CN110452892A

CN110452892A - A kind of lipase of macro gene source, encoding gene, carrier, engineering bacteria and the application in lutein preparation

Info

Publication number: CN110452892A
Application number: CN201910904220.XA
Authority: CN
Inventors: 谢恬; 王秋岩; 张路路; 许新德; 拜重阳; 陈铸恺; 徐瑶; 蒋太斗; 王铎; 沈一丁; 林舒妮
Original assignee: Hangzhou Normal University
Current assignee: Zhejiang Keming biomedical Co., Ltd; Zhejiang Medicine Co Ltd Xinchang Pharmaceutical Factory
Priority date: 2019-09-24
Filing date: 2019-09-24
Publication date: 2019-11-15
Anticipated expiration: 2039-09-24
Also published as: CN110452892B

Abstract

A kind of lipase of macro gene source of the present invention, encoding gene, carrier, engineering bacteria and the application in lutein preparation, belong to gene engineering technology field.The amino acid sequence of the lipase HsLIP1 of macro gene source is as shown in SEQ ID NO.1.The gene of encoding lipase HsLIP1.The nucleotide sequence of gene is as shown in SEQ ID NO.2.A kind of recombinant vector containing the gene.A kind of genetic engineering bacterium converted from the recombinant vector.Application of the gene in preparation and reorganization lipase HsLIP1.Application of the lipase HsLIP1 in lutein preparation.The invention has the following advantages that lipase HsLIP1 is easy to prokaryotic expression, lipase of the present invention can efficiently be produced using recombinant vector, bacterial strain and preparation method；Moreover, the preparation method simple and effective of enzyme of the present invention, is easy to great expression, it is suitable for industrialized production.

Description

A kind of lipase of macro gene source, encoding gene, carrier, engineering bacteria and in leaf Huang Application in element preparation

Technical field

The invention belongs to gene engineering technology fields, and in particular to a kind of lipase of macro gene source, encoding gene, load Body, engineering bacteria and the application in lutein preparation.

Background technique

Lutein belongs to carotenoid, includes multiple conjugated double bonds, these conjugated double bonds do not give only color spy Sign, also make its in disease prevention and cure have stronger anti-oxidation function, such as prevent or prevent as cancer, artery sclerosis, cataract, Color spot is degenerated and other class diseases.The method for obtaining a large amount of lutein crystals is mass produced at present to be mentioned from natural resources It takes, separate and purifying obtains.Lutein class in plant is often and some C12-C18 long chain fatty acids such as myristic acid, oil The esterifications such as acid, linoleic acid and palmitinic acid form monoesters or the form of dibasic acid esters exists.It is at present to utilize highly basic with most methods Soap solution lutein ester, then it is recrystallized to give lutein.However this method has following disadvantage: using a large amount of highly basic；Process It is middle to generate a large amount of waste water；Using arrive some toxic organic solvents；The yield of lutein is lower.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a kind of special ester linkage hydrolyzing Enzyme is defined as capableing of the enzyme of hydrolysis of long chain fatty acid esters, be widely present in various animals, plant and microorganism (including mould, Yeast and bacterium) in, microorganism is the main source of current industrial lipase.Using lipase in two-phase system (i.e. oil-water Interface) this characteristic of the hydrolysis of ester bond can be catalyzed by lutein fatty acid ester (lutein ester) hydrolysis release lutein crystal.Rouge Fat enzyme hydrolysis condition is mild, high-efficient, and bottom line uses organic solvent, and purification step is succinct, therefore the enzyme is preparing leaf Flavine field of crystals has wide application space.

Metagenomics analysis does not need to cultivate all microorganisms of environment, it can be directly by thin to environment Born of the same parents crack, and then separate and recover DNA from matrix and cell fragment, and the Nomenclature Composition and Structure of Complexes of microbiologic population is analyzed with this, So that the research and development that script is more than 99% uncultured microorganisms genetic resources becomes reality, develop to the utmost The potential of environmental microorganism resource.

Summary of the invention

It is an object of the invention to disclose a kind of lipase of macro gene source, encoding gene, carrier, engineering bacteria and Application in lutein preparation.

The purpose of the present invention is what is be achieved through the following technical solutions:

A kind of lipase HsLIP1 of macro gene source, wherein the amino of the lipase HsLIP1 of the macro gene source Acid sequence is as shown in SEQ ID NO.1.

A kind of gene of lipase HsLIP1 described in coding above-mentioned technical proposal.

Gene as described in the above technical scheme, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

A kind of recombinant vector containing gene described in above-mentioned technical proposal.

The recombinant vector according to the above technical scheme, wherein the recombinant vector is the gene by encoding HsLIP1 Complementary connect of viscosity is carried out with pET-28a carrier to obtain.

A kind of genetic engineering bacterium that the recombinant vector as described in above-mentioned technical proposal converts.

The genetic engineering bacterium according to the above technical scheme, wherein the genetic engineering bacterium is by the recombination matter Grain conversion is obtained into E.coli BL21 competent cell.

Application of the gene described in above-mentioned technical proposal in preparation and reorganization lipase HsLIP1.

