CN110904064A

CN110904064A - Gene sequence of fructosyl transferase and preparation method and application thereof

Info

Publication number: CN110904064A
Application number: CN201911099638.4A
Authority: CN
Inventors: 梁智群; 陈桂光; 韩苏苏; 曾伟; 叶童; 冷硕
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2020-03-24

Abstract

The invention discloses a gene sequence of fructosyltransferase and a preparation method and application thereof, belonging to the technical field of food biology. The invention takes the genome of Aspergillus oryzae ZT65 as a template, obtains the coding gene 1872 bp fragment of the fructose transferase by designing primers, gene amplification and sequence determination, and determines the nucleotide sequence and amino acid sequence thereof. The invention designs a pair of primers for amplifying fructosyltransferase gene segments by a PCR method, amplifies specific segments by using the primers, obtains a coding sequence of the Aspergillus oryzae fructosyltransferase gene after sequencing, and the sequence is verified to be a brand new gene sequence.

Description

Gene sequence of fructosyl transferase and preparation method and application thereof

Technical Field

The invention relates to the technical field of food biology, in particular to a fructosyltransferase gene sequence and a preparation method and application thereof.

Background

The fructo-oligosaccharide is also called oligofructose or kestose family oligosaccharide, the molecular formula is G-F-Fn, n is 1-3 (G is glucose, F is fructose), the fructo-oligosaccharide is fructo-oligosaccharide formed by connecting 1-3 fructosyl groups on fructose residues of sucrose molecules through β -1-2 glycosidic bonds, i.e. kestose, kestotetraose, kestopentaose and mixtures thereof.

High-purity and high-yield fructo-oligosaccharide can be obtained by utilizing a microbial fructo-oligosaccharide production enzyme (fructosyltransferase), and the fructosyltransferase is an enzyme with fructosyltransferase activity and can act on sucrose to catalyze and obtain oligosaccharides such as kestose and the like. In the research aspect of fructosyltransferase, at present, the domestic main focus is on the aspects of wild bacteria screening, property determination and the like. The Wanglimei and the like research the enzymatic properties and substrate specificity of aspergillus fructosyltransferase, and the fructosyltransferase has the highest activity under the conditions of pH 5.5 and 30 ℃. Zhengjinyun et al investigated the enzymatic catalytic activities of Invertase (INV) and Fructose Transferase (FTS) purified from Aspergillus niger AS0023 at different sucrose concentrations. In the future research process, the discovery and discovery of an excellent fructosyltransferase gene with excellent catalytic performance is particularly important.

Disclosure of Invention

The invention aims to provide a gene sequence of fructosyltransferase and a preparation method and application thereof, the invention takes the genome of Aspergillus oryzae ZT65 as a template, obtains a coding gene segment of the fructosyltransferase by designing primers, gene amplification and sequence determination, and determines the nucleotide sequence and amino acid sequence of the fructosyltransferase.

The invention is realized by the following technical scheme:

the invention provides a fructosyltransferase, the nucleotide sequence of which is SEQ ID NO: 1; the amino acid sequence is SEQID NO: 2.

the preparation method of the fructosyltransferase comprises the following steps: a1872 bp fragment of a coding gene of the fructosyltransferase is obtained by designing primers, gene amplification and sequence determination by taking a genome of Aspergillus oryzae ZT65 as a template.

The primers comprise a forward primer and a reverse primer, wherein the forward primer P1: 5'-ATGAGGCTCTCAACCGC-3', respectively; reverse primer P2: 5'-TTAGCCAATGCCTTGT-3' are provided.

The gene amplification is PCR amplification, the PCR conditions are that initial denaturation is carried out for 5 minutes at 95 ℃, denaturation is carried out for 30s at 95 ℃, annealing is carried out for 30s at 54 ℃, extension is carried out for 2 min at 72 ℃, 30 cycles are carried out, a PCR product is obtained, the PCR product is connected with pMD 18-TVector overnight at 16 ℃, the connection product is transformed into DH5 α competent cells, selective LB plate containing aminobenzyl is used for culture at 37 ℃, white spots are selected, LB liquid culture medium containing ampicillin is inoculated, shaking culture is carried out overnight at 37 ℃, plasmids are extracted and identified by double enzyme digestion of BamH I and Hind III, and positive bacteria liquid is sent to the Shanghai engineering for sequencing.

