CN107400636A - Sucrase gene and expression and application thereof - Google Patents

Sucrase gene and expression and application thereof Download PDF

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CN107400636A
CN107400636A CN201710682389.6A CN201710682389A CN107400636A CN 107400636 A CN107400636 A CN 107400636A CN 201710682389 A CN201710682389 A CN 201710682389A CN 107400636 A CN107400636 A CN 107400636A
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gene
seq
sucrose
asp
invertase
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CN107400636B (en
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方泽民
肖亚中
孙秋影
房伟
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Anhui University
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase

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Abstract

The invention discloses a sucrase gene and expression and application thereof, wherein the sucrase gene has a sequence shown as SEQ ID No. 1, and the sucrase is polypeptide with an amino acid sequence shown as SEQ ID No. 2; the invention also discloses an expression plasmid and a strain containing the sucrase. The sucrase is derived from soil microorganisms, has good stability within the pH range of 5.5-6.0, has a half-life period of 90 minutes at 45 ℃, can efficiently hydrolyze sucrose, has good tolerance to sucrose, and can be applied to preparation of high-concentration fructose syrup.

Description

A kind of saccharase gene and its expression and application
Technical field
The present invention relates to biological technical field, specifically a kind of sucrose enzyme coding gene and its expression, and the sugarcane The application of carbohydrase albumen industrially.
Background technology
Invertase (Invertase, EC.3.2.1.26) also referred to as invertase or saccharase (β- Fructofuranosidase), the non reducing end beta-D-fructofuranose glycosidic bond hydrolysis of beta-D-fructofuranose glycosides end can be catalyzed, Can not only catalysing sucrose be hydrolyzed to equimolar fructose and glucose, also can catalysing raffinose hydrolysis generation melibiose and fructose. Classify according to amino acid sequence similarity, invertase can be classified as 32 families (GH32) in glycoside hydrolase, GH68 and GH100(http://www.cazy.org/).Wherein, GH32 families invertase has typical N- and C- ends conserved domain, The significant structure Field Numbers of its Pfam are respectively PF0251 and PF08244.
Invertase is widely present in fungi.In recent years, the sucrose of some fungi source particularly yeast sources should For industrial production.For example, never belonged in the yeast of source discovery sucrose enzyme coding gene together, part sucrose zymoprotein is also The characterization analysis of carry out system, including Pichia anomala, Arxula adeninivorans, Saccharomyces The invertase in the sources such as cerevisiae etc..In addition, Aspergillus belongs to the presence that invertase is have also discovered in fungi. Gongronella sp. belong to Cunninghamellaceae (Family Cunninghamellaceae) fungi.At present, it is true to the category The research of bacterium focuses primarily upon synthesis and the glycosides hydrolase such as β-grape of the encoding gene, chitosan of its chitosan The research of glycosidase etc..However, so far, the report of source saccharase gene is also belonged to without Gongronella.
The content of the invention
The present invention is to avoid the weak point present in above-mentioned prior art, there is provided a kind of invertase is (entitled GspInv) and the engineered strain of the saccharase gene is expressed.
The strain classification is named as Pichia pastoris/pPIC9K-GspInv, is preserved in Chinese Typical Representative culture guarantor Tibetan center, deposit number are CCTCC M 2017438..
The invertase of the present invention, its primary source are derive specifically from Gongronella in middle soil sources microorganism Sp., its gene, there is one of following nucleotide sequence：
(1) the SEQ ID No in sequence table：1；
(2) the SEQ ID No in sequence table：1 is substituted, lacks or adds one or several nucleotides and the identical work(of coding The nucleotide sequence of energy protein.
(3) SEQ ID No in polynucleotide：The polynucleotide sequence of 2 protein sequences；
A kind of invertase, there are one of following amino acid sequences：
(1) the SEQ IDNo in sequence table：2；
(2) the SEQ IDNo in sequence table：2 are substituted, lack or add one or several amino residues and encode identical The amino acid sequence of functional protein.
The present invention also aims to provide the recombinant expression carrier containing above-mentioned saccharase gene.
The present invention also aims to provide the Host Strains containing above-mentioned saccharase gene.
