese Academy of Sciences, 100101)) in clone obtain one kind
Novel gene, its albumen encoded have fructosyltransferaseactivity activity.Genome nucleotide sequence through the sequencing identification gene
For SEQ ID NO：1, cDNA sequence is SEQ ID NO：3, encoding amino acid sequence is SEQ ID NO：2 albumen, the albumen
It is a kind of transfructosylase with fructosyltransferaseactivity activity.The present inventor further retrieves discovery, and the gene order exists
It is not enrolled in GenBank, belongs to a kind of gene identification.On this basis, the present inventor is further in Escherichia coli and ferment
The gene is expressed in female bacterium, functional verification is carried out to it, so as to complete the present invention.
The nucleotide sequence for encoding above-mentioned transfructosylase is cloned into structure restructuring table in expression vector by the present inventor
Up to carrier.Expression vector wherein used includes, but not limited to pET-22b such as, but not limited to, secreted expression carrier,
PGAPZA or pGAPZ α A, these carriers can be commercially available from Reagent Company.
Specifically, the present inventor by the nucleotide sequence for the encoding fructose based transferase cloned from aspergillus niger 10 according to
Common molecular cloning process is cloned into commercially available pET-22b, pGAPZA or pGAPZ α A carriers, constructs recombinant expression carrier
PET22b-cFTS, pET22b-cFTSW, pGAPZA-cFTS and pGAPZ α A-cFTSW.Wherein pET22b-cFTS and pET22b-
CFTSW is used to be expressed in Escherichia coli, pGAPZA-cFTS and pGAPZ α A-cFTSW are used to carry out table in saccharomycete
Reach.The zymoprotein with fructosyltransferaseactivity activity can be obtained after expression, is referred to as transfructosylase.
In preferred embodiments, there is provided a kind of method for preparing transfructosylase, methods described include following steps
Suddenly：
(1) clone or composite coding SEQ ID NO：The nucleotide sequence of albumen shown in 2, and be cloned into it is appropriate
In expression vector, structure contains coding SEQ ID NO：The recombinant expression carrier of the nucleotide sequence of albumen shown in 2；
(2) recombinant expression carrier that step (1) obtains is transferred in appropriate host cell, cultivated, express SEQ
ID NO：Albumen shown in 2.
It should be appreciated by those skilled in the art that the above method also includes purification step, purifying obtains the fructose of higher degree
Based transferase.
It should be appreciated by those skilled in the art that selection excretion vector carrys out the transfructosylase of the construction expression present invention
Recombinant expression carrier, it is therefore an objective to target protein is secreted into culture supernatant, rather than form inclusion body, the training obtained from
Supporting thing supernatant inherently has preferable fructosyltransferaseactivity activity, and it is i.e. available to pass through simple purification step
Target protein with fructosyltransferaseactivity activity, and not pass through the complex process of denaturation purifying and renaturation.
It should be appreciated by those skilled in the art that when refer to the present invention " transfructosylase " when, the term except including
By SEQ ID NO：Outside the albumen of 2 amino acid sequence composition, in addition to by SEQ ID NO：2 amino acid sequence passes through one
Have and SEQ ID NO obtained from individual or multiple 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing or insertion：2 amino acid sequence is same or similar
Fructosyltransferaseactivity activity albumen.
Therefore, the present invention provides following：
1. a kind of transfructosylase, it is：
(1) by SEQ ID NO：The albumen of 2 amino acid sequence composition；Or
(2) by SEQ ID NO：2 amino acid sequence obtains by one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing or insertion
Have and SEQ ID NO：The albumen of the 2 same or analogous fructosyltransferaseactivity activity of amino acid sequence.
2. the polynucleotide sequence of the transfructosylase described in coding the 1st.
3. according to the polynucleotide sequence described in the 2nd, it is shown in following (1) or (2)：
(1)SEQ ID NO：Nucleotide sequence shown in 1 or 3 or 4；
(2) nucleotide sequence hybridization and coding limited under strict conditions with (1) has fructosyltransferaseactivity activity
The nucleotide sequence of protein.
4. the recombinant expression carrier of the polynucleotide sequence containing the encoding fructose based transferase described in the 2nd or 3.
5. according to the expression vector described in the 4th, it is characterised in that the expression vector is excretion vector.