The Aspergillus oryzae ZT65 has a preservation number of CCTCC NO of M2014422, a preservation date of 2014, 9 and 17 days, and the preservation unit is as follows: china center for type culture Collection, collection address: wuhan, Wuhan university. The Aspergillus oryzae ZT65 can be used in production of fructosyltransferase preparation.

The nucleotide sequence of the fructosyltransferase obtained by the invention can be further used for cloning and expressing the fructosyltransferase gene, and can also be used for biological preparation of functional fructo-oligosaccharide after the fructosyltransferase is obtained by recombinant expression.

The invention has the beneficial effects that:

1. the invention takes the genome of Aspergillus oryzae ZT65 as a template, obtains the coding gene 1872 bp fragment of the fructose transferase by designing primers, gene amplification and sequence determination, and determines the nucleotide sequence and amino acid sequence thereof. The invention designs a pair of primers for amplifying fructosyltransferase gene segments by a PCR method, amplifies specific segments by using the primers, obtains a coding sequence of the Aspergillus oryzae fructosyltransferase gene after sequencing, and the sequence is verified to be a brand new gene sequence.

2. The fructosyltransferase has a wide pH stability range, and is stable under the conditions of pH 4.0-11.0 through determination.

3. The fructosyl transferase of the invention has high catalytic efficiency: the Km and kcat are respectively 307 mmol L by determination^-1And 1.962 × 10³min^-1。

4. The fructosyltransferase of the invention has high fructo-oligosaccharide conversion rate, and the concentration of a converted substance is 63.9% (w/w) by determination.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 a fructosyltransferase genomic DNA sequence consisting of 1872 bases;

SEQ ID NO: 2 fructosyltransferase consisting of 593 amino acid residues.

Drawings

FIG. 1 shows the result of PCR amplification of fructosyltransferase according to the present invention;

FIG. 2 shows the results of pH stability range detection of fructosyltransferase according to the present invention;

FIG. 3 shows the result of detecting the catalytic efficiency of fructosyltransferase according to the present invention;

FIG. 4 shows the results of the measurement of high fructooligosaccharide conversion of the fructosyltransferase of the present invention.

Detailed Description

In order to make the technical solutions and advantages of the present invention clearer, the following describes the technical solutions of the present invention clearly and completely in combination with the embodiments of the present invention.

Example 1

The preparation method of the fructosyl transferase comprises the following steps: a1872 bp fragment of a coding gene of the fructosyltransferase is obtained by designing primers, gene amplification and sequence determination by taking a genome of Aspergillus oryzae ZT65 as a template.

The gene amplification is PCR amplification, the PCR conditions are that initial denaturation is carried out for 5 minutes at 95 ℃, denaturation is carried out for 30s at 95 ℃, annealing is carried out for 30s at 54 ℃, extension is carried out for 2 min at 72 ℃, 30 cycles are carried out, a PCR product is obtained (the result is shown in figure 1), the PCR product and pMD 18-T Vector are connected overnight at 16 ℃, the connection product is transformed into DH5 α competent cells, selective LB plate containing aminobenzyl is used for culture at 37 ℃, white spots are selected, LB liquid culture medium containing ampicillin is inoculated, shaking culture is carried out overnight at 37 ℃, plasmids are extracted and identified by double enzyme digestion of BamH I and HindIII, and positive bacteria liquid is sent to the Shanghai engineering for sequencing.

Example 2

Determination of optimum pH and pH stability of fructosyltransferase:

a fructosyltransferase isolated and purified from Aspergillus oryzae ZT65 fermentation broth was enzymatically reacted at different pH conditions to determine the optimum pH. The substrate was subjected to fructosyltransferase enzyme activity assay at 55 ℃ in 0.2 mol/L phosphate buffer at various pH. The result shows that the enzyme activity is highest when the optimum pH value of the fructosyl transferase is 6. Fructosyltransferases were treated in the above various pH buffers at 25 ℃ for 24h to investigate the pH stability of the enzyme. The result shows that the fructosyltransferase has high stability between pH 4.0-11.0, and the enzyme activity is more than 80% after treatment for 24 hours (the detection result is shown in figure 2). This indicates that the fructosyltransferase has a wide pH stability range.

