CN112266906B

CN112266906B - High-temperature-resistant acid lipase LIP and gene and application thereof

Info

Publication number: CN112266906B
Application number: CN202011217199.5A
Authority: CN
Inventors: 吴培均; 罗建杰; 李富伟; 李兆勇
Original assignee: Beijing Crvab Bio Tech Co ltd
Current assignee: Beijing Crvab Bio Tech Co ltd
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2022-04-12
Anticipated expiration: 2040-11-04
Also published as: CN112266906A

Abstract

The invention relates to the field of genetic engineering, in particular to high-temperature-resistant acid lipase LIP, a gene thereof and application thereof. The invention provides a lipase LIP derived from Aspergillus niger CICC 2103, the amino acid sequence of which is shown in SEQ ID NO.1 or 2, and provides a coding gene lipL75 for coding the lipase. The lipase of the invention has the following properties: the optimum pH value is 4.5, the optimum temperature is 55 ℃, and the thermal stability is better under the condition of 70 ℃. The high-temperature resistant acid lipase LIP provided by the invention is used as a novel enzyme preparation and can be applied to the industries of feed, food, brewing, baking and the like.

Description

High-temperature-resistant acid lipase LIP and gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to high-temperature-resistant acid lipase LIP, a gene thereof, a recombinant vector containing the gene and application.

Background

Lipase EC 31.1.3, also called a glyceride hydrolase, catalyzes the hydrolysis of triglycerides to fatty acids and diglycerides or monoglycerides or glycerol. Microbial lipases, also known as triacylglycerol acylhydrolases, refer to enzymes that break down or synthesize the triglyceride ester bonds formed by higher fatty acids and glycerol. Lipases are a special class of ester bond hydrolases, which act only on heterogeneous systems, i.e. only on the oil-water interface, and have a significant catalytic effect on substrate hydrolysis only when the substrate is in a particulate, small polymeric dispersion state or in emulsified particles. The optimal conditions for lipase action are strongly dependent on the source of the enzyme. The optimum conditions for the microbial lipase are pH7.0, temperature 37 deg.C, and can be stimulated by calcium ion and low concentration bile salt. The optimal condition of the animal lipase is pH9.0, temperature 37 deg.C, and can be activated by calcium oleate, albumin and bile salt. Lipases are widely found in nature, and almost all animal organs contain lipases, which are also found in many plants, bacteria and fungi. There are three methods for producing lipases: extraction, chemical synthesis and fermentation. The chemical synthesis method is still in research stage at present due to the limitation of conditions such as experimental technology, and the extraction method is greatly limited due to less organs and tissues of animals and plants, while the microbial fermentation method is the main method for producing lipase.

Microbial lipases generally do not require cofactors for their activity, but divalent cations, such as calcium ions, often stimulate enzyme activity because calcium ions promote the formation of long-chain fatty acid calcium salts. In addition, the activity of lipase is also strongly inhibited by heavy metal ions such as cobalt ions, nickel ions and tin ions, but slightly inhibited by zinc ions and magnesium ions. Gupta et al (2001) classify microbial lipases into three major classes, namely non-specific lipases, specific selective lipases and fatty acid-specific lipases based on substrate specificity. Non-specific lipofectin acts on triglyceride randomly, and triglyceride is hydrolyzed into lipofectin and glycerin. In contrast, specific lipases are 1.3-position specific lipoidases, which hydrolyze only the triglyceride C1 and C3 ester bonds, thereby producing free fatty acids, 1.2-diglycerides, 2, 3-diglycerides and 2-monoglycerides. Bacterial extracellular lipases are specific lipases. The third class of lipases comprises fatty acid-specific lipases which exhibit a pronounced preferential hydrolysis of fatty acids.

Although many researchers have achieved satisfactory results in obtaining good quality lipases through microbial screening. However, the production of lipase by genetically engineered bacteria will dominate in the future, since engineered bacteria will enable the production of suitable lipase with significant properties for specific application areas. Although the lipase is sold in a commodity, the application of the lipase is limited due to low yield and high production cost. Therefore, the development of lipases with high enzyme activity, excellent properties and low production cost is an urgent need.

Disclosure of Invention

The invention aims to provide a high-temperature-resistant acid lipase which can be efficiently applied.

It is still another object of the present invention to provide a gene encoding the above high temperature resistant acid lipase.

Another object of the present invention is to provide a recombinant vector comprising the above gene.

