Mutant lipase

Abstract

The present invention relates to a mutant lipase. The present invention further relates to a gene encoding the lipase, a vector containing the gene, and a host cell. The present invention also relates to uses of the lipase.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, more particularly to variant lipases, genes encoding said enzymes, vectors and host cells containing said genes, and uses of said enzymes. [Background technology]

[0002] Diesel, an important petroleum product, accounts for a large share of the fuel mix in each country and is an important power fuel. The global trend toward dieselization of automobiles is accelerating, and demand for diesel is expected to grow even more in the future. As petroleum resources become increasingly depleted and people's environmental awareness increases, countries around the world are actively developing diesel as a fuel alternative. Since the 1990s, biodiesel has attracted attention from various countries for its excellent environmental protection properties. Currently, the global annual production of new cars is approximately 50 million, and the global vehicle fleet (including motorcycles) is approximately 750 million. With the rapid development of the automobile industry, the use of gasoline and diesel has increased along with the increase in the number of automobiles, causing problems such as automobile exhaust pollution.

[0003] It is well known that diesel molecules are composed of approximately 15 carbon chains. Research has shown that vegetable oil molecules generally have 14 to 18 carbon chains, which is similar to the number of carbon atoms in a diesel molecule. Therefore, biodiesel is a new type of fuel processed from renewable vegetable oils such as rapeseed. Compared to traditional diesel, biodiesel has the following unparalleled properties: (1) excellent environmental protection. Biodiesel can reduce atmospheric toxic substances by 90%, reduce cancer rates by 94%, and reduce carbon monoxide emissions by approximately 10% compared to diesel. (2) excellent engine starting performance at low temperatures. (3) excellent lubrication. (4) high safety performance. (5) good fuel economy. (6) renewable properties. Biodiesel combustion releases far less carbon dioxide than the carbon dioxide absorbed during plant growth, thereby mitigating global warming caused by carbon dioxide emissions, a major environmental problem harmful to humanity. Therefore, biodiesel is truly green diesel.

[0004] Currently, the United States, Europe, and some Asian countries and regions are beginning to establish commercial biodiesel production bases, and biodiesel is widely used as an alternative fuel. This is mainly due to the availability of soybeans in the United States, rapeseed in Europe, and palm oil in Asia. The sustainable development of these agricultural industries requires finding ways to consume large amounts of vegetable oil outside of the food and chemical industries, ensuring legal biodiesel consumption, and promoting the development of the biodiesel industry through subsidies.

[0005] Currently, biodiesel is mainly produced by chemical methods, namely, animal and vegetable oils and low-carbon alcohols such as methanol or ethanol are transesterified under high temperature (230-250°C) conditions using an acid or alkali catalyst to produce the corresponding fatty acid methyl esters or ethyl esters, which are then washed and dried to obtain biodiesel. The methanol or ethanol can be recycled in the production process, and the production equipment is the same as that used for general petroleum production. Approximately 10% of glycerol is produced as a by-product during the production process.

[0006] The main problem with current biodiesel is its high cost. Statistics show that 75% of the cost of biodiesel production is raw material costs. Therefore, the use of cheaper raw materials and improved conversion to reduce costs are key to whether biodiesel can be made practical. The United States has begun research into high-oil plants through genetic engineering techniques. Japan uses industrial waste and used frying oil. Europe cultivates oil-rich crops on land unsuitable for food cultivation. Asia uses waste oil and oil processing by-products for production.

[0007] However, chemical synthesis of biodiesel has several drawbacks: the process is complicated, an excess of alcohol is required, subsequent alcohol recovery equipment is required, which consumes a lot of energy, the unsaturated fatty acids in the fat are easily degraded at high temperatures, resulting in a dark color, the recovery of the esterified product is difficult and expensive, and waste alkali is discharged during the production process.

[0008] To address these issues, bioenzymatic biodiesel synthesis has been studied. This involves using animal fats and low-carbon alcohols for transesterification with lipase to produce the corresponding fatty acid methyl esters and ethyl esters. Enzymatic biodiesel synthesis has the advantages of mild conditions, low alcohol loading, and no pollutant emissions. However, the main drawbacks are the enzyme's low thermostability, the toxicity of short-chain alcohols to the enzyme, and its short lifespan. The by-products, glycerol and water, are difficult to recover and inhibit product formation. Furthermore, glycerol is toxic to immobilized enzymes, shortening their lifespan.

[0009] The sequence of Thermomyces duPontii lipase (TDL) was published in 2012. The protein is 291 amino acids long, with a 22-amino acid signal peptide and a 269-amino acid mature peptide. It has a typical lipase triplex catalytic domain structure of Ser168-Asp223-His280. TDL has a high preference for hydrolyzing C8 triglycerides at 50°C and pH 9.0. The conversion rate of biodiesel produced from waste cooking oil (WCO) is 91.6%.

