CN104450645A - Lipase, and encoding gene and application of lipase - Google Patents
Lipase, and encoding gene and application of lipase Download PDFInfo
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- CN104450645A CN104450645A CN201410695438.6A CN201410695438A CN104450645A CN 104450645 A CN104450645 A CN 104450645A CN 201410695438 A CN201410695438 A CN 201410695438A CN 104450645 A CN104450645 A CN 104450645A
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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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The invention discloses a novel lipase ThiLip, and an encoding gene and application of the lipase ThiLip. The protein ThiLip is one of proteins having amino acid residue sequences: 1) a protein having the amino acid residue sequence of SEQ ID No.2 in a sequence table; 2) a lipopotein having hydrolytic activity and formed in such a way that the amino acid residue sequence of SEQ ID No.2 in a sequence table is subjected to the substitution, deletion and/or addition of one or more amino acid residues. The protein and the encoding gene of the protein can be used for the food processing industry, the pharmaceuticals and cosmetics preparing industry, and the washing industry.
Description
Technical field
The invention belongs to genetically engineered field, be specifically related to a kind of Lipase protein and encoding gene and its thereof and apply.
Background technology
Lipase (Lipase) is the common class fat hydrolase of occurring in nature, is prevalent in animals and plants and microorganism.Although the nucleotide sequence difference of the lipase of different sources is larger, but there is the α/β folding enzymes structure of high similarity in three-dimensional structure, the reaction mechanism of serine hydrolase is followed in catalysis, and namely catalyst structure domain comprises Serine-histidine-glutamic acid (or aspartic acid) triplet active sites and is G-X-S-X-G at the conserved sequence of serine proteinase activities location proximate.Lipase protein molecule stable, catalyzed reaction simply, do not rely on the features such as cofactor, substrate spectrum be wide.
In wine industry, lipase can improve the content of ester class fragrance matter in white wine, accelerates the molecular balance of various acid alcohol ester in white wine, shorten the storage aging time, regulate content and the ratio of various acid alcohol ester in white wine, vinosity is stored aromatic strongly fragrant, sweet refreshing ice-cold, smell coordination is full, and pleasant impression is long; Quality liquor product rate significantly improves.
Summary of the invention
The invention provides a kind of lipase ThiLip albumen and encoding gene thereof and its application, described lipase ThiLip dietary protein origin in osculant thermophilic actinomycete (
thermoactinomyces intermedius, CICC 23799).
An object of the present invention is to provide a kind of albumen, is following 1) or 2) albumen:
1) protein of the composition of the aminoacid sequence shown in SEQ ID № .2 in sequence table;
2) amino acid residue sequence of the SEQ ID № .2 in sequence table had the protein of ester-type hydrolysis activity through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
In sequence table, the aminoacid sequence shown in SEQ ID № .2 is made up of 275 amino-acid residues.
Above-mentioned 1) and 2) in lipase ThiLip albumen can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned 1) and 2) in the encoding gene of lipase ThiLip albumen by the DNA sequence dna shown in SEQ ID № .1 in sequence table is lacked the codon of one or several amino-acid residue, and/or to obtain after the missense mutation carrying out one or several base pair.
The nucleic acid molecule of described lipase ThiLip albumen of encoding also belongs to protection scope of the present invention.
Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA, hnRNA or tRNA etc.
When described nucleic acid molecule is DNA, its sequence is one of following nucleotide sequence:
1) nucleotide sequence of SEQ ID №: 1 1-828 position in sequence table;
2) polynucleotide sequence of SEQ ID №: 2 protein sequence in polynucleotide;
3) nucleotide sequence that the DNA sequence dna that can limit with SEQ ID in sequence table №: 1 under high high stringency conditions is hybridized;
4) with 1) or 2) or 3) DNA sequence dna that limits has more than 90% homology, and coding identical function protein DNA sequence; Concrete, described homology is more than 95%; Concrete is more than 96% again; Concrete is more than 97% again; Concrete is more than 98% again; Concrete is more than 99% again.