Application of the lipase HsLIP1 described in above-mentioned technical proposal in lutein preparation, wherein lipase HsLIP1 Refer to that lipase HsLIP1 discharges the anti-of lutein in the hydrolysis of catalysis lutein extract substrate preparing the application in lutein Application in answering.The technical program Lutein medicinal extract substrate contains fatty acid ester of lutein.

Application of the lipase HsLIP1 as described in the above technical scheme in lutein preparation, wherein the lutein Medicinal extract substrate is the monoesters or dibasic acid esters that C12-C18 long chain fatty acid is formed.Preferably the C12-C18 long chain fatty acids are Myristic acid, oleic acid, linoleic acid or palmitinic acid.

The lipase HsLIP1 of macro gene source in the present invention, amino acid sequence is as shown in SEQ ID NO.1, it may be assumed that HG FTGWGREEMFGFKYWGGVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFG RTYPGLLPELKRGGRIHIIAHSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIATPHDG TTLVNMVDFTDRFFDLQKAVLEAAAAASNVPYTSQVYDFKLDQWGLRRQPGESFDHYFERLKRSPVWTSTDTARYD LSVSGAEKLDQWVQASPNTYYLSFSTERTYRGALTGNHYPELGMNAFSAVVCAPFLGPYRNPTLGIDDRWLENDGI VNTVSMNGPKRGSSDRIVPYDGTL。

Heretofore described lipase HsLIP1 has pH tolerance range extensively and metal ion Ni²⁺、Na⁺And Mg²⁺And have Solvent dimethyl sulfoxide (DMSO) and ethylene glycol have the features such as certain promotion to the hydrolysing activity of the lipase.

Due to the particularity of amino acid sequence, the piece of any peptide albumen containing amino acid sequence shown in SEQ ID NO.1 Section or its variant, such as its examples of conservative variations, bioactive fragment or derivative, as long as the segment of the peptide albumen or peptide protein variant With aforementioned amino acid sequences homology 95% or more, the column of the scope of the present invention are belonged to.The specific change can wrap Include the missing of amino acid, insertion or replacement in amino acid sequence；Wherein, the conservative of variant is sexually revised, the amino replaced Acid has structure similar with original acid or chemical property, such as replaces isoleucine with leucine, and variant can also have non-guarantor It keeps and sexually revises, such as replace glycine with tryptophan.The segment of peptide albumen of the present invention, derivative or the like refer to substantially Keep the identical biological function of lipase of the present invention or active peptide albumen, can be following state: (I) one or More amino acid is guarded or nonconserved amino acid residues (preferably conservative amino acid residues) replace, and replace Amino acid, which can be, may not be by genetic codon coding；(II) some group on one or more amino acid residues Replaced by other groups；(III) mature peptide albumen and another compound (for example extend the compound of peptide protein half-life, such as Polyethylene glycol) fusion；(IV) the peptide protein sequence that additional amino acid sequence is integrated into mature peptide albumen and is formed (is such as used to Purify the sequence or proprotein sequence of this peptide albumen).

The peptide albumen can be recombinant protein, native protein or synthetic proteins, can be the product of pure natural purifying, or Be chemical synthesis product, or using recombinant technique from protokaryon or eucaryon host (such as: bacterium, yeast, higher plant, insect And mammalian cell) in generate.According to host used in recombinant production scheme, peptide albumen of the invention can be glycosylation 's.Peptide albumen of the invention can also include or not include the methionine residues originated.

The invention further relates to the genes for encoding the lipase HsLIP1.

Specifically, the gene nucleotide series can be as shown in SEQ ID NO.2, it may be assumed that

CATGGGTTTA CCGGATGGGG ACGAGAGGAA ATGTTTGGAT TCAAGTATTG GGGCGGCGTG CGCGGCGATA TCGAACAATG GCTGAACGAC AACGGTTATC GAACGTATAC GCTGGCGGTC GGACCGCTCT CGAGCAACTG GGACCGGGCG TGTGAAGCGT ATGCTCAGCT TGTCGGCGGG ACGGTCGATT ATGGGGCAGC CCATGCGGCA AAGCACGGCC ATGCGCGGTT TGGCCGCACT TATCCCGGCC TGTTGCCGGA ATTGAAAAGG GGTGGCCGCA TCCATATCAT CGCCCACAGC CAAGGGGGGC AGACGGCCCG CATGCTTGTC TCGCTCCTAG AGAACGGAAG CCAAGAAGAG CGGGAGTACG CCAAGGCGCA TAACGTGTCG TTGTCACCGT TGTTTGAAGG TGGACATCAT TTTGTGTTGA GTGTGACGAC CATCGCCACT CCTCATGACG GGACGACGCT TGTCAACATG GTTGATTTCA CCGATCGCTT TTTTGACTTG CAAAAAGCGG TGTTGGAAGC GGCGGCTGCC GCCAGCAACG TGCCGTACAC GAGTCAAGTA TACGATTTTA AGCTCGACCA ATGGGGACTG CGCCGCCAGC CGGGTGAATC GTTCGACCAT TATTTTGAAC GGCTCAAGCG CTCCCCTGTT TGGACGTCCA CAGATACCGC CCGCTACGAT TTATCCGTTT CCGGAGCTGA GAAGTTGGAT CAATGGGTGC AAGCAAGCCC GAATACGTAT TATTTGAGTT TCTCTACAGA ACGGACGTAT CGCGGAGCGC TCACAGGCAA CCATTATCCC GAACTCGGAA TGAATGCATT CAGCGCGGTC GTATGCGCTC CGTTTCTCGG TCCGTACCGC AATCCGACGC TCGGCATTGA CGACCGATGG TTGGAGAACG ATGGCATTGT CAATACGGTT TCCATGAACG GTCCAAAGCG TGGATCAAGC GATCGGATCG TGCCGTATGA CGGGACGTTG。