The present invention also aims to provide the preparation method of above-mentioned sucrose zymoprotein, following steps are specifically included：
(1) by inoculating strain in BMGY fluid nutrient mediums, 28 DEG C, under the conditions of 200rpm/min culture to OD600=2~ 6；Thalline is centrifuged, cell is resuspended to OD with BMMY fluid nutrient mediums600=1.0, adding methanol every 24h makes wherein methanol concentration For 0.5%；Zymotic fluid after induction is separated by filtration to obtain supernatant, is invertase crude preparation；
(2) crude preparation is put into ultrafiltration container, be concentrated by ultrafiltration under the conditions of 4~10 DEG C on magnetic stirring apparatus, so Concentrate is dissolved in phosphate buffer solution afterwards, after centrifuging supernatant, with DEAE Sepharose ff ion exchange columns Purifying, elution buffer is 0.3M (NH4)2SO4, washed with 5%, 10%, 20%, 30%, 40%, 60%, 80%, 100% gradient De-, flow velocity 1.0mL/min steady to baseline is rushed in elution every time, the final sucrose zymoprotein for obtaining purifying.
Below by taking expression plasmid carrier pPIC9K, host strain Pichia pastoris GS115 as an example, specific introduce contains There is the construction method of the recombinant strains of saccharase gene of the present invention, specifically include following steps：
(1) be template by the cDNA library sequence obtained from Gongronella sp.mRNA reverse transcriptions, using P1 and P2 as Primer enters performing PCR amplification, and it is invertase cDNA of the present invention to obtain pcr amplification product；The primer is：
P1：5′-CCTAGGATGGTGCTTGCTGATCCT-3′
P2：5′-GCGGCCGCTCAAGGGCGATTGAACG-3′
(2) pcr amplification product obtained by step (1) is connected with pEASY-T1simple carriers, obtains connection product；Will be even Thing of practicing midwifery converts Escherichia coli Trans1-T1 competent cells, screening positive clone, carries out sequence analysis；It is correct to select sequence Clone extract plasmid, obtain containing saccharase gene (GspInv) pEASY-T1simple recombinant plasmids；
(3) with Not I and pEASY-T1simple recombinant plasmids and pPIC9K plasmids obtained by the digestion steps (2) of Bln I；Enzyme Carrier pPIC9K and saccharase gene GspInv fragments T after cutting4DNA ligase connects, and obtains connection product；Connection production Thing carries out single endonuclease digestion to recombinant plasmid GspInv-pPIC9K with restriction enzyme Sac I, after the single endonuclease digestion product recovery of acquisition Electroporated Pichia pastoris GS115 protoplasm somatocytes, screening positive clone, are obtained containing gene of the present invention GspInv engineered strain Pichia pastoris/pPIC9K-GspInv.
Brief description of the drawings
Fig. 1 is the electrophoresis pattern of pcr amplification product of the present invention；
1 and 2 be pcr amplification product in Fig. 1；M is molecular weight standard.
Fig. 2 is that the present invention expresses and purified the electrophoresis pattern for obtaining target protein.
A is SDS-PAGE collection of illustrative plates in Fig. 2, and M is standard molecular weight；B is native-PAGE collection of illustrative plates, and blob of viscose is placed after electrophoresis 45 DEG C of incubation 30min in final concentration of 200mmol/L sucrose solutions, are incubated after terminating and rinse gel by remaining sugarcane with clear water Sugar juice removes, and is developed the color in the NaOH solution for the 0.2% benzyltriphenylphosphonium chloride tetrazole that gel is placed in after boiling.
Optimal pH, pH stability, optimum temperature and the temperature that the GspInv that Fig. 3 is determined when being using sucrose as substrate is catalyzed are steady It is qualitative.
A is optimal pH in Fig. 3, and b is pH stability, and c is optimum temperature, and d is temperature stability
Fig. 4 is hydrolysis abilities of the GspInv to various concentrations sucrose.
Bacterial strain Pichia pastoris/pPIC9K-GspInv in the specific embodiment of the invention have been sent to Chinese Typical Representative Culture collection (China Center for Type Culture Collection, CCTCC) preservation.Bacterial strain preservation is compiled Number it is CCTCC M 2017438.
Embodiment
With reference to embodiment, the invention will be further described, but it is understood that these embodiments not limit the present invention Scope.Meanwhile the implementation in the following example, it is this area conventional reagent and conventional method unless otherwise instructed.