[0010] In the applicant's patent application CN2016112256785, the inventors obtained the TDL mutant EP14. At a protein concentration of 20 mg / ml, a dosage of 0.5%, and a reaction time of 48 hours, the biodiesel conversion rate reached 93%, but did not meet the biodiesel standard of 96.5% conversion rate.

[0011] Therefore, there remains a need in the art for new lipases with high conversion rates for biodiesel conversion. Summary of the Invention

[0012]

[0013] In this study, we used the amino acid sequence of EP14 (CN2016112256785) as the starting sequence and introduced seven mutations (D27R, G38A, D96E, D111A, G163K, D254S, and A256T) by site-directed mutagenesis to obtain mutant TDLm2. TDLm2 was incubated at 35°C in 80% methanol for 6 hours, and the residual enzyme activity increased from 7.7% of that of EP14 to 72%.

[0014] The biodiesel reaction was carried out using fatty acids as the substrate at a protein concentration of 20 mg / ml, a loading of 0.45%, and a reaction time of 24 hours, achieving a conversion rate of 94%.

[0015] Three mutations, L86I, I93L, and M95F, were introduced into TDLm2 by site-directed mutagenesis to obtain mutant LM. Four mutations, L86I, I93L, M95F, and L211F, were introduced into TDLm2 to obtain mutant LMB.

[0016] Biodiesel reactions were performed using fatty substances as substrates at a protein concentration of 20 mg / ml and a loading of 0.45%. The conversion rate reached 94% after 8 hours of LM reaction. After 24 hours of reaction, the conversion rate reached 96.5%. After 8 hours of reaction, the conversion rate reached 95% for LMB reaction. After 24 hours of reaction, the conversion rate reached 96.5%.

[0017] Starting from LMB, the point mutation F95W was introduced to obtain the mutant LMBW, and the point mutation F95Y was introduced to obtain the mutant LMBY.

[0018] Biodiesel reactions were carried out using fatty substances as substrates at a protein concentration of 20 mg / ml and a loading of 0.45%. The conversion rate for LMBW reached 92% after 8 hours of reaction. After 24 hours of reaction, the conversion rate reached 96.6%. After 8 hours of reaction for LMBY, the conversion rate reached 96%. After 24 hours of reaction, the conversion rate reached 96.6%.

[0019] The biodiesel conversion rates of the mutants TDLm2, LM, LMB, LMBW, and LMBY obtained by the present invention are significantly improved, and the amount of enzyme used and the reaction time can be reduced, thereby reducing the cost of biodiesel production.

[0020] Specifically, the present invention relates to the following aspects:

[0021] In one aspect, the invention relates to a lipase comprising an amino acid sequence having a mutation relative to SEQ ID NO: 1 at one or more amino acid positions corresponding to positions 27, 38, 96, 111, 163, 254, and 256 of SEQ ID NO: 1. In one embodiment, the mutation is one or more of D27R, G38A, D96E, D111A, G163K, D254S, and A256T.

[0022] In one embodiment, the lipase of the invention further comprises additional mutations at one or more amino acid positions corresponding to positions 86, 93, 95, and 211 of SEQ ID NO: 1. In one embodiment, the additional mutation is at position 95 of SEQ ID NO: 1 and is one or more of L86I, I93L, and L211F. In one embodiment, the additional mutations are one or more of L86I, I93L, and M95F; L86I, I93L, M95F, and L211F; L86I, I93L, M95W, and L211F; or L86I, I93L, M95Y, and L211F.

[0023] In one embodiment, the lipase of the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, and 10, or a sequence having at least 90%, or at least 95%, or at least 99% sequence identity thereto.

[0024] As used herein, "percent identity" refers to the degree to which two optimally aligned DNA or protein segments do not change over a comparison window of components, such as nucleotide or amino acid sequences. The "identity fraction" of a comparison segment between a test sequence and a reference sequence is the number of identical components shared by the two comparison segment sequences over the comparison window divided by the total number of sequence components in the reference segment, which is the smaller of the complete test sequence and the complete reference sequence. The "percent identity" ("% identity") is the percent identity multiplied by 100. In another aspect, the present invention relates to a method for producing a lipase variant, comprising introducing one or more mutations at amino acid positions of a starting lipase corresponding to positions 27, 38, 96, 111, 163, 254, and 256 of SEQ ID NO:1. In one embodiment, the mutations are one or more of D27R, G38A, D96E, D111A, G163K, D254S, and A256T. In one embodiment, the method further comprises introducing one or more mutations at amino acid positions of the starting lipase corresponding to positions 86, 93, 95, and 211 of SEQ ID NO:1. In one embodiment, the mutations are one or more of: L86I, I93L, and M95F; L86I, I93L, M95F, and L211F; L86I, I93L, M95W, and L211F; or L86I, I93L, M95Y, and L211F. In one embodiment, the sequence of the starting lipase is as set forth in SEQ ID NO:1.