Due to the particularity of nucleotide sequence, the variant of polynucleotides shown in any SEQ ID NO.2, as long as it is more with this Nucleotide has 70% or more homology, belongs to the column of the scope of the present invention.The variant of the polynucleotides refers to one kind The polynucleotide sequence changed with one or more nucleotide.The variant of this polynucleotides can make raw displacement variant or The variant of non-life, including substitution variants, Deletion variants and insertion variant.As known in the art, allelic variant It is the alternative forms of a polynucleotides, it may be substitution, missing or the insertion of a polynucleotides, but not from substantial Change the function of the peptide albumen of its coding.

Of the invention be characterized by provides shown in amino acid sequence shown in SEQ ID NO.1 and SEQ ID NO.2 Nucleotide sequence, in the case where the known amino acid sequence and nucleotide sequence, the amino acid sequence and nucleotide sequence Acquisition and the acquisition of relevant carriers, host cell, are obvious to those skilled in the art.

The invention further relates to the recombinant vector containing the gene and gene works converted from the recombinant vector Journey bacterium.

The invention further relates to application of the gene in preparation and reorganization lipase HsLIP1.Specifically, the application Are as follows: building contains the recombinant vector of the encoding gene, the recombinant vector is transferred in Escherichia coli, the recombination of acquisition Engineering bacteria carries out Fiber differentiation, and the isolated somatic cells containing recombinant lipase of culture solution, somatic cells are broken through cell, divide The recombinant lipase HsLIP1 is obtained from purifying.

The experiment proved that lipase HsLIP1 of the present invention expresses yield height in host strain, such as big in angstrom Xi Shi It is overexpressed in enterobacteria, the solubility expression of HsLIP1 is high.Using p-nitrophenol rouge as substrate, under the conditions of pH8.0,50 DEG C, The enzyme activity highest of HsLIP1, the enzyme activity under optimum condition are 9.37U/mL, are incubated for after 480min and protect in 35 DEG C of water-bath Hold 87% relative surplus vigor.The hydrolysis of HsLIP1 catalysis lutein extract (fatty acid ester of lutein) of 50ug 30min discharges 0.537ug lutein, metal ion Ni²⁺、Na⁺And Mg²⁺And organic solvent dimethyl sulfoxide (DMSO) and second two Alcohol has certain promotion to the hydrolysing activity of HsLIP1.

Lipase HsLIP1 of the invention has high expression quantity, high activity, substrate specificity, while lipase of the present invention HsLIP1 can be catalyzed ester linkage hydrolyzing in lutein extract and release lutein and corresponding C12-C18 long chain fatty acids as such as meat Myristic acid, oleic acid, linoleic acid and palmitinic acid etc..

The preparation method and zymetology characterization of the lipase HsLIP1 of high expression, high activity of the invention:

(1), the acquisition of lipase HsLIP1 full length gene of the present invention.Lipase degenerate primer is designed, with this experiment Room acquisition soil (water) sample include Ningbo, Dongtou, Xiangshan, balcony, TAIHU LAKE, Xinchang, Zhejiang Province, Xi'an nearby loess plateau, The macro genome DNA of soil (water) sample extraction of eight different regions such as Shenzhen Repulse Bay laterite is template, and clone obtains macro The new enzyme of genome 1, obtains overall length lipase gene, i.e. lipase HsLIP1, and lipase nucleotide shown in SEQIDNO.2 is complete Long sequence.

(2), the building of the expression vector system containing target gene.Extremely by cloning lipase gene described in step (1) Expression vector, such as pET-28a.

(3), the recombinant vector of enzyme HsLIP1 gene fatty in step (2) is transferred in heterogenous expression host cell, such as (Escherichiacoli) BL21 cultivates recombinant host cell under conditions suitable for the expression.

(4), the lipase of heretofore described high expression, high activity is isolated and purified out from the culture of step (3) HsLIP1。

(5), by method produced above, further zymetology characteristic present, including most thermophilic are carried out to the lipase of acquisition Degree, thermal stability, optimum pH, heavy metal ion solvent and tolerance of organic solvent etc..

The invention further relates to the lipase HsLIP1 to discharge lutein in the hydrolysis of catalysis lutein extract Using.

The lutein extract substrate includes some C12-C18 long chain fatty acids such as myristic acid, oleic acid, linoleic acid and palm fibre The monoesters or dibasic acid esters that the esterifications such as palmitic acid acid are formed.