(1), the structure of the Yeast expression bacterial strain containing saccharase gene of the present invention
1st, the activation of Gongronella sp. bacterial strains
On the Gongronella sp.W5 pickings of slant preservation to CPDA inclined-planes, 28 DEG C of quiescent cultures 3 days are to activate bacterium Strain.
2nd, the preparation and sterilizing of culture medium
Prepare fermentation medium：15g sucrose, 1.5g DL- asparagines, 0.5g MgSO4·7H2O, 0.1g Na2HPO4·12H2O, 0.01g CaCl2, 0.01g FeSO4·7H2O, 0.0275g adenine, 50 μ g VB1, 115 DEG C of sterilizings 20min is standby.
3rd, prepared by Gongronella sp.W5 bacterial strains mRNA
5 pieces of the 4mm Gongronella sp.W5 mycelium of picking diameter activation, it is inoculated into the fermentation medium of sterilizing In, 28 DEG C, 120rpm shaken cultivation 2d, decompression filter obtain mycelium, be stored in -70 DEG C it is standby.
4th, GspInv cDNA clones
The mycelium of acquisition is ground using liquid nitrogen, and is used according to RNeasy Plant Mini Kit (Qiagen) Specification extracted total RNA.CDNA reverse transcriptions are prepared using PrimeScript RT reagent kit (TaKaRa) and following drawn Thing, experimental implementation are carried out according to kit operation instructions.
P1：5′-CCTAGGATGGTGCTTGCTGATCCT-3′
P2：5′-GCGGCCGCTCAAGGGCGATTGAACG-3′
The cDNA accordings to the form below of acquisition enter performing PCR reaction：
PCR amplification conditions are 58 DEG C of annealing 30s, 72 DEG C of extension 2kb/ of 94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, Tm Min totally 30 circulations, finally extend 10min at 72 DEG C, and obtained fragment is carried out into 1% Ago-Gel detection, as a result sees Fig. 1. The nucleotide sequence of obtained invertase such as SEQ ID No:Shown in 1, its amino acid sequence such as SEQ ID No:Shown in 2.
5th, the structure of expression vector
40ng pcr amplification products will be obtained in step 4, add 10ng pEASY-T1simple carriers (Transgen), It is immediately placed on ice after 25 DEG C of reaction 15min.The heat-shock transformed Escherichia coli Trans1-T1 competent cells of connection product, are obtained Transformant sequence verification whether be mutated；Select sequence and correctly clone extraction plasmid, acquisition contains saccharase gene (GspInv) pEASY-T1simple recombinant plasmids；Recombinated with the pEASY-T1simple obtained by Not I and the double digestions of Bln I Plasmid and pPIC9K carriers, then use T4GspInv after digestion is connected by DNA ligase with expression plasmid carrier, is established such as Lower digestion system：20ng pPIC9K carriers, 60ng GspInv endonuclease bamhis, 10 × ligation of 3ul buffer, 1ul T4 DNA ligase (TaKaRa), moisturizing to 30ul, 16 DEG C of connection 8h, obtain connection product；Connection product restriction enzyme Sac I carries out single endonuclease digestion to recombinant plasmid GspInv-pPIC9K, electricity conversion Host Strains Pichia pastoris after recovery Whether GS115 protoplasm somatocytes, obtained transformant sequence verification are mutated, and select the correct transformant of sequence and obtain containing this Invention gene GspInv engineered strain Pichia pastoris/pPIC9K-GspInv.
(2) expression and protein purification, containing sucrase genetic engineering bacteria of the present invention
The engineered strain Pichia pastoris/pPIC9K-GspInv of the gene obtained in (one) are inoculated in containing 5mL In the test tube of BMGY fluid nutrient mediums, inoculum concentration 1%, 28 DEG C, 200rpm/min, it is incubated overnight；1mL overnight cultures are taken to turn It is connected in 100mL BMGY triangular flask, 28 DEG C, 200rpm/min, shaken cultivation to OD600=2~6 (UNICO UV2102 purples Outer visible spectrophotometer, using BMGY culture mediums as blank)；Thalline is centrifuged, cell is resuspended to OD with BMMY fluid nutrient mediums600 =1.0, and in 28 DEG C, continue to cultivate under the conditions of 200rpm/min, methanol is added every 24h, makes the wherein methanol volumetric concentration be 0.5% is expressed with inducible protein.
