WO2015003655A1 - Escherichia f6 expressing lipase, and f6 lipase and production and application thereof - Google Patents

Abstract

Isolated Escherichia F6 expressing lipase, and F6 lipase and production and application thereof, especially for use in biodiesel production.

Description

 Escherichia coli expressing F6, F6 lipase and its production and application

 The present invention relates to microorganisms, enzymatic engineering, to lipases and their production and use. Background technique:

 Lipase (triglyceride acyl hydrolase, EC 3.1.1.3) catalyzes the hydrolysis and synthesis of esters (esters synthesized from long-chain fatty acids and glycerol) (Petersen M, Daniel R. 2006. Purification and characterization of an extracellular lipase from Clostridium) Tetanomorphum. J Microbiol Biotechnol 22: 431-435). In addition to the basic function of hydrolyzing carboxyl ester bonds, lipases can also catalyze esterification (formation of esters;), transesterification, transesterification (exchange of ester bonds;) (Hawm F, Aamer AS, Abdul H 2006. Industrial application of microbial lipases. Enzyme and Microb Technol 39:235-25 P). Since lipases have enormous potential applications in industrial, biomedical, bioenergy, and medical research, there are already a large number of studies on lipases.

 Lipase is ubiquitous in nature and can be produced from different plants, animals and microorganisms. (Gt/pta R, Namita G, Pooja R. 2004. Bacterial Upases: An overview of production, purification, Appl Microbiol Biotechnol 64:763-781). Although a variety of lipases have been obtained from plants and animals, the lipase produced by bacteria is mostly extracellular lipase, which can be secreted into the medium through the extracellular membrane. Among them, this is particularly advantageous in industrial applications, and therefore, the production of lipase by microorganisms, especially bacteria, is increasingly favored (Royter M. 2006. Cloning and Characterization of Thermostable Lipases from Thermophilic Anaerobic Bacteria [Dissertation] .Hamburg : Technische Mikrobiologie, Technische Universitat Hamburg).

 A large number of lipase producing strains have been discovered, but only a few wild-type or recombinant lipases can be used in commercial applications (Jaeger KE, Ransae S, Djikstra BW, Colson C, van Heuvel M, Misset O. 1994. Bacterial lipases . FEMS Microbiol Rev 15:29-63·)).

Due to the biodegradability, renewable, non-toxic, low-emission and environmentally friendly biodiesel, biodiesel has recently received worldwide attention (Leca M, Tcacenco L, Micutz M, Staicu T. 2010. Optimization of Biodiesel production by transesterification of vegetable oils using lipases. Romanian Biotechnological Letters 15(5): 5618-5630). Since biodiesel is produced from renewable resources such as vegetable oils, it is considered to be a CO ₂ neutral, biodegradable oil that contributes to the conservation of fossil fuels. Compared with conventional diesel fuel, its combustion pollution emissions are greatly reduced (Ra/aeZ C, Giandra V, Keiko W, Marco A. 2008. Enzymatic synthesis of biodiesel from transesterification reactions of vegetable oils and short chain alcohols. J Am Oil Chem Soc 85: 925-930).

 Industrially, biodiesel can be produced by esterification of vegetable oils and short-chain alcohols under basic or acidic catalyst conditions, wherein the short-chain alcohol is typically methanol. The resulting reaction product is a mixture of the desired esters, monoglycerides, glycidyl esters, glycerin, water, and a catalyst. This process consumes more energy than an enzyme-mediated process. Due to the presence of soap by-products, the separation and purification of chemically prepared biodiesel requires a more complicated step than the enzymatically prepared biodiesel. Based on this, the use of biocatalysts is an attractive option because enzymatically synthesized biodiesel can be used without purification (Ra/aeZ C, Giandra V, Keiko W, Marco A. 2008. Enzymatic synthesis of biodiesel from Transesterification reactions of vegetable oils and short chain alcohols. J Am Oil Chem Soc 85: 925-930).

 By using a lipase as a biocatalyst, a triglyceride derived from a vegetable oil can be converted into a fatty acid alkyl ester by a transesterification reaction. Enzyme treatment can be carried out under mild conditions, which reduces energy consumption and reduces the cost of waste disposal. At the same time, the immobilized enzyme can be reused, which reduces the cost and makes the enzyme treatment process economical and practical (/gor N, Susana L, Marly C, Otavio L, Marcio F, Marta A. 2011. Enzymatic Biodiesel synthesis using a byproduct obtained from palm oil refining. Enzyme Research). Summary of the invention:

 In summary, the present invention mainly includes the following contents:

 In a first aspect, the present invention provides an isolated lipase-expressing strain designated as Escherichia F6, deposited at the General Microbiology Center of the China Collection of Microorganisms and Cultures, under the accession number CGMCC No. 7507.

 In a second aspect, the invention provides a lipase having an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: l or a conservative variant thereof.

In a third aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a lipase, the amino acid sequence of the lipase being selected from the amino acid sequence shown in SEQ ID NO: 1, or a conservative variant thereof; The nucleotide sequence is preferably as shown at 1 to 1842 of SEQ ID NO: 2; the nucleic acid sequence of the nucleic acid molecule is preferably as shown in SEQ ID NO: 2. In a fourth aspect, the present invention provides a recombinant cell comprising the nucleic acid molecule of the third aspect, preferably, the recombinant cell is selected from the group consisting of Escherichia coli containing the nucleic acid molecule, a yeast containing the nucleic acid molecule, and Escherichia F6 containing the nucleic acid molecule deposited at the General Microbiology Center of the China Collection of Microorganisms and Cultures, CGMCC No. 7507.

 In a fifth aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule of the third aspect, preferably, the recombinant vector is a plasmid, preferably, for example, pWB201 comprising the nucleic acid molecule constructed from pUC19, pWB204 containing the nucleic acid molecule constructed from pRK415, or pWB210 containing the nucleic acid molecule constructed from pColdTF.

 In a sixth aspect, the present invention provides a method for producing a lipase, comprising: cultivating the strain of the first aspect and/or the recombinant cell of the fourth aspect, such that the strain or cell produces lipase, and harvests fat. Preferably, the method comprises adding a lipase inducing agent to the medium, preferably the inducer is a triglyceride, and the preferred triglyceride is olive oil, palm oil, coconut oil, soybean oil, palm kernel oil.

 The harvesting may include isolating and collecting a culture supernatant containing a lipase secreted or released to the extracellular, thereby obtaining a lipase containing lipase; preferably, the method further comprises the lipase solution Concentrate.

 In a seventh aspect, the present invention provides a lipase prepared by the method of the sixth aspect.

