Aspergillus niger ethyl lactate lipase, coding gene, method and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses aspergillus niger ethyl lactate lipase, a coding gene, a method and application of the aspergillus niger ethyl lactate lipase, and the amino acid sequence of the aspergillus niger ethyl lactate lipase is shown as SEQ ID NO.1 or SEQ ID NO.2. The invention belongs to a genetic engineering technology, and aims to obtain the aspergillus niger lipase which is defined as the aspergillus niger lipase ANI138064 and the aspergillus niger lipase ANI12344024. The lipase disclosed by the invention can be used for synthesizing ethyl lactate and ethyl acetate in a pure mouldy bran system containing ethanol, lactic acid and acetic acid. The lipase ANI138064 and ANI12344024 recombinant strains disclosed by the invention are used for catalytically synthesizing important flavor esters (ethyl lactate and ethyl acetate) in a water-phase system for brewing white spirit, and have a relatively good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular to an Aspergillus niger lactate ethyl lipase, a coding gene, a method and an application thereof. Background Art

[0002] Chinese liquor is one of the world's six most famous distilled liquors along with brandy, whiskey, vodka, rum and gin, and is also the oldest distilled liquor in the world. Liquor is mainly ester-flavored, and ethyl acetate, ethyl lactate, ethyl butyrate and ethyl caproate are the four major esters in liquor, and their content determines the style and quality of liquor. However, the complex open solid-state fermentation system of liquor leads to its poor production stability, which affects the content of flavor esters such as ethyl lactate in the base liquor, and thus affects the quality of the base liquor. Therefore, it is of great significance to understand the synthesis pathway of ethyl lactate in the wine brewing system, which is not only conducive to stabilizing the production quality, but also conducive to strengthening the synthesis of ethyl lactate, which is helpful for the production of high-ester flavored liquor.

[0003] There are two main pathways for the synthesis of esters in the process of liquor brewing. The first is the spontaneous esterification reaction of acid and alcohol. This process is a dynamic reversible reaction. During long-term storage of base liquor, the reaction will proceed in the direction of ester hydrolysis, thereby reducing the ester content in the base liquor. The second is biosynthesis or enzyme-mediated ester synthesis, which mainly includes the hemiacetal dehydrogenase pathway, the monooxygenase pathway, the alcohol acyltransferase pathway and the esterification pathway. Among them, the hemiacetal dehydrogenase pathway and the monooxygenase pathway use aldehydes and ketones as substrates, respectively. However, the liquor fermentation process is mainly anaerobic fermentation, and only a small amount of aldehydes and ketones are produced during anaerobic fermentation. Therefore, these two pathways contribute less to the synthesis of esters. The catalytic reaction of alcohol acyltransferase is the main pathway for yeast to synthesize esters such as ethyl acetate, while medium- and long-chain fatty acid esters such as ethyl butyrate and ethyl hexanoate are synthesized by relying on the esterification enzyme catalytic system.

[0004] At present, the esterification enzymes widely used in the synthesis of fatty acid esters are: esterase, cutinase, and lipase. Patent Publication No.: CN104031898B. This patent publication uses carboxylesterase for the synthesis of butyl butyrate. By optimizing the concentration of butyric acid and ethanol, the conversion rate is increased to 92%. In addition, esterase can also synthesize other ester compounds such as isopentyl acetate and ethyl hexanoate, and has important application potential in the synthesis of flavor esters. (Food chemistry 2016131-136) This article uses cutinase for the synthesis of fatty acid esters in an organic phase system. The ester yields of C3-C8 acids and C1-C6 alcohols reach 98% and 95%, respectively. Cutinase shows a wide range of chain length selectivity. Under the conditions of cutinase concentration of 50U / ml, 40℃, and water content of 0.5%, the highest yield of ethyl hexanoate reaches 99.2%. It can be seen that cutinase can efficiently catalyze the synthesis of ethyl hexanoate while tolerating a certain water content. In an organic phase system, the combination of acetic acid and hexanoic acid is relatively easier. The hydroxyl activity of ethanol is relatively high. When reacting with hexanoic acid, it can quickly carry out nucleophilic attack, which accelerates the reaction rate and improves the synthesis efficiency. Among the four major esters of liquor, ethyl lactate is the most special. Its precursors are lactic acid and ethanol. Lactic acid is a carboxylic acid containing hydroxyl groups and has good hydrophilicity. Therefore, it is generally believed that the synthesis of ethyl lactate mainly depends on the spontaneous esterification reaction of acid and alcohol. The research on esterification enzymes is mostly concentrated in the organic phase system, while the liquor brewing system belongs to the aqueous phase system. There are few reports on the exploration of liquor esterification enzymes and their catalytic mechanism for synthesizing esters under aqueous conditions. Therefore, studying esterification enzymes in aqueous phase systems can provide a deep understanding of the formation mechanism of ethyl lactate during liquor fermentation.