[0025] In another aspect, the present invention relates to a nucleic acid molecule selected from the group consisting of (a) a nucleotide sequence encoding the above-described lipase, or a sequence having at least 90%, at least 95%, or at least 99% sequence identity thereto; and (b) a nucleotide sequence complementary to the nucleotide sequence set forth in (a), which may be partially complementary or fully complementary. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence selected from SEQ ID NOs: 3, 5, 7, 9, and 11, or a sequence having at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0026] Those skilled in the art will understand that due to the degeneracy of the genetic code, a variety of different nucleotide sequences can encode the same enzyme. Furthermore, those skilled in the art will understand that they can use routine techniques to substitute nucleotides that do not affect the enzyme activity encoded by the nucleotide sequences of the invention, and that this may reflect the codon bias of any particular host organism used to express the enzymes of the invention.

[0027] The present invention also provides a vector comprising the nucleic acid molecule and a host cell comprising the nucleic acid molecule or the vector.

[0028] "Vector" refers to an extrachromosomal element that normally carries genes, which are not part of the central metabolism of the cell and are often in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genome-integrating sequences, phage, or nucleotide sequences from any source, linear or circular, single- or double-stranded DNA or RNA, where many of the nucleotide sequences have been joined or recombined into specific constructs capable of introducing into cells promoter fragments and DNA sequences for selected gene products, along with appropriate 3' untranslated sequences.

[0029] The lipase-encoding gene and gene product of the present invention can be expressed in heterologous host cells, such as bacterial cells, fungal cells, e.g., yeast cells, mammalian cells, insect cells, and plant cells. Heterologous host cells for expressing the nucleic acid molecules of the present invention can be microbial hosts of the fungal or bacterial families, and can grow over a wide range of temperatures, pH, and solvent tolerances. For example, it is contemplated that any bacteria, yeast, and filamentous fungi can be suitable hosts for expressing the nucleic acid molecules of the present invention.Examples of host strains are bacterial, fungal or yeast species, such as Pichia, Aspergillus, Trichoderma, Saccharomyces, Phaffia, Kluyveromyces, Yarrowia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Zymo Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacterium Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus s), Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus species, but are not limited to these. In some embodiments, the host cell is a fungal cell.In one embodiment, the host cell is Pichia pastoris.

[0030] Vectors useful for transforming the above-mentioned host cells are well known in the art. Typically, a vector contains a sequence that directs the transcription and translation of the relevant gene, a selectable marker, and a sequence that allows autonomous replication or chromosomal integration. A suitable vector contains a 5' region of the gene that controls transcription initiation and a 3' region of the DNA fragment that controls transcription termination.

[0031] In one aspect, the present invention also relates to a method for producing a lipase comprising expressing a nucleic acid molecule encoding a lipase of the present invention in a host cell and recovering the resulting polypeptide.

[0032] A variety of culture techniques can be used to produce the enzymes of the invention. For example, large-scale production of specific gene products from recombinant microbial hosts can be carried out in batch, fed-batch, and continuous culture techniques.

[0033] Batch and fed-batch culture methods are commonly used and well known in the art, examples of which can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989)) and Deshpande, Mukund V., (Appl. Biochem. Biotechnol., 36: 227-234 (1992)).

[0034] Commercial production of the enzymes of the present invention can also be carried out by continuous culture. Continuous culture is an open system in which conditioned medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous culture generally maintains cells at a constant high liquid density, where the cells are primarily in logarithmic growth phase. Alternatively, continuous culture can be carried out with immobilized cells, where carbon and nutrients are continuously added and valuable products, by-products, or waste products are continuously removed from the cell pellet. Cell immobilization can be carried out using a wide variety of solid supports made of natural and / or synthetic materials.

[0035] Recovery of the desired enzyme from batch fermentation, fed-batch fermentation, or continuous culture can be achieved by any method known to those skilled in the art. For example, when the enzyme is produced intracellularly, the cell slurry is separated from the culture medium by centrifugation or membrane filtration, and optionally washed with water or an aqueous buffer solution of the desired pH. The cell slurry is then suspended in an aqueous buffer solution of the desired pH and homogenized to produce a cell extract containing the desired enzyme.