 In an eighth aspect, the present invention provides a lipase solution prepared by the method of the sixth aspect. The ninth aspect, the present invention provides a method for producing a biodiesel, characterized in that the strain of the first aspect, the lipase of the second aspect, the lipase of the eighth aspect, the recombinant cell of the fourth aspect Or the lipase solution of the ninth aspect as a catalyst.

 Oil palm (Zae gi« iee ^) fruit surface or oil palm fruit circumference, due to its oily environment, is a potential source for the separation of lipase-expressing strains. We successfully isolated a strain expressing lipase from the oil palm fruit, named Escherichia. 6( «"/1⁄2^^ ?6), referred to herein as F6, and the F6-derived lipase is named F6 lipase (F61ip), and the lipolytic activity expressed by F6 is called F6 lipase activity. The invented Escherichia F6 strain was deposited on April 22, 2013 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCCX No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, 100101), the deposit number For CGMCC No. 7507.

 The present invention provides a method for producing a lipase comprising culturing the F6 isolated according to the present invention to produce a lipase, and harvesting the lipase produced by the bacteria. F6 secretes the produced lipase into the extracellular medium. In one embodiment, the harvesting comprises separating the supernatant from the culture, thereby obtaining a lipase solution containing lipase having lipase activity. Alternatively, the producer can be subjected to cell disruption to release more lipase.

In one embodiment, the lipase solution of the present invention is concentrated to increase fat Lipase activity of the enzyme solution. The enzyme solution of the present invention can be used as it is. The lipase can be further isolated and purified from the enzyme solution of the present invention by various methods known in the art of enzyme engineering, thereby obtaining the F6 lipase of the present invention.

 In one embodiment, the present invention adds a lipase inducing agent to the culture medium. The present inventors have found that the inducer significantly increases the enzyme activity in the culture supernatant, thereby indicating that the inducer significantly increases the enzyme yield in the supernatant. Examples of the inducer are triglyceride oils such as olive oil, palm oil, coconut oil, soybean oil, and palm kernel oil. Pogori et al reported that triglyceride is a commonly used lipase inducer and is widely used to enhance the secretion of lipase; adding 2% soybean oil or olive oil can enhance the secretion of R/^^ lipase (尸ogo N, Cheikhyoussef A, Xu Y, Wang D. 2008. Production and biochemical characterization of an extracellular lipase from Rhizopus chinensis CCTCC M20102L Biotechnology 7(4): 710-717. In one embodiment of the present invention, 1-2 % (vA's olive oil promotes increased lipase activity in the medium.

 A method for measuring lipase activity is, for example, p-nitrophenol palmitate (; pNPP) method. One unit of lipase activity is defined as: The amount of enzyme consumed by pNPP to release 1 μηιοΐ p-nitrophenol per minute at pH 8.0, 37 °C. The method includes:

 1) Drawing of standard curve

 1. Weigh 0.11 g of pNP dissolved in 50 ml of isopropanol to prepare a 20 mM stock solution. When using it to make a standard curve, it was diluted at a ratio of 10:100 to obtain a 2.0 mM pNP solution. When making standard curves and conducting experiments on samples such as culture supernatants, make sure all reaction conditions and reaction volumes are the same.

 2. Mix all the solutions together as shown in the table below. Incubate for 15 min at 37 °C and add 2 ml of 95% ethanol. After centrifugation at 7000 rpm for 2 min, the absorbance value A410 of the supernatant at 410 nm was measured (when using spectrophotometry, water was added as a blank control;). Record the A410 value of each sample and make a standard curve with the formula: Y=ax+b (Y: pNPP concentration; X: A410; a, b: reaction factor)

Reaction buffer: 0.05 mol/L NaH ₂ PO ₄ -Na ₂ HPO ₄ (pH 8.0), 0.01% (m/v) Arabian tree Glue and 0.23% (; m/v) sodium deoxycholate. These ingredients were stirred and dissolved and stored at 4 °C.

 (2) Detection of lipase activity

 Material:

1. Reaction buffer: 0.05 mol/L NaH ₂ PO ₄ -Na ₂ HPO ₄ (pH 8.0), 0.01% (m/v) gum arabic and 0.23% (m/v) sodium deoxycholate. These ingredients Stir and dissolve, and store at 4 °C.

 2. Solution A: 30 mg pNPP (Sigma) was dissolved in 10 ml of isopropanol. Store at 4 °C.

 Method:

 2 ml of solution A and 18 ml of reaction buffer were mixed at a mixing ratio of 1:9. The substrate solution was ready to use and preheated at 37 ° C for 3 min and loaded into 6 test tubes in 2.4 ml to detect lipase activity. One culture tube was added with the supernatant of the culture 100 μl, and the other tube was added with the boiled supernatant as a control (approximately 5 min for boiling, and placed on an ice bath to cool the sample;), after incubation at 37 ° C for 15 min, immediately add 2 ml. The reaction was terminated with 95% ethanol. The A410 absorbance of the supernatant was measured by centrifugation at 7000 rpm for 2 min. Record the A410 value of each sample (when using spectrophotometry, add water as a blank control;), calculate the lipase activity using the following formula.

 Lipase activity (U/ml) = =(axAOD+b)xl0xnxl/15

 AOD : A-A0 (absorbance of sample and control at 410 nm, respectively)

 a,b: formula factor of pNP standard curve

 10: Convert ΙΟΟμΙ^ sample to 1ml sample volume

 n: dilution factor

 1/15: Reaction time 15min, converted to lmin

 A method for measuring lipase activity is, for example, a pH STAT method. For example, an olive oil emulsion containing water, 1% (v/v) olive oil (Sigma, USA) and 1% gum arabic was prepared by stirring at 15,000-20000 rpm for 2 minutes. Lipase activity was measured by the pH STAT method at 40 °C. Adjust the pH of the emulsion substrate to 8 before adding the enzyme solution. The reaction was started after the addition of 5 μl of the enzyme. To maintain a 5-minute reaction time, the enthalpy value was 8 and titration of free fatty acids was adjusted by 10 mM NaOH. Lipase The rate at which olive oil is hydrolyzed to free fatty acids is determined by 902 Titrando (Metrohm Switzerland). Catalytic release per minute Ιμηιοΐ The amount of enzyme consumed by free fatty acids is defined as an enzyme unit.