Above-mentioned high high stringency conditions can be with 6 × SSC, the solution of 0.5% SDS, and hybridize at 65 DEG C, then use 2 × SSC, 0.1% SDS and 1 × SSC, 0.1% SDS respectively washes film once.
Wherein, SEQ ID №: 1 in sequence table is made up of 828 Nucleotide, its open reading frame (ORF) is from 5 ' end 1-828 position Nucleotide, the protein shown in SEQ ID №: 2 in polynucleotide, i.e. lipase ThiLip albumen of the present invention.
Recombinant vectors containing above-mentioned nucleic acid molecule, expression cassette, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Described recombinant expression vector can use existing expression vector establishment.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any one enhancement type, composing type, organizing specific type or inducible promoter before its transcription initiation Nucleotide, they can be used alone or are combined with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer.For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, as being added in enzyme or the gene (gus gene, GFP gene, luciferase genes etc.) of luminophor, the antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance or the chemical resistance reagent marker gene (as anti-weedkiller gene) etc. expressing in plant and can produce colour-change.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.
Described recombinant expression vector is specially and the nucleic acid fragment with nucleotide sequence shown in SEQ ID №: 1 in sequence table is inserted pET28a carrier
bamhI and
hindobtain between III digestion site.
The primer pair of encoding gene total length of the present invention or its any fragment of increasing also belongs to the scope of protection of the invention.
Another object of the present invention is to provide albumen of the present invention, encoding gene and contains the application of the recombinant vectors of described encoding gene, expression cassette, transgenic cell line or recombinant bacterium.
An also object of the present invention is to provide albumen of the present invention, encoding gene and the recombinant vectors containing described encoding gene, expression cassette, transgenic cell line or the application of recombinant bacterium in ester hydrolysis class.
Another object of the present invention is to provide albumen of the present invention, encoding gene and the recombinant vectors containing described encoding gene, expression cassette, transgenic cell line or the application of recombinant bacterium in Hydrolysis of Olive Oil.
Novel fatty zymoprotein provided by the invention has ester-type hydrolysis activity, and the suitableeest operative temperature is 50 DEG C, and Optimun pH is 8.Novel fatty zymoprotein provided by the invention and encoding gene thereof are in food-processing, and medical cosmetic preparation and washing industry have important application potential.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of Novel fatty zymoprotein ThiLip expression and purification; Lane1: the recombinant bacterium crude enzyme liquid before purifying; Lane2: the recombinant protein solution after ni-sepharose purification.
Fig. 2 is the specific activity of novel lipase ThiLip under condition of different temperatures.
Fig. 3 is the specific activity of novel lipase ThiLip under condition of different pH.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Osculant thermophilic actinomycete (
thermoactinomyces intermedius, CICC 23799) be located away from and pull well high-temperature daqu down, be preserved in Chinese industrial Microbiological Culture Collection administrative center.
Embodiment 1, lipase gene
thilipacquisition
Extraction osculant thermophilic actinomycete (
thermoactinomyces intermedius, CICC 23799) STb gene and purifying, with the genomic dna of this purifying for template carries out pcr amplification, pcr amplification primer is as follows:
Upstream primer: 5'-GCG
gGATCCcAAACGGATGAAGACATC-3'(contains
bamhI restriction enzyme site)
Downstream primer: 5'-GCG
aAGCTTtGCTCCAAGGGCATCATAAAC-3'(contains
hindiII restriction enzyme site)
The PCR primer obtained sends to order-checking.Sequencing result shows, the nucleic acid fragment that above-mentioned pcr amplification obtains comprises restriction enzyme site and has the nucleic acid fragment of nucleotide sequence shown in SEQ ID №: 1 in sequence table; Nucleotide sequence shown in SEQ ID №: 1 is 828bp altogether, wherein encode head of district 828bp, this coding region sequence as shown in 1-828 position Nucleotide in SEQ ID №: 1 in sequence table, the aminoacid sequence shown in SEQ ID №: 2 in polynucleotide, totally 257 amino-acid residues.This had the nucleic acid fragment called after of nucleotide sequence shown in SEQ ID №: 1 in sequence table
thilip.