Another object of the present invention is to provide a recombinant strain comprising the above gene.

Another purpose of the invention is to provide a genetic engineering method for preparing the high-temperature-resistant acid lipase.

The invention also aims to provide application of the high-temperature-resistant acid lipase.

The invention clones a new lipase gene from Aspergillus niger CICC 2103, the coded lipase has higher enzyme activity under acidic and neutral conditions, the optimum temperature is 55 ℃, the optimum pH is 4.5, and the lipase has better thermal stability under the condition of 70 ℃.

The invention provides a high-temperature resistant acid lipase LIP, the amino acid sequence of which is shown in SEQ ID NO. 1.

SEQ ID NO.1：

Wherein, the enzyme gene codes 287 amino acids and a stop codon, and the N end 19 amino acids are the predicted signal peptide sequence 'MFSGRFGVLL TALAALSAA' (SEQ ID NO. 3). Thus, the theoretical molecular weight of mature lipase LIP is 29.5kDa, and its amino acid sequence is shown in SEQ ID NO. 2:

the lipase LIP of the invention has better thermal stability, higher activity in a slightly acidic and neutral range, the optimum reaction pH value of 4.5 and the optimum reaction temperature of 55 ℃.

The present invention provides a gene encoding the lipase lipL 75. Specifically, the base sequence of the gene is shown as SEQ ID NO. 4:

the invention separates and clones lipase gene LipL75 by PCR method, and the whole sequence analysis result shows that the total length of Lipase LIP structural gene LipL75 is 873 bp. Wherein, the base sequence of the signal peptide is as follows: atgttttctg gtagatttgg tgttttgttg actgctttgg ctgctttgtc tgctgct (SEQ ID NO. 6). Therefore, the mature lipase LIP gene is 816bp in length, and the base sequence of the mature lipase LIP gene is shown as SEQ ID NO. 5:

BLAST alignments of the LipL75 sequence and the deduced amino acid sequence of the lipase gene, which has 72% identity to the lipase amino acid sequence from Aspergillus niger CBS 513.88, were performed in GenBank. Description the lipase LIP of the present invention is a novel lipase.

The invention also provides a recombinant vector containing the lipase gene LipL75, preferably pPIC-LipL 75. The lipase gene of the present invention is inserted between suitable restriction enzyme sites of an expression vector after signal peptides are removed, so that the nucleotide sequence is operably linked with an expression regulatory sequence. As a most preferred embodiment of the present invention, it is preferred that the lipase gene of the present invention is inserted between the EcoR I and Not I restriction sites on the plasmid pPIC9 so that the nucleotide sequence is located downstream of and under the control of the AOX1 promoter to give a recombinant lipase expression plasmid pPIC9-LipL 75.

The invention also provides a recombinant strain containing the lipase gene LipL75, wherein the recombinant strain is preferably escherichia coli, yeast (pichia pastoris, rhodosporidium toruloides and the like), bacillus or lactobacillus containing the lipase gene LipL75, and further preferably can be a recombinant pichia pastoris strain GS115/LipL 75.

The invention also provides a method for preparing lipase LIP, which comprises the following steps:

1) transforming host cells by using the recombinant vector to obtain a recombinant strain;

2) culturing the recombinant strain, and inducing the expression of the recombinant lipase;

3) recovering and purifying the expressed lipase LIP.

Preferably, the host cell is a Pichia cell, a beer yeast cell or a red wintersweet yeast cell, and the recombinant expression plasmid is transformed into a Pichia cell (Pichia pastoris) GS115 to obtain a recombinant strain GS115/LipL 75.

The invention also provides applications of the high-temperature-resistant acid lipase LIP, the high-temperature-resistant acid lipase gene LipL75, the recombinant vector containing the high-temperature-resistant acid lipase gene LipL75 and the recombinant strain, and particularly the applications in the fields of feed, food, washing, brewing, baking and the like.

The invention firstly aims to overcome the defects of the prior art and provide a novel lipase which has excellent properties and is suitable for being applied to feeds, foods, washing, brewing and baking. The lipase disclosed by the invention has the most suitable pH value of 4.5, belongs to acid lipase, is more suitable for being used in animal feed, and has higher enzyme activity at the pH value of 4-7; the pH stability is good. Has better thermal stability at 70 ℃, solves the problem of poor heat resistance of the prior lipase, and has better thermal stability and storage stability.