Using the transposon mutation and the genome walking method, the present invention successfully isolated the full length of the F6 lipase gene. DNA sequence analysis indicated that the gene was 1845 bp in length and its sequence was as shown in SEQ ID NO: 2, wherein the position 1-1842 was CDS, and the amino acid sequence shown in SEQ ID NO: 1 was obtained, and the molecular weight was 64512 Da. Based on this, the present invention provides a lipase, designated as F6 lipase, whose amino acid sequence is selected from the group consisting of:

1) the amino acid sequence shown as SEQ ID NO: l, or

 2) A conservative variant of the amino acid sequence shown in SEQ ID ΝΟ: 1.

 The "conservative variant" refers to a "conservative" change compared to the parent sequence, such as substitutions, insertions, deletions or combinations thereof, such as amino acid substitutions having similar structural or chemical properties, provided that the conservative change is not Destroy the biological nature of the parent. Computer programs such as alanine scanning and DNASTAR software, which are well known in the art, can be used to direct or determine conservative variants of a given parental sequence. For the purposes of the present invention, the conservative variants still exhibit F6 lipase activity.

 F6 lipase is a member of the 1.3 lipase family. 1.3 The lipase family is a member of the Gram-negative bacterial population of true lipases with different protein secretion systems (T 1 S S). Enzymatic properties indicate that F6 lipase is active under neutral or alkaline conditions (pH 8.0-8.5), optimal temperature is 25-35 °C, and over a wide temperature range of 20 °C - 80 ° Stability under C. It also exhibits good stability in organic solvents (e.g., ethanol and methanol;) at a concentration of 10-20% (up to 50% or more;). F6 lipase is effective in hydrolyzing various vegetable oils. These properties make F6 lipase an ideal enzyme for biodiesel and other fields, and can efficiently convert triglycerides to biodiesel (fatty acid alkyl esters;).

 The invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a F6 lipase. In one embodiment, the nucleotide sequence encoding the F6 lipase is shown as position 1 - 1842 of SEQ ID NO: 2. In one embodiment, the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO: 2, and another embodiment is shown in position 1 - 1842 of SEQ ID NO: 2.

 In order to obtain higher lipase activity, the present invention constructs a large number of expression vectors, such as a common vector in recombinant technology, and performs recombinant expression of F6 lipase in homologous and heterologous hosts, and obtains the positive expression of the lipase. . The expression of F6 lipase in a homologous host, F6, gives us a convenient system for the production of the F6 lipase original host. The lipase of the F6 system is easily secreted directly into the medium, which provides convenience for separation and purification. The wild type F6 strain can be used as a non-transgenic reference system. Recombinant expression in the F6 strain provides higher enzyme activity and lipase yield. Moreover, the present invention also tests a large number of heterologous hosts, such as common hosts in recombinant techniques, such as Escherichia coli, yeast, etc., for example, in Pichia piwton, 卩 卩 酉 ( (Sacc wra yc^ Recombinant expression of F6 lipase was constructed in cereW ae).

 Based on this, the present invention provides a recombinant cell comprising a nucleic acid molecule encoding a F6 lipase. For example, the host cell may be selected from the group consisting of F6, Escherichia coli, yeast, and the like isolated according to the present invention.

The invention also provides a series of recombinant vectors comprising a nucleic acid molecule encoding a F6 lipase. The recombinant vector can be constructed using various commonly used vectors in the field of recombinant technology, such as pUC 19, pRK415 and pColdTF Wait. Examples of the recombinant vector of the present invention include: pWB201 containing the F6 lipase coding sequence constructed by pUC19, pWB204 containing the F6 lipase coding sequence constructed by pRK415, and F6 lipase containing pColdTF as described in the Examples below. The coding sequence of pWB210.

 The present invention provides a method for producing a lipase by recombinant techniques, which comprises culturing a recombinant cell of the present invention to produce a lipase, and harvesting a lipase produced by the cell. Some host cells, such as F6 and yeast cells, secrete the produced lipase into extracellular medium. In one embodiment, the harvesting comprises separating the supernatant from the culture, thereby obtaining a lipase solution containing lipase having lipase activity. Alternatively, the producer can be subjected to cell disruption to release more lipase. When the selected host (e.g., E. coli;) does not secrete the product extracellularly, cell disruption becomes a necessary step. These adjustments are well known to those skilled in the art.

 In one embodiment, the lipase solution of the present invention is concentrated to increase the lipase activity of the lipase solution. The enzyme solution of the present invention can be used as it is. The F6 lipase can be further isolated and purified from the enzyme solution of the present invention by methods known in the art of enzyme engineering.

In one embodiment, the present invention adds a lipase inducing agent to the culture medium. The present inventors have found that the inducer significantly increases the enzyme activity in the culture supernatant, thereby indicating that the inducer significantly increases the enzyme yield in the supernatant. Examples of the inducer are triglyceride oils such as olive oil, palm oil, coconut oil, soybean oil, and palm kernel oil. In one of the examples, the addition of 1-2% (v/ _v ) of olive oil promotes the lipase activity in the medium.

 The present invention also provides a method for producing biodiesel using the lipase-expressing strain or lipase of the present invention. In one embodiment, the method produces biodiesel from palm oil and methanol. In one embodiment, the lipase may be in the form of a lipase solution produced by the method of the invention as described hereinbefore, especially a concentrated lipase solution. BRIEF DESCRIPTION OF THE DRAWINGS:

 Figure 1 : Lipase activity produced by F6 in LB medium and LB medium containing olive oil in one of the examples.

 Figure 2: Sequence of the primers used in the examples.

 Figure 3: Alignment between F6 lipase and Serratia marcescens (; ABI 13521) lipase sequences. Figure 4: Amino acid sequence alignment of F6 lipase, S. arc^ce fSM), R/H'zo t/cw /^(RM), and Thermomyces lanuginosus (TL) lipase. The boxes indicate the oxygen anion hole "RG" and the lipase active site "GHSLGG".

Figure 5: In one of the examples, Rhodamine B's agar plate (; left;) and TCN plate (; right;) Positive expression of TOP 10/pWB201.

 Fig. 6: In one of the examples, the recombinant expression of F6 lipase in the recombinant strain BL21/pWB210 of Escherichia coli showed the enzyme activity measured by the PH STAT method.

 Figure 7: Optimal temperature (A), thermal stability (B) and optimal pH (C) for F6 lipase.

 Figure 8: Stability of F6 lipase in methanol and ethanol, with the abscissa of methanol and ethanol.

 Figure 9: Substrate specificity of F6 lipase for different vegetable oils.

 Figure 10: Substrate specificity of F6 lipase to triglyceride.

 Figure 11: HPLC analysis of the hydrolysis products of tricaprylin with F6 lipase (F6), Rhizomucor miehei lipase (Rm) and palatase® (Novo), respectively, and tricaprylin as a control.