[0036] The present invention also relates to compositions comprising fermentation broths, fermentation supernatants, and / or fermentation concentrates of the lysophospholipases of the present invention or the host cells of the present invention. The enzyme compositions of the present invention can be in any form suitable for use, such as crude fermentation broths with or without cell removal, cell lysates with or without cell debris, semi-purified or purified enzyme compositions, or host cells as the enzyme source. The enzyme compositions can be dry powders or granules, dust-free granules, liquids, stabilized liquids, or stabilized protected enzymes. Liquid enzyme compositions can be stabilized by established methods, such as the addition of stabilizers such as sugars, sugar alcohols, or other polyols and / or lactic acid or other organic acids. The enzyme composition of the present invention may also contain other lipases, such as Lipex Evity 200L, Lipozyme 435, Lipase A "Amano" 6, Lipase AY "Amano" 30SD, Lipase G "Amano" 50, Lipase R "Amano", Lipase DF "Amano" 15, Lipase MER "Amano", and Newlase F.

[0037] The present invention further relates to a fermentation broth, fermentation supernatant, or fermentation concentrate of the host cells of the present invention.

[0038] The present invention also relates to the use of the lipase of the present invention in the conversion of fatty materials to biodiesel. When the lipase of the present invention is used in biodiesel conversion, the amount of enzyme used and the reaction time can be reduced compared to prior art lipases, thereby reducing the cost of biodiesel production.

[0039] The present invention also relates to a method for producing biodiesel, comprising incubating a feedstock, a low-carbon alcohol, and a fermentation broth, fermentation supernatant, and / or fermentation concentrate of the lipase or the composition or host cell of the present invention together to produce the corresponding fatty acid esters. In one embodiment, the feedstock is selected from oils of aquatic plants such as oil crops, wild oil plants, and recombinant microalgae, animal fats, and oils from waste cooking oil, and the low-carbon alcohol is selected from methanol and ethanol. [Brief explanation of the drawings]

[0040] [Figure 1] FIG. 1 shows the results of a comparison of the methanol tolerance between TDLm2 of the present invention and EP14 of the prior art.

[0041] [Figure 2] Figure 2 shows the results of measuring the concentrations of PTDLm2, PLM, PLMB, PLMBW, and PLMBY in the fermentation broth using a modified Bradford protein concentration measurement kit. DETAILED DESCRIPTION OF THE INVENTION

[0042] Experimental materials

[0043] 1. Experimental strains and plasmids

[0044] Strain: Pichia pastoris M316 (prepared in the applicant's previous research, see accession number CGMCC19221, patent application number WO2020135763A1).

[0045] Plasmid: pET-pAOm-plc plasmid, synthesized by Shanghai Sangon Biotech. The synthesis steps were as follows: pAO815 vector sequence was prepared, and the coding sequence of the PLC mutant sequence in the "Highly efficient zinc-independent phospholipase C mutant" expression cassette of patent CN201680072289.5 (sequence number 7 of CN201680072289.5, Xaa is His) was prepared and ligated into the pET-28C vector.

[0046] The coding sequence of the PLC mutant sequence in the above-mentioned patent CN201680072289.5 "Highly efficient zinc-independent phospholipase C mutant" is as follows: tggtcagctgaggacaagcataaggaaggtgtgaatagtcacttatggatcgtgaaccgtgccattgatataatgtctaggaatacaactctggttaagcaagatagagttgctcaattgaatgaatggcgtacagagctagagaatggcatctacgctgctgatcatgaaaacccctattacg atgacagtaccttcgcttctcacttttacgatccagacaacggaaagacatatcccattcgccaagcaagctaaggagactggagctaagtacttcaagttggctggagagtcatacaagaataaagacatgaagcaggccttcttttatcttgggttgtcattgcattatttgggcgatgtc aaccaacctatgcatgccgcatcctttacggacctgtcctatccacagggttttcactccaagtacgagaactttgtcgatactattaaagacaactacaaagttaccgatgggaacggatattggaattggaaaggcaccaaccctgaagaatggattcacggtgcagcagtagttgcaaaac aggactactctggaattgtcaatgacaataccaaagattggtttgtgaaagccgcagtctcccaggaatatgcagataaatggagagctgaagttacacctatgactggtaaacgactaatggatgcccaaagagttactgctggttacattcaattatggttcgacacttacggtgacaggtaa (Sequence number 12)

[0047] 2. Culture media and solutions

[0048] YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose.

[0049] YPD solid medium: LB liquid medium plus 2% agar.