Example 3

Fructosyltransferase kinetic parameter determination:

using sucrose as substrate at different concentrations, at 0.1M phosphateThe enzyme activity was measured at 55 ℃ in a buffer system at pH 5.5, and kinetic parameters at 55 ℃ were calculated (as shown in FIG. 3). The Km and kcat are respectively 307 mmol L by determination when sucrose is taken as a substrate^-1And 1.962 × 10³min^-1。

Example 4

Fructosyltransferase catalyzes sucrose to produce fructooligosaccharides:

the fructosyltransferase is adopted to catalyze the substrate sucrose (500g/L), the catalytic treatment is carried out for 12h under the conditions of 55 ℃ and pH 6.0, and the concentration of the fructo-oligosaccharide in the reaction system is determined by HPLC. The content of fructo-oligosaccharide can reach 63.9% (w/w) by measurement analysis calculation (as shown in figure 4).

Sequence listing

<110> Guangxi university

<120> gene sequence of fructosyl transferase and preparation method and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>1782

<212>DNA

<213> fructosyl transferase (2 Ambystoma latex x Ambystoma jeffersonia)

<400>1

atgaagctct caaccgcgag tgccttggtc accagccagg cggcctacgc tgcctccgcc 60

atcgattaca acgcagctcc cccaaacctt tcgactttgg ccaatggctc tctgtatgac 120

acgtggagac ctagagctca tatcctccca ccaaacggcc gaattggtga cccgtgtggc 180

cattacactg atcctgacac cggtctgttc cacgttggct tcctgtacaa tggcagcggt 240

atcgcaggcg ctacgaccga tgatatggtc agattccgtg atctgaatcc caacggaagt 300

caattcatca cgcctggtgg caagaatgat cctgtagcgg tctttgatgg ctctgtaatc 360

cctaagggaa ttgacggcaa gcccacccta ctctacacct ccgtcacatc actccctatc 420

cattggtcaa tcccctacaa cccaggagcc gaaacgcaat ccttggccgt cacatccaac 480

ggcggtcgca attttaccaa gctcgatcgt ccaccggtca ttcccctccc tccgtccgac 540

agcgatgtga ccgcgttccg tgacccctac gctttccaaa gcccggagtt ggacgctgcc 600

gccgacagcg ccccgggtac ctggtacaca gccatctctg gtggtgtcca cgaagatggt 660

cctggtcagt tcctctatcg tcaggaccag aaggaaatga gcctcgagag ctgggagtat 720

cttggtttgt ggtggcagga gaaggtcaac acgacctggg gtaacggcga ctgggccgga 780

ggatggggct tcaacttcga gaccggtaac gtcttcggtc tgaacgagga agggtacagc 840

gtcgatggtg agatgttcat gaccttgggt actgagggat ccggaactcc tattgtgtcc 900

caggtatcat ccattcacga tatgctgtgg gctgctggca acgtctctaa caacggaaat 960

gtcactttca ccccaactat ggccggtgtc ttcgactggg gtgcttctgg ctatgctgcc 1020

gctggtcata ttctgcccgc aacctctcag gtgtccacaa agagtggcgc ccccgaccgt 1080

ttcatctcgt ttgtctggtt gaccggagac ttgttcgagc aggccaaggg ttaccccact 1140

tcgcaacaaa actgggttgg cacccttctg cttcctcgcg agctgcacat caagaccatc 1200

tccaacgtgg ttgataatga gcttgcccgg gaagagggat catcttggcg cgtagagcgt 1260

ggccagtctg gcattgagct gaagaccctg ggaattgata ttgctcggga gacgcgtgaa 1320

gctctcatgt ctggaccgaa gatcactgag cccgagcgca catcgaagga ggctggcctc 1380

gtgcctttcc aggtttcccc gaccaccaag ttccacgtcc tgaccgccca gctgtctttc 1440

cctcgctctg cccgtaattc tgatctccag gccggattcc aagtgctgtc gtctgacctc 1500

gaaagcacca ctatctacta ccagttctcc aatgagtcaa tcatcgtcga ccgcagcaac 1560

accagtgctg ccgcgaagac caccaatgga atcgtcagca ccaatgagtc tggacgtctc 1620

cggttgttcg atttgcaggg cgatgtccag gaaattgaga ctctggacct cacggtcgtt 1680

gtggataact ctgtcctcga gatctatgcc aatggacgtt ttgccctgag tacttgggct 1740

cggtatgtat tctgtgatgg tcgacaacaa ggcattggct aa 1782

<210>2

<211>593

<212>PRT

<213> fructosyl transferase (2 Ambystoma latex x Ambystoma jeffersonia)