 Figure 12: Molecular properties of F6 lipase. Left panel A: TCN-Zymograms, in which lanes 1 and 2 are F6 lipases recombinantly expressed by the homologous host F6/pWB204, and lane 3 is a high molecular weight pre-stained SDS-PAGE standard (HMW), purchased from Bio- Right; B: SDS-PAGE, where the first lane is a low molecular weight pre-stained SDS-PAGE standard (LMW), purchased from Bio-rad, and the second lane is a lipase produced by fermentation of wild strain F6, 3rd The fourth lane is the F6 lipase obtained by recombinant expression of the homologous host F6/pWB204; the arrow indicates the ~66 kDa band.

 Figure 13: Results of transesterification of palm oil under F6 lipase catalysis in one of the examples. detailed description:

 The features, effects, and advantages of the technical solutions of the present invention are exemplified below. The examples do not constitute a limitation of the embodiments, scope and effect of the invention. The medium formula used in the experiment:

 LB (Luria Broth) agar:

 Tryptone 1%

 Yeast cream 0.50%

 NaCl 1%

 Bacterial Agar Powder 1.50% TCN (tricaprylin) Agar:

 Preparation of lOx gum arabic solution:

 Arabian gum 10%

NaCl 200 nM CaCl 50 mM

Preparation of LB medium:

Tryptone 3g

Yeast extract 1.5 g

 NaCl 1.5 g

Agar 4.5 g

Distilled water 270 mL

Preparation of TCN solution:

Caprylic glyceryl triglyceride 3 mL

 10χ gum arabic solution 27 mL

TCN agar medium:

 1. Prepare TCN emulsion by mixing TCN solution with a stirrer.

 2. Place TCN solution and LB medium in autoclave for sterilization

 3. Cool the LB medium to 50 °C.

 4. Thoroughly mix the TCN emulsion with the LB medium.

 5. Pour the mixture into a Petri dish

M9 medium agar (per liter)

Na ₂ HPO ₄ 6 g

KH ₂ PO ₄ 3 g

NH ₄ C1 lg

NaCl 0.5 gram

Bacterial agar powder 20 gram

Glucose solution 10 ml

MgSO ₄ .7H ₂ O solution 1 ml

Thiamine hydrochloride solution 1 ml

CaCl ₂ solution 1 ml glucose solution:

 D-glucose 20 g

Add glucose to distilled water, let the final volume be 100 mL, mix thoroughly, put into the autoclave, 120 ° C, 15 min, 15 psi.

MgSO^I^O solution:

MgSO ₄ .7H ₂ O 246.5 g

Add MgSO ₄ .7H ₂ O to distilled water, let a final volume of 1 L, mix thoroughly, and place in an autoclave at 120 ° C, 15 min, 15 psi. Thiamine hydrochloride solution:

 Thiamine hydrochloride 10 mg

 Add thiamine hydrochloride to distilled water: let the final volume be lOOmL, mix thoroughly, filter and sterilize,

CaCl _z solution:

CaCl ₂ 14.7 g

Add CaCl ₂ to distilled water to a final volume of 1 L, mix thoroughly, and place in an autoclave at 120 ° C for 15 min at 15 psi.

Preparation of M9 medium:

1. The ingredients other than MgSO ₄ .7H ₂ O, glucose, thiamine hydrochloride, and CaCl ₂ in the formulation were solution prepared in distilled water, and then the volume was adjusted to 987 ml.

 2. Mix completely.

 3. Place in an autoclave at 120 ° C for 15 min at 15 psi.

 4. Cool to room temperature

5. In a sterile environment, add sterile MgSO ₄ .7H ₂ O solution, glucose solution, thiamine hydrochloride solution, CaC12 solution. Preparation of olive oil emulsion:

 Olive oil 15 ml

 2% PVA (polyvinyl alcohol) 50 mL

 Emulsified at 10,000 rpm for 3 min, allowed to stand for 5 min, repeated stirring for 3 min, placed in an autoclave, 115 ° C, 30 min. Preparation of Rhodamine B solution:

 Rhodamine B 0.1 g

Distilled water 100 ml After the dissolution is complete, it is sterilized by filtration.

M9+ olive oil + rhodamine B medium configuration:

 M9 medium 1 L

 Olive Oil Emulsion 120 mL

 Rhodamine B solution 10 mL Example 1 : Isolation and identification of lipase-expressing strains from oil palm fruit circumference

 Sample collection: Oil palm fruit was obtained from Tania Selatan's oil palm plantation in South Sumatra, Indonesia.

Isolation and screening of lipase-producing microorganisms: The palm fruit was peeled, and the peeled peel was crushed in a 0.85% NaCl solution and applied to a rhodamine B agar plate. Rhodamine B agar plates were used to rapidly screen for lipase-expressing strains (3⁄4t^er G & Jaeger KE. 1987. Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53·211 - 21 ). The formulation of Rhodamine B agar medium is: 0. 1% (NH ₄ ) ₂ SO ₄ , 0.1% K ₂ HPO ₄ , 0.5% KC1, 0.05% MgSO ₄ -7H ₂ O, 0.01% FeSO ₄ -7H ₂ O, 0.5% yeast extract, 0.5% tryptone, 2% Qiongyue, olive oil lotion (2% polyvinyl alcohol: olive oil = 3: 1), containing 0.001% rhodamine B. After incubation at 30 ° C for 48 hours (h), bacterial colonies of orange fluorescent halo were observed by ultraviolet irradiation, and the colonies were identified as colonies having lipase activity (Kouker & Jaeger 1987, supra). Clones with lipase activity were collected and streaked on a new Rhodamine B plate. Thus, a batch of clones was successfully isolated from the oil palm fruit, and the strain with the highest lipase activity was identified. For F6.

 The 16S rRNA gene was sequenced by Genetic Analyzer 3 130 (Applied Biosystems, Applied Biosystem), and the gene sequence of the 16s rRNA of F6 is shown in SEQ ID NO: 3. The obtained sequence was aligned with the NCBI-GenBank database sequence using BLAST software, and the results showed that the F6 strain and Hermann's Escherichia (/1⁄2 'c/»'a /1⁄2r a ) [Accession number: HQ407263] had 97.8. % similarity. The strain was named as Escherichia coli F6 ( ^/1⁄2 'c/»'a F6), referred to as F6 in this paper, and deposited with the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC) on April 22, 2013. ), the deposit number is CGMCC No. 7507. Example 2: Isolation and Sequence Analysis of F6 Lipase Gene

 From the transposon mutation and the genome walking PCR method, we successfully obtained the full length of the lipase F6 gene from the F6 isolate.