The zymotic fluid of 8 days will be induced by the way that supernatant is collected by centrifugation.Then supernatant is put into Millipore ultrafiltration cups In, it is concentrated by ultrafiltration at 4 DEG C on magnetic stirring apparatus to the 1/50 of original volume, and pass through SDS-PAGE and native-PAGE Purity of protein is detected, pure protein is finally obtained, as a result sees Fig. 2.
Using sucrose as substrate, the invertase Protein G spInv of purifying optimal pH is 5.0, and enzyme is in the range of pH5.5-6.0 There is higher stability, more than the 60% of protoenzyme vigor is able to maintain that after being incubated 5h.Using ABTS as substrate, GspInv 30 DEG C- Catalytic activity can be shown in the range of 60 DEG C, the half-life period of enzyme is 90min when its optimum temperature is 45 DEG C, 45 DEG C, as a result sees figure 3。
(3), the DEAE-Sepharose FF cation exchange chromatographies containing sucrose zymoprotein of the present invention
All reagents use deionized water dissolving in chromatography process, go the removal of impurity through 0.22 μm of membrane filtration, then be placed in super Ultrasonic 1h is to remove bubble removing in sound cleaning device.Specifically purification step is：
1) start AKTA Purifier systems, open Unicorn softwares, A, B pump are all put into deionized water, start Pump washbasic programs are by system flush one time.
2) flow velocity is turned down to 0.5mL/min, and DEAE Sepharose FF ion exchange columns are loaded into purification system.
3) A, B pump are respectively put into less salt and high-salt buffer, regulation flow velocity first uses high-salt buffer to 1mL/min Anion-exchange column is rinsed, until baseline is steady, then is rinsed with low salt buffer steady to baseline.
4) sample centrifuged will be dialysed with constant flow pump loading is used after 0.22 μm of membrane filtration, loading volume is generally post 50mL concentrates are gone up in the 1-5% of bed volume, this experiment altogether.
5) gradient elution is used, by gradually increasing ionic strength, so that each group combined with ion exchange resin Divide and be eluted.Elution buffer in this experiment is 0.3M (NH4) 2SO4, with 5%, 10%, 20%, 30%, 40%, 60%, 80%, flow velocity 1.0mL/min steady to baseline, automatic fraction collector are rushed in 100% gradient elution, every time elution Often pipe collects 1.0mL, and the component that collecting has laccase activity carries out Native-PAGE and SDS-PAGE detections, merges respective components, Collect pure enzyme.
(4), detected containing sucrose zymoprotein sucrose hydrolysis ability of the present invention
Under the conditions of pH5.0,45 DEG C, be separately added into final concentration of 0 in reaction system, 50,300,600,900,1200, 1500th, 1800 and 2000mmol/L sucrose solution, GspInv enzyme activities are determined.As a result show, sucrose concentration be 300mM with Under, GspInv hydrolysing activity increases with the increase of sucrose concentration；Continue to increase with sucrose concentration, GspInv water Solution vigor is on a declining curve, however, when sucrose concentration reaches 1000mmol/L, vigor also retains more than 70%, zymoprotein ratio Vigor is 1500U/mg, illustrates that GspInv has preferable sucrose hydrolysis activity, and has high resistance characteristics to sucrose, can be used In the preparation of high concentration fructose syrup, Fig. 4 is as a result seen.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments The present invention is described in detail, it will be understood by those within the art that：It still can be to foregoing each implementation Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these modification or Replace, the essence of appropriate technical solution is departed from the spirit and scope of various embodiments of the present invention technical scheme.
<110>University of Anhui
<120>A kind of saccharase gene and its expression and application
<130> ...
