CN107400636B - Sucrase gene and expression and application thereof - Google Patents

Sucrase gene and expression and application thereof Download PDF

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CN107400636B
CN107400636B CN201710682389.6A CN201710682389A CN107400636B CN 107400636 B CN107400636 B CN 107400636B CN 201710682389 A CN201710682389 A CN 201710682389A CN 107400636 B CN107400636 B CN 107400636B
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方泽民
肖亚中
孙秋影
房伟
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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

Sucrase gene and expression and application thereof
Technical Field
The invention relates to the field of biotechnology, in particular to a sucrase encoding gene and expression thereof, and industrial application of sucrase protein.
Background
Sucrases (Invertase, EC.3.2.1.26), also known as sucrose invertases or β -fructofuranosidases, catalyze the hydrolysis of the terminal non-reducing β -D-fructofuranoside linkages of β -D-fructofuranoside, and not only catalyze the hydrolysis of sucrose to equimolar fructose and glucose, but also raffinose to melibiose and fructose. Sucrases can be classified into 32 families of glycoside hydrolases (GH32), GH68, and GH100(http:// www.cazy.org /), according to amino acid sequence similarity classification. Among them, the sucrases of the GH32 family have typical N-and C-terminal conserved domains, and the Pfam marker domains are numbered PF0251 and PF08244, respectively.
Sucrases are widely present in fungi. In recent years, sucrose of some fungal origin, in particular of yeast origin, has been used for industrial production. For example, sucrase encoding genes have been found in yeast of various genera, and some sucrase proteins have also been subjected to systematic enzymatic property analysis, including sucrases derived from Pichia anomala, Arxula adeninivorans, Saccharomyces cerevisiae, and the like. In addition to this, sucrases are also found in fungi of the genus Aspergillus. Gongronella sp. belongs to the Family Cunninghamellaceae (Family Cunninghamellaceae) fungi. Currently, the research on the fungi of the genus is mainly focused on the coding gene of chitosan, the synthesis of chitosan, and the research on partial glycoside hydrolase such as beta-glucosidase. However, there has been no report on a sucrase gene derived from Gongronella so far.
Disclosure of Invention
In order to avoid the defects of the prior art, the invention provides a sucrase (named as Gsplnv) and an engineering strain for expressing the sucrase gene.
The strain is classified and named as Pichia pastoris/pPIC9K-GspINv, is preserved in China center for type culture Collection with the preservation number of CCTCC M2017438. .
The invertase is originally derived from a soil-derived microorganism, particularly from Gongronella sp, and the gene of the invertase has one of the following nucleotide sequences:
(1) SEQ ID No: 1;
(2) SEQ ID No:1 nucleotide sequence which is substituted, deleted or added with one or more nucleotides and encodes the same functional protein.
(3) Encoding SEQ ID No: 2a polynucleotide sequence of a protein sequence;
a sucrase having one of the following amino acid sequences:
(1) SEQ ID NO: 2;
(2) SEQ ID NO:2 amino acid sequence which is substituted, deleted or added with one or more amino acid residues and encodes the same functional protein.
The present invention also provides a recombinant expression vector containing the sucrase gene.
The present invention also aims to provide a host bacterium containing the above-mentioned sucrase gene.
The invention also aims to provide a preparation method of the sucrase protein, which specifically comprises the following steps:
(1) inoculating strain in BMGY liquid medium, culturing at 28 deg.C and 200rpm/min to OD6002-6; centrifuging the cells, resuspending the cells to OD in BMMY liquid medium600Adding methanol every 24h to make the methanol concentration be 0.5%; filtering and separating the induced fermentation liquor to obtain supernatant, namely a crude sucrase preparation;
(2) putting the crude preparation into an ultrafiltration container, performing ultrafiltration concentration on a magnetic stirrer at 4-10 ℃, dissolving the concentrated solution into a phosphate buffer solution, centrifugally separating supernatant, and purifying by using a DEAE Sepharose ff ion exchange column, wherein the elution buffer solution is 0.3M (NH)4)2SO4Gradient elution is carried out by 5%, 10%, 20%, 30%, 40%, 60%, 80% and 100%, each elution is flushed until the baseline is stable, the flow rate is 1.0mL/min, and finally the purified sucrase protein is obtained.