We inactivated the transposon mutation and labeled the lipase gene from F6. Transposon of F6 The mutant was obtained by parental hybridization of the donor E. coli S 17_aPIR (pUTmini Tn5 sp/sm ^R , purchased from Biomedal, Sevilla, Spain;) and the recipient F6 strain. When the transposon was randomly inserted into the F6 lipase gene, the F6 strain lost its lipase activity on the plates of M9 medium + olive oil + rhodamine B and selected antibiotics (gimycin and streptomycin; Can no longer produce orange fluorescent halo, but can grow in ^! ! ^ Medium. White colonies were picked, and F6 lipase mutants (lip-, sp/sm ^R ) were further verified based on 16s rRNA homology analysis, loss of lipase activity, and spectinomycin/streptomycin resistance phenotype, due to transposition Insertion of the daughter on the lipase gene, which has lost the ability to produce lipase.

Transposon mutations can be used to map the F6 lipase gene. The sp/sm ^R gene fragment inserted into the chromosome is a marker for mapping the lipase gene. The full length of the lipase gene sequence was obtained by the genome walking PCR method (Clontech) using the primers shown in Figure 2 (SEQ ID NO: 4-16). The full length of the lipase gene sequence was analyzed by the Geneious Pro 5.3.3 program.

 The sequencing results showed that the full length of the F6 lipase gene DNA sequence was 1845 bp (SEQ ID NO: 2) encoding 614 amino acids (SEQ ID NO: 1). The molecular weight of the protein was 64,512 Da, F6 lipase sequence and viscosity. Serrate marcescens SM6 extracellular lipase LipA (GenBank: U 1 1258) has a similarity of 79.6 % at the DNA level, and Serratia marcescens (GenBank: ABI 13521) lipase at the amino acid level There is a 7.7% similarity (Figure 3).

Based on the alignment with the Serratia marcescens SM6 sequence, the GAGGA sequence was found 7 bases upstream of the AUG start codon, which is similar to a typical ribosome binding site. We also found that the termination region is rich in the GC region and a 9 bp sequence was found downstream of the termination region UAA stop codon. The amino acid sequence of F6 comprises multiple repeats of a 9 amino acid residue (GGXGXDXXX) which is rich in glycine and aspartic acid. Although the function of this region is not clear, it is speculated that these amino residue sequences are Ca ²⁺ binding sites. It has been reported that most proteins containing this sequence are secreted into the culture medium by a special mechanism (Jaeger et al. 1999; Long et al. 2007; Shibatani et al. 2000).

Sequence alignment revealed that the amino acid sequence comprises a consensus sequence of a lipase active site comprising a catalytic triplet Ser-Asp-His. Based on the alignment with the S. marcescens lipase sequence, we predicted that the F6 lipase also has the same GHSLGG active site containing six amino acids, this active site and Serratia marcescens Lipases and other filamentous fungi (eg Rhizomucor miehei, Thermomyces /imt/g^iiwi^) share the same active site sequence 歹lj (Fig. 4) (Table 1) (Bell PJL, Sunna A, Gibbs MD, Curach NC) , Nevalainen H, Bergquist PL. 2002. Prospecting for novel lipase genes using PCR. Microbiol 148:2283-2291 ; Ryu HS, Kim HK, Choi WC, Kim MH, Park SY, Han NS, Oh TK, Lee JK. 2006. New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely Appl Microbiol Biotechnol 70:321-326) Table 1: Comparison of F6 lipase active site and oxyanion region with other lipases Lipase source accession number Oxygen anion region active site

 F6 - IGIAFRG GHSLGG

 Serratia marcescens AAA81002 IGISFRG GHSLGG

 (Serratia (GenBank)

 Marcescens)

 Pseudomonas aeruginosa AAF87594 IGVSFRG GHSLGG

 (Pseudomonas (GenBank)

 Brassicacearum )

 Thermophilic fungus 059952 IVLSFRG GHSLGG

 (Thermomyces (UniProt/Swis

 Lanuginosus) s-Prot)

 Fusarium oxysporum Q02351 IVVSVRG GHSLGG

 (Fusarium (UniProt/Swis

 Heterosporum) s-Prot)

 Mucor miehei P19515 IYIVFRG GHSLGG

 (Rhizomucor (UniProt/Swis

 Miehei) s-Prot)

 Luminescent bacteria AY527197 YVIAIRG GHSKGG

 Lipolyticum( Photob (GenBank)

 Acterium

Lipolyticum) Based on sequence analysis and characterization, we learned that F6 lipase is a member of the 1.3 lipase family {Arpigny JL, Jaeger KE. 1999. Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177-183) . The enzyme family is a member of the Gram-negative bacterial true lipase population. The lipases in this population are reported to be some of the lipases from Serratia marcescens and Pseudomonas fluorescens. 1.3 The lipase family and the 1.1 lipase family or the 1.2 lipase family exhibit very small amino acid sequence similarities (; <20%). The lipase family, and other families, differ in amino acid sequence and secretion machinery, such as the type I secretion system (; T 1 SS;). The secretion of this system contains only one step, secreted directly from the cytoplasm to the extracellular medium, without any intermediate periplasmic processes. In the unfolded or partially folded state, the enzyme of the 1.3 lipase family is directly secreted into the extracellular medium and folded outside the cell. This process does not require the assistance of any molecular chaperone (Awgtow Wj'aj'a C, Kanaya S. 2006. Famili 1.3 lipase: bacterial lipases secreted by the type I secretion system. Cell Mol Life Sci 63: 2804-2817) _a Example 3 : Recombinant expression of the F6 lipase gene

Recombinant expression in heterologous hosts The genomic DNA of F6 was isolated using the Wizard® Genomic DNA Purification Kit (Promega) as a template, using ButLip-3end2 (SEQ ID NO: 17) and ButLip-ATGl (SEQ ID NO: 18) as primers, using Phusion® high fidelity. DNA polymerase (FINNZYMES) PCR amplified the 1845 bp F6 lipase gene (SEQ ID NO: 2). The PCR fragments at the ends of the two blunt ends were ligated into the pUC19 vector. Validation of transformation was performed using M13 primer pairs (SEQ ID NO: 23-24) and nucleotide sequencing (ABI 3130 Genetic Analyzer). The pUC19 plasmid clone carrying the F6 lipase gene insert was designated pWB201.