[0050] BMMY-Olive Oil Screening Medium: Component A: Yeast Nitrogen Base (YNB) containing 1% yeast extract, 2% peptone, 1.34% ammonium sulfate and no amino acids, 1% glycerol, 4 x 10 -5 % D-biotin (added after sterilization), 0.1M citric acid-sodium citrate buffer pH 6.6, 2% agar. Component B: Olive oil substrate solution: Take 150ml of 4% PVA solution, add 50ml of olive oil, and emulsify in a high-speed homogenizer at 8000 rpm for 3 minutes, rest for 1 minute, and then emulsify for 3 minutes to prepare the substrate solution. Mix 100ml of sterilized Component A with 12ml of Component B, add 1ml of 0.1% rhodamine B and 2% methanol.

[0051] BMGY liquid medium: Yeast nitrogen base (YNB) containing 1% yeast extract, 2% peptone, 1.34% ammonium sulfate, and no amino acids, 1% glycerol, 4 × 10 -5 % D-biotin, 0.1 M citric acid-sodium citrate buffer pH 6.6.

[0052] BMMY liquid medium: Yeast nitrogen base (YNB) containing 1% yeast extract, 2% peptone, 1.34% ammonium sulfate, and no amino acids, 2% methanol, 4 × 10 -5 % D-biotin, 0.1 M citric acid-sodium citrate buffer pH 6.6.

[0053] A modified Bradford protein concentration determination kit was purchased from Shanghai Sangon Biotech Co,Ltd.

[0054] PCR enzyme: PrimeSTAR® HS DNA Polymerase (purchased from TakaraBio (Dalian) Co., Ltd.). [Example]

[0055] Example 1: Expression of mutant TDLm2 in Pichia pastoris and preparation of enzyme solution

[0056] The amino acid sequence of the mature peptide of EP14 in CN2016112256785 is as follows: DVSQDLFDQFNLFAQYSAAAYCAKNNDAPAGAIVTCRGSICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSLENWIGNINMDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGGALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPReLGYSHSSPEYWITSGTLVPVRRRDIVKVEGIDSTDGNNQPNTPDIAAHFWYFGSIGTCL (EP14, SEQ ID NO: 1)

[0057] Seven mutation sites, D27R, G38A, D96E, D111A, G163K, D254S, and A256T, were introduced into SEQ ID NO: 1 to obtain the amino acid sequence. DVSQDLFDQFNLFAQYSAAAYCAKNNRAPAGAIVTCRASICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSLENWIGNINMELKGIDDICSGCKGHAGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGGALATVAGASLRGNKYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYWITSGTLVPVRRRDIVKVEGIDSTDGNNQPNTPSITAHFWYFGSIGTCL (TDLm2, SEQ ID NO: 2)

[0058] The DNA sequence was designed based on the codon preferences of Pichia pastoris, and the a-factor prepropeptide sequence (derived from the DNA sequence of the commercially available vector pPIC9K) was added. The following DNA coding sequence was obtained:

[0059]

[0060] This DNA sequence was sent to Suzhou Genewiz Biotechnology Co, Ltd. for complete gene synthesis and cloned into pET-pAOm-plc vector via SacII and EcoRI sites to obtain plasmid p-TDLm2.

[0061] p-TDLm2 was linearized with SalI, and competent cells of Pichia pastoris GS115 were prepared using the LiAC method. The linearized p-TDLm2 fragment was then transformed into competent GS115 cells by electroporation. Transformants were plated onto MGYS plates and cultured at 30°C for 3 days. Multiple single clones on the plates were picked on BMMY-olive oil screening plates, and the most active positive clone was selected and designated PTDLm2.

[0062] The PTDLm2 and EP14 strains (the EP14 strain was constructed using the same construction method as the PTDLm2 strain, but using the coding sequence for the EP14 enzyme (SEQ ID NO: 6 in patent application CN201611225678.5)) were first activated in liquid YPD and inoculated into BMGY. After overnight cultivation at 30°C and 220 rpm, the strains were transferred to BMGY medium (initial OD600 = 6) and initially induced with 2% methanol. After 24 and 32 hours, 1% methanol was added, and after 48 and 56 hours, 1% methanol was added. After 72 hours, samples were collected and concentrated using an ultrafiltration tube with a 10 kD molecular weight cutoff to a protein concentration of 30 mg / ml.

[0063] The enzyme solutions of pTDLm2 and EP14 were adjusted to 30 mg / ml. After incubation in 80% methanol at 35°C for 6 hours, the residual enzyme activity was measured. The experiment was performed in duplicate and repeated twice. The enzyme activity of the untreated enzyme solution was used as a control, and its relative enzyme activity was recorded as 100%. The enzyme activity measurement method is as follows.