The following takes an expression plasmid vector pPIC9K and a host strain Pichia pastoris GS115 as examples, and specifically introduces a construction method of a recombinant expression strain containing the sucrase gene of the invention, and the construction method specifically comprises the following steps:
(1) performing PCR amplification by taking a cDNA library sequence obtained by mRNA reverse transcription from Gongronella sp as a template and taking P1 and P2 as primers to obtain a PCR amplification product, namely the sucrase cDNA; the primer is as follows:
P1：5′-CCTAGGATGGTGCTTGCTGATCCT-3′
P2：5′-GCGGCCGCTCAAGGGCGATTGAACG-3′
(2) connecting the PCR amplification product obtained in the step (1) with a pEASY-T1simple vector to obtain a connection product; transforming the ligation product into escherichia coli Trans1-T1 competent cells, screening positive clones, and performing sequence analysis; selecting a clone with a correct sequence to extract a plasmid, and obtaining a pEASY-T1simple recombinant plasmid containing a sucrase gene (Gspinv);
(3) digesting the pEASY-T1simple recombinant plasmid and the pPIC9K plasmid obtained in the step (2) by using Not I and BlnI; the digested vector pPIC9K and sucrase gene Gspinnv fragment are treated with T4DNA ligase is connected to obtain a connection product; the ligation product is subjected to single enzyme digestion on recombinant plasmid GspInv-pPIC9K by using restriction enzyme SacI, the obtained single enzyme digestion product is recovered and then electrically shocked to transform a Pichia pastoris GS115 protoplast cell, and positive clones are screened, so that the engineering strain Pichia pastoris/pPIC9K-GspInv containing the gene GspInv is obtained.
Drawings
FIG. 1 is an electropherogram of the PCR amplification product of the present invention;
1 and 2 in FIG. 1 are PCR amplification products; m is a molecular weight standard.
FIG. 2 is an electropherogram of a target protein expressed and purified according to the present invention.
In FIG. 2, a is an SDS-PAGE pattern, and M is a standard molecular weight; b is native-PAGE, the gel block after electrophoresis is placed in a sucrose solution with the final concentration of 200mmol/L for incubation at 45 ℃ for 30min, the gel is washed by clear water after the incubation is finished to remove the residual sucrose solution, and the gel is placed in a boiled NaOH solution of 0.2 percent of triphenyltetrazolium chloride for color development.
Fig. 3 shows the pH optimum, pH stability, temperature optimum and temperature stability of GspInv catalysis measured with sucrose as substrate.
In FIG. 3, a is the optimum pH, b is the pH stability, c is the optimum temperature, and d is the temperature stability
FIG. 4 shows the ability of Gsplnv to hydrolyze sucrose at different concentrations.
The strain Pichia pastoris/pPIC9K-GspINv in the specific embodiment of the invention is sent to China Center for Type Culture Collection (CCTCC) for preservation. The strain preservation number is CCTCC M2017438.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. Meanwhile, the implementation methods in the following examples are all conventional reagents and conventional methods in the field unless otherwise specified.
(I) construction of Yeast expression Strain containing the sucrase Gene of the present invention
1. Activation of Gongronella sp
Gongronella sp.W5 deposited on the slant was picked up on a CPDA slant and cultured statically at 28 ℃ for 3 days to activate the strain.
2. Preparation and sterilization of culture medium
Preparing a fermentation medium: 15g sucrose, 1.5g DL-asparagine, 0.5g MgSO4·7H2O，0.1gNa2HPO4·12H2O，0.01g CaCl2，0.01g FeSO4·7H2O, 0.0275g adenine, 50 μ g VB1Sterilizing at 115 deg.C for 20 min.
3. Gongronella sp.W5 strain mRNA preparation
Selecting 5 pieces of 4mm Gongronella sp.W5 mycelium with activated diameter, inoculating into sterilized fermentation medium, performing shake culture at 28 deg.C and 120rpm for 2d, vacuum filtering to obtain mycelium, and storing at-70 deg.C.
4. Gsplnv cDNA clone
The obtained mycelia were ground using liquid nitrogen and total RNA was extracted according to RNeasy Plant Mini Kit (Qiagen) using instructions. Preparation of cDNA reverse transcription PrimeScript RT reagent kit (TaKaRa) and the following primers were used, and the experimental procedures were performed according to the kit instructions.