The present invention devises conserved positions primer, and then expand in macro genome according to the fatty enzyme sequence in database Increase and homologous fragment, construct the new enzyme (HsLIP1) comprising macro genome homologous fragment, function characterization has been carried out to it, and It applies it in rouge hydrolysis.Using p-nitrophenol rouge as substrate, enzyme activity of the enzyme under the conditions of pH8.0,50 DEG C Highest, the enzyme activity under optimum condition are 9.37U/mL, be incubated in 35 DEG C of water-bath keep after 480min 87% it is relatively surplus Remaining vigor.The hydrolysis 30min of the HsLIP1 catalysis lutein extract of 50ug discharges 0.537ug lutein, metal ion Ni² ⁺、Na⁺And Mg²⁺And organic solvent dimethyl sulfoxide (DMSO) and ethylene glycol have one to the hydrolysing activity of lipase HsLIP1 Determine facilitation.

The invention has the following advantages:

Lipase HsLIP1 of the invention is easy to prokaryotic expression, can be using recombinant vector, bacterial strain described in this patent And preparation method efficiently produces lipase of the present invention；Moreover, the preparation method simple and effective of enzyme of the present invention, is easy to great expression, Suitable for industrialized production.

Detailed description of the invention:

1, Fig. 1 is the agarose gel electrophoresis figure of macro genome DNA.

2, Fig. 2 is the agarose gel electrophoresis figure of macro Genomic PCR products.

3, Fig. 3 is the agarose gel electrophoresis figure of colony identification PCR product.

4, Fig. 4 is lipase inducing expression result SDS-PAGE electrophoresis；

5, Fig. 5 is lipase purification result SDS-PAGE electrophoresis；

6, Fig. 6 is p-nitrophenol standard curve；

7, Fig. 7 is the reaction of lipase different temperatures；

8, Fig. 8 is fatty enzyme heat stability reaction；

9, Fig. 9 is the reaction of lipase difference pH value；

10, Figure 10 is lutein standard curve；

11, Figure 11 is that lipase reacts the different temperatures of lutein extract；

12, Figure 12 is that lipase reacts the different pH value of lutein extract；

13, Figure 13 is that lipase reacts the different metal ions of lutein extract；

14, Figure 14 is that lipase reacts the different organic solvents of lutein extract；

Specific embodiment:

In order to make the technical solution of the present invention easy to understand, the present invention is further retouched combined with specific embodiments below It states, but protection scope of the present invention is not limited to that:

Experimental method in the present invention is conventional method unless otherwise instructed, for details, reference can be made to " MolecularCloning:ALaboratoryManual " (SambrookandRussell, ed.2001).

Bacillus coli DH 5 alpha, BL21 used in the embodiment of the present invention (DE3) are purchased from TransGen Biotech company； Primer synthesis is completed by Shanghai JaRa biotechnology Co., Ltd；Sequence work is by raw work bioengineering (Shanghai) stock Part Co., Ltd completes.

Embodiment 1:The building of the recombinant bacterium of the acquisition of lipase HsLIP1 and fatty enzyme HsLIP1:

It 1, include that Ningbo, Dongtou, Xiangshan, balcony, TAIHU LAKE, Zhejiang are new from the field environment of different geographical all over China Eight different regions such as loess plateau, Shenzhen Repulse Bay laterite near prosperous, Xi'an, acquire 10 soil (water) samples, wherein 3 altogether Number soil sample derives from Xiangshan soil.Soil constitution is complicated, and the macro genome DNA of extraction contains the humic of brownish black or black The impurity such as acid, they have great inhibiting effect to subsequent reactions such as PCR, it is therefore desirable to effectively carry out to humic acid in soil Removal.Pedotheque humic acid is effectively removed using Humic acid-Be-Gone A kit, the soil for recycling precipitating can be straight It connects and is extracted for DNA.Pedotheque gene is extracted using Soil Genomic DNAKit soil genome DNA extracting reagent kit Group DNA, the DNA solution of collection, agarose gel electrophoresis detection, electrophoretogram is as shown in Figure 1, column M:15,000DNA marker； Arrange M ': 10,000DNA marker；It arranges 1-10:Metagenomic DNA (macro genome DNA)；And use Ago-Gel DNA QIAquick Gel Extraction Kit (Generay, Shanghai) recycles DNA, saves in -20 DEG C.

2, basis source is in the higher lipase gene BTL2 of activity (the GenBank number of G.thermocatenulatus For ACCESSIONX95309) and from the lipase gene bacterium T6 (EMBL of Geobacillus stearothermophilus Accession number AF429311.1), it carries out sequence alignment analysis and searches conserved region sequence, design primer.

The PCR amplification primer sequence of the present invention of table 1

Using macro genome DNA as template, PCR amplification is carried out with high fidelity enzyme, it is as follows to prepare reaction system:

After above-mentioned system mixes, setting program is as follows:

After PCR amplification, PCR product is subjected to agarose gel electrophoresis detection, voltage stabilization and current stabilization electrophoresis apparatus is set when detection The voltage 120V set, electric current 120mA, time 30min；Agarose gel electrophoresis figure as shown in Fig. 2, wherein M be marker, Column 1 are PCR product, and purpose product size is 1000bp or so, and cutting correct position and recycling containing purpose product glue obtains To the nucleotide sequence of SEQ ID NO.2, concrete operation step is detailed in the slim Ago-Gel DNA QIAquick Gel Extraction Kit of GENEray (Generay, Shanghai) specification.

3, the purpose product DNA of previous step purification and recovery is connected to pUCm-T carrier, reaction system is as follows:

16 DEG C of water bath with thermostatic control connections are placed on overnight by the mixing of above-mentioned system.