[0005] Patent Publication No.: CN201610281015.9. This patent disclosure uses freeze-dried cutinase powder in the organic phase to synthesize short-chain aromatic esters, and the highest esterification rates of ethyl caproate, ethyl butyrate, ethyl acetate, and ethyl lactate are 98.3%, 95.5%, 92.8%, and 88.1%, respectively. The recombinant cutinase was further applied to the solid-state fermentation experiment of light-fragrance liquor to catalyze the synthesis of short-chain fatty acid esters such as ethyl acetate, ethyl butyrate, ethyl caprylate, and ethyl caproate, which significantly increased the content of ethyl lactate and ethyl acetate in liquor. Although there are reports on bacterial agents or crude enzyme preparations that enhance the synthesis of ethyl lactate, there are few reports on the role of lipase in the production of medium- and short-chain fatty acid esters such as ethyl lactate and ethyl acetate.

[0006] Aspergillus niger is a very important brewing microorganism in the process of liquor brewing. It has a close relationship with the utilization of raw material nutrients and the synthesis of flavor esters. The exploration and application of the catalytic synthesis of ethyl lactate in Aspergillus niger not only helps to enrich the understanding of the catalytic mechanism of liquor esterification enzymes, but also provides new materials and ideas for the green biomanufacturing and process optimization of liquor flavor. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an Aspergillus niger lactate ethyl lipase, an encoding gene, a method and an application thereof.

[0008] The technical solution adopted by the present invention to solve the technical problem is:

[0009] A Aspergillus niger ethyl lactate lipase, the amino acid sequence of the Aspergillus niger ethyl lactate lipase is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0010] The gene encoding the Aspergillus niger lactate ethyl lipase as described above.

[0011] Furthermore, the sequence of the coding gene is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0012] Use of the Aspergillus niger ethyl lactate lipase as described above in catalyzing the synthesis of ethyl lactate and / or ethyl acetate.

[0013] Furthermore, the ethyl lactate synthesized by the Aspergillus niger lactate ethyl lipase is significantly higher than that of the wild type, with an increase of more than 20%; the ethyl acetate synthesized by the Aspergillus niger lactate ethyl lipase is more than 70% higher than that of the wild type.

[0014] The use of the Aspergillus niger ethyl lactate lipase as described above in the preparation of ester aroma liquid for wine and / or white wine containing ethyl lactate and / or ethyl acetate.

[0015] A recombinant strain of Aspergillus niger lipase comprising the Aspergillus niger lactate ethyl lipase as described above.

[0016] Furthermore, the Aspergillus niger lipase recombinant strain is obtained by PEG-mediated transformation;

[0017] Alternatively, the esterification ability of the recombinant strain of Aspergillus niger lipase under pure bran koji conditions is increased by more than 20%;

[0018] Alternatively, the saccharification ability of the recombinant Aspergillus niger lipase strain catalyzing the synthesis of ethyl lactate and ethyl acetate in a pure bran koji system is significantly higher than that of the wild-type Aspergillus niger under the pure bran koji conditions.

[0019] The method for constructing the above-mentioned Aspergillus niger lipase recombinant strain comprises the following steps:

[0020] The Aspergillus niger lactate ethyl lipase repair template was transformed into Aspergillus niger protoplasts through PEG6000 and cultured at 30°C for 3-5 days, and the single clone was verified by PCR strain verification.

[0021] The method for catalyzing the synthesis of ethyl lactate and ethyl acetate using the above-mentioned Aspergillus niger lipase recombinant strain comprises the following steps:

[0022] The recombinant strain of Aspergillus niger lipase is inoculated into a 65% bran medium and cultured at 30°C for 48 hours; the bran with mycelium is placed at 37°C for drying and shaken to be broken up to obtain the first-class seeds; after cooling, the first-class seeds are inoculated into an 85% bran medium, shaken well, and cultured at 30°C for 40 hours, and when the mycelium is fully covered and the bran is connected into a cake shape, the bottle is buckled to separate the bran from the bottom and continue to culture for 24 hours before the bottle is taken out; after the bottle is taken out, it is placed in a cowhide bag, quickly dried at 37-40°C to make its water content less than 12%, and fully broken up to obtain ethyl lactate and ethyl acetate;

[0023] The formula of 65% bran medium per 100 g is: 100 g bran, 65 mL water;

[0024] The formula of 85% bran medium per 100 g is: 100 g bran, 85 mL water.