<400>2

Met Lys Leu Ser Thr Ala Ser Ala Leu Val Thr Ser Gln Ala Ala Tyr

1 5 10 15

Ala Ala Ser AlaIle Asp Tyr Asn Ala Ala Pro Pro Asn Leu Ser Thr

20 25 30

Leu Ala Asn Gly Ser Leu Tyr Asp Thr Trp Arg Pro Arg Ala His Ile

35 40 45

Leu Pro Pro Asn Gly Arg Ile Gly Asp Pro Cys Gly His Tyr Thr Asp

50 55 60

Pro Asp Thr Gly Leu Phe His Val Gly Phe Leu Tyr Asn Gly Ser Gly

65 70 75 80

Ile Ala Gly Ala Thr Thr Asp Asp Met Val Arg Phe Arg Asp Leu Asn

85 90 95

Pro Asn Gly Ser Gln Phe Ile Thr Pro Gly Gly Lys Asn Asp Pro Val

100 105 110

Ala Val Phe Asp Gly Ser Val Ile Pro Lys Gly Ile Asp Gly Lys Pro

115 120 125

Thr Leu Leu Tyr Thr Ser Val Thr Ser Leu Pro Ile His Trp Ser Ile

130 135 140

Pro Tyr Asn Pro Gly Ala Glu Thr Gln Ser Leu Ala Val Thr Ser Asn

145 150 155 160

Gly Gly Arg Asn Phe Thr Lys Leu Asp Arg Pro Pro Val Ile Pro Leu

165 170 175

Pro Pro Ser Asp Ser Asp ValThr Ala Phe Arg Asp Pro Tyr Ala Phe

180 185 190

Gln Ser Pro Glu Leu Asp Ala Ala Ala Asp Ser Ala Pro Gly Thr Trp

195 200 205

Tyr Thr Ala Ile Ser Gly Gly Val His Glu Asp Gly Pro Gly Gln Phe

210 215 220

Leu Tyr Arg Gln Asp Gln Lys Glu Met Ser Leu Glu Ser Trp Glu Tyr

225 230 235 240

Leu Gly Leu Trp Trp Gln Glu Lys Val Asn Thr Thr Trp Gly Asn Gly

245 250 255

Asp Trp Ala Gly Gly Trp Gly Phe Asn Phe Glu Thr Gly Asn Val Phe

260 265 270

Gly Leu Asn Glu Glu Gly Tyr Ser Val Asp Gly Glu Met Phe Met Thr

275 280 285

Leu Gly Thr Glu Gly Ser Gly Thr Pro Ile Val Ser Gln Val Ser Ser

290 295 300

Ile His Asp Met Leu Trp Ala Ala Gly Asn Val Ser Asn Asn Gly Asn

305 310 315 320

Val Thr Phe Thr Pro Thr Met Ala Gly Val Phe Asp Trp Gly Ala Ser

325 330 335

Gly Tyr Ala Ala Ala Gly His Ile LeuPro Ala Thr Ser Gln Val Ser

340 345 350

Thr Lys Ser Gly Ala Pro Asp Arg Phe Ile Ser Phe Val Trp Leu Thr

355 360 365

Gly Asp Leu Phe Glu Gln Ala Lys Gly Tyr Pro Thr Ser Gln Gln Asn

370 375 380

Trp Val Gly Thr Leu Leu Leu Pro Arg Glu Leu His Ile Lys Thr Ile

385 390 395 400

Ser Asn Val Val Asp Asn Glu Leu Ala Arg Glu Glu Gly Ser Ser Trp

405 410 415

Arg Val Glu Arg Gly Gln Ser Gly Ile Glu Leu Lys Thr Leu Gly Ile

420 425 430

Asp Ile Ala Arg Glu Thr Arg Glu Ala Leu Met Ser Gly Pro Lys Ile

435 440 445

Thr Glu Pro Glu Arg Thr Ser Lys Glu Ala Gly Leu Val Pro Phe Gln

450 455 460

Val Ser Pro Thr Thr Lys Phe His Val Leu Thr Ala Gln Leu Ser Phe

465 470 475 480

Pro Arg Ser Ala Arg Asn Ser Asp Leu Gln Ala Gly Phe Gln Val Leu

485 490 495

Ser Ser Asp Leu Glu Ser Thr Thr Ile TyrTyr Gln Phe Ser Asn Glu