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 1761
<212> DNA
<213> Gongronella
<400> 1
atggtgcttg ctgatccttt taccaacatg caagactcct ttggcaagat tgagactggt 60
tatggcgcca acgacgcgga tttcgtcaaa tatcgcccaa aattccacgt cattggccct 120
gtcaattgga tgaacgatcc atcggcgccc tactttgatg gcacgcatta ccacatctat 180
tatcagcaca atccctatgc tcaaatttgg ggcaacatga cgtggggcca tgccacctcg 240
acagatctcc tccattggca agatcagcca tttgcgcttt atcctaattc gacttgggac 300
atggagggcg catttgacgg cacatgcatg gcggaaaagg gatatcaagg caagacgacg 360
ttgatctata cggcggtcag ctacagcggc ccgacgtacg tcaatggcca agagaagcag 420
gtgcttgcgg tgacggacga caatggcaat tcgtggacac gcatcaagcc tgtgatcaat 480
ggcccgcccg acaattttgt ggtcaatggc ttccgcgacc cttatttgtt ccgctccgcg 540
tcgtttgacg cgttcctcaa catgcccacc gcgccagatt cgatctacat gacggtgtcg 600
tcgggtatcc cagataaggg cggcagactc tggctgtacc actcgtccga ttgggaaaat 660
tgggatttcc atggtccatt cctgtttcac ccgccaaatc acgtcgcgtc gcccaaccca 720
gcattttacg gcaatgacgg cgtcaattgg gaagttgcgt cgtattttga actccctgat 780
ccttctggtg gcgattcttg gcacattgca acatggggct cgcaaggtgg acgtaatgga 840
gaagattttg accattgggc gctctggact gctggacagc aattggacat gtcgatgccg 900
gacaccaacg aggttgcgaa gccgacgcca cgaattcacg aaaccatgag cggcgtgtat 960
gattggggca tcagttacgc gcaaatggcc ttcaaggatg caaaggatcc caatggaaga 1020
tacctcgccg tcggatgggt tcaggatgat gtcattgctc ccgccactag cgccaacagg 1080
tggaatggtg tccttggtct ctaccgcgag ctctttatcc aagaaatcaa tggaattgac 1140
gcttctgacc cattgctgca agagggctac gcgagctggg tgtatgacgc gtccacaaac 1200
aaagtcaaga ccctcggcat gcgtccgtta cccgaatacg cgtccatgcg cggcaccggt 1260
gtgtggtcac tgcccaaaaa taccaagaaa accgacctca gcaatcttgc catccccatt 1320
gactcgaccc acgtggaaat tgacgccgtc atcgccctgg acaccaattc cgaccccatc 1380
agcttcgttg tgcgccaatc tgcaaaggaa gagaccgtca tcacctatat tccttcccaa 1440
ggcaatatct tcgtcaatcg caccgcatcc acctcgcgtt ccgacctgtg gcgcacatcc 1500
gatgaaacgc acgcgcttcc gctcttccgt gtcaacaatg gcggcctcaa tggcaccggt 1560
ggccttgagc ccttgcatct gcgcgtcttt gtcgacaatt cgctgattga ggtctttgca 1620
aacgaccgct acgccgtgtc cacgcgtatc tacccggatg acgccgatgc cactagaatg 1680
gcattgcgtg cgccagcaaa tgtcaagatc acaagcctga acgtctaccc aatcactgac 1740
agtgcgttca atcgcccttg a 1761
<210> 2
<211> 586
<212> PRT
<213> Gongronella
<400> 2
Met Val Leu Ala Asp Pro Phe Thr Asn Met Gln Asp Ser Phe Gly Lys
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Ala Pro Tyr Phe Asp Gly Thr His Tyr His Ile Tyr Tyr Gln His Asn
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Pro Tyr Ala Gln Ile Trp Gly Asn Met Thr Trp Gly His Ala Thr Ser
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Thr Asp Leu Leu His Trp Gln Asp Gln Pro Phe Ala Leu Tyr Pro Asn
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Ser Thr Trp Asp Met Glu Gly Ala Phe Asp Gly Thr Cys Met Ala Glu
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Lys Gly Tyr Gln Gly Lys Thr Thr Leu Ile Tyr Thr Ala Val Ser Tyr
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Ser Gly Pro Thr Tyr Val Asn Gly Gln Glu Lys Gln Val Leu Ala Val
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Thr Asp Asp Asn Gly Asn Ser Trp Thr Arg Ile Lys Pro Val Ile Asn
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Arg Leu Trp Leu Tyr His Ser Ser Asp Trp Glu Asn Trp Asp Phe His
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Gly Pro Phe Leu Phe His Pro Pro Asn His Val Ala Ser Pro Asn Pro
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Ala Phe Tyr Gly Asn Asp Gly Val Asn Trp Glu Val Ala Ser Tyr Phe
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Glu Leu Pro Asp Pro Ser Gly Gly Asp Ser Trp His Ile Ala Thr Trp
260 265 270
Gly Ser Gln Gly Gly Arg Asn Gly Glu Asp Phe Asp His Trp Ala Leu
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Trp Thr Ala Gly Gln Gln Leu Asp Met Ser Met Pro Asp Thr Asn Glu
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Val Ala Lys Pro Thr Pro Arg Ile His Glu Thr Met Ser Gly Val Tyr