E.coli DH5 α competent cell is prepared according to CaCl2 method, overnight recombinant plasmid transformed will be connected to DH5 α sense By in state cell, specific steps are as follows:

(1) 10 μ L connection products, ice is added in the 100 μ LDH5 α competent cells (thawing on ice) taken out to -80 DEG C of refrigerators Bathe 30min；

(2) 42 DEG C of heat shock 90s；

(3) it is placed on rapidly on ice, continues ice bath 2min；

(4) LB liquid medium of 500 μ L non-resistants is added, 37 DEG C, 200rpm shakes bacterium 1h；

(5) 100 μ L bacterium solutions is taken to be uniformly coated on the Amp of the blue hickie screening of IPTG containing 0.1mM and 20 μ g/mL X-gal On the LB solid plate of resistance, first plate forward direction is placed, 37 DEG C of culture 1h, rear be inverted is incubated overnight.

After cultivating 16h, single bacterium colony is grown on LB solid plate, the white monoclonal colonies on picking LB solid plate, It is added in 10 μ L sterile waters, 2 μ L is taken to carry out PCR positive identification, bacterium colony PCR reaction system is as follows:

After above-mentioned system mixes, setting PCR response procedures are as follows:

3 μ LPCR amplified productions are taken to carry out agarose gel electrophoresis, whether verifying purpose DNA fragmentation is already inserted into, and detection is inserted The segment clone for entering the size of segment, and selecting suitable size is sent to gene sequencing company and (holds up the new industry biotechnology of section in Beijing Hangzhou sequencing portion, Co., Ltd) it is sequenced, the agarose gel electrophoresis figure of colony identification PCR product is as shown in figure 3, wherein M It is PCR product for marker, 1~column of column 3；Sequencing primer is M13:

M13F:CGCCAGGGTTTTCCCAGTCACGAC

M13R:CACACAGGAAACAGCTATGAC.

By the operation of front, we have been obtained wild type lipase gene (MetlipXS) overall length, are named as MetlipXS identifies sequencing to get being the full length sequence of lipase recombinant bacterium shown in SEQ ID NO.1 to sequence.

Remaining 8 μ L bacterium solution is added in LB culture medium of the 1mL containing Amp and cultivates, and 37 DEG C, 200rpm is incubated overnight, and uses Beijing Quan Shijin plasmid extraction kit EasyPure PlasmidMiniPrep Kit extracts cultured recombinant plasmid respectively and contains There is the pUCm-T plasmid of HsLIP1 target dna.

4, using EcoR I and Hind III enzyme by pET-28a plasmid and pUCm-T plasmid containing HsLIP1 target dna Double digestion is carried out to generate the cohesive terminus,cohesive termini of corresponding complementary.Digestion system is as follows:

37 DEG C of constant temperature endonuclease reaction 15min are placed on by the mixing of above-mentioned system, digestion products are subjected to Ago-Gel electricity Swimming detection.Glue in correct position cuts and recycles, and concrete operation step is detailed in slim Ago-Gel DNA QIAquick Gel Extraction Kit (Generay, Shanghai) specification.

The HsLIP1 gene recycled through same digestion is subjected to sticky complementary connection with pET-28a carrier, linked system is such as Under:

25 DEG C of water bath with thermostatic control connection reaction 20min are placed on by the mixing of above-mentioned system, obtain HsLIP1 gene and pET-28a The recombinant plasmid of carrier.

5, bacterium solution is uniformly coated on containing 0.1mM by recombinant plasmid transformed into E.coli BL21 competent cell IPTG contains on the LB solid plate for the kanamycins Kan resistance for emulsifying opaque glyceride substrate screening, just by plate first To placement, 37 DEG C of culture 1h, to absorb excessive liquid, inversion is incubated overnight, and obtaining having the bacterium colony of transparent circle is to recombinate Bacterium E.coli BL21/pET28a-HsLIP1.

There is picking the bacterium colony of transparent circle to carry out bacterium colony PCR identification, primer T7.The method of bacterium colony PCR is same to be contained The positive identification of the pUCm-T plasmid of HsLIP1 target dna.It chooses suitable size cloned sequence and serves the raw work sequencing in sea, sequencing is drawn Object is T7.The primer is purchased from Beijing Qing Kexin industry Bioisystech Co., Ltd, sequencing portion, Hangzhou, T7 sequence are as follows:

T7:TAATACGACTCACTATAGGG

T7t:GCTAGTTATTGCTCAGCGG

So far, the Escherichia coli heterogenous expression system of lipase HsLIP1, that is, E.coliBL21/pET28a/HsLIP1 fat The building of expression of enzymes system is completed.