[0064] 150 ml of 4% PVA solution was taken, 50 ml of olive oil was added, and the mixture was emulsified in a high-speed homogenizer at 8000 rpm for 3 minutes. After a 1-minute pause, the mixture was emulsified for another 3 minutes to prepare the substrate solution (this solution must be used immediately after preparation). JPEG2025542305000001.jpg160147

[0065] The residual enzyme activities of pTDLm2 and EP14 after incubation in the presence of 80% methanol at 35°C for 6 hours are shown in Figure 1.

[0066] Example 2: TDLm2 biodiesel reaction test

[0067] Biodiesel Production Method

[0068] 1. Raw materials: fatty substances (50-60% palmitic acid, 40-50% palmitic acid methyl ester; specifically, palmitic acid (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and palmitic acid methyl ester (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were mixed in a ratio of 5-6:4-5. In the following example, the specific fatty substance used was obtained by mixing them in a ratio of 6:4. Methanol was purchased from Sinopharm Group.

[0069] Reaction system and reaction conditions

[0070] Initial reaction system: 1 ml of fatty substance + 150 ul of methanol + 4.5 ul of enzyme solution (protein concentration was 20 mg / ml) + 30 ul of water

[0071] After the methanol and fatty substances were mixed uniformly, the enzyme solution and water were added. The sample was placed in a metal bath and incubated at 35°C and 1000 rpm. After 2.5 hours of reaction, 100 μL of methanol was added. After 4.5 hours of reaction, 50 μL of methanol was added. The acid value was measured after 8 and 24 hours of reaction, and the free fatty acid (FFA) % was calculated.

[0072] 2. FFA Test Method: The oil sample must be liquid and homogeneously mixed before measurement.

[0073] 3. Approximately 0.2 g of oil sample was weighed accurately to 0.001 g and placed in a 50 mL centrifuge tube.

[0074] 4. 10 mL of neutral isopropanol was added (a few drops of 1% phenolphthalein indicator were added). The oil sample was completely dissolved by shaking (and heating if necessary).

[0075] 5. Titrate with 0.05 mol / L potassium hydroxide standard solution while shaking until a pink color appears. If the color does not disappear within 30 seconds, this is the titration endpoint.

[0076] 6. The amount of potassium hydroxide consumed in the titration (V) was recorded and the result was calculated.

[0077] Presentation of analytical results Acid value=V×c×56.1 / m

[0078] During the ceremony,

[0079] V - the amount of potassium hydroxide solution used, ml;

[0080] c - the exact concentration of the potassium hydroxide standard solution used, in mol / L;

[0081] M - mass of the sample, g;

[0082] 56.1 - Molar mass of potassium hydroxide, g / mol... Ch.

[0083] FFA% = Acid value / 2 x 100%

[0084] The biodiesel production results for TDLm2 and EP14 are shown in the table below. [Table 1]

[0085] It can be seen that TDLm2 of the present invention significantly improves the biodiesel conversion rate compared to EP14.

[0086] Example 3: Expression of mutant LM and LMB in Pichia pastoris and preparation of enzyme solution

[0087] The amino acid sequence of the mature peptide of TDLm2 was introduced at three mutation sites, L86I, I93L, and M95F, to obtain the following amino acid sequence: DVSQDLFDQFNLFAQYSAAAYCAKNNRAPAGAIVTCRASICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSIENWIGNLNFELKGIDDICSGCKGHAGFTSSWRSVANTLTQQVQNAVRE HPDYRVVFTGHSLGGALATVAGASLRGNKYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYWITSGTLVPVRRRDIVKVEGIDSTDGNNQPNTPSITAHFWYFGSIGTCL (LM, SEQ ID NO: 4)

[0088] The DNA sequence was designed based on the codon preferences of Pichia pastoris, and the a-factor prepropeptide sequence (derived from the DNA sequence of the commercially available vector pPIC9K) was added. The following DNA sequence was obtained:

[0089]

[0090] This DNA sequence was sent to Suzhou Genewiz Biotechnology Co, Ltd. for complete gene synthesis and cloned into pET-pAOm-plc vector via SacII and EcoRI sites to obtain plasmid p-LM.

[0091] The amino acid sequence of the mature peptide of TDLm2 was introduced at four mutation sites, L86I, I93L, M95F, and L211F, to obtain the following amino acid sequence: DVSQDLFDQFNLFAQYSAAAYCAKNNRAPAGAIVTCRASICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSIENWIGNLNFELKGIDDICSGCKGHAGFTSSWRSVANTLTQQVQNAVREHPDYRV VFTGHSLGGALATVAGASLRGNKYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWITSGTLVPVRRRDIVKVEGIDSTDGNNQPNTPSITAHFWYFGSIGTCL(LMB, SEQ ID NO: 6)

[0092] The DNA sequence was designed based on the codon preferences of Pichia pastoris, and the a-factor prepropeptide sequence (derived from the DNA sequence of the commercially available vector pPIC9K) was added. The following DNA sequence was obtained:

[0093]

[0094] This DNA sequence was sent to Suzhou Genewiz Biotechnology Co, Ltd. for complete gene synthesis and cloned into pET-pAOm-plc vector via SacII and EcoRI sites to obtain plasmid p-LMB.