 Then, the competent E. coli TOP10 host was transformed with pWB201, and the transformant TOP10/pWB201 was obtained and cultured on a rhodamine B agar plate and a tricaprilyn (TCN) plate. The F6 recombinant (clone #5 in Figure 5) on the Rhodamine B agar plate showed an orange fluorescent halo under UV light, and a transparent circle around the clone of the TCN plate. This indicates that the present invention successfully achieved recombinant expression of F6 lipase in a heterologous host. Recombinant expression in homologous hosts

 Enzyme digestion of plasmid pWB201 by Hindlll and Sacl restriction enzymes to obtain F6 lipase gene

(SEQ ID NO: 2). The F6 lipase gene (SEQ ID NO: 2) was then integrated into plasmid pRK415 (purchased from Korea veterinary culture collection) and transformed into E. coli TOP10. Transformants were confirmed by M13 primer pairs (SEQ ID NO: 23-24) and nucleic acid sequencing (ABI 3130 Genetic Analyzer). The clone of the pRK415 plasmid carrying the F6 lipase gene insert was designated pWB204.

F6 lipase was expressed in a homologous host (F6) by a donor (E. coli TOP/pWB 204), a helper plasmid E. _C oli/pRK2013 (purchased from Beijing Lebo Biotechnology Co., Ltd.) and a receptor (F6) The three parental crosses between the two are completed. At the later stage of logarithmic culture of the donor, helper plasmid and recipient cells, they were centrifuged, rinsed, and resuspended in LB medium. 25 μl of the mixed cells were dropped onto a 0.45 μl sterile filter paper placed on LB medium. Hybridization at 30 ° C for 3-4 h, the cells in the filter paper were resuspended in 0.85% NaCl solution and transferred to selective M9 medium + olive oil + rhodamine containing tetracycline (10μ ₈ /ηι1) On the tablet. These plates were incubated at 30 ° C, and within 2-3 days, the transforming binder F6/pWB 204 showed an orange fluorescent halo. Transformants were verified by PCR using the M13 primer pair (SEQ ID NO: 23-24). This example demonstrates that the present invention successfully achieves recombinant expression of F6 lipase in a homologous host. Example 4: Production of lipase from wild type F6

The isolated F6 wild strain (CGMCC No. 7507) was inoculated into 10 ml of LB medium, followed by After that, it was cultured overnight at 30 ° C and 175 rpm. The overnight culture solution was taken and inoculated into 50 ml of LB culture solution at a 2% inoculation amount, followed by incubation at 30 ° C, 175 rpm for 24 hours. This strain secretes the produced lipase into the medium. Then, the culture solution was centrifuged at 100 rpm, 4 ° C, and 10 min to remove the cells, and the culture supernatant was taken to be a lipase solution containing F6 lipase.

 Lipase activity was measured spectrophotometrically using p-nitrophenyl palmitate (p-NPP). One unit of lipase activity is defined as the amount of lipase required to release 对μηιοΐ of p-nitrophenol per minute at pH 8.0, 40 °C. The enzyme activity of the F6 lipase solution obtained above was 3 U/ml.

 The above fermentation was repeated, and 2% (; v/v) olive oil was added as an inducer to 50 ml of LB production medium. As shown in Fig. 1, the activity of the obtained F6 lipase solution was increased by more than 4 times to 14 U/ml. Adding 2% olive oil can trigger an increase in lipase activity in the medium, which may be due to the bio-emulsifier produced by the F6 strain.

 The induced F6 lipase enzyme solution was concentrated by a centrifugal filter (Amicon® Ultra-15 (Millipore, USA) molecular weight cutoff of 30,000 MW). 15 ml of the enzyme solution was added to an Amicon Ultra tube and rotated at 4000 rpm, 25 °C for 15-25 min until concentration to 200-500 μΐ. The lipase activity of the concentrated lipase was measured by pH STAT. The results showed that the enzyme activity of the F6 lipase concentrate was 173 U/ml. Example 5: Production of F6 lipase by recombinant method

Production of F6 lipase in a homologous host

 The F6/pWB204 constructed in Example 3 was introduced into 10 ml of LB medium and incubated at 30 ° C, 175 rpm overnight. The overnight culture was taken and inoculated into 50 ml of LB medium containing tetracycline antibiotic (10 g/ml) at 2% inoculation and incubated at 30-40 ° C, 175 rpm for 24 h. The culture solution was centrifuged at 1000 rpm, 4 ° C, and 10 min to obtain a lipase solution containing F6 lipase. The pH STAT assay showed that the extracellular enzyme activity in the enzyme solution reached 15-20 U/ml.

 The above obtained F6 lipase enzyme solution was passed through a centrifugal filter (Amicon® Ultra-15 (Millipore,

USA) The molecular weight cutoff is 30,000 MW) for concentration. 15 ml of the enzyme solution was added to an Amicon Ultra tube and rotated at 4000 rpm, 25 ° C for 15-25 min until concentration to 200-500 μΐ. According to the pH STAT method, the lipase activity after concentration was 700 U/ml. Production of F6 lipase in a heterologous host

Genomic DNA of F6 isolated using the Wizard® Genomic DNA Purification Kit (Promega), using the resulting genomic DNA as a template, using ButLip-3endl-BamHI (SEQ ID NO: 20) WoButLip-ATGl-Ndel (SEQ ID NO: 19) was a primer, and a 1845 bp F6 lipase gene (SEQ ID NO: 2) was PCR-amplified using Phusion® high-fidelity DNA polymerase (FINNZYMES). The PCR fragment was ligated into pColdTF vector (Takara Bio Inc, Japan) and transformed into E. coli TOP 10 . Transformants were confirmed by pCold-TF-F1 and pCold R primer pairs (SEQ ID NO: 21-22) and nucleic acid sequencing (ABI 3 130 Genetic Analyze-r). The clone of the pColdTF plasmid carrying the F6 lipase gene insert was designated pWB210. This plasmid was subsequently transformed into the expression host BL21 (; DE3).

 E. coli BL21 (DE3) containing pWB210 was added to LB medium, and incubated overnight at 30 ° C, 175 rpm with shaking. The overnight culture solution was taken and inoculated into 50 ml of LB medium containing tetracycline antibiotic (10 g/ml) in a 2% inoculum, and incubated at 37 ° C, 175 rpm for 2-3 h until the OD600 reached 0.5-0.6. The culture was then placed at 4- 15 °C for 15-30 min, then IPTG was added to a final concentration of 0.5-lmM, followed by incubation at 4-15 °C, 150-200 rpm for 5-24 h. The cells were centrifuged at 1000 rpm, 4 ° C, lOmin, resuspended in 100 mM Tris-HCl (pH 8), and sonicated. The fermentation supernatant showed lipase activity, and the recombinantly expressed F6 lipase aggregated in a soluble form in the cells. The recombinantly expressed F6 lipase was collected by centrifugation at 10,000 rpm, 4 ° C, and 10 min. According to the pH STAT method, the enzyme activity of the enzyme solution reached 641.3 U/ml, as shown in Fig. 6.

