CN104450645B - A kind of lipase and its encoding gene and its application - Google Patents
A kind of lipase and its encoding gene and its application Download PDFInfo
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- CN104450645B CN104450645B CN201410695438.6A CN201410695438A CN104450645B CN 104450645 B CN104450645 B CN 104450645B CN 201410695438 A CN201410695438 A CN 201410695438A CN 104450645 B CN104450645 B CN 104450645B
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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 kind of novel lipase ThiLip and its encoding gene and application.Albumen ThiLip, is the protein with one of following amino acid residue sequences：1）The amino acid residue sequence of the SEQ ID № .2 in sequence table；2）By the amino acid residue sequence of the SEQ ID № .2 in sequence table by the albumen of the substitution of one or several amino acid residues and/or missing and/or addition with ester-type hydrolysis activity.Albumen of the invention and its encoding gene can be used on food processing, and medical cosmetic is prepared and washing industry.
Description
Technical field
The invention belongs to genetic engineering field, and in particular to a kind of Lipase protein and its encoding gene and its application.
Background technology
Lipase（Lipase）It is a common class fat hydrolase in nature, is prevalent in animals and plants and microorganism
In.Although the nucleotide sequence difference of the lipase of separate sources has high similitude than larger in three-dimensional structure
α/β fold enzymatic structure, catalysis follow the reaction mechanism of serine hydrolase, i.e. catalyst structure domain comprising serine-histidine-
Glutamic acid（Or aspartic acid）Triplet active sites and serine proteinase activities location proximate conserved sequence be G-X-S-
X-G.Lipase protein molecule stabilization, catalytic reaction is simple, be independent of confactor, the features such as substrate spectrum is wide.
In brewing industry, lipase can improve the content of esters fragrance matter in white wine, accelerate various acid alcohols in white wine
The reaction balance of ester, shortens the storage aging time, and the content and ratio of various acid alcohol esters, make vinosity cellar aroma flavoring dense in regulation white wine
Strongly fragrant, sweet refreshing ice-cold, smell coordination is full, pleasant impression is long；Quality liquor product rate is significantly improved.
The content of the invention
The invention provides a kind of lipase ThiLip albumen and its encoding gene and its application, the lipase ThiLip
Albumen source is in osculant thermophilic actinomycete（Thermoactinomyces intermedius, CICC 23799）.
It is following 1 it is an object of the present invention to provide a kind of albumen）Or 2）Albumen：
1）The protein of the amino acid sequence composition shown in the SEQ ID № .2 in sequence table；
2）By amino acid residue sequence the taking by one or several amino acid residues of the SEQ ID № .2 in sequence table
Generation and/or missing and/or addition and with ester-type hydrolysis activity protein.
Amino acid sequence in sequence table shown in SEQ ID № .2 is made up of 275 amino acid residues.
Above-mentioned 1）With 2）In lipase ThiLip albumen can be artificial synthesized, also can first synthesize its encoding gene, then carry out
Biological expression is obtained.Above-mentioned 1）With 2）In the encoding gene of lipase ThiLip albumen can be by by SEQ ID in sequence table
DNA sequence dna shown in № .1 lacks the codon of one or several amino acid residues, and/or carries out one or several base-pairs
Obtained after missense mutation.
The nucleic acid molecules for encoding the lipase ThiLip albumen fall within protection scope of the present invention.
The nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also be
RNA, such as mRNA, hnRNA or tRNA.
When the nucleic acid molecules are DNA, its sequence is one of following nucleotide sequence：
1）SEQ ID № in sequence table：1 nucleotide sequence of 1-828；
2）SEQ ID № in polynucleotide：The polynucleotide sequence of 2 protein sequences；
3）Can be with SEQ ID № in sequence table under high stringency conditions high：The nucleotide sequence of the 1 DNA sequence dna hybridization for limiting；
4）With 1）Or 2）Or 3）The DNA sequence dna of restriction has more than 90% homology, and encodes identical function protein
DNA sequence dna；Specifically, the homology is more than 95%；Specific again is more than 96%；Specific again is more than 97%；It is specific again
Be more than 98%；Specific again is more than 99%.
Above-mentioned high stringency conditions high can be the solution of 0.5% SDS with 6 × SSC, hybridize at 65 DEG C, then with 2 × SSC,
0.1% SDS and 1 × SSC, 0.1% SDS respectively washes film once.
Wherein, the SEQ ID № in sequence table：1 is made up of 828 nucleotides, its open reading frame（ORF）It is from 5 '
The nucleotides of end 1-828, SEQ ID № in polynucleotide：Protein shown in 2, i.e., lipase of the present invention
ThiLip albumen.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned nucleic acid molecules fall within of the invention
Protection domain.
The recombinant vector can be recombinant expression carrier, or recombinant cloning vector.