Drawings

FIG. 1 shows the optimum pH of the recombinant lipase of the present invention.

FIG. 2 shows the pH stability of the recombinant lipase of the present invention.

FIG. 3 shows the optimum temperature of the recombinant lipase of the present invention.

FIG. 4 shows the thermostability of the recombinant lipase of the present invention.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier: the new lipase LIP is separated and obtained from Aspergillus niger CICC 2103 (purchased from China center for culture Collection of Industrial microorganisms, No. 32 Tokyo Luo, Tokyo, Chaoyang, Beijing, China research institute for food fermentation industry, 100027) with the preservation number of CICC No. 2103. The pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents: the endonuclease was purchased from TaKaRa, and the ligase was purchased from Invitrogen. The soluble starch was purchased from Sigma, and all others were made from domestic reagents (all available from general Biochemical Co.).

3. Culture medium:

(1) the Aspergillus niger CICC 2103 culture medium is a PDB culture medium: 1000mL of potato juice, 10g of glucose and 25g of agar, and the pH is natural.

(2) Coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH Natural).

(3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V).

(4) BMMY medium: the components were identical to BMGY, pH4.0, except that 0.5% methanol was used instead of glycerol.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (fourth edition) J. SammBruker, or according to the kit and product instructions.

EXAMPLE 1 enrichment culture of Aspergillus niger CICC 2103

Culturing Aspergillus niger in enrichment medium (NH)₄)₂SO₄ 5g/L，KH₂PO₄ 1g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 0.01g/L，CaCl₂0.2g/L, 20g/L soluble starch, pH 4.5), diluted conventionally and plated onto enzyme-producing medium ((NH)₄)₂SO₄ 5g/L，KH₂PO₄ 1g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 0.01g/L，CaCl₂0.2g/L, 2% of soluble starch, 1.5% of agarose and pH 4.5), culturing for 3-4 d at 30 ℃, selecting colonies, streaking and separating on a culture medium plate, and repeating the streaking and separating process for 3 times to purify the strain.

Example 2 cloning of the Gene encoding the Lipase of Aspergillus niger CICC 2103

Extracting Aspergillus niger CICC 2103 genome DNA:

filtering mycelium of liquid culture for 3 days with sterile filter paper, placing into mortar, grinding with liquid nitrogen, adding 5mL lysate, placing the ground solution into 50mL centrifuge tube, cracking in 65 deg.C water bath for 120min, mixing once every 10min, and centrifuging at 4 deg.C at 12000rpm for 5 min. Extracting the supernatant in phenol/chloroform to remove impurity proteins, adding equal volume of isopropanol into the supernatant, standing at-20 deg.C for 20min, and centrifuging at 12000rpm at 4 deg.C for 10 min. Discarding the supernatant, washing the precipitate with 70% ethanol twice, vacuum drying, adding appropriate amount of TE to dissolve, and standing at-20 deg.C for use.

PCR amplification was performed using Aspergillus niger CICC 2103 total DNA as template. The PCR reaction parameters are as follows: denaturation at 95 deg.C for 5 min; then denaturation at 94 ℃ for 30sec, annealing at 45 ℃ for 30sec, extension at 72 ℃ for 1min, and heat preservation at 72 ℃ for 10min after 30 cycles. The fragment of the gene was recovered and ligated with pEASY-T3 vector for sequencing.

Example 3 preparation of recombinant Lipase

Carrying out double enzyme digestion (EcoR I + Not I) on the expression vector pPIC9, carrying out double enzyme digestion (EcoR I + Not I) on a gene LipL75 coding lipase, cutting out a gene fragment coding mature lipase, connecting the gene fragment with the expression vector pPIC9, obtaining a recombinant plasmid pPIC-LipL75 containing an Aspergillus niger CICC 2103 lipase gene LipL75, transforming Pichia pastoris GS115, and obtaining a recombinant Pichia pastoris strain GS115/LipL 75.

The GS115 strain containing the recombinant plasmid was inoculated into 300mL of BMGY culture medium, subjected to shaking culture at 30 ℃ and 250rpm for 48 hours, and centrifuged to collect the cells. Then resuspended in 150mL BMMY medium and cultured with shaking at 30 ℃ and 250 rpm. After 72h induction, the supernatant was collected by centrifugation. The lipase activity was measured. The expression level of the recombinant lipase was 9600U/mL. The recombinant lipase is expressed in pichia pastoris, and the specific activity of the recombinant lipase is 6890U/mg.