 The F6 lipase enzyme solution obtained above was concentrated by a centrifugal filter (Amicon® Ultra-15 (Millipore, USA) molecular weight cutoff of 30,000 MW). 15 ml of enzyme solution was added to the Amicon Ultra tube and spun at 4000 rpm, 25 V for 15-25 min until concentration to 200-500 μΐ. According to the pH STAT method, the enzyme activity of the concentrate was 4394.4 U/ml, as shown in Fig. 6. Example 6: Characterization of lipase

Optimum temperature and thermal stability: The enzyme solution used was the unconcentrated F6 lipase solution produced by the homologous host F6/pWB204 of Example 5. To determine the optimal temperature for lipase, at different temperatures (20 ° C - 90 ° C), in the reaction buffer (0.05 mol / L NaH ₂ PO ₄ -Na ₂ HPO ₄ , 0.01% (m / v) In the gum arabic and 0.23% (m/v) sodium deoxycholate, pH 8.0, the optimum temperature of the lipase enzyme activity was detected by the pNPP method. Figure 7 (A) shows the relative enzyme activity of the enzyme activity at the optimum temperature for the enzyme activity at different temperatures.

In order to detect the thermal stability of lipase, the lipase solution was reacted in a reaction buffer (0.05 mol/L NaH ₂ PO ₄ -Na ₂ HPO ₄ - 0.01% at different temperatures (20 ° C - 90 ° C)). m/v) gum arabic and 0.23%

(m/v) sodium deoxycholate, pH 8.0) was incubated for 30 minutes, and then the enzyme activity of the lipase was measured by the pNPP method. The definition of 1 unit of lipase activity is: At pH 8.0, 40 ° C, the release of Ι μηιοΐ of p-nitrophenol per minute requires the amount of lipase. Figure 7 (B) shows the relative enzyme activity of the enzyme activity after incubation compared to the enzyme activity before incubation. Optimal pH: The enzyme solution used was the concentrated F6 lipase solution produced by the homologous host F6/pWB204 in Example 5. The pNPP method was used to detect the lipase activity at different pH between pH 6.5-9.0, using sodium phosphate buffer (pH 6.5-7.5, 0.1 M), and Tris HCl (pH 8.0-9.0, 0.1 M, respectively). ) Prepare pNPP reaction buffer. Figure 7 (C) shows the relative enzyme activity of the enzyme activity at the optimal pH at different pHs.

As shown in Fig. 7, the experimental results show that the F6 lipase has high activity under neutral or slightly alkaline conditions (; pH 8.0-8.5) and room temperature conditions (25-35 °C). At 50 °C, the lipase activity decreased to below 40% and remained stable at 20 °C and 80 °C (Fig. 7). Solvent stability: The solvent stability of F6 lipase was examined using F6 lipase enzyme solution (173 U/ml) produced and concentrated in F6 wild-type strain. The lipase was incubated at 30 ° C in a reaction buffer containing different final concentrations of organic solvents (methanol and ethanol) (0.05 mol/L NaH ₂ PO ₄ -Na ₂ HPO ₄ , 0.01% (m/v) gum arabic And 0.23% (m/v) sodium deoxycholate, pH 8.0) for 30 min, wherein the final concentration of organic solvent ranged from 10-50% (v/v). The stability of the incubated lipase was measured by the pNPP method. The relative activity was calculated by comparison of lipases that were not incubated in an organic solvent as a control. F6 lipase also has good enzyme stability (>50%) in 10-20% (v/v) concentration of organic solvent (ethanol and methanol) (Fig. 8). The organic solvent stability of F6 lipase makes this enzyme ideal for applications such as biodiesel. Substrate specificity: The substrate specificity of F6 lipase was examined using F6 lipase enzyme solution (700 U/ml) produced by FBB204 transformation and transformed with pWB204. Substrate specific detection of enzymes acting on triglycerides using pH STAT (pH 8): tributyrin (C4), tricaprylin (C8), triolein (C 18) and vegetable oil (; palm kernel oil, olive oil, palm oil, corn oil, soybean oil, coconut oil, sunflower oil, rapeseed oil, rice bran oil;). A lipase unit is defined as: the amount of enzyme required to produce Ιμηιοΐ fatty acids per minute (Ryw HS, Kim HK, Choi WC, Kim MH, Park SY, Han NS, Oh TK, Lee JK. 2006. New cold-adapted lipase From Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipase. Appl Microbiol Biotechnol 70:321 -326).

An important property of lipase is its substrate specificity for triglycerides. At pH 8, 40 °C, F6 lipase can effectively hydrolyze various vegetable oils (palm kernel oil, olive oil, palm oil, corn oil, soybean oil, coconut oil, sunflower oil, rapeseed oil, rice bran oil) ;). Among these experimental oils, palm kernel oil and Coconut oil is relatively poorly hydrolyzed (Figure 9). We believe that this is because the main fatty acid component of palm kernel oil and coconut oil is medium chain fatty acid (C 12). This result was compared with the triglyceride substrate specificity (triolein, tributyrin, tricaprylin) we have analyzed (Fig. 10), and it was found that F6 lipase to tricaprylin (C8) , medium chain fatty acids) have the lowest activity. In general, F6 lipase has a broad substrate specificity, and although it tends to hydrolyze the triglyceride of the short-chain fatty acid, it can also hydrolyze the long-chain fatty acid triglyceride. Position specificity: The site specificity of F6 lipase was examined by F6 lipase enzyme solution (700 U/ml) produced and transformed with pWB204 after transformation with pWB204. The position specificity of the lipase was determined by high performance liquid chromatography (HPLCXAgilent 1200 series) to detect the hydrolysis product obtained by hydrolysis of tricaprylin (C8). The detection conditions for high performance liquid chromatography are in accordance with the AOCS official method Cd l ld-96 (2009). The reaction mixture contained 1 ml of tricaprylin and 4 ml of F6 enzyme (total enzyme 100 units, diluted with Tris-HCl buffer (0.1 M pH 8.0)). Incubate at 40 ° C, 150 RPM for 16 h. The reaction product was extracted with 5 mL of a n-hexane/isopropanol (90:10) solution. The product after the reaction was then analyzed by HPLC.