The recombinant expression carrier can use existing expression vector establishment.The expression vector can also include foreign gene
3 ' ends untranslated region, i.e., the DNA fragmentation comprising polyadenylation signals and any other participation mRNA processing or gene expression.Institute
State the 3 ' ends that the bootable polyadenylic acid of polyadenylation signals is added to mRNA precursor.Use the gene constructed recombinant expression carrier
When, any enhanced, composing type, organizing specific type or inducible promoter can be added before its transcription initiation nucleotides,
They can be used alone or are used in combination with other promoters；Additionally, using gene constructed recombinant expression carrier of the invention
When, it is also possible to use enhancer, including translational enhancer or transcriptional enhancer.For the ease of entering to transgenic plant cells or plant
Row identification and screening, can be processed to plant expression vector used, and color change can be produced as being added in be expressed in plant
The gene of enzyme or luminophor（Gus gene, GFP genes, luciferase genes etc.）, resistant antibiotic marker
（Gentamicin label, kanamycins label etc.）Or anti-chemical reagent marker gene（Such as anti-herbicide gene）Deng.From
The security consideration of genetically modified plants, can be not added with any selected marker, directly screen transformed plant with adverse circumstance.
The recombinant expression carrier specially will be with SEQ ID № in sequence table：The nucleic acid fragment of nucleotide sequence shown in 1
Insertion pET28a carriersBamHI andHindObtained between III digestion site.
The primer pair for expanding encoding gene total length of the present invention or its any fragment falls within the scope of protection of the invention.
Weight it is a further object to provide albumen of the present invention, encoding gene and containing the encoding gene
The application of group carrier, expression cassette, transgenic cell line or recombinant bacterium.
It is of the invention that offer albumen of the present invention, encoding gene and the weight containing the encoding gene are provided
Group carrier, the application of expression cassette, transgenic cell line or recombinant bacterium in esters are hydrolyzed.
Weight it is also another object of the present invention to provide albumen of the present invention, encoding gene and containing the encoding gene
The application of group carrier, expression cassette, transgenic cell line or recombinant bacterium in Hydrolysis of Olive Oil.
The Novel fatty zymoprotein that the present invention is provided has ester-type hydrolysis activity, and most suitable operative temperature is 50 DEG C, most suitable
Action pH value is 8.Novel fatty zymoprotein that the present invention is provided and its encoding gene in food processing, medical cosmetic prepare with
And washing industry has important application potential.
Brief description of the drawings
Fig. 1 is the SDS-PAGE that Novel fatty zymoprotein ThiLip is expressed and purified；lane1：Weight before purification
Group bacterium crude enzyme liquid；lane2：Recombinant protein solution after ni-sepharose purification.
Fig. 2 is specific activities of the novel lipase ThiLip under condition of different temperatures.
Fig. 3 is specific activities of the novel lipase ThiLip under condition of different pH.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Quantitative test in following examples, is respectively provided with three repetitions and tests, results averaged.
Osculant thermophilic actinomycete（Thermoactinomyces intermedius, CICC 23799）It is located away from and pulls down
Well high-temperature daqu, is preserved in Chinese industrial Microbiological Culture Collection administrative center.
Embodiment 1, lipase geneThilipAcquisition
Extract osculant thermophilic actinomycete（Thermoactinomyces intermedius, CICC 23799）STb gene
And purify, entering performing PCR amplification by template of the genomic DNA of the purifying, pcr amplification primer thing is as follows：
Sense primer：5'- GCGGGATCCCAAACGGATGAAGACATC-3'（ContainBamHI restriction enzyme sites）
Anti-sense primer：5'-GCGAAGCTTTGCTCCAAGGGCATCATAAAC-3'（ContainHindIII restriction enzyme sites）
The PCR primer for obtaining sends to sequencing.Sequencing result shows that the nucleic acid fragment that above-mentioned PCR amplifications are obtained includes digestion
Site and with SEQ ID № in sequence table：The nucleic acid fragment of nucleotide sequence shown in 1；SEQ ID №：Nucleotides sequence shown in 1
The common 828bp of row, wherein coding head of district 828bp, SEQ ID № in the coding region sequence such as sequence table：1-828 nucleosides in 1
Shown in sour, SEQ ID № in polynucleotide：Amino acid sequence shown in 2, totally 257 amino acid residues.This had into sequence
SEQ ID № in table：The nucleic acid fragment of nucleotide sequence shown in 1 is named asThilip。
Also can be prepared by artificial synthesized with SEQ ID № in sequence table：The nucleic acid piece of nucleotide sequence shown in 1
Section.
Embodiment 2, lipase geneThilipFunctional verification
（One）The structure of recombinant plasmid pET- ThiLip
1st, restriction enzyme is usedBamHI andHindThe pcr amplification product that III double digestions embodiment 1 is obtained, obtains
Digestion products.
2nd, restriction enzyme is usedBamHI andHindIII double digestion plasmids pET28a（It is biological purchased from the full formula gold in Beijing
Technology Co., Ltd.）, reclaim the carrier framework of about 5300bp.