500 505 510

Ser Ile Ile Val Asp Arg Ser Asn Thr Ser Ala Ala Ala Lys Thr Thr

515 520 525

Asn Gly Ile Val Ser Thr Asn Glu Ser Gly Arg Leu Arg Leu Phe Asp

530 535 540

Leu Gln Gly Asp Val Gln Glu Ile Glu Thr Leu Asp Leu Thr Val Val

545 550 555 560

Val Asp Asn Ser Val Leu Glu Ile Tyr Ala Asn Gly Arg Phe Ala Leu

565 570 575

Ser Thr Trp Ala Arg Tyr Val Phe Cys Asp Gly Arg Gln Gln Gly Ile

580 585 590

Gly

Claims

1. A fructosyltransferase, wherein: the nucleotide sequence is SEQ ID NO: 1.

2. the fructosyltransferase of claim 1, wherein: the amino acid sequence is SEQ ID NO: 2.

3. the fructosyltransferase of claim 1 or 2, wherein: the preparation method comprises the following steps: a1872 bp fragment of a coding gene of the fructosyltransferase is obtained by designing primers, gene amplification and sequence determination by taking a genome of Aspergillus oryzae ZT65 as a template.

4. The fructosyltransferase of claim 3, wherein: the primers comprise a forward primer and a reverse primer, wherein the forward primer P1: 5'-ATGAGGCTCTCAACCGC-3', respectively; reverse primer P2: 5'-TTAGCCAATGCCTTGT-3' are provided.

5. The fructosyltransferase of claim 3, wherein the gene amplification is PCR amplification under the conditions of initial denaturation at 95 ℃ for 5 minutes, denaturation at 95 ℃ for 30 seconds, annealing at 54 ℃ for 30 seconds, extension at 72 ℃ for 2 min for 30 cycles to obtain PCR products, overnight ligation of the PCR products with pMD 18-T Vector at 16 ℃, transformation of the ligation products into DH5 α competent cells, culture with benzyl-containing selective LB plates at 37 ℃, selection of white spots, inoculation of ampicillin-containing LB liquid medium, shake culture at 37 ℃ overnight, extraction of plasmids identified by double digestion with BamHI and HindIII, and marine sequencing of positive bacteria.

6. The fructosyltransferase of claim 3, wherein: the Aspergillus oryzae ZT65 has a preservation number of CCTCC NO of M2014422, a preservation date of 2014, 9 and 17 days, and the preservation unit is as follows: china center for type culture Collection, collection address: wuhan, Wuhan university.

7. Encoding the fructosyltransferase of claim 1 for the clonal expression of a fructosyltransferase gene.

8. Use of the fructosyltransferase of claim 1 for the biological preparation of functional fructooligosaccharides.

9. Use of an Aspergillus oryzae ZT65 of claim 4 in the production of a fructosyltransferase formulation.