CN110452892B - Macro-gene-derived lipase, encoding gene, vector, engineering bacterium and application of macro-gene-derived lipase in preparation of lutein - Google Patents

Abstract

The invention relates to a macro-gene-derived lipase, a coding gene, a vector, an engineering bacterium and application thereof in preparation of lutein, belonging to the technical field of genetic engineering. The amino acid sequence of the macro-gene derived lipase HsLIP1 is shown in SEQ ID NO. 1. A gene encoding lipase HsLIP 1. The nucleotide sequence of the gene is shown in SEQ ID NO. 2. A recombinant vector containing the gene. A genetically engineered bacterium obtained by transforming the recombinant vector. The application of the gene in preparing recombinant lipase HsLIP 1. The lipase HsLIP1 is applied to the preparation of lutein. The invention has the following advantages: the lipase HsLIP1 is easy for prokaryotic expression, and the lipase can be efficiently produced by adopting a recombinant vector, a strain and a preparation method; moreover, the preparation method of the enzyme is simple, convenient and efficient, is easy for mass expression, and is suitable for industrial production.

Description

Macro-gene-derived lipase, encoding gene, vector, engineering bacterium and application of macro-gene-derived lipase in preparation of lutein

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a macro-gene-derived lipase, an encoding gene, a vector, an engineering bacterium and application in preparation of lutein.

Background

Xanthophyll belongs to carotenoid, and contains a plurality of conjugated double bonds, and the conjugated double bonds not only endow color characteristics, but also have strong antioxidant function in disease prevention and treatment, such as prevention and treatment of cancers, arteriosclerosis, cataract, color spot degeneration and other diseases. The method for obtaining a large amount of lutein crystals by large-scale production is to extract, separate and purify the lutein crystals from natural resources at present. Xanthophylls in plants tend to be present in the form of mono-or diesters esterified with some C12-C18 long chain fatty acids such as myristic acid, oleic acid, linoleic acid and palmitic acid. Currently, the most widely used method is to saponify lutein ester with strong alkali and then recrystallize to obtain lutein. However, this method has several disadvantages: a large amount of strong base is used; a large amount of wastewater is generated in the process; some toxic organic solvents are used; the yield of lutein is low.

Lipases (EC 3.1.1.3) are a special class of ester bond hydrolases, defined as enzymes capable of hydrolyzing long-chain fatty acid esters, widely found in various animals, plants and microorganisms (including molds, yeasts and bacteria), which are the major sources of industrial lipases today. The lutein fatty acid ester (lutein ester) is hydrolyzed to release lutein crystals by utilizing the characteristic that lipase can catalyze the hydrolysis of ester bonds in a two-phase system (namely an oil-water interface). The lipase has mild hydrolysis condition, high efficiency, minimal use of organic solvent and simple refining steps, so the lipase has wide application space in the field of preparing lutein crystals.

The metagenomics analysis does not need to culture all microorganisms in the environment, can directly analyze the composition and the structure of a microbial community by cracking environmental cells and then separating and recovering DNA from matrixes and cell debris, so that the research and the development of over 99 percent of the genetic resources of non-culturable microorganisms originally become reality, and the potential of the environmental microbial resources is developed to the utmost extent.

Disclosure of Invention

The invention aims to disclose a macro-gene-derived lipase, a coding gene, a vector, an engineering bacterium and application in preparation of lutein.

The purpose of the invention is realized by the following technical scheme:

a macro-gene derived lipase HsLIP1, wherein the amino acid sequence of the macro-gene derived lipase HsLIP1 is shown in SEQ ID No. 1.

A gene for coding the lipase HsLIP1 in the technical scheme.

The gene according to the above technical scheme, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

A recombinant vector containing the gene of the technical scheme.

The recombinant vector according to the above technical scheme, wherein the recombinant vector is obtained by performing cohesive complementation connection of a gene encoding HsLIP1 and a pET-28a vector.

A genetically engineered bacterium obtained by transforming the recombinant vector in the technical scheme.

The genetically engineered bacterium according to the technical scheme is obtained by transforming the recombinant plasmid into E.coli BL21 competent cells.

The gene of the technical scheme is applied to the preparation of recombinant lipase HsLIP 1.

The application of the lipase HsLIP1 in the preparation of lutein in the technical scheme is that the application of the lipase HsLIP1 in the preparation of lutein refers to the application of the lipase HsLIP1 in a reaction of catalyzing hydrolysis of a lutein extract substrate to release lutein. The lutein extract substrate in the technical scheme contains lutein fatty acid ester.