6. according to the expression vector described in the 4th, wherein expression vector used is selected from pET-22b, pGAPZA or pGAPZ
αA。
7. the polynucleotide sequence containing the encoding fructose based transferase described in the 2nd or the recombination expression described in the 4th
The recombinant host cell of carrier.
8. according to the recombinant host cell described in the 7th, the recombinant host cell is transgenic cell line or gene work
Journey bacterium, for example, the Escherichia coli of restructuring or Pichia pastoris.
9. the polynucleotide sequence of the encoding fructose based transferase described in the 2nd, recombinant expression carrier described in the 4th or
Application of the recombinant host cell in the production of transfructosylase preparation described in 7th or 8.
10. a kind of method for preparing the transfructosylase described in the 1st, methods described comprise the steps：Cultivate the 7th
Or the recombinant host cell described in 8, obtain the transfructosylase described in the 1st.
11. application of the transfructosylase described in the 1st in fructooligosaccharide is prepared, the application include：With sugarcane
Sugar is substrate, in reaction system add the 1st described in transfructosylase or the 7th or 8 described in recombinant host cell
Culture supernatants, fructooligosaccharide is obtained after enzyme reaction, the fructooligosaccharide includes ketose, Nystose and sugarcane
Fruit pentasaccharides and its mixture.
12. a kind of method for preparing fructooligosaccharide, methods described include：Using sucrose as substrate, add in reaction system
Enter the culture supernatants of transfructosylase described in the 1st or the recombinant host cell described in the 7th or 8, after enzyme reaction
Fructooligosaccharide is obtained, the fructooligosaccharide includes ketose, Nystose and GF4 and its mixture.
Brief description of the drawings
From detailed description below in conjunction with the accompanying drawings, features described above of the invention and advantage will be apparent from, wherein：
Fig. 1 shows (A) recombinant expression carrier pET22b-cFTS physical map；(B) recombinant expression carrier pET22b-
CFTSW physical map；
Fig. 2 shows (A) recombinant expression carrier pGAPZA-cFTS physical map；(B) recombinant expression carrier pGAPZ α A-
CFTSW physical map；
Fig. 3 shows HPLC collection of illustrative plates (G, the glucose for turning glycosyl product；S, sucrose；GF2, ketose；GF3, sugarcane fruit four
Sugar).
Sequence explanation
SEQ ID NO：1 transfructosylase genomic dna sequence, it is contained in sequence by 1941 base compositions
1 introne, for the 1767th to 1820 of 5 ' ends
SEQ ID NO：The amino acid sequence that 2 transfructosylases derive, is made up of 628 amino acid residues, from nitrogen end
1-15th amino acids residue is N-terminal signal peptide sequence
SEQ ID NO：The cDNA base sequences of 3 transfructosylases, there is N-terminal signal peptide sequence, by 1887 bases
Composition, abbreviation cFTS
SEQ ID NO：4 cFTSW sequences, that is, eliminate the N-terminal signal peptide sequence of 15 amino acid and not comprising last
The nucleotide sequence of terminator codon
SEQ ID NO：The N-terminal signal peptide sequence of 5 transfructosylases
Embodiment
The present invention is further described referring to specific embodiment, it will be appreciated by those skilled in the art that this hair
It is bright to be not limited to these specific embodiments.
The present invention is expanded on further by the following examples, described embodiment is exemplary, be only used for explaining
The present invention, and be not construed as limiting the claims.The test method of unreceipted actual conditions in the following example, unless separately
It is described, embodiments of the invention are by using the biography in the fields such as the molecular biology within the limit of power of those skilled in the art
System technology.In addition, unless otherwise indicated, herein, nucleic acid is write from left to right with 5 ' to 3 ' direction, and amino acid sequence is then
From left to right write with the direction of aminoterminal to c-terminus.It is substantially all and enters according to the condition described in common molecular cloning handbook
Row operation.
Comprise the following steps that：
(1) transfructosylase gene is cloned；
(2) expression vector containing transfructosylase gene is built；
(3) expression vector transformation receptor bacterium；
The expression vector is excretion vector, and preferably pET-22b (is purchased from Novagen), pGAPZA or pGAPZ α A
(both carriers are purchased from Invitrogen).
The recipient bacterium is Escherichia coli (E.coli BL21) or Pichia pastoris.
(4) recipient bacterium of culture conversion, obtains transfructosylase.