EXAMPLE 4 Properties of recombinant Lipase LIP

1. The optimum pH and pH stability of the recombinant lipase LIP were determined as follows:

the recombinant lipase of example 3 was subjected to enzymatic reactions at different pH to determine its optimum pH. The lipase activity of the substrate is measured by using 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution with different pH values at 55 ℃. The results (FIG. 1) show that recombinant LIP has an optimum pH of 4.5 and a relative enzyme activity of 50% or more at pH4.0 to 5.0. The lipase was treated in the above-mentioned buffers with various pH values at 37 ℃ for 60min, and then the enzyme activity was measured in a buffer system at pH4.5 at 55 ℃ to examine the pH resistance of the enzyme. The results (FIG. 2) show that the lipase is stable at pH 4.0-7.0, and the residual enzyme activity after 60min treatment in the pH range is more than 80%, which indicates that the lipase has better pH stability in the acidic and neutral ranges.

2. The optimum temperature and thermal stability of lipase were determined as follows:

the optimum temperature of lipase was determined by performing enzymatic reactions in a citrate-disodium phosphate buffer (pH 4.5) buffer system at various temperatures. The temperature tolerance is determined by treating lipase at different temperatures for different times and then determining the enzyme activity at 55 ℃. The results of the measurement of the optimum temperature for the enzyme reaction (FIG. 3) showed that the optimum temperature was 55 ℃. The enzyme thermostability test showed (FIG. 4) that LIP has good thermostability, and can maintain more than 80% of enzyme activity after being incubated at 70 ℃ for 2 h.

3. The effect of different metal ion chemical reagents on the activity of LIP enzyme was determined as follows:

adding different metal ions and chemical reagents with different concentrations into an enzymatic reaction system to research the enzyme activity of the enzymatic reaction systemSexual influence, final concentrations of the various species were 1 and 5 mmol/L. The enzyme activity was measured at 55 ℃ and pH 4.5. The results show that the activity of most of the ion and chemical agents is not obviously changed at the concentration of 1mmol, and only SDS weakly inhibits the activity. When Cu²⁺，Ag⁺And when the concentration of beta-mercaptoethanol is 5mmol, LIP enzyme activity can be partially inhibited, and the activity is completely lost by 5mmol SDS.

4. The lipase anti-pepsin and trypsin abilities were determined as follows:

0.1mg/mL pepsin was prepared using pH2.0 KCl-HCl buffer, and 0.1mg/mL trypsin was prepared using pH7.0 Tris-HCl buffer. 0.5mL of pepsin is added into 0.5mL of purified enzyme solution diluted by pH2.0 KCl-HCl buffer solution, 0.5mL of trypsin is added into 0.5mL of purified enzyme solution diluted by pH7.0 Tris-HCl buffer solution and mixed, protease/lipase (w/w) is approximately equal to 0.1, the sample is taken after the temperature is kept at 37 ℃ for 180min, and the enzyme activity is measured under the conditions of pH4.5 and 55 ℃. Experimental results show that after lipase LIP is treated by pepsin and trypsin for 180min, the enzyme activity of LIP treated by trypsin is improved by 19% compared with that before treatment, and the lipase activity of LIP treated by pepsin is improved by 30% compared with that before treatment.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Beijing Kogyo Bo Biotech Co., Ltd

<120> high-temperature-resistant acid lipase LIP, gene thereof and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 287

<212> PRT

<213> Aspergillus niger CICC 2103(Aspergillus niger)