The application of the lipase HsLIP1 in the preparation of lutein according to the technical scheme is characterized in that a lutein extract substrate is a monoester or diester formed by esterification of C12-C18 long-chain fatty acid. Preferably, the C12-C18 long-chain fatty acid is myristic acid, oleic acid, linoleic acid or palmitic acid.

The amino acid sequence of the macro-gene-derived lipase HsLIP1 is shown in SEQ ID No.1, namely: HGFTGWGREEMFGFKYWGGVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRGGRIHIIAHSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLEAAAAASNVPYTSQVYDFKLDQWGLRRQPGESFDHYFERLKRSPVWTSTDTARYDLSVSGAEKLDQWVQASPNTYYLSFSTERTYRGALTGNHYPELGMNAFSAVVCAPFLGPYRNPTLGIDDRWLENDGIVNTVSMNGPKRGSSDRIVPYDGTL are provided.

The lipase HsLIP1 has wide pH tolerance range and metal ion Ni²⁺、Na⁺And Mg²⁺And the organic solvents of dimethyl sulfoxide (DMSO) and glycol have certain promotion effect on the hydrolytic activity of the lipase.

Due to the specificity of the amino acid sequence, any fragment of the peptide protein containing the amino acid sequence shown in SEQ ID NO.1 or its variants, such as conservative variants, bioactive fragments or derivatives thereof, as long as the homology of the fragment of the peptide protein or the variant of the peptide protein with the aforementioned amino acid sequence is more than 95%, falls within the protection scope of the present invention. Particular such alterations may include deletions, insertions or substitutions of amino acids in the amino acid sequence; where conservative changes to a variant are made, the substituted amino acid has similar structural or chemical properties as the original amino acid, e.g., replacement of isoleucine with leucine, and the variant may also have non-conservative changes, e.g., replacement of glycine with tryptophan. The fragment, derivative or analogue of the peptide protein of the present invention refers to a peptide protein that substantially retains the same biological function or activity as the lipase of the present invention, and may be the following: (ii) (i) one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and the substituted amino acid may or may not be encoded by the genetic code; (ii) one or more of the amino acid residues is substituted with another group; (III) the mature peptide protein is fused to another compound (such as a compound that extends the half-life of the peptide protein, e.g., polyethylene glycol); (IV) peptide protein sequences formed by fusing additional amino acid sequences to the mature peptide protein (e.g., sequences used to purify the peptide protein or proprotein sequence).

The peptide protein may be a recombinant, natural or synthetic protein, may be a pure natural purified product, or may be a chemically synthesized product, or may be produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). The peptide proteins of the invention may be glycosylated depending on the host used in the recombinant production scheme. The peptide proteins of the present invention may or may not also include an initial methionine residue.

The invention also relates to a gene encoding the lipase HsLIP 1.

Specifically, the nucleotide sequence of the gene can be shown as SEQ ID NO.2, namely:

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 specificity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.2 is within the scope of the present invention as long as it has more than 70% homology with the polynucleotide. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotide may be either degenerate variants or non-degenerate variants, including substitution, deletion and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, without substantially altering the function of the peptide protein encoded thereby.

The gist of the present invention is to provide the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2, and under the condition that the amino acid sequence and the nucleotide sequence are known, the obtaining of the amino acid sequence and the nucleotide sequence, and the obtaining of related vectors and host cells are all obvious to those skilled in the art.

The invention also relates to a recombinant vector containing the gene and a genetic engineering bacterium obtained by transforming the recombinant vector.

The invention also relates to application of the gene in preparation of recombinant lipase HsLIP 1. Specifically, the application is as follows: constructing a recombinant vector containing the coding gene, transferring the recombinant vector into escherichia coli, carrying out induction culture on the obtained recombinant gene engineering bacteria, separating culture solution to obtain thallus cells containing the recombinant lipase, and crushing, separating and purifying the thallus cells to obtain the recombinant lipase HsLIP 1.

Experiments prove that the lipase HsLIP1 has high expression yield in host bacteria, such as inOver-expression in Escherichia coli, and soluble expression of HsLIP1 was high. The method is characterized in that p-nitrophenol ester is used as a substrate, the enzyme activity of HsLIP1 is highest under the conditions of pH8.0 and 50 ℃, the enzyme activity under the optimal condition is 9.37U/mL, and 87% of relative residual activity is kept after incubation for 480min in water bath at 35 ℃. 50ug of HsLIP1 catalyze the hydrolysis reaction of lutein extract (lutein fatty acid ester) for 30min to release 0.537ug of lutein, metal ion Ni²⁺、Na⁺And Mg²⁺And organic solvents dimethyl sulfoxide (DMSO) and ethylene glycol have certain promotion effect on the hydrolysis activity of HsLIP 1.