Embodiment 2:The inducing expression of recombinant bacterium and the purifying of lipase HsLIP1:

Contain kanamycins in the ratio inoculation recombinant bacterium E.coli BL21/pET28a-HsLIP1 to 100mL of 1:100 The LB liquid medium of (50mg/mL), 37 DEG C, 200rpm culture, shaking table shaken cultivation to OD₆₀₀=0.6, IPTG is added to end Concentration is that 0.4mM is induced, and IPTG is not added as not inducing control in another set, 25 DEG C, 180rpm inducing expression 14h.It will For the bacterium solution induced overnight under the conditions of 4 DEG C, 6000rpm is centrifuged 5min, abandons supernatant.With 10mL PBS (pH 7.4) buffer by bacterium Body is resuspended, continues to be centrifuged 5min under equal conditions, abandons supernatant and collects thallus.PBS (the pH for adding 10mL to contain 2mM beta -mercaptoethanol 7.4) thallus is resuspended.Mixed liquor will be resuspended to be placed in ice-water bath, carry out clasmatosis 25min using Ultrasonic Cell Disruptor.Ultrasound is broken Broken instrument parameter setting is as follows: ultrasonic power 300W, ultrasonic time 3s, interval time 3s.In 4 DEG C, 12000rpm after the completion of broken Under the conditions of be centrifuged 15min, the supernatant of collection is crude enzyme liquid.

After the crude enzyme liquid of collection to be crossed to 0.45 μm of water system membrane filtration, purification and recovery is carried out with Ni-NTA Purification Resin, Obtain the heretofore described pure enzyme of lipase HsLIP1；Affinity chromatography purifies the whole process of destination protein in 4 DEG C of layers It is carried out in analysis cabinet, the specific steps are as follows:

(1) it is bled off after balancing nickel column 1h using the 20mM Tris-HCl buffer (pH 7.4) of 10 times of volumes；

(2) filtered crude enzyme liquid is splined in column, in conjunction with the enzyme solution after the combination bled off after 1h inside column；

(3) column first is washed with sample-loading buffer, then washes away foreign protein with the 20mM imidazole buffer of 20 times of volumes, finally with 10 250mM imidazole buffer again elutes destination protein, collects eluent.

It dialyses in 4 DEG C of refrigerators, the eluent finally collected is placed in bag filter, be placed in 20mM Tris-HCl In buffer (pH 7.4), the buffer renewed after magnetic agitation 6h is repeated 3 times；Enzyme solution in bag filter is collected, it is as pure The lipase enzyme solution of change.The detection of SDS-PAGE protein electrophoresis is carried out to the sample of protein purification, analyzes the purifying of Lipase protein Situation.

The buffer that purification process is used:

1) sample-loading buffer: Tris 20Mm (Ph7.4), NaCl 250mM；

2) imidazoles 20mM imidazole buffer: is added in sample-loading buffer to 20mM；

3) imidazoles 250mM imidazole buffer: is added in sample-loading buffer to 250mM；

Recombinant bacterium is crushed after centrifugation through IPTG inducing expression, analyzes through SDS-PAGE protein electrophoresis, have in 40kD or so One apparent protein band coincide with expected purpose band, lipase inducing expression result SDS-PAGE electrophoresis such as Fig. 4 It is shown, wherein M: protein low molecular weight label；1: not inducing bacterium solution；2: induction bacterium solution；3: induction supernatant of bacteria solution.The lipase Expressing quantity is relatively high, and the solubility expression of HsLIP1 is high.Protein purification result is as shown in figure 5, wherein M: low point of protein Son amount label；1: sample efflux；2: loading efflux；3:20mM imidazole elution；4:250mM imidazole elution.

Embodiment 3:It is measured using vigor of the para-nitrophenol method in colorimetric method to lipase:

Using p-NPP (p-nitrophenol palmitate) as substrate, lipase can hydrolyze p-nitrophenol palmitate, It generates with coloured p-nitrophenol, p-nitrophenol has characteristic absorption peak at 410nm.Absorbance and p-nitrophenol amount In good linear relationship, therefore lipase activity can be calculated according to absorbance of the reaction solution at 410nm.

Reaction mechanism is as shown in the figure:

Formulas I is the reaction equation of lipase hydrolysis p-nitrophenol palmitate

Measuring lipase activity using p-NPP as substrate, the specific operation method is as follows:

P-nitrophenyl phenol solution (6mM): weighing 0.0835g p-nitrophenol (p-NP), first molten with 95% ethyl alcohol of 1mL Solution, then 100mL is settled to distilled water.

Nitrophenol palmitate solution: 30mg p-nitrophenol palmitate (p-NPP) is weighed, is settled to isopropanol 10mL。

(1) drafting of standard curve: be separately added into centrifuge tube 10 μ L of p-NP solution, 15 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L are separately added into 50mM Tris-HCl (pH 8.0) buffer to 2mL.Then 10% trichloroacetic acid of 0.5mL is added in every pipe 10% sodium carbonate liquor of solution and 0.5mL, rocking is uniformly mixed it, and extinction is measured at 410nm using spectrophotometer Value draws enzymatic activity standard curve

(2) it measures lipase activity: 0.1mLp-NPP substrate solution and 1.8mL 50mM being added into the test tube of 15mL Tris-HCl (pH 8.0) buffer, 50 DEG C of water bath with thermostatic control 5min.0.1mL enzyme solution is added in experimental group, and rock makes its mixing immediately Uniformly, 5mL 0.5M solution of trichloroacetic acid is added immediately after 50 DEG C of water bath with thermostatic control 10min and terminates reaction, adds 5.25mL The colour developing of 0.5M sodium hydroxide solution, (blank group adds the absorption value for the p-nitrophenol that measurement enzymatic generates under 410nm wavelength Enter 0.1mL deionized water instead of 0.1mL enzyme solution).