[0095] p-LM and p-LMB were linearized with SalI, and competent cells of Pichia pastoris GS115 were prepared using the LiAC method. The linearized p-LM and p-LMB fragments were then transformed into competent GS115 cells by electroporation. Transformants were plated onto MGYS plates and cultured at 30°C for 3 days. Multiple single clones on the plates were picked on BMMY-olive oil screening plates, and the most active positive clones were selected and designated PLM and PLMB.

[0096] The PLM and PLMB strains were first activated in liquid YPD, inoculated into BMGY, and grown overnight at 30°C and 220 rpm. The cultures were then transferred to BMGY medium (initial OD600: 6) and initially induced with 2% methanol. 1% methanol was added after 24 and 32 hours, respectively, and 1% methanol was added after 48 and 56 hours. After 72 hours, the sample was concentrated using an ultrafiltration tube with a 10 kD molecular weight cutoff to a protein concentration of 30 mg / ml.

[0097] Example 4: Biodiesel reaction test of LM and LMB

[0098] The method for producing biodiesel is as described in Example 2.

[0099] The biodiesel production results for LM and LMB are shown in the table below. [Table 2]

[0100] It can be seen that the LM and LMB of the present invention have a higher biodiesel conversion rate compared to TDLm2.

[0101] Example 5: Expression of mutants LMBW and LMBY in Pichia pastoris and preparation of enzyme solution

[0102] The amino acid sequence of the mature peptide of LMB was obtained by introducing the mutation F95W at amino acid position 95, resulting in the following amino acid sequence: DVSQDLFDQFNLFAQYSAAAYCAKNNRAPAGAIVTCRASICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSIENWIGNLNWELKGIDDICSGCKGHAGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGGALATVAGASLRGNKYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWITSGTLVPVRRRDIVKVEGIDSTDGNNQPNTPSITAHFWYFGSIGTCL (LMBW, SEQ ID NO: 8)

[0103] The DNA sequence was designed based on the codon preferences of Pichia pastoris, and the a-factor prepropeptide sequence (derived from the DNA sequence of the commercially available vector pPIC9K) was added. The following DNA coding sequence was obtained:

[0104]

[0105] This DNA sequence was sent to Suzhou Genewiz Biotechnology Co, Ltd. for complete gene synthesis and cloned into pET-pAOm-plc vector via SacII and EcoRI sites to obtain plasmid p-LMBW.

[0106] The amino acid sequence of the mature peptide of LMB was obtained by introducing the mutation F95Y at amino acid position 95, resulting in the following amino acid sequence: DVSQDLFDQFNLFAQYSAAAYCAKNNRAPAGAIVTCRASICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSIENWIGNLNYELKGIDDICSGCKGHAGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGGALATVAGASLRGNKYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWITSGTLVPVRRRDIVKVEGIDSTDGNNQPNTPSITAHFWYFGSIGTCL (LMBY, SEQ ID NO: 10)

[0107] The DNA sequence was designed based on the codon preferences of Pichia pastoris, and the a-factor prepropeptide sequence (derived from the DNA sequence of the commercially available vector pPIC9K) was added. The following DNA sequence was obtained:

[0108]

[0109] This DNA sequence was sent to Suzhou Genewiz Biotechnology Co, Ltd. for complete gene synthesis and cloned into pET-pAOm-plc vector via SacII and EcoRI sites to obtain plasmid p-LMBY.

[0110] p-LMBW and p-LMBY were linearized with SalI, and competent cells of Pichia pastoris GS115 were prepared using the LiAC method. The linearized p-LMBW and p-LMBY fragments were then transformed into competent GS115 cells by electroporation. Transformants were plated onto MGYS plates and cultured at 30°C for 3 days. Multiple single clones on the plates were picked on BMMY-olive oil screening plates, and the most active positive clones were selected and designated PLMBW and PLMBY.

[0111] PTDLm2, PLM, PLMB, PLMBW, and PLMBY strains were first activated in liquid YPD, inoculated into BMGY, and grown overnight at 30°C and 220 rpm. Then, the strains were transferred to BMGY medium (initial OD600 = 6) and initially induced with 2% methanol. After 24 and 32 hours, 1% methanol was added, and after 48 and 56 hours, 1% methanol was added. After 72 hours, the sample was concentrated using an ultrafiltration tube with a 10 kD molecular weight cutoff to a protein concentration of 30 mg / ml.