P1：5′-CCTAGGATGGTGCTTGCTGATCCT-3′
P2：5′-GCGGCCGCTCAAGGGCGATTGAACG-3′
The obtained cDNA was subjected to PCR reaction according to the following Table:
Figure BDA0001375837740000041
the PCR amplification conditions were pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at Tm 58 ℃ for 30s, extension at 72 ℃ for 2kb/min for 30 cycles, and finally extension at 72 ℃ for 10min, and the obtained fragments were subjected to 1% agarose gel detection, the results are shown in FIG. 1. The obtained sucrase has a nucleotide sequence shown as SEQ ID No. 1 and an amino acid sequence shown as SEQ ID No. 2.
5. Construction of expression vectors
40ng of the PCR amplification product obtained in step 4 was added to 10ng of pEASY-T1simple vector (Transgen), reacted at 25 ℃ for 15min and immediately placed on ice. The ligation product is thermally shocked to transform escherichia coli Trans1-T1 competent cells, and sequencing of the obtained transformant verifies whether mutation exists; selecting a clone with a correct sequence to extract a plasmid, and obtaining a pEASY-T1simple recombinant plasmid containing a sucrase gene (Gspinv); the resulting pEASY-T1simple recombinant plasmid and pPIC9K vector were double digested with Not I and BlnI, followed by T4The DNA ligase connects the digested GspINv with an expression plasmid vector to establish an enzyme digestion system comprising 20ng of pPIC9K vector, 60ng of GspINv digestion fragment, 3ul of 10 × ligation buffer, 1ul of T4DNA ligase (TaKaRa), water supplementing to 30ul, connecting for 8h at 16 ℃ to obtain a connection product, performing single enzyme digestion on the connection product by using restriction enzyme Sac I to recombinant plasmid GspINv-pPIC9K, electrically transforming a host bacterium Pichia pastoris GS115 protoplast cell after recovery, verifying whether the obtained transformant is mutated by sequencing, and selecting a transformant with a correct sequence to obtain an engineering strain Pichia pastoris/pPIC9K-GspINv containing the gene GspINv.
(II) expression and protein purification of the sucrase genetic engineering bacteria of the invention
Inoculating the engineered strain Pichia pastoris/pPIC9K-GspINv of the gene obtained in the step (I) into a test tube containing a 5mLBMGY liquid culture medium, wherein the inoculation amount is 1%, the temperature is 28 ℃, the rpm is 200rpm/min, and the overnight culture is carried out; transferring 1mL of overnight culture into 100mL BMGY triangular flask, culturing at 28 deg.C and 200rpm/min with shaking to OD6002-6 (UNICO UV2102 UV visible spectrophotometer, BMGY medium blank); centrifuging the cells, resuspending the cells to OD in BMMY liquid medium600Culturing at 28 deg.C and 200rpm/min, adding methanol every 24 hr to make methanol volume concentration 0.5% to induce proteinAnd (4) expressing.
The fermentation broth after 8 days of induction was centrifuged to collect the supernatant. The supernatant was then placed in a Millipore cuvette, concentrated by ultrafiltration on a magnetic stirrer at 4 ℃ to 1/50 in bulk and the purity of the protein was checked by SDS-PAGE and native-PAGE to obtain pure protein, the results of which are shown in fig. 2.
Sucrose is taken as a substrate, the optimum pH value of the purified sucrase protein Gspinv is 5.0, the enzyme has higher stability within the pH range of 5.5-6.0, and more than 60% of the activity of the proenzyme can be maintained after incubation for 5 h. ABTS is taken as a substrate, GspINv can show catalytic activity within the range of 30-60 ℃, the optimal temperature is 45 ℃, the half-life period of enzyme at 45 ℃ is 90min, and the result is shown in figure 3.
(III) DEAE-Sepharose FF ion exchange chromatography purification containing the sucrase protein
All reagents in the chromatography process are dissolved by deionized water, impurities are removed by filtering through a filter membrane of 0.22 mu m, and then the obtained product is placed in an ultrasonic cleaning instrument for ultrasonic treatment for 1 hour to remove bubbles. The specific purification steps are as follows:
1) the AKTA Purifier system was started, the Unicorn software was turned on, the A, B pumps were all placed in deionized water, and the Pump washbasic program was started to flush the system through.
2) The flow rate was reduced to 0.5mL/min, and a DEAE Sepharose FF ion exchange column was loaded into the purification system.