Lipase defines the enzyme activity of p-NPP: under the conditions of 50 DEG C, pH 8.0, every 1min discharges 1 μm of ol p-nitrophenol Required enzyme amount.

Enzyme activity calculation formula:

U is sample enzyme activity (U/mL), and C is p-NP concentration (μm ol/mL), and n is extension rate, and V is the volume of reaction solution (mL), V' is the volume (mL) of enzyme solution, and T is reaction time (min).

The result shows that p-nitrophenol concentration and light absorption value are in good linear relationship, such as Fig. 6, p-nitrophenol standard Curve are as follows: y=0.1553x+0.0209, R²=0.9985

The light absorption value A measured at 410nm₄₁₀Be 1.218, according to calibration curve formula calculate the ratio work of lipase is 9.37U/mL。

Embodiment 4:The optimal reactive temperature and thermal stability analysis of lipase HsLIP1

Using the PBS of pH 8.0 as buffer, reacted in a water bath in the range of 30-80 DEG C with 10 DEG C of interval respectively 10min measures kinetic curve under ultraviolet specrophotometer 410nm wavelength.By Purification of Lipase sample distinguish 40 DEG C, 50 DEG C and After being incubated for 30,60,90,120,150,180 and 240min under the conditions of 60 DEG C, using p-NPP as substrate under optimum condition, rouge is measured Fat enzyme residual activity analyzes influence of the temperature to stability of lipase.

The reaction of lipase different temperatures value is as shown in Figure 7.Optimal reactive temperature of the lipase in the buffer of pH 7.4 It is 50 DEG C, within the temperature range of 40-60 DEG C, the vigor of lipase is maintained at 80% or more, under conditions of 80 DEG C, still protects Hold 46% enzyme activity.For the thermal stability for measuring lipase, pure enzyme solution is incubated in the buffer of pH 8.0, different time The relative surplus activity for measuring them, as a result as shown in figure 8, after 50 DEG C of processing 180min, the vigor residue 80% of lipase More than, under conditions of 65 DEG C, the ratio of lipase activity decline is very fast, and after handling 240min, the vigor of lipase only has 20% Remaining vigor, and under the conditions of 35 DEG C, the activity of lipase is had little effect.

Embodiment 5:The optimal reaction pH value of lipase HsLIP1 is analyzed

To measure lipase optimal pH, the 50mM buffer for preparing pH 5.0-10.0 is as follows: Glycine-HCl buffer (pH 5.0-6.0), Tris-HCl/NaOH buffer (pH 7.0-8.0), Tris-HCl buffer (pH 9.0), Glycine- NaOH buffer (pH 10.0).Respectively in different pH buffers, according to the system of embodiment 4, reacted in 37 DEG C of water-baths 10min, enzyme kinetics curve of the measurement lipase to substrate p-NPP under ultraviolet specrophotometer 410nm wavelength.

Influence of the different pH value to enzymatic reaction as shown in figure 9, lipase pH value be enzyme activity in 8.0 buffers most Height, pH value be 10.0 buffer in, still maintain 55% vigor, when buffer pH value 7.0 and it is following when, The vigor of lipase starts rapidly to decline, and under conditions of pH value is 5.0, relative activity only has 23%.

Embodiment 6:Lutein Specification Curve of Increasing

The chloroform that 10mg lutein standard items are dissolved in 10ml is accurately weighed, -80 DEG C save and (use in two weeks).It takes 1ml lutein chloroform soln is dissolved in 10ml normal propyl alcohol, forms the lutein normal propyl alcohol solution of 0.1mg/ml.Take 10 respectively, 20,30,40,50,100,200,300,400ul lutein normal propyl alcohol solution constant volume is in 1000ul n-hexane.

The instrumental conditions of HPLC are as follows:

Chromatographic column: Agilent HC-C18 column (4.6mm × 250mm, 5 μm)；

Mobile phase: acetonitrile, 0.45 μm of membrane filtration, ultrasonic degassing 15min；

Detection wavelength: 446nm；

Flow velocity: 0.6ml/min；

Column temperature: 28 DEG C；

Sample volume: 20 μ l.

It is measured by machine in the standard liquid series of preparation, using peak area as ordinate (Y), concentration is abscissa (X) drafting Standard curve is shown in Figure 10.Lutein concentration and peak area are in good linear relationship, regression equation are as follows: Y=77680X- 35777, R²=0.9978.

Embodiment 7:Lipase HsLIP1 is catalyzed the optimal reactive temperature analysis of lutein extract

Lutein extract solution: weighing 10mg lutein extract (total lutein content 15.8%), mixed with 8mL normal propyl alcohol It closes, is warming up to 65 DEG C, stirring is until form a kind of medicinal extract solution of uniform flow.

It is measurement lipase to the optimal reactive temperature of lutein extract, exists respectively in Tris buffer (pH 7.4) To measure lipase under 10 DEG C of interval to the enzyme kinetics curve of substrate lutein extract in the range of 30-80 DEG C. Be added 350 μ l Tris buffers (pH 7.4) into EP pipe respectively, then plus 50 μ l medicinal extract solution mix, finally plus 100 μ l enzymes, It mixes, respectively at 30,40,50,60,70,80 DEG C after water bath with thermostatic control 30min, 1ml n-hexane, whirlpool mixing is added immediately 10s is centrifuged 1min, recycling upper organic phase 1ml or so.It is surveyed with upper machine after the organic membrane filter filtering of 0.45 μm of syringe-type Fixed, the method for HPLC is the same as embodiment 6.