Also the nucleic acid fragment with nucleotide sequence shown in SEQ ID №: 1 in sequence table is prepared by synthetic.
Embodiment 2, lipase gene
thilipfunctional verification
(1) structure of recombinant plasmid pET-ThiLip
1, restriction enzyme is used
bamhI and
hindthe pcr amplification product that III double digestion embodiment 1 obtains, obtains digestion products.
2, restriction enzyme is used
bamhI and
hindiII double digestion plasmid pET28a(is purchased from Beijing Quanshijin Biotechnology Co., Ltd), reclaim the carrier framework of about 5300bp.
3, the digestion products of step 1 is connected with the carrier framework of step 2 under the effect of T4 DNA ligase, obtains recombinant plasmid; The exactness of sequence verification sequence; Sequencing result shows that gained plasmid for inserting the nucleic acid fragment with nucleotide sequence shown in SEQ ID №: 1 in sequence table between the Bam HI and Hind III digestion site of plasmid pET28a, by this plasmid called after pET-ThiLip.
(2) expression and purification of recombinant protein
Plasmid pET-ThiLip step () obtained is by chemical transformation transformation of E. coli BL21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd), at the LB agar plate containing 50mg/mL kantlex, transformant is screened, obtain recombinant bacterium pET-ThiLip/
e. colibL21 (DE3).The recombinant bacterium bacterial strain in contrast that plasmid pET28a transformation of E. coli BL21 (DE3) obtains.
By recombinant bacterium pET-ThiLip/
e. colibL21 (DE3) is inoculated in 100mL LB liquid nutrient medium (containing 50mg/mL kantlex), and 37 DEG C of shaking culture are to OD
600reach about 0.6, adding final concentration is 0.5 mM IPTG, 25 DEG C of overnight induction.6000rpm collects thalline in centrifugal 10 minutes, adds 100 mM Tris-HCl damping fluids (pH 8.0) according to 1:5, ultrasonic disruption cell 10 minutes (200w, ultrasonic 2s stop 3s).12000 rpm remove cell debris in centrifugal 10 minutes, obtain crude enzyme liquid.
Crude enzyme liquid is passed through ProteinIsoTM Ni-NTA Resin(purchased from Beijing Quanshijin Biotechnology Co., Ltd) carry out purifying.After crude enzyme liquid flows through chromatography column completely, use 5mL washings to rinse unconjugated foreign protein in nickel post, then combine more weak albumen with in 10mL rinsing liquid rinsing nickel post.5ml elutriant is finally used to be eluted by target protein, collect elutriant, add 25mL 100 mM Tris-HCl damping fluid (pH 8.0), use the protein concentration pipe of 10000Da, centrifugal 30 minutes of 3000 rpm, remove unnecessary imidazoles, obtain the elutriant concentrated, the lipase Thilip that SDS-PAGE electrophoresis detection obtains, detected result is shown in Fig. 1.
(3) property testing of novel lipase ThiLip
The vigor of lipase is defined as 1g solid enzyme powder (or 1 mL liquid enzymes), and at the suitableeest temperature and pH value condition, the titratable lipid acid that 1 minute hydrolysis substrate produces 1 μm of ol is 1 enzyme activity unit, represents with u/g (or u/mL).
Take sweet oil as substrate, according to the method for GB/T 23535-2009, measure the enzyme activity of novel lipase under differing temps (10 DEG C-80 DEG C) and different pH value (pH 3-10) condition.Fig. 2 is the specific activity of novel lipase ThiLip under condition of different temperatures.Fig. 3 is the specific activity of novel lipase ThiLip under condition of different pH.Result shows: the optimum temperuture of recombinant protein is 50 DEG C, and optimum pH is 8.