3rd, the carrier framework of the digestion products of step 1 and step 2 is attached in the presence of T4 DNA ligases, is obtained
To recombinant plasmid；The correctness of sequence verification sequence；Sequencing result show gained plasmid be plasmid pET28a Bam HI and
Insertion has SEQ ID № in sequence table between Hind III digestions site：The nucleic acid fragment of nucleotide sequence shown in 1, by this
Plasmid is named as pET-ThiLip.
（Two）The expression and purification of recombinant protein
By step（One）The plasmid pET-ThiLip for obtaining converts e. coli bl21 (DE3) by chemical transformation（Purchase
From Beijing Quanshijin Biotechnology Co., Ltd）, transformant is sieved in the LB agar plates containing 50mg/mL kanamycins
Choosing, obtains recombinant bacterium pET-ThiLip/E. coliBL21 (DE3).Plasmid pET28a converts e. coli bl21 (DE3) and obtains
The recombinant bacterium for arriving is used as control strain.
By recombinant bacterium pET-ThiLip/E. coliBL21 (DE3) is inoculated into 100mL LB fluid nutrient mediums（Containing 50mg/
ML kanamycins）In, 37 DEG C of shaken cultivations to OD6000.6 or so is reached, final concentration of 0.5 mM IPTG, 25 DEG C of inductions is added
Overnight.6000rpm is centrifuged 10 minutes collects thallines, according to 1:5 add 100 mM Tris-HCl buffer solutions（pH 8.0）, surpass
Sonication cell 10 minutes（200w, ultrasonic 2s, stop 3s）.12000 rpm are centrifuged 10 minutes removal cell fragments, obtain
Crude enzyme liquid.
Crude enzyme liquid is passed through into ProteinIsoTM Ni-NTA Resin（Purchased from Beijing Quanshijin Biotechnology Co., Ltd）
Purified.After crude enzyme liquid flows completely through chromatographic column, the foreign protein being not associated with nickel post is rinsed using 5mL cleaning solutions, then use
Weaker albumen is combined in 10mL rinsing liquids rinsing nickel post.Destination protein is eluted using 5ml eluents finally, is collected
Eluent, adds the mM Tris-HCl buffer solutions of 25mL 100（pH 8.0）, use the protein concentration pipe of 10000Da, 3000
Rpm is centrifuged 30 minutes, removes unnecessary imidazoles, the eluent for being concentrated, the lipase that SDS-PAGE electrophoresis detections are obtained
Thilip, testing result is shown in Fig. 1.
（Three）The property of novel lipase ThiLip is determined
The vigor of lipase is defined as 1g solid enzyme powders（Or 1 mL liquid enzymes）, at most suitable temperature and pH value condition,
Hydrolysis substrate produces the titratable aliphatic acid of 1 μm of ol to be 1 enzyme activity unit within 1 minute, with u/g（Or u/mL）Represent.
With olive oil as substrate, according to the method for GB/T 23535-2009, novel lipase is determined in different temperatures（10
℃-80℃）And different pH value（pH 3-10）Under the conditions of enzyme activity.Fig. 2 is novel lipase ThiLip in different temperatures bar
Specific activity under part.Fig. 3 is specific activities of the novel lipase ThiLip under condition of different pH.Result shows：Recombinant protein
Optimum temperature is 50 DEG C, and optimum pH is 8.
In sum, the Novel fatty zymoprotein that the present invention is provided has ester-type hydrolysis activity, and most suitable operative temperature is
50 DEG C, Optimun pH is 8.In food processing, prepared by medical cosmetic and washing industry has important application potential.
Nucleotides and amino acid sequence table
<110>China National Academy of Food ＆ Fermentation Industries
<120>A kind of lipase and its encoding gene and its application
<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 (6)
1. a kind of albumen, it is characterised in that：Amino acid sequence group of the albumen as shown in the SEQ ID № .2 in sequence table
Into, and with ester-type hydrolysis activity.
2. the encoding gene of albumen described in claim 1, it is characterised in that：The encoding gene is SEQ ID in polynucleotide
№：The polynucleotide sequence of 2 protein sequences.
3. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the encoding gene described in claim 2, it is described
Recombinant vector is recombinant expression carrier or recombinant cloning vector.
4. the encoding gene described in the albumen and claim 2 described in claim 1 and the recombinant vector described in claim 3,
The application of expression cassette, transgenic cell line or recombinant bacterium.
5. the encoding gene described in the albumen and claim 2 described in claim 1 and the recombinant vector described in claim 3,
The application of expression cassette, transgenic cell line or recombinant bacterium in esters are hydrolyzed.
6. the encoding gene described in the albumen and claim 2 described in claim 1 and the recombinant vector described in claim 3,
The application of expression cassette, transgenic cell line or recombinant bacterium in Hydrolysis of Olive Oil.
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