The reaction of lipase different temperatures value is as shown in figure 11.The optimum temperature of HsLIP1 is 50 DEG C.In pH value at 40-50 DEG C Under conditions of, HsLIP1 shows preferable activity.

Embodiment 8:Lipase HsLIP1 is catalyzed the optimal reaction pH value analysis of lutein extract

Respectively in pH 5.0-11.0 buffer, according to embodiment of the method 7, under the conditions of temperature is 50 DEG C, measurement is different Enzyme kinetics curve of the lipase to substrate lutein extract under pH.With the organic membrane filter of 0.45 μm of syringe-type Upper machine measurement after filtering.

The reaction of lipase different temperatures is as shown in figure 12.The optimal pH of HsLIP1 is 8.0, range of the pH value in 7.0-9.0 Interior, HsLIP1 shows preferable activity.We provide with temperature that as 50 DEG C, pH value is the lipase activity that measures under the conditions of 8.0 Property is 100.

Embodiment 9:Different metal ions are catalyzed the active influence of lutein extract to lipase HsLIP1

Contain 10mM different metal ions LiCl, KCl, NaCl, CuCl respectively₂、CaCl₂、BaCl₂、FeSO₄、MgSO₄、 NiSO₄、AlCl₃In buffer, system in Tris buffer (pH 8.0) at 50 DEG C according to embodiment 7 is measured in different gold Enzyme kinetics curve of the lipase to substrate lutein extract under category ion.With the organic filter membrane mistake of 0.45 μm of syringe-type Upper machine measurement after filter filtering.

Different metal ions are as shown in figure 13 to lipase hydrolysis activity influence.The condition existing for 10mM metal ion Under, wherein Na+, Ni+, Ba2+, Mg2+ and Ca2+ have promotion to make the hydrolysing activity of lipase HsLIP1 catalysis lutein extract With wherein Ni2+ is most obvious to facilitation of the fat enzyme HsLIP1 to the hydrolysing activity of lutein ester, the output increased of lutein 151%.We provide exist in no metal ion, and temperature is 50 DEG C, the lipase activity that pH value measures under conditions of being 8.0 Property is 100.

Embodiment 10:Different organic solvents are on the active influence of lipase HsLIP1

Contain in 25% different organic solvents buffer respectively, according to implementation at 50 DEG C in Tris buffer (pH 8.0) The system of example 7, the measurement enzyme kinetics curve of lipase to substrate lutein extract under different organic solvents.With Upper machine measurement after the organic membrane filter filtering of 0.45 μm of syringe-type.

Different organic solvents are as shown in figure 14 to lipase hydrolysis activity influence.The condition existing for 25% organic solvent Under, lipase is all very high to the tolerance of different organic solvents, and DMSO and ethylene glycol are catalyzed lutein to lipase HsLIP1 Ester hydrolysis activity has certain promotion.We provide exist in no organic solvent, and temperature is 50 DEG C, pH value is 8.0 Under the conditions of the lipase active that measures be 100.

The above, only presently preferred embodiments of the present invention, not to the present invention make in any form with substantial limit System, all those skilled in the art, without departing from the scope of the present invention, when using disclosed above skill Art content, and the equivalent variations for a little variation, modification and evolution made, are equivalent embodiment of the invention；Meanwhile it is all according to According to the variation, modification and evolution of substantial technological any equivalent variations to the above embodiments of the invention, this is still fallen within In the range of the technical solution of invention.

Sequence table

<110>Hangzhou Pedagogic University

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Claims

1. a kind of lipase HsLIP1 of macro gene source, it is characterised in that: the lipase HsLIP1's of the macro gene source Amino acid sequence is as shown in SEQ ID NO.1.

2. the gene of lipase HsLIP1 described in coding claim 1 a kind of.

3. gene as claimed in claim 2, it is characterised in that: the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

4. a kind of recombinant vector containing gene described in Claims 2 or 3.

5. recombinant vector according to claim 4, it is characterised in that: the recombinant vector is the gene by encoding HsLIP1 Complementary connect of viscosity is carried out with pET-28a carrier to obtain.

6. a kind of genetic engineering bacterium converted by the recombinant vector of claim 4 or 5.

7. genetic engineering bacterium according to claim 6, it is characterised in that: the genetic engineering bacterium is by recombinant plasmid transformed It is obtained into E.coliBL21 competent cell.

8. application of the gene described in claim 2 or 3 in preparation and reorganization lipase HsLIP1.

9. application of the lipase HsLIP1 described in claim 1 in lutein preparation, it is characterised in that: lipase HsLIP1 Refer to that lipase HsLIP1 discharges the anti-of lutein in the hydrolysis of catalysis lutein extract substrate preparing the application in lutein Application in answering.

10. application of the lipase HsLIP1 as claimed in claim 9 in lutein preparation, it is characterised in that: the leaf is yellow Plain medicinal extract substrate is the monoesters or dibasic acid esters that C12-C18 long chain fatty acid is formed；The preferred C12-C18 long chain fatty acids For myristic acid, oleic acid, linoleic acid or palmitinic acid.