Transfructosylase enzyme activity determination method：
Taken with reference to Wang Limei etc. (2006, food and biotechnology journal 25 (2)) the crude enzyme liquid 0.5ml that suitably dilutes with
25% sucrose solution (is prepared) 2ml in 0.1M pH5.0 phosphoric acid-citrate buffer and is well mixed, and 1 is digested at 45 DEG C
Hour, 100 DEG C are boiled 15 minutes enzymolysis reactions.Transfructosylase is lived by determining the Portugal released in enzymolysis process
The content of grape sugar (G) and reduced sugar (R), caused quantity of fructose (F) and the fruit being transferred in enzymolysis process are calculated according to the following formula
Sugared (Fc) amount.
F=R—G
Fc=G—F=2G-R
Under the same conditions, the sucrose solution to add inactivation enzyme liquid is used as control.Under these conditions, with per minute turn
The enzyme amount moved needed for 1 μm of ol fructose is a fructose-transferring enzyme unit of activity.
Material and reagent：
Restriction enzyme used, PCR reagent are purchased from NEB companies；RNA is extracted and reverse transcription reagent box is purchased from Promega
Company；T4DNA ligases, DNA Marker etc. are purchased from precious biotech firm；PEASY-B carriers, competent escherichia coli cell DH5
For α, BL21, PCR purification kit purchased from full formula gold, primer is purchased from Hua Da gene, and DNA and plasmid extraction kit are purchased from Tiangeng life
Thing；Electrotransfer is purchased from Bio-Rad companies；YPD and YPD-Zeocin prepares according to Invitrogen companies operation manual；
Other reagents are the AR bought both at home and abroad.
Embodiment 1：The clone of aspergillus niger transfructosylase gene
Extract (the CGMCC3.316, purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms of aspergillus niger 10
The heart (CGMCC, BeiJing, China Chaoyang District great Tun roads institute of microbiology of the Chinese Academy of Sciences, 100101)) genomic DNA.Design primer enters
Performing PCR reacts, and aspergillus niger transfructosylase full genome coded sequence (FTS) is obtained, wherein primer sequence used is as follows：
sense primer：
5′-TAGGCGGATCCCATGAAGCTTCAAACGGCTTC-3′
anti-sense primer：
5′-AGGGCGGA ATTCTTA AGACTGACGATCCGGC-3′
The total serum IgE of aspergillus niger 10 is extracted, and reverse transcription is into cDNA.Design primer enters performing PCR reaction, obtains aspergillus niger fructosyl
Transferase cDNA coded sequences (cFTS, are SEQ ID NO：3), such as similarly hereinafter transfructosylase DNA draws primer sequence used
Thing sequence.
The structure of pET22b-cFTS carriers
Primer sequence used is the same as transfructosylase DNA sequence dna primer.
Transfructosylase cDNA sequence (the SEQ ID NO expanded with BamH I and EcoR I double digestions：And pET22b 3)
(being purchased from Novagen).Purpose fragment through digestion is connected overnight with carrier pET22b with T4DNA ligases, and heat shock method conversion is big
Intestines competent cell, select positive strain extraction plasmid and obtain recombinant plasmid pET22b-cFTS.
Embodiment 2：The structure of pET22b-cFTSW carriers
Primer sequence is as follows：
cffs-p11：
5’-CTCGGAATTCAGCCTCTCCTTCCATGCAGAC-3’
cffs-p2w：
5’-TAGCGGCCGCAGACTGACGATCCGGCCAAG-3’
The transfructosylase nucleotide sequence for expanding to obtain using above-mentioned primer is named as cFTSW, its sequence is shown in SEQ
ID NO：4.
cFTSW(SEQ ID NO：4) with cFTS (SEQ ID NO：3) difference is as follows：
CFTS is the cDNA sequence for including itself signal peptide (preceding ten five amino acid of protein amino acid sequence), by 1887
Individual base composition；CFTSW is the cDNA sequence not comprising native signal peptide, by 1839 bases (not including last terminator)
Composition.
CFTSW sequences (the SEQ ID NO expanded with EcoR I and Not I double digestions：4) (it is purchased from carrier pET22b
Novagen).Purpose fragment through digestion is connected overnight with carrier pET22b with T4DNA ligases, heat shock method conversion large intestine impression
State cell, select positive strain extraction plasmid and obtain recombinant plasmid pET22b-cFTSW.