 The results are shown in Figure 11. We predict that the first peak is the peak of the triglyceride, and the second peak is

The 1,3-glycolide, the third peak is 1,2-glycolide, the fourth peak is 1-monoglyceride, and the fifth peak is 2-monoglyceride. As shown in Figure 11, a sample of the product obtained from F6 lipase and a Mucor mimosa lipase derived from Wilmar Shanghai (No. Rm) and a commercial Mucor mimosa lipase from Novozyme (Novo: Novo) (Palatase®) The product samples are identical and have the same 1,3-site specificity. Molecular weight

 The molecular weight of F6 lipase was determined by SDS-PAGE and zyymograms experiments. The SDS-PAGE experiment was carried out using a polyacrylamide gel (12%) as described by Laemmli (Laemmli, UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685). The protein was stained using Coomassie Brilliant Blue R-250. The gel for zymograms was washed with 50 mM Tris-HCl (pH 8.0) containing 1% triton X-100 for 10 min, then a second time with 50 mM Tris-HCl (pH 8.0) containing 0.1% triton X-100. ) Shake for 10 min, then rinse with distilled water for 10 minutes. The activity of the revived protein was determined by attaching a gel to a caproic acid agar plate and incubating at 37 ° C for 3 h.

The F6 lipase solution was added to a polyacrylamide gel for separation and zymography analysis. The TCN zymogram shows the formation of a single unique band in each lane (Fig. 12A;). The molecular weight of the enzyme was estimated to be approximately 66 kDa by standard band size and zymogram results (Figures 12A and B). Left panel A: TCN-Zymograms, in which lanes 1 and 2 are recombinant F6 lipases expressed by homologous hosts, and lane 3 is high molecular weight. Pre-stained SDS-PAGE standard (HMW), purchased from Bio-rad; right panel B: SDS-PAGE, the first lane is low molecular weight pre-stained SDS-PAGE standard (LMW), purchased from Bio-rad, Lane 2 is a lipase produced by fermentation of wild strain F6, and lanes 3 and 4 are recombinant F6 lipase expressed by a homologous host; the arrow indicates a ~66 kDa band. Example 7: Application of F6 lipase in biodiesel

 Our preliminary research indicates that F6 lipase is suitable for use as a biodiesel catalyst. F6 lipase is methanol tolerant and is stable in up to 20% (v/v) methanol.

 For the preliminary experiment of F6 biodiesel application, the F6 lipase concentrate (4300 U/ml) produced by the heterologous recombinant BL21/pWB210 in Example 5 was diluted to 2000 U/mL with Tris-HCl buffer (0.1 M pH 8.0). , used as a catalyst.

 The transesterification reaction was carried out in a 100 ml glass vial. The reaction mixture was placed in a vial comprising: 4 ml of palm oil, 1 ml of methanol and 2 ml of lipase catalyst (2000 U/mL) or other catalyst. The reaction mixture was then placed at 400 rpm and incubated at 37 ° C for 24 h. Ethanol was added stepwise (Oh and 6 h, 500 μl each). Biodiesel, ethanol and water were separated by centrifugation at 13,000 rpm for 2 min.

 The product was visualized by thin layer chromatography (TLC) strips: The ΙΟΟμΙ sample was diluted in 0.5 ml of n-hexane. A diluted sample of ΙΟμΙ was applied to a silica gel 60 F254 thin layer chromatography plate (Merck). N-hexane:ethyl acetate:acetic acid (90:10:1) was used as a developing solvent, and a methanol solution containing 1% sulfuric acid was used as a color developing agent. After the developer was sprayed on the developed silica gel plate, the plate was heated at 150 ° C for 20 minutes.

 The TLC results are shown in Figure 13. The first lane is the positive control, which is the product obtained by producing the biodiesel with the chemical catalyst sodium methoxide; the second lane is the palm oil as the substrate; the third lane is the commercial enzyme Lipozyme TL 100L ( ; Novozymes;) product obtained as a catalyst; the fourth lane is a product obtained by using the F6 lipase of the present invention as a catalyst.

 As shown in the figure, as compared with the results of conversion of known catalysts, the F6 lipase of the present invention is capable of efficiently converting a triglyceride (e.g., palm oil;) into biodiesel. The catalytic activity of the F6 lipase of the present invention is higher than that of the commercially available Lipozyme TL 100 L, which indicates that the F6 lipase of the present invention is a highly efficient biodiesel catalyst, indicating that it has good industrial application value. And prospects.

Claims

1. An isolated lipase-expressing strain deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee under the accession number CGMCC No. 7507.  2. A lipase whose amino acid sequence is selected from:  l) the amino acid sequence shown as SEQ ID NO: l, or  2) A conservative variant of the amino acid sequence set forth in SEQ ID NO: 1.  3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a lipase, the amino acid sequence of the lipase being selected from the amino acid sequence set forth in SEQ ID NO: 1 or a conservative variant thereof; Preferably, it is as shown in 1-1842 of SEQ ID NO: 2; the nucleic acid sequence of the nucleic acid molecule is preferably as shown in SEQ ID NO: 2.  A recombinant cell comprising the nucleic acid molecule according to claim 3, wherein the recombinant cell is selected from the group consisting of Escherichia coli containing the nucleic acid molecule, a yeast containing the nucleic acid molecule, and a deposit containing the nucleic acid molecule. Escherichia coli F6 of CGMCC No. 7507 is deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee.  5. A recombinant vector comprising the nucleic acid molecule of claim 3. Preferably, the recombinant vector is a plasmid, preferably pWB201 containing the nucleic acid molecule constructed from pUC19, and the nucleic acid molecule is constructed from PRK415. pWB204, pWB210 containing the nucleic acid molecule constructed from pColdTF.  6. A method for producing a lipase, comprising: cultivating the strain of claim 1 and/or the recombinant cell of claim 4 such that the strain or cell produces a lipase, harvesting a lipase; preferably, The method comprises adding a lipase inducing agent to the medium, preferably the inducer is a triglyceride, and the preferred triglyceride is olive oil, palm oil, coconut oil, soybean oil, palm kernel oil.  The method according to claim 6, wherein the harvesting comprises isolating and collecting a culture supernatant containing a lipase secreted or released to the extracellular, thereby obtaining a lipase containing lipase; preferably, The method also includes concentrating the lipase solution.  8. A lipase prepared by the method of claim 6 or 7.  A lipase solution obtained by the method of claim 7.  A method for producing biodiesel, characterized by using the strain according to claim 1, the lipase of claim 2, the lipase of claim 8, the recombinant cell of claim 4 or the method of claim 9. The lipase solution acts as a catalyst.