[0112] The shake flask fermentation broths of PTDLm2, PLM, PLMB, PLMBW, and PLMBY were collected and their protein concentrations were measured using a modified Bradford protein concentration kit. The protein concentrations of the fermentation broths for each strain are shown in Figure 2.

[0113] The shake flask fermentation protein yields of LMBW and LMBY were 0.461 mg / ml and 0.498 mg / ml, respectively, which were 84% and 99% higher than those of TDLm2 (0.25 mg / ml) and 29% and 39% higher than those of LMB (0.358 mg / ml).

[0114] Example 6: Biodiesel reaction test of LMBW and LMBY

[0115] The method for producing biodiesel is as described in Example 2.

[0116] The biodiesel production results for LMBW and LMBY are shown in the table below. [Table 3]

[0117] After 24 hours of reaction, the FFA% of both LMBW and LMBY could be reduced to 3.4%, which meets the criteria for biodiesel. Therefore, LMBW and LMBY have the advantage of cost reduction compared with TDLM2, LM, and LMB.

Claims

1. a lipase comprising an amino acid sequence having a mutation relative to SEQ ID NO: 1 at one or more amino acid positions corresponding to positions 27, 38, 96, 111, 163, 254, and 256 of SEQ ID NO: 1; Preferably, the lipase wherein the mutations are one or more of D27R, G38A, D96E, D111A, G163K, D254S, and A256T.

2. 10. The lipase of claim 1, further comprising additional mutations at one or more amino acid positions corresponding to positions 86, 93, 95, and 211 of SEQ ID NO:1; Preferably, the lipase wherein the additional mutations are one or more of L86I, I93L, and M95F; L86I, I93L, M95F, and L211F; L86I, I93L, M95W, and L211F, or L86I, I93L, M95Y, and L211F.

3. 3. The lipase of claim 1 or 2, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, and 10, or a sequence having at least 90%, or at least 95%, or at least 99% sequence identity thereto.

4. (a) a nucleotide sequence encoding the lipase of claim 1, or a sequence having at least 90%, at least 95%, or at least 99% sequence identity thereto; and (b) a nucleic acid molecule comprising a nucleotide sequence complementary to the nucleotide sequence of (a), preferably a nucleotide sequence selected from SEQ ID NOs: 3, 5, 7, 9, and 11, or a sequence having at least 90%, or at least 95%, or at least 99% sequence identity thereto.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. 7. The host cell of claim 6, wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a mammalian cell, an insect cell, and a plant cell, preferably a fungal cell, more preferably a Pichia pastoris cell.

8. A method for producing a lipase, comprising expressing a nucleic acid molecule encoding the lipase of any one of claims 1 to 3 in a host cell and recovering the resulting polypeptide.

9. A composition comprising a fermentation broth, a fermentation supernatant, and / or a fermentation concentrate of a lipase according to any one of claims 1 to 3 or a host cell according to claim 6 or 7.

10. A fermentation broth, fermentation supernatant, or fermentation concentrate of the host cell of claim 6 or 7.

11. Use of a lipase according to any one of claims 1 to 3, or a composition according to claim 9, or a fermentation broth, fermentation supernatant and / or fermentation concentrate of a host cell according to claim 6 or 7 in the conversion of fatty substances into biodiesel.

12. 1. A method for producing a lipase variant, comprising: introducing one or more mutations at amino acid positions of the starting lipase corresponding to positions 27, 38, 96, 111, 163, 254, and 256 of SEQ ID NO:1; Preferably, the mutations are one or more of D27R, G38A, D96E, D111A, G163K, D254S, and A256T; Optionally, further comprising introducing one or more mutations at amino acid positions of the starting lipase corresponding to positions 86, 93, 95, and 211 of SEQ ID NO:1; Preferably, the mutations are one or more of: L86I, I93L, and M95F; L86I, I93L, M95F, and L211F; L86I, I93L, M95Y, and L211F; or L86I, I93L, M95W, and L211F, Preferably, the sequence of the starting lipase is the sequence shown in SEQ ID NO:

1.

13. 1. A method for producing biodiesel, comprising: The method comprises incubating a raw material oil, a low-carbon alcohol, and the lipase according to any one of claims 1 to 3, or the composition according to claim 9, or a culture medium, culture supernatant, and / or culture concentrate of the host cell according to claim 6 or 7, to produce the corresponding fatty acid ester, Preferably, the feedstock oil is selected from oils of aquatic plants such as oil crops, wild oil plants, and recombinant microalgae, animal fats, and waste cooking oil, and the low-carbon alcohol is selected from methanol and ethanol.

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