Embodiment 3：The structure of pGAPZA-cFTS carriers
Primer sequence is as follows：
ffs-p1：
5′-CTCGGAATTCATGAAGCTTCAAACGGCTTC-3′
cffs-p2：
5′-TAGCGGCCGCTTAAGACTGACGATCCGGC-3′
High-fidelity Tag enzymatic amplification transfructosylases cDNA sequence (SEQ ID NO：3), while spread cultivation containing pGAPZA matter
Grain (being purchased from Invitrogen) coli strain simultaneously extracts pGAPZA plasmids.The fructose of EcoR I and Not I double digestions amplification
Based transferase cDNA sequence (SEQ ID NO：3) purpose fragment through digestion is connected overnight with carrier pGAPZA, T4DNA ligase
With carrier, competent escherichia coli cell is converted, positive strain extraction plasmid is selected and obtains recombinant plasmid pGAPZA-cFTS.
Embodiment 4：The structure of pGAPZ α A-cFTSW carriers
Primer used is as follows：
cffs-pl：
5′-CTCGGAATTCGCCTCTCCTTCCATGCAGAC-3′
cffs-p2w：
5′-TAGCGGCCGCAGACTGACGATCCGGCCAAG-3′
High-fidelity Tag enzymatic amplifications are free of transfructosylase cDNA sequence (cFTSW, the SEQ ID of its own signal peptide sequence
NO：, while the spread cultivation coli strain containing pGAPZ α A plasmids (being purchased from Invitrogen) and upgrading grain 4).EcoR I and
The cFTSW sequences of Not I double digestions amplification connect purpose fragment and load through digestion overnight with pGAPZ α A, T4DNA ligases
Body, competent escherichia coli cell is converted, select positive strain extraction plasmid and obtain recombinant plasmid pGAPZA-cFTSW.
Embodiment 5：PET22b-cFTS, pET22b-cFTSW recon convert
The positive strain of restructuring extracts plasmid after spreading cultivation, heat-shock transformed e. coli bl21 expresses bacterial strain.
Embodiment 6：PGAPZA-cFTS, the conversion of pGAPZ α A-cFTSW recons
The positive strain of restructuring extracts plasmid after spreading cultivation, and is linear plasmid with BspH I single endonuclease digestions, and total amount is that 5-10ug enters
Row electricity turns, and being transferred to Pichia pastoris GSll5 competent cells, (Pichia pastoris GSll5 is purchased from Invitrogen, and its competent cell is pressed
Prepared by more solito).Electroporation is arranged to voltage 1500v, electric capacity 25uF, the Ω of resistance 200.
Embodiment 7：The screening of recon and Molecular
400ul LB culture mediums are added after heat-shock transformed e. coli bl21, coating ammonia benzyl concentration is after 37 DEG C of recoveries
100ug/ml LB flat boards.Using the single bacterium colony grown as template, performing PCR reaction is entered with gene specific primer, obtained correspondingly sized
Band is defined as positive strain.
1ml sorbitol solutions are added after electricity conversion Pichia pastoris GS115, coating Zeocin concentration is after 30 DEG C of recoveries
100ug/ml YPD flat boards, the single bacterium colony inoculation YPD fluid nutrient medium cultures grown, take supernatant to determine transfructosylase
Activity, the culture supernatant of the positive Pichia pastoris bacterium colony of antibiotic Zeocin screenings detect fructosyltransferaseactivity activity,
Because the ability of each bacterial strain expression transfructosylase has difference, the high engineered strain of fructosyltransferaseactivity activity can be chosen and used
In further research.
Embodiment 8：PET22b-cFTS, pET22b-cFTSW recon positive strain shake flask fermentation
Picking positive strain single bacterium colony, 5ml Tube propagations are stayed overnight.Take in 1ml to 50ml LB triangular flasks, 37 DEG C,
250rpm, bacterium 2.5-3h is shaken, be placed in 5 minutes on ice, (thalline solubility is OD6000.6-0.8) adds 10mM IPTG50-100ul
Induction, 25 DEG C, concussion and cultivate.Enzyme activity (that is, taking culture supernatant to survey enzyme activity), enzyme activity after 24 hours are surveyed in sampling at regular intervals
Power reaches 1.68U/ml.