In sum, Novel fatty zymoprotein provided by the invention has ester-type hydrolysis activity, and the suitableeest operative temperature is 50 DEG C, and Optimun pH is 8.In food-processing, medical cosmetic preparation and washing industry have important application potential.
Nucleotide and aminoacid sequence table
<110> China National Academy of Food & Fermentation Industries
<120> lipase and encoding gene thereof are applied with it
<130> 20151112
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 828
<212> DNA
<213>
Thermoactinomyces intermedius，CICC 23799
<400> 1
atgtctgcct tccgatttat tttcagaata ttcatgattt cggtgttgac actggctcct 60
tgtctcttcg gggtctcatc cgcccatgcc caaacggatg aagacatcct gaaagaagcc 120
gctttgtcac ctcccggcgc caatcttccc gattgcaagc cgagcgagga acatccttat 180
ccggtcatcc tggtgccggg cacttttgaa agcatggcat ttaactggag caaactgtca 240
ccccttttgg cgaagaaagg ttattgcgta tatgctttaa actacggcat cacccatctc 300
gacttcagta ccggaccgat tgaaaaatct gccgaagaat tgaaagcgtt tgtcgatcag 360
gtgttggagc aaaccggcgc ggagaaagtt tccattgtcg ggcacagcca gggaggcatg 420
atgccgcgct actatatcaa atacctgggc ggggcggaaa aagtggaaga cctgatcgga 480
ctggctccat ccaaccacgg aaccatcggg gttctcggca ttgaacccat tatgattctt 540
ccgggtcttt tgggatgcac ggcatgcgat cagcaaaaaa ccggctctga cttcctgaat 600
gatttaaacg ccggcgatga aaccccggga gatgtttcat acaccgtgat cagcaccaaa 660
tacgatgaaa ttgtcgttcc ctacacctcc gcctttttgg atggacccga agaccaagtg 720
tccaatatta cgattcagga tcaagtgccg gctgatctgg cggctcacct cggtatcgcc 780
tttgattccc atgcatatga attcgtttat gatgcccttg gagcatga 828
<210> 2
<211> 275
<212> PRT
<213>
Thermoactinomyces intermedius，CICC 23799
<400> 2
Met Ser Ala Phe Arg Phe Ile Phe Arg Ile Phe Met Ile Ser Val Leu
1 5 10 15
Thr Leu Ala Pro Cys Leu Phe Gly Val Ser Ser Ala His Ala Gln Thr
20 25 30
Asp Glu Asp Ile Leu Lys Glu Ala Ala Leu Ser Pro Pro Gly Ala Asn
35 40 45
Leu Pro Asp Cys Lys Pro Ser Glu Glu His Pro Tyr Pro Val Ile Leu
50 55 60
Val Pro Gly Thr Phe Glu Ser Met Ala Phe Asn Trp Ser Lys Leu Ser
65 70 75 80
Pro Leu Leu Ala Lys Lys Gly Tyr Cys Val Tyr Ala Leu Asn Tyr Gly
85 90 95
Ile Thr His Leu Asp Phe Ser Thr Gly Pro Ile Glu Lys Ser Ala Glu
100 105 110
Glu Leu Lys Ala Phe Val Asp Gln Val Leu Glu Gln Thr Gly Ala Glu
115 120 125
Lys Val Ser Ile Val Gly His Ser Gln Gly Gly Met Met Pro Arg Tyr
130 135 140
Tyr Ile Lys Tyr Leu Gly Gly Ala Glu Lys Val Glu Asp Leu Ile Gly
145 150 155 160
Leu Ala Pro Ser Asn His Gly Thr Ile Gly Val Leu Gly Ile Glu Pro
165 170 175
Ile Met Ile Leu Pro Gly Leu Leu Gly Cys Thr Ala Cys Asp Gln Gln
180 185 190
Lys Thr Gly Ser Asp Phe Leu Asn Asp Leu Asn Ala Gly Asp Glu Thr
195 200 205
Pro Gly Asp Val Ser Tyr Thr Val Ile Ser Thr Lys Tyr Asp Glu Ile
210 215 220
Val Val Pro Tyr Thr Ser Ala Phe Leu Asp Gly Pro Glu Asp Gln Val
225 230 235 240
Ser Asn Ile Thr Ile Gln Asp Gln Val Pro Ala Asp Leu Ala Ala His
245 250 255
Leu Gly Ile Ala Phe Asp Ser His Ala Tyr Glu Phe Val Tyr Asp Ala
260 265 270
Leu Gly Ala
275
Claims (8)
1. an albumen is following 1) or 2) albumen:
1) protein of the composition of the aminoacid sequence shown in SEQ ID № .2 in sequence table;
2) amino acid residue sequence of the SEQ ID № .2 in sequence table had the protein of ester-type hydrolysis activity through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
2. the encoding gene of albumen described in claim 1.