3) The A, B pump was placed into low salt and high salt buffers, respectively, the flow rate was adjusted to 1mL/min, the anion exchange column was washed with high salt buffer until the baseline leveled off, and then washed with low salt buffer until the baseline leveled off.
4) The dialyzed and centrifuged sample is filtered by a 0.22 mu m filter membrane and then loaded by a constant flow pump, the loading volume is generally 1-5% of the volume of the column bed, and 50mL of concentrated solution is totally loaded in the experiment.
5) Gradient elution is used, whereby the fractions bound to the ion exchange resin are eluted by gradually increasing the ionic strength. The elution buffer solution used in the experiment is 0.3M (NH4)2SO4, the elution is performed in a gradient manner by 5%, 10%, 20%, 30%, 40%, 60%, 80% and 100%, the elution is performed until the baseline is stable in each time, the flow rate is 1.0mL/min, 1.0mL is collected by each tube of an automatic partial collector, components with laccase activity are collected, Native-PAGE and SDS-PAGE are performed for detection, corresponding components are combined, and pure enzyme is collected.
(IV) detection of sucrose hydrolysis Capacity of the sucrase protein of the present invention
Sucrose solutions with final concentrations of 0, 50, 300, 600, 900, 1200, 1500, 1800 and 2000mmol/L are respectively added into the reaction system under the conditions of pH5.0 and 45 ℃ to determine the enzyme activity of Gsplnv. The results show that the hydrolytic activity of Gsplnv increases with increasing sucrose concentration at sucrose concentrations below 300 mM; with the continuous increase of the sucrose concentration, the hydrolysis activity of the GspINv is in a descending trend, however, when the sucrose concentration reaches 1000mmol/L, the activity is still more than 70%, and the specific activity of the enzyme protein is 1500U/mg, which indicates that the GspINv has better sucrose hydrolysis activity and high tolerance characteristic to sucrose, and can be used for preparing high-concentration fructose syrup, and the result is shown in FIG. 4.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Figure BDA0001375837740000061
Figure BDA0001375837740000071
Figure BDA0001375837740000081
Figure BDA0001375837740000091
Figure BDA0001375837740000101
Figure BDA0001375837740000111
Figure BDA0001375837740000121
Figure BDA0001375837740000131
Figure BDA0001375837740000141
<110> university of Anhui
<120> sucrase gene and expression and application thereof
<130>...
<160>2
<170>PatentIn version 3.1
<210>1
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<212>DNA
<213>Gongronella
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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 ccgctccgcg540
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
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Pro Tyr Ala Gln Ile Trp Gly Asn Met Thr Trp Gly His Ala Thr Ser
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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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Claims (7)

1. An engineering bacterium for expressing sucrase gene, characterized in that the strain is classified and named
Pichia pastoris/pPIC9K-GspINv, which is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2017438, the strain contains a sucrase gene which is resistant to sucrose at high concentrations.
2. A sucrase gene characterized in that the nucleotide sequence of the sucrase gene is shown in SEQ ID No:1 is shown.
3. A sucrase, wherein the amino acid sequence of sucrase is shown in SEQ ID No:2, respectively.
4. A recombinant expression vector containing the sucrase gene of claim 2.
5. A recombinant expression host bacterium comprising the sucrase gene of claim 2.
6. A method for preparing the sucrase of claim 3, comprising the steps of: (1) inoculating the engineering bacteria of claim 1 into BMGY liquid medium, and culturing at 28 ℃ and 200rpm until OD600 is 2-6; centrifuging thallus, and resuspending cells with BMMY liquid culture medium until OD600 is 1.0, and adding methanol every 24h to make methanol concentration 0.5%; filtering and separating the induced fermentation liquor to obtain supernatant, namely a crude sucrase preparation;
(2) putting the crude preparation into an ultrafiltration container, performing ultrafiltration concentration on a magnetic stirrer at 4-10 ℃, dissolving the concentrated solution into a phosphate buffer solution, centrifugally separating supernatant, and purifying by using a DEAE Sepharose ff ion exchange column, wherein the elution buffer solution is 0.3M (NH)4)2SO4Gradient elution is carried out by 5%, 10%, 20%, 30%, 40%, 60%, 80% and 100%, each elution is flushed until the baseline is stable, the flow rate is 1.0mL/min, and finally the purified sucrase protein is obtained.
7. Use of the sucrase gene as claimed in claim 2 for the preparation of high-concentration fructose syrup.
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