<400> 1

Met Phe Ser Gly Arg Phe Gly Val Leu Leu Thr Ala Leu Ala Ala Leu

1 5 10 15

Ser Ala Ala Ala Pro Thr Pro Ser Val Ser Leu Thr Val Arg Thr Ser

20 25 30

Thr Leu Asp Glu Leu Gln Leu Phe Ser Gln Trp Ser Ala Ala Trp Tyr

35 40 45

Cys Ser Asn Asn Ile Asp Ser Asp Asp Ser Asn Val Thr Cys Ala Cys

50 55 60

Pro Ser Val Thr Cys Asp Glu Glu Ala Ser Thr Lys Met Leu Leu Glu

65 70 75 80

Phe Asp Leu Asn Phe Gly Thr Asn Gly Thr Ala Gly Phe Leu Ala Ala

85 90 95

Asp Asn Thr Asn Lys Arg Leu Phe Arg Gly Val Val Ala Ser Ser Thr

100 105 110

Ile Lys Ala Asp Asn Trp Ile Leu Asp Phe Asn Leu Gln Asp Asp Ile

115 120 125

Asp Leu Cys Thr Gly Cys Lys Val His Thr Gly Phe Trp Lys Ala Trp

130 135 140

Glu Ala Ala Ala Asp Asn Leu Thr Ser Lys Ile Lys Ser Ala Met Ser

145 150 155 160

Thr Tyr Ser Gly Tyr Thr Leu Tyr Phe Thr Gly His Ser Leu Gly Gly

165 170 175

Ala Leu Ala Thr Leu Gly Ala Thr Val Leu Arg Asn Asp Gly Tyr Ser

180 185 190

Val Glu Leu Tyr Thr Tyr Gly Cys Pro Arg Val Gly Asn Tyr Ala Leu

195 200 205

Ala Glu His Ile Thr Ser Gly Ala Ser Gln Gly Asn Phe Arg Val Leu

210 215 220

Asn Thr His Asp Ile Val Pro Arg Leu Pro Asp Phe Pro Met Gly Phe

225 230 235 240

Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Asp Ile Glu Glu Gly Ile

245 250 255

Leu Ile Asn Ser Thr Ala Gly Asn Ala Ala Gly Glu Thr Val Leu Val

260 265 270

Leu Leu Trp Ala His Tyr Phe Phe Ala Ile Cys Leu Ser Glu Leu

275 280 285

<210> 2

<211> 268

<212> PRT

<213> Aspergillus niger CICC 2103(Aspergillus niger)

<400> 2

Ala Pro Thr Pro Ser Val Ser Leu Thr Val Arg Thr Ser Thr Leu Asp

1 5 10 15

Glu Leu Gln Leu Phe Ser Gln Trp Ser Ala Ala Trp Tyr Cys Ser Asn

20 25 30

Asn Ile Asp Ser Asp Asp Ser Asn Val Thr Cys Ala Cys Pro Ser Val

35 40 45

Thr Cys Asp Glu Glu Ala Ser Thr Lys Met Leu Leu Glu Phe Asp Leu

50 55 60

Asn Phe Gly Thr Asn Gly Thr Ala Gly Phe Leu Ala Ala Asp Asn Thr

65 70 75 80

Asn Lys Arg Leu Phe Arg Gly Val Val Ala Ser Ser Thr Ile Lys Ala

85 90 95

Asp Asn Trp Ile Leu Asp Phe Asn Leu Gln Asp Asp Ile Asp Leu Cys

100 105 110

Thr Gly Cys Lys Val His Thr Gly Phe Trp Lys Ala Trp Glu Ala Ala

115 120 125

Ala Asp Asn Leu Thr Ser Lys Ile Lys Ser Ala Met Ser Thr Tyr Ser

130 135 140

Gly Tyr Thr Leu Tyr Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala

145 150 155 160

Thr Leu Gly Ala Thr Val Leu Arg Asn Asp Gly Tyr Ser Val Glu Leu

165 170 175

Tyr Thr Tyr Gly Cys Pro Arg Val Gly Asn Tyr Ala Leu Ala Glu His

180 185 190

Ile Thr Ser Gly Ala Ser Gln Gly Asn Phe Arg Val Leu Asn Thr His

195 200 205

Asp Ile Val Pro Arg Leu Pro Asp Phe Pro Met Gly Phe Ser Gln Pro

210 215 220

Ser Pro Glu Tyr Trp Ile Thr Asp Ile Glu Glu Gly Ile Leu Ile Asn

225 230 235 240

Ser Thr Ala Gly Asn Ala Ala Gly Glu Thr Val Leu Val Leu Leu Trp

245 250 255

Ala His Tyr Phe Phe Ala Ile Cys Leu Ser Glu Leu

260 265

<210> 3

<211> 19

<212> PRT

<213> Aspergillus niger CICC 2103(Aspergillus niger)

<400> 3

Met Phe Ser Gly Arg Phe Gly Val Leu Leu Thr Ala Leu Ala Ala Leu

1 5 10 15

Ser Ala Ala

<210> 4

<211> 873

<212> DNA

<213> Aspergillus niger CICC 2103(Aspergillus niger)