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Asp Trp Gly Ile Ser Tyr Ala Gln Met Ala Phe Lys Asp Ala Lys Asp
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Pro Asn Gly Arg Tyr Leu Ala Val Gly Trp Val Gln Asp Asp Val Ile
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Ala Pro Ala Thr Ser Ala Asn Arg Trp Asn Gly Val Leu Gly Leu Tyr
355 360 365
Arg Glu Leu Phe Ile Gln Glu Ile Asn Gly Ile Asp Ala Ser Asp Pro
370 375 380
Leu Leu Gln Glu Gly Tyr Ala Ser Trp Val Tyr Asp Ala Ser Thr Asn
385 390 395 400
Lys Val Lys Thr Leu Gly Met Arg Pro Leu Pro Glu Tyr Ala Ser Met
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Arg Gly Thr Gly Val Trp Ser Leu Pro Lys Asn Thr Lys Lys Thr Asp
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Trp Arg Thr Ser Asp Glu Thr His Ala Leu Pro Leu Phe Arg Val Asn
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Asn Gly Gly Leu Asn Gly Thr Gly Gly Leu Glu Pro Leu His Leu Arg
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Val Phe Val Asp Asn Ser Leu Ile Glu Val Phe Ala Asn Asp Arg Tyr
530 535 540
Ala Val Ser Thr Arg Ile Tyr Pro Asp Asp Ala Asp Ala Thr Arg Met
545 550 555 560
Ala Leu Arg Ala Pro Ala Asn Val Lys Ile Thr Ser Leu Asn Val Tyr
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Pro Ile Thr Asp Ser Ala Phe Asn Arg Pro
580 585

Claims (8)

1. a kind of engineering bacteria for expressing saccharase gene, it is characterised in that strain classification is named as Pichia pastoris/ PPIC9K-GspInv, is preserved in China typical culture collection center, and deposit number is CCTCC M 2017438, the bacterial strain In the saccharase gene containing enduring high-concentration sucrose.
A kind of 2. saccharase gene, it is characterised in that the gene order such as SEQ ID No：Shown in 1.
3. the gene of invertase as claimed in claim 2, it is characterised in that the gene order is by SEQ ID No：1 institute The nucleotide sequence shown still has former SEQ ID No after substituting or lacking or add one or more nucleotides：Base shown in 1 Because of function identical sequence.
4. a kind of invertase, it is characterised in that the invertase has one of following amino acid sequences：
(1) the SEQ ID No in sequence table：2；
(2) the SEQ ID No in sequence table：2 are substituted, lack or add one or several amino residues and coding identical function The amino acid sequence of protein.
5. the recombinant expression carrier containing the saccharase gene described in claim 2.
6. the recombination expression Host Strains containing saccharase gene described in claim 2.
7. the preparation method of the recombinant sucrose zymoprotein obtained is expressed by claim 4, it is characterised in that including following step Suddenly：
(1) by inoculating strain in BMGY fluid nutrient mediums, 28 DEG C, under the conditions of 200rpm/min culture to OD600=2~6；From Heart thalline, cell is resuspended to OD with BMMY fluid nutrient mediums600=1.0, adding methanol every 24h makes the wherein methanol concentration be 0.5%；Zymotic fluid after induction is separated by filtration to obtain supernatant, is invertase crude preparation；
(2) crude preparation is put into ultrafiltration container, be concentrated by ultrafiltration under the conditions of 4~10 DEG C on magnetic stirring apparatus, then will Concentrate is dissolved in phosphate buffer solution, pure with DEAE Sepharose ff ion exchange columns after centrifuging supernatant Change, elution buffer is 0.3M (NH4)2SO4, washed with 5%, 10%, 20%, 30%, 40%, 60%, 80%, 100% gradient De-, flow velocity 1.0mL/min steady to baseline is rushed in elution every time, the final sucrose zymoprotein for obtaining purifying.
8. application of the saccharase gene in high concentration fructose syrup is prepared described in claim 1,2 or 6.
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