3. encoding gene according to claim 2, is characterized in that: described encoding gene has one of following nucleotide sequence:
1) nucleotide sequence of SEQ ID №: 1 1-828 position in sequence table;
2) polynucleotide sequence of SEQ ID №: 2 protein sequence in polynucleotide;
3) nucleotide sequence that the DNA sequence dna that can limit with SEQ ID in sequence table №: 1 under high high stringency conditions is hybridized;
4) with 1) or 2) or 3) DNA sequence dna that limits has more than 90% homology, and coding identical function protein DNA sequence; Concrete, described homology is more than 95%; Concrete is more than 96% again; Concrete is more than 97% again; Concrete is more than 98% again; Concrete is more than 99% again.
4. the recombinant vectors containing the encoding gene described in Claims 2 or 3, expression cassette, transgenic cell line or recombinant bacterium; Described recombinant vectors is specially recombinant expression vector or recombinant cloning vector.
5. the primer pair of encoding gene total length or its any fragment described in Claims 2 or 3 of increasing.
6. the application of albumen according to claim 1 and the arbitrary described encoding gene of claim 2-3 and recombinant vectors according to claim 4, expression cassette, transgenic cell line or recombinant bacterium.
7. albumen according to claim 1 and the arbitrary described encoding gene of claim 2-3 and the application in ester hydrolysis class of recombinant vectors according to claim 4, expression cassette, transgenic cell line or recombinant bacterium.
8. albumen according to claim 1 and the arbitrary described encoding gene of claim 2-3 and the application in Hydrolysis of Olive Oil of recombinant vectors according to claim 4, expression cassette, transgenic cell line or recombinant bacterium.
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| CN104450645A true CN104450645A (en) | 2015-03-25 |
| CN104450645B CN104450645B (en) | 2017-06-16 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151214A (en) * | 2021-04-26 | 2021-07-23 | 中国农业科学院生物技术研究所 | Protein PnlipA with lipase activity and gene and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423837A (en) * | 2008-11-07 | 2009-05-06 | 中国科学院上海有机化学研究所 | Biological synthesis gene cluster of siomycin |
| CN103555636A (en) * | 2013-11-14 | 2014-02-05 | 新疆农业科学院微生物应用研究所 | Streptomyces violaceorubidus and application thereof |
-
2014
- 2014-11-27 CN CN201410695438.6A patent/CN104450645B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423837A (en) * | 2008-11-07 | 2009-05-06 | 中国科学院上海有机化学研究所 | Biological synthesis gene cluster of siomycin |
| CN103555636A (en) * | 2013-11-14 | 2014-02-05 | 新疆农业科学院微生物应用研究所 | Streptomyces violaceorubidus and application thereof |
Non-Patent Citations (1)
| Title |
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| 周晓云等: "链霉菌碱性脂肪酶的研究", 《应用与环境生物学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151214A (en) * | 2021-04-26 | 2021-07-23 | 中国农业科学院生物技术研究所 | Protein PnlipA with lipase activity and gene and application thereof |
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