<400> 4

atgttttctg gtagatttgg tgttttgttg actgctttgg ctgctttgtc tgctgctgct 60

ccaactccat ctgtttcttt gactgttaga nnnacttcta ctttggatga attgcaattg 120

ttttctcaat ggtctgctgc ttggtattgt tctaataata ttgattctga tgattctaat 180

gttacttgtg cttgtccatc tgttacttgt gatnnngaag aagcttctac taaaatgttg 240

ttggaatttg atttgaattt tggtactaat ggtactgctg gttttttggc tgctgataat 300

actaataaaa gattgtttag aggtgttgtt gcttcttcta ctattaaagc tgataattgg 360

attttggatt ttaatttgca agatgatatt gatttgtgta ctggttgtaa agttcatact 420

ggtttttgga aagcttggga agctgctgct gataatttga cttctaaaat taaatctgct 480

atgtctactt attctggtta tactttgtat tttactggtc attctttggg tggtgctttg 540

gctactttgg gtgctactgt tttgagaaat gatggttatt ctgttgaatt gtatacttat 600

ggttgtccaa gagttggtaa ttatgctttg gctgaacata ttacttctgg tgcttctcaa 660

ggtaatttta gagttttgaa tactcatgat attgttccaa gattgccaga ttttccaatg 720

ggtttttctc aaccatctcc agaatattgg attactnnnn nngatattga agaaggtatt 780

ttgattaatt ctactgctgg taatgctgct ggtgaaactg ttttggtttt gttgtgggct 840

cattattttt ttgctatttg tttgtctgaa ttg 873

<210> 5

<211> 816

<212> DNA

<213> Aspergillus niger CICC 2103(Aspergillus niger)

<400> 5

gctccaactc catctgtttc tttgactgtt agannnactt ctactttgga tgaattgcaa 60

ttgttttctc aatggtctgc tgcttggtat tgttctaata atattgattc tgatgattct 120

aatgttactt gtgcttgtcc atctgttact tgtgatnnng aagaagcttc tactaaaatg 180

ttgttggaat ttgatttgaa ttttggtact aatggtactg ctggtttttt ggctgctgat 240

aatactaata aaagattgtt tagaggtgtt gttgcttctt ctactattaa agctgataat 300

tggattttgg attttaattt gcaagatgat attgatttgt gtactggttg taaagttcat 360

actggttttt ggaaagcttg ggaagctgct gctgataatt tgacttctaa aattaaatct 420

gctatgtcta cttattctgg ttatactttg tattttactg gtcattcttt gggtggtgct 480

ttggctactt tgggtgctac tgttttgaga aatgatggtt attctgttga attgtatact 540

tatggttgtc caagagttgg taattatgct ttggctgaac atattacttc tggtgcttct 600

caaggtaatt ttagagtttt gaatactcat gatattgttc caagattgcc agattttcca 660

atgggttttt ctcaaccatc tccagaatat tggattactn nnnnngatat tgaagaaggt 720

attttgatta attctactgc tggtaatgct gctggtgaaa ctgttttggt tttgttgtgg 780

gctcattatt tttttgctat ttgtttgtct gaattg 816

<210> 6

<211> 57

<212> DNA

<213> Aspergillus niger CICC 2103(Aspergillus niger)

<400> 6

atgttttctg gtagatttgg tgttttgttg actgctttgg ctgctttgtc tgctgct 57

Claims

1. A high temperature resistant acid lipase LIP is characterized in that the amino acid sequence of the high temperature resistant acid lipase LIP is shown as SEQ ID NO.1 or SEQ ID NO. 2.

2. A thermostable acid lipase gene lipL75, encoding the thermostable acid lipase LIP of claim 1.

3. The high-temperature-resistant acid lipase gene LipL75 as claimed in claim 2, wherein its base sequence is shown in SEQ ID NO.4 or SEQ ID NO. 5.

4. A recombinant vector comprising the thermostable acid lipase gene LipL75 as claimed in claim 2 or 3.

5. A recombinant strain comprising the thermostable acid lipase gene LipL75 as claimed in claim 2 or 3.

6. A method for preparing high temperature resistant acid lipase LIP is characterized by comprising the following steps:

1) transforming a host cell with the recombinant vector of claim 4 to obtain a recombinant strain;

3) recovering and purifying the expressed high temperature resistant acid lipase LIP.