Embodiment 9：PGAPZA-cFTS, pGAPZ α A-cFTSW recon positive strain shake flask fermentations
With reference to Invitrogen companies operation manual, picking positive strain single bacterium colony is seeded in 5ml YPD culture mediums, and 28
DEG C, 250rpm shaking table cultures are stayed overnight, and are transferred in 1-4% ratio in 50ml YPD culture medium triangular flasks, the same terms culture,
Every 12 hours, enzyme activity (that is, taking culture supernatant to survey enzyme activity) was surveyed in sampling, and after cultivating 48 hours, enzymatic activities reach highest, are
508U/ml.
Embodiment 10：The enzymolysis product analysis of Yeast engineering bacteria production transfructosylase
Using 25% sucrose as substrate, transfructosylase content is every gram of sucrose of 8 unit, pH5.0, is reacted at 45 DEG C, often
Every a period of time, (for example, 20 minutes, 2 hours, 12 hours, 24 hours after reaction) takes appropriate reactant mixture to carry out product point
Analysis.HPLC analysis shows, the ketose (GF2) occurred first in product, Nystose (GF3), is finally GF4.Instead
FOS highest in liquid is answered to account for the 58% of gross mass.Fig. 3 shows the HPLC collection of illustrative plates for turning glycosyl product, spectrum data with reaction solution
It is 58% consistent to account for gross mass for FOS highest, and sugarcane is may occur in which without display GF4, reaction extension in the HPLC collection of illustrative plates
Fruit pentasaccharides.
It should be understood that although with reference to its exemplary embodiment, particularly shown and description is carried out to the present invention,
It should be understood by those skilled in the art that without departing substantially from by spirit of the invention as defined in the claims and model
Under conditions of enclosing, the change of various forms and details can be carried out wherein, can carry out any combination of various embodiments.
Claims (10)
1. a kind of transfructosylase, it is by SEQ ID NO：The albumen of amino acid sequence composition shown in 2.
2. encode the polynucleotide sequence of the transfructosylase described in claim 1.
3. polynucleotide sequence according to claim 2, it is SEQ ID NO：Nucleotide sequence shown in 1 or 3 or 4.
4. the recombinant expression carrier of the polynucleotide sequence containing the encoding fructose based transferase described in Claims 2 or 3.
5. expression vector according to claim 4, it is characterised in that the expression vector is excretion vector.
6. expression vector according to claim 4, wherein expression vector used is selected from pET-22b, pGAPZA or pGAPZ
αA。
7. the restructuring described in polynucleotide sequence or claim 4 containing the encoding fructose based transferase described in claim 2
The recombination engineering cell of expression vector.
8. recombination engineering cell according to claim 7, its Escherichia coli or Pichia pastoris for restructuring.
9. the polynucleotide sequence of the encoding fructose based transferase described in claim 2, the recombination expression described in claim 4 carries
Application of the recombination engineering cell in the production of transfructosylase preparation described in body or claim 7 or 8.
10. a kind of method for preparing the transfructosylase described in claim 1, methods described comprise the steps：Culture power
Profit requires the recombination engineering cell described in 7 or 8, obtains the transfructosylase described in claim 1.
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| 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 |
| CN113234609A (en) * | 2015-09-18 | 2021-08-10 | 上海交通大学 | Special strain for synthesizing fructo-oligosaccharide and method for synthesizing fructo-oligosaccharide by using special strain |
| CN105441512B (en) * | 2016-01-20 | 2019-02-19 | 天津科技大学 | A kind of method and its enzyme preparation preparing oligofructose |
| 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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|---|---|---|---|---|
| CN1335402A (en) * | 2001-08-12 | 2002-02-13 | 广西大学 | Production process of cane-fruit oligosaccharide with immobilized fructose-base transferase |
| CN102127574A (en) * | 2010-11-18 | 2011-07-20 | 山东文远生物技术有限公司 | Method for producing fructooligosaccharide by using transfructosylase |
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|---|---|---|---|---|
| CN1335402A (en) * | 2001-08-12 | 2002-02-13 | 广西大学 | Production process of cane-fruit oligosaccharide with immobilized fructose-base transferase |
| CN102127574A (en) * | 2010-11-18 | 2011-07-20 | 山东文远生物技术有限公司 | Method for producing fructooligosaccharide by using transfructosylase |
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