[0025] The advantages and positive effects achieved by the present invention are:

[0026] 1. The present invention belongs to genetic engineering technology, and obtains Aspergillus niger lipase, which is defined as Aspergillus niger lipase ANI_1_58064 and Aspergillus niger lipase ANI_1_2344024. The lipase of the present invention can synthesize ethyl lactate and ethyl acetate in a pure bran koji system containing ethanol, lactic acid and acetic acid. The lipase ANI_1_58064 and ANI_1_2344024 recombinant strains of the present invention are used to catalyze the synthesis of important flavor esters (ethyl lactate and ethyl acetate) in the aqueous phase system of liquor brewing, and have good industrial application prospects.

[0027] 2. In view of the fact that there is little research on the catalytic synthesis of ethyl lactate using the biosynthetic esterification enzyme pathway, the lipase recombinant strain of the present invention can catalyze the synthesis of ethyl lactate and ethyl acetate in the aqueous phase. The lipase recombinant strain of the present invention can be used to catalyze the synthesis of important flavor esters ethyl lactate and ethyl acetate in the brewing of beverage wine, and has good prospects for industrial application.

[0028] 3. The Aspergillus niger lactate ethyl lipase of the present invention has a certain promoting effect on the saccharifying enzyme activity of lipase ANI_1_58064 and ANI_1_2344024 while increasing the amount of ethyl lactate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The figure is a graph showing the PCR amplification results of the lipase ANI_1_58064 and ANI_1_2344024 encoding genes in Example 1 of the present invention; wherein, M: 5000Marker; Lane 1: using the Aspergillus niger genome as a template, primers SEQ ID No.11 and SEQ IDNo.12 were used for PCR amplification to obtain a target band of 1257bp; Lane 2: using the Aspergillus niger genome as a template, primers SEQ IDNo.13 and SEQ IDNo.14 were used for PCR amplification to obtain a target band of 1259bp; Lane 3: using the Aspergillus niger genome as a template, primers SEQ IDNo.15 and SEQ IDNo.16 were used for PCR amplification to obtain a target band of 1788bp; Lane 4: using the Aspergillus niger genome as a template, primers SEQ IDNo.17 and SEQ IDNo.18 were used for PCR amplification to obtain a target band of 1720bp; Lane 5: using the Aspergillus niger genome as a template, primers SEQ IDNo.19 and SEQ ID No.20 was amplified by PCR to obtain the target band of 783 bp;

[0030] Figure 2 This is a verification diagram of the genome of the recombinant strains ANI_1_58064 and ANI_1_2344024 of Example 2 of the present invention; wherein, M: 5000Marker; Lane 1: Using the genome of the recombinant strain ANI_1_58064 as a template, verification primers SEQ ID No.21 and SEQ ID No.22 were used for PCR amplification to obtain a target band of 1793bp; Lane 3: Using the genome of the recombinant strain ANI_1_2344024 as a template, verification primers SEQ ID No.23 and SEQ ID No.24 were used for PCR amplification to obtain a target band of 1765bp; Lane 5: Using the genome of the recombinant strain ANI_1_1202034 as a template, verification primers SEQ ID No.25 and SEQ ID No.26 were used for PCR amplification to obtain a target band of 2225bp; Lane 7: Using the genome of the recombinant strain ANI_1_2588074 as a template, verification primers SEQ ID No.27 and SEQ ID No.28 was used for PCR amplification to obtain a target band of 2226 bp; Lane 9: Using the genome of the recombinant strain ANI_1_582104 as a template, the verification primers SEQ ID No.29 and SEQ ID No.30 were used for PCR amplification to obtain a target band of 1231 bp; Lanes 2, 4, 6, 8, and 10: No bands were obtained by PCR amplification using the Aspergillus niger genome as a template;

[0031] Figure 3This is a graph showing the determination of the content of ethyl lactate and ethyl acetate synthesized by the lipase ANI_1_58064 and ANI_1_244024 recombinant strains in Example 4 of the present invention. a, b, c, and d represent the significance level (0.01 <P<0.05);

[0032] Figure 4 The graphs for the determination of the saccharification capacity of the lipase ANI_1_58064 and ANI_1_2344024 recombinant strains in Example 5 of the present invention, a, b, c, d represent the significance level (0.01 <P<0.05)。 DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0034] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0035] A Aspergillus niger ethyl lactate lipase, the amino acid sequence of the Aspergillus niger ethyl lactate lipase is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0036] The gene encoding the Aspergillus niger lactate ethyl lipase as described above.

[0037] Preferably, the sequence of the coding gene is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0038] Use of the Aspergillus niger ethyl lactate lipase as described above in catalyzing the synthesis of ethyl lactate and / or ethyl acetate.

[0039] Preferably, the ethyl lactate synthesized by the Aspergillus niger lactate ethyl lipase is significantly higher than that of the wild type, with an increase of more than 20%; the ethyl acetate synthesized by the Aspergillus niger lactate ethyl lipase is more than 70