The lipase HsLIP1 has high expression, high activity and substrate specificity, and simultaneously, the lipase HsLIP1 can catalyze ester bonds in lutein extract to hydrolyze so as to release lutein and corresponding C12-C18 long-chain fatty acids such as myristic acid, oleic acid, linoleic acid, palmitic acid and the like.

The preparation method and the enzymological characterization of the high-expression and high-activity lipase HsLIP1 of the invention are as follows:

(1) and obtaining the whole length of the lipase HsLIP1 gene. Designing a lipase degenerate primer, taking metagenome DNA extracted from soil (water) samples collected in a laboratory, including Ningbo, cave head, Xiangshan, Tiantai, Taihu lake water, Zhejiang Xinchang, loess plateau near Western Ann, Shenzhen shallow bay red soil and other eight different regions of soil (water) samples as templates, cloning to obtain 1 metagenome new enzymes, and obtaining full-length lipase genes, namely lipase HsLIP1 and a lipase nucleotide full-length sequence shown by SEQIDNO.2.

(2) And constructing an expression vector system containing the target gene. Cloning the lipase gene described in step (1) into an expression vector, such as pET-28 a.

(3) Transferring the recombinant vector containing the lipase HsLIP1 gene in the step (2) into a heterologous expression host cell, such as (Escherichia coli) BL21, and culturing the recombinant host cell under the condition suitable for expression.

(4) And (3) separating and purifying the high-expression and high-activity lipase HsLIP1 from the culture of the step (3).

(5) And carrying out further enzymological characteristic characterization on the obtained lipase by the preparation method, wherein the characteristics comprise optimum temperature, heat stability, optimum pH value, heavy metal ion solvent and organic solvent tolerance and the like.

The invention also relates to application of the lipase HsLIP1 in catalyzing hydrolysis reaction of lutein extract to release lutein.

The lutein extract substrate contains some monoester or diester formed by esterification of C12-C18 long chain fatty acid such as myristic acid, oleic acid, linoleic acid and palmitic acid.

According to the invention, a conservative site primer is designed according to a lipase sequence in a database, so that a homologous fragment is amplified in a metagenome, a new enzyme (HsLIP1) containing the metagenome homologous fragment is constructed, the function characterization is carried out on the new enzyme, and the new enzyme is applied to a lipolysis reaction. P-nitrophenol ester is used as a substrate, the enzyme has the highest enzyme activity under the conditions of pH8.0 and 50 ℃, the enzyme activity under the optimal condition is 9.37U/mL, and 87 percent of relative residual activity is kept after incubation for 480min in water bath at 35 ℃. 50ug of HsLIP1 catalyze the hydrolysis reaction of the lutein extract for 30min to release 0.537ug of lutein and metal ions Ni^{2 +}、Na⁺And Mg²⁺And the organic solvents dimethyl sulfoxide (DMSO) and glycol have certain promotion effect on the hydrolysis activity of the lipase HsLIP 1.

The invention has the following beneficial effects:

the lipase HsLIP1 is easy for prokaryotic expression, and the recombinant vector, the strain and the preparation method disclosed by the patent can be adopted to efficiently produce the lipase; moreover, the preparation method of the enzyme is simple, convenient and efficient, is easy for mass expression, and is suitable for industrial production.

Description of the drawings:

1. FIG. 1 is an agarose gel electrophoresis image of metagenomic DNA.

2. FIG. 2 is an agarose gel electrophoresis image of the metagenomic PCR product.

3. FIG. 3 is an agarose gel electrophoresis image of the colony-identifying PCR product.

4. FIG. 4 is an SDS-PAGE electrophoresis of the lipase-induced expression;

5. FIG. 5 is an SDS-PAGE electrophoresis of the lipase purification results;

6. FIG. 6 is a standard curve for p-nitrophenol;

7. FIG. 7 shows the reaction of lipase at different temperatures;

8. FIG. 8 shows a lipase thermostability reaction;

9. FIG. 9 shows different pH reactions of lipase;

10. FIG. 10 is a standard curve for lutein;

11. FIG. 11 shows different temperature reactions of lipase on lutein extract;

12. FIG. 12 shows the reaction of lipase on different pH values of lutein extract;

13. FIG. 13 shows the reaction of lipase on different metal ions of lutein extract;

14. FIG. 14 shows the reaction of lipase on different organic solvents of lutein extract;

the specific implementation mode is as follows:

in order to facilitate understanding of the technical solutions of the present invention, the following further describes the present invention with reference to specific examples, but the scope of the present invention is not limited thereto:

the experimental procedures used in the present invention are conventional unless otherwise specified, and can be specifically referred to "molecular cloning: AlaboratoryManual" (Sambrookand Russell, ed.2001).

Coli DH5 α, BL21(DE3) used in the examples of the present invention were purchased from TransGen Biotech Co; the primer synthesis is completed by Shanghai Czeri bioengineering technology, Inc.; the sequencing work was performed by Biotechnology engineering (Shanghai) Inc.

Example 1:obtaining lipase HsLIP1 and constructing a recombinant strain containing the lipase HsLIP 1:

1. the method comprises the steps of collecting 10 soil (water) samples from eight different regions of wild environments of different regions in China, including Ningbo, cave head, Xiangshan, Tiantai, Taihu lake water, Xinchang Zhejiang, loess plateau near Western Ann, Shenzhen shallow bay red soil and the like, wherein the No. 3 soil sample is derived from Xiangshan soil. The soil components are complex, the extracted metagenome DNA contains brown black or black humic acid and other impurities, and the impurities have great inhibition effect on subsequent reactions such as PCR (polymerase chain reaction) and the like, so that the humic acid in the soil needs to be effectively removed. The Humic acid-Be-Gone A kit is adopted to effectively remove Humic acid in a soil sample, and the recovered and precipitated soil can Be directly used for DNA extraction. Extracting the Genomic DNA of the Soil sample by using a Soil Genomic DNA extraction kit of Soil Genomic DNA of Soil; 10,000DNA markers; metagenomic DNA (Metagenomic DNA) in columns 1-10; and DNA was recovered using an agarose gel DNA recovery kit (general, Shanghai) and stored at-20 ℃.

2. Based on the lipase gene BTL2 (accession number: ACCESSION 95309) with higher activity derived from G.thermocatenulatus and the lipase gene bacterium T6(EMBL accession number: AF429311.1) derived from Geobacillus stearothermophilus, sequence comparison analysis is carried out to search the sequence of a conserved region, and a primer is designed.

TABLE 1 PCR amplification primer sequences of the invention

Using metagenome DNA as a template, and performing PCR amplification by using high-fidelity enzyme, wherein a prepared reaction system comprises the following steps:

after the above system is mixed uniformly, the setting procedure is as follows:

after the PCR amplification is finished, carrying out agarose gel electrophoresis detection on the PCR product, wherein the voltage set by a voltage-stabilizing and current-stabilizing electrophoresis apparatus is 120V, the current is 120mA, and the time is 30 min; the agarose gel electrophoresis pattern is shown in FIG. 2, wherein M is marker, column 1 is PCR product, the size of the target product is about 1000bp, the gel containing the target product at the correct position is cut and recovered to obtain the nucleotide sequence of SEQ ID NO.2, and the specific operation steps are described in the specification of GENERAY thin agarose gel DNA recovery kit (in general, Shanghai).

3. The DNA of the target product recovered by the previous step is ligated to a pUCm-T vector in the following reaction system:

mixing the above systems, and placing in a constant temperature water bath at 16 ℃ for connection overnight.

Coli DH5 alpha competent cells were prepared according to the CaCl2 method, and overnight ligated recombinant plasmids were transformed into DH5 alpha competent cells, with the following specific procedures:

(1) add 10. mu.L of ligation product to 100. mu.LDH 5. alpha. competent cells (thawed on ice) removed from a-80 ℃ freezer and ice-bathed for 30 min;

(2) heat shock at 42 ℃ for 90 s;

(3) rapidly placing on ice, and continuously ice-cooling for 2 min;

(4) adding 500 μ L of nonresistant LB liquid medium, shaking at 37 deg.C and 200rpm for 1 h;

(5) 100 mu L of the bacterial liquid is evenly coated on an Amp resistant LB solid plate containing 0.1mM IPTG and 20 mu g/mL X-gal of blue-white spot screening, the plate is firstly placed in the positive direction, cultured for 1h at 37 ℃, and then inverted and cultured overnight.

After culturing for 16h, a single colony grows on an LB solid plate, a white monoclonal colony on the LB solid plate is picked and added into 10 mu L of sterile water, 2 mu L of the white monoclonal colony is taken for PCR positive identification, and the colony PCR reaction system is as follows:

after the system is mixed uniformly, the PCR reaction program is set as follows:

taking a 3 mu LPCR amplification product to perform agarose gel electrophoresis, verifying whether a target DNA fragment is inserted, detecting the size of the inserted fragment, selecting a fragment with a proper size to clone and sending the fragment to a gene sequencing company (Hangzhou sequencing department of Beijing Optimalaceae New Biotechnology, Inc.) for sequencing, wherein an agarose gel electrophoresis chart of a colony identification PCR product is shown in figure 3, wherein M is marker, and columns 1-3 are PCR products; the sequencing primer was M13:

M13F：CGCCAGGGTTTTCCCAGTCACGAC

M13R：CACACAGGAAACAGCTATGAC。

through the above operations, we have obtained the full length of the wild-type lipase gene (MetlipXS), named MetlipXS, and identified and sequenced to obtain the full length sequence of the lipase recombinant strain with the sequence shown as SEQ ID NO. 1.

The remaining 8. mu.L of the bacterial solution was cultured in 1mL of LB medium containing Amp at 37 ℃ and 200rpm overnight, and the cultured recombinant plasmid, i.e., pUCm-T plasmid containing target DNA of HsLIP1, was extracted with the Beijing all-purpose gold plasmid extraction Kit EasyPure plasmid MiniPrep Kit, respectively.

4. The pET-28a plasmid and the pUCm-T plasmid containing the HsLIP1 target DNA were double digested using EcoR I and Hind III enzymes to generate the corresponding complementary cohesive ends. The enzyme digestion system is as follows:

mixing the above systems, performing enzyme digestion reaction at 37 deg.C for 15min, and performing agarose gel electrophoresis detection on the enzyme digestion product. The gel in the correct position is cut and recovered, and the specific procedures are described in detail in the thin agarose gel DNA recovery kit (general, Shanghai).

The HsLIP1 gene recovered by the same enzyme digestion and pET-28a vector are subjected to viscosity complementary connection, and the connection system is as follows:

and mixing the above systems uniformly, placing the mixture in a thermostatic water bath at 25 ℃ for connection reaction for 20min to obtain the recombinant plasmid of the HsLIP1 gene and the pET-28a vector.

5. The recombinant plasmid is transformed into E.coli BL21 competent cells, the bacterial liquid is evenly coated on an LB solid plate containing 0.1mM IPTG and kanamycin Kan resistance screened by emulsified opaque glyceride substrates, the plate is placed in the positive direction, the culture is carried out for 1h at 37 ℃ to absorb excessive liquid, the culture is carried out in an inverted overnight manner, and a bacterial colony with a transparent ring, namely the recombinant bacterium E.coli BL21/pET28a-HsLIP1, is obtained.

Colonies with clearing circles were picked for colony PCR identification with primer T7. The colony PCR method was the same as the positive identification of the pUCm-T plasmid containing the target DNA of HsLIP 1. And selecting a proper size of clone fragment and sending the clone fragment to Shanghai worker for sequencing, wherein a sequencing primer is T7. The primer is purchased from Hangzhou sequencing department of New Biotechnology Limited of Beijing Ongkogaku, and the T7 sequence is as follows:

T7:TAATACGACTCACTATAGGG

T7t:GCTAGTTATTGCTCAGCGG

so far, the construction of an escherichia coli heterologous expression system of lipase HsLIP1, namely an E.coli BL21/pET28a/HsLIP1 lipase expression system is completed.

Example 2:induced expression of recombinant bacteria and purification of lipase HsLIP 1:

inoculating recombinant bacteria E.coli BL21/pET28a-HsLIP1 to 100mL LB liquid medium containing kanamycin (50mg/mL) according to the ratio of 1:100, culturing at 37 ℃ and 200rpm, and shaking and culturing to OD by a shaker₆₀₀When IPTG was added to a final concentration of 0.4mM for induction, and another set was induced without IPTG as an uninduced control at 25 ℃ and 180rpm for 14 hours. The overnight induced bacterial liquid was centrifuged at 6000rpm for 5min at 4 ℃ and the supernatant was discarded. The cells were weighed with 10mL PBS (pH7.4) bufferSuspending, continuing to centrifuge for 5min under the same condition, discarding the supernatant and collecting the thallus. The cells were resuspended in 10mL of PBS (pH7.4) containing 2 mM. beta. -mercaptoethanol. Placing the re-suspended mixed solution in ice water bath, and performing cell disruption for 25min by using an ultrasonic disruptor. The parameters of the ultrasonic crusher are set as follows: ultrasonic power 300W, ultrasonic time 3s and interval time 3 s. After the crushing is finished, centrifuging for 15min at 4 ℃ and 12000rpm, and collecting the supernatant which is the crude enzyme solution.

Filtering the collected crude enzyme solution by a 0.45 mu m water system filter membrane, and then purifying and recycling the crude enzyme solution by using Ni-NTA purification resin to obtain the lipase HsLIP1 pure enzyme in the invention; the whole process of purifying the target protein by the nickel column affinity chromatography is carried out in a chromatography cabinet at 4 ℃, and the specific steps are as follows:

(1) the nickel column was equilibrated with 10 volumes of 20mM Tris-HCl buffer (pH7.4) for 1h and then discharged;

(2) loading the filtered crude enzyme solution into a column, combining for 1h, and then discharging the combined enzyme solution in the combined column;

(3) eluting the column with a loading buffer solution, eluting with 20 times the volume of 20mM imidazole buffer solution to remove foreign proteins, eluting the target protein with 10 times of 250mM imidazole buffer solution, and collecting the eluate.

Dialyzing in refrigerator at 4 deg.C, placing the eluate in dialysis bag, placing in 20mM Tris-HCl buffer solution (pH7.4), magnetically stirring for 6 hr, replacing with new buffer solution, and repeating for 3 times; collecting enzyme liquid in the dialysis bag, namely purified lipase enzyme liquid. And (3) carrying out SDS-PAGE protein electrophoresis detection on the protein purified samples, and analyzing the purification condition of the lipase protein.

Buffer used for purification:

1) loading buffer solution: tris 20Mm (Ph7.4), NaCl 250 mM;

2)20mM imidazole buffer: imidazole is added to the loading buffer to 20 mM;

3)250mM imidazole buffer: imidazole is added to the loading buffer to 250 mM;

the recombinant bacteria are induced to express by IPTG, after crushing and centrifugation, an obvious protein band is formed around 40kD through SDS-PAGE protein electrophoresis analysis, the protein band is matched with an expected band, and an SDS-PAGE electrophoresis image of a lipase induced expression result is shown in figure 4, wherein M: protein low molecular weight markers; 1: non-induced bacteria liquid; 2: inducing bacterial liquid; 3: inducing bacterial liquid supernatant. The lipase has high protein expression level, and high soluble expression of HsLIP 1. The protein purification results are shown in fig. 5, where M: protein low molecular weight markers; 1: a sample effluent; 2: loading and discharging liquid; 3: 20mM imidazole eluent; 4: 250mM imidazole eluent.

Example 3:the lipase activity was determined by the p-nitrophenol method in a colorimetric assay:

with p-NPP (p-nitrophenol palmitate) as a substrate, lipase can hydrolyze p-nitrophenol palmitate to generate colored p-nitrophenol, and the p-nitrophenol has a characteristic absorption peak at 410 nm. The absorbance and the p-nitrophenol amount have good linear relation, so that the lipase activity can be calculated according to the absorbance of the reaction liquid at 410 nm.

The reaction mechanism is shown in the figure:

formula I is a reaction equation of lipase hydrolyzing p-nitrophenol palmitate

The specific operation method for determining the lipase activity by taking p-NPP as a substrate is as follows:

p-nitrophenol solution (6 mM): 0.0835g of p-nitrophenol (p-NP) were weighed out, dissolved in 1mL of 95% ethanol and made up to 100mL with distilled water.

Nitrophenol palmitate solution: 30mg of p-nitrophenol palmitate (p-NPP) was weighed out and made up to 10mL with isopropanol.

(1) Drawing a standard curve: 10. mu.L, 15. mu.L, 20. mu.L, 30. mu.L, 40. mu.L and 50. mu.L of p-NP solution were added to the centrifuge tube, and 50mM Tris-HCl (pH 8.0) buffer was added to the centrifuge tube to 2 mL. Then, 0.5mL of 10% trichloroacetic acid solution and 0.5mL of 10% sodium carbonate solution were added to each tube, mixed well by shaking, and the absorbance at 410nm was measured using a spectrophotometer to draw a standard curve for the enzyme activity.

(2) And (3) measuring lipase activity: to a 15mL test tube were added 0.1mLp-NPP substrate solution and 1.8mL 50mM Tris-HCl (pH 8.0) buffer, and the mixture was incubated at 50 ℃ for 5 min. 0.1mL of enzyme solution is added into the experimental group, the mixture is immediately shaken to be uniformly mixed, 5mL of 0.5M trichloroacetic acid solution is immediately added after 10min of constant-temperature water bath at 50 ℃ to stop the reaction, 5.25mL of 0.5M sodium hydroxide solution is added to carry out color development, and the absorption value of the p-nitrophenol generated by enzyme catalysis is measured at the wavelength of 410nm (0.1 mL of deionized water is added into the blank group to replace 0.1mL of enzyme solution).

The enzyme activity of lipase on p-NPP is defined as the amount of enzyme required to release 1. mu. mol of p-nitrophenol per 1min at 50 ℃ and pH 8.0.

The enzyme activity calculation formula is as follows:

u is sample enzyme activity (U/mL), C is p-NP concentration (mu mol/mL), n is dilution factor, V is volume of reaction solution (mL), V' is volume of enzyme solution (mL), and T is reaction time (min).

The result shows that the concentration of the p-nitrophenol and the light absorption value have a good linear relationship, and as shown in fig. 6, the standard curve of the p-nitrophenol is as follows: 0.1553x +0.0209, R²＝0.9985

Absorbance A measured at 410nm₄₁₀The specific activity was 1.218, and the lipase activity was 9.37U/mL as calculated according to the standard curve equation.

Example 4:optimum reaction temperature and thermal stability analysis of Lipase HsLIP1

PBS with pH of 8.0 is used as buffer solution, reaction is carried out in water bath for 10min at the interval of 10 ℃ within the range of 30-80 ℃, and the kinetic curve is determined by an ultraviolet spectrophotometer under the wavelength of 410 nm. And (3) incubating the purified lipase sample for 30, 60, 90, 120, 150, 180 and 240min at 40 ℃, 50 ℃ and 60 ℃ respectively, determining the residual activity of the lipase by using p-NPP as a substrate under the optimal condition, and analyzing the influence of temperature on the stability of the lipase.

The different temperature values of the lipase are reflected in figure 7. The optimum reaction temperature of the lipase in a buffer solution with the pH value of 7.4 is 50 ℃, the activity of the lipase is kept above 80 percent in the temperature range of 40-60 ℃, and the activity of the lipase is still kept 46 percent under the condition of 80 ℃. To determine the thermostability of lipase, the pure enzyme solution was incubated in a buffer solution with pH8.0 and their relative residual activities were determined at different times, and the results are shown in FIG. 8. the activity of lipase remained more than 80% after treatment at 50 ℃ for 180min, the activity of lipase decreased faster at 65 ℃, the activity of lipase only remained 20% after treatment for 240min, and the activity of lipase had little effect at 35 ℃.

Example 5:pH analysis of Lipase HsLIP1 optimum reaction

To determine the optimum pH for the lipase, 50mM buffer pH 5.0-10.0 was prepared 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 the example 4, the reaction is carried out for 10min in water bath at 37 ℃, and the enzymatic reaction kinetic curve of the lipase to the substrate p-NPP is measured under the wavelength of 410nm by an ultraviolet spectrophotometer.

The effect of different pH values on the enzymatic reaction is shown in FIG. 9, in which the lipase activity is highest in the buffer solution with pH value of 8.0, and still maintains 55% of the activity in the buffer solution with pH value of 10.0, and when the pH value of the buffer solution is 7.0 or below, the lipase activity begins to decrease rapidly, and under the condition of pH value of 5.0, the relative activity is only 23%.

Example 6:drawing a lutein standard curve

10mg of lutein standard was accurately weighed out and dissolved in 10ml of chloroform and stored at-80 deg.C (for use over two weeks). 1ml of lutein trichloromethane solution is dissolved in 10ml of n-propanol to form a lutein n-propanol solution of 0.1 mg/ml. 10, 20, 30, 40, 50, 100, 200, 300 and 400ul of lutein n-propanol solution are respectively taken and added into 1000ul of n-hexane.

The instrumental analysis conditions for HPLC were as follows:

a chromatographic column: agilent HC-C18 column (4.6 mm. times.250 mm,5 μm);

mobile phase: filtering with 0.45 μm filter membrane, and ultrasonic degassing for 15 min;

detection wavelength: 446 nm;

flow rate: 0.6 ml/min;

column temperature: 28 ℃;

sample introduction amount: 20 μ l.

The prepared standard solution series is subjected to on-machine determination, and a standard curve is drawn by taking the peak area as the ordinate (Y) and the concentration as the abscissa (X), and is shown in figure 10. The lutein concentration and the peak area have a good linear relation, and the regression equation is as follows: 77680X-35777, R²＝0.9978。

Example 7:optimum reaction temperature analysis of lutein extract catalyzed by lipase HsLIP1

Lutein extract solution: 10mg of lutein extract (total lutein content 15.8%) is weighed and mixed with 8mL of n-propanol, heated to 65 ℃ and stirred until a uniform fluid extract solution is formed.

To determine the optimum reaction temperature of lipase for lutein extract, the enzymatic reaction kinetics curves of lipase for substrate lutein extract were determined in Tris buffer (pH7.4) at intervals of 10 ℃ in the range of 30-80 ℃ respectively. Respectively adding 350 μ l Tris buffer solution (pH7.4) into an EP tube, adding 50 μ l extract solution, mixing, finally adding 100 μ l enzyme, mixing, respectively performing constant temperature water bath at 30, 40, 50, 60, 70 and 80 deg.C for 30min, immediately adding 1ml n-hexane, vortex mixing for 10s, centrifuging for 1min, and recovering 1ml upper organic phase. The resulting mixture was filtered through a 0.45-. mu.m syringe-type organic membrane filter and then subjected to the HPLC method as in example 6.

The different temperature values of the lipase are reflected in FIG. 11. The optimum temperature for HsLIP1 was 50 ℃. HsLIP1 showed good activity at pH 40-50 deg.C.

Example 8:optimum reaction pH value analysis of lutein extract catalyzed by lipase HsLIP1

The kinetics of the enzymatic reaction of lipase on the substrate lutein extract at different pH was determined in pH 5.0-11.0 buffer at 50 deg.C according to method example 7. Filtering with 0.45 μm syringe type organic filter membrane filter, and testing on machine.

The reaction of lipase at different temperatures is shown in FIG. 12. HsLIP1 has an optimum pH of 8.0 and a pH in the range of 7.0-9.0, and HsLIP1 shows a good activity. We stipulate that the lipase activity measured at a temperature of 50 ℃ and a pH of 8.0 is 100.

Example 9:influence of different metal ions on activity of lutein extract catalyzed by lipase HsLIP1

Respectively contains 10mM different metal ions LiCl, KCl, NaCl and CuCl₂、CaCl₂、BaCl₂、FeSO₄、MgSO₄、NiSO₄、AlCl₃The kinetics of the enzymatic reaction of lipase on the substrate lutein extract at different metal ions was determined in buffer, in Tris buffer (pH 8.0), at 50 ℃ following the system of example 7. Filtering with 0.45 μm syringe type organic filter membrane filter, and testing on machine.

The effect of different metal ions on the lipase hydrolysis activity is shown in FIG. 13. Under the condition of the existence of 10mM metal ions, Na +, Ni +, Ba2+, Mg2+ and Ca2+ have the promotion effect on the hydrolysis activity of lutein extract catalyzed by lipase HsLIP1, wherein the promotion effect of Ni2+ on the hydrolysis activity of lutein ester by lipase HsLIP1 is most obvious, and the yield of lutein is improved by 151%. We specify a lipase activity of 100 measured in the absence of metal ions at a temperature of 50 ℃ and a pH of 8.0.

Example 10:effect of different organic solvents on Lipase HsLIP1 Activity

The kinetics of the enzymatic reaction of lipase on the substrate lutein extract in different organic solvents were determined in 25% different organic solvent buffers at 50 ℃ in Tris buffer (pH 8.0) according to the system of example 7. Filtering with 0.45 μm syringe type organic filter membrane filter, and testing on machine.

The effect of different organic solvents on lipase hydrolytic activity is shown in figure 14. Under the condition of the existence of 25% of organic solvent, the lipase has higher tolerance to different organic solvents, and DMSO and glycol have certain promotion effect on the hydrolysis activity of lutein ester catalyzed by lipase HsLIP 1. We stipulate that the lipase activity measured in the absence of an organic solvent at a temperature of 50 ℃ and a pH of 8.0 is 100.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims; meanwhile, any equivalent changes, modifications and variations of the above embodiments according to the essential technology of the present invention are within the scope of the technical solution of the present invention.

Sequence listing

<110> university of teachers in Hangzhou

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Claims (11)

1. A macrogene-derived lipase, HsLIP1, characterized by: the amino acid sequence of the macro-gene derived lipase HsLIP1 is shown in SEQ ID NO. 1.

2. A gene encoding the lipase HsLIP1 of claim 1.

3. The gene of claim 2, wherein: the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

4. A recombinant vector comprising the gene of claim 2 or 3.

5. The recombinant vector according to claim 4, wherein: the recombinant vector is obtained by performing cohesive complementary connection on a gene coding HsLIP1 and a pET-28a vector.

6. A genetically engineered bacterium transformed with the recombinant vector of claim 4 or 5.

7. The genetically engineered bacterium of claim 6, wherein: the genetically engineered bacterium is transformed from a recombinant vector toE.coliBL21 competent cells.

8. Use of the gene of claim 2 or 3 for the preparation of recombinant lipase HsLIP 1.

9. The use of the lipase HsLIP1 of claim 1 in the preparation of lutein, wherein: the application of lipase HsLIP1 in preparing lutein refers to the application of lipase HsLIP1 in catalyzing the hydrolysis of lutein extract substrate to release lutein.

10. The use of the lipase HsLIP1 in the preparation of lutein according to claim 9, wherein: the lutein extract substrate is monoester or diester formed by esterification of C12-C18 long-chain fatty acid.

11. The use of the lipase HsLIP1 in the preparation of lutein according to claim 10, wherein: the C12-C18 long-chain fatty acid is myristic acid, oleic acid, linoleic acid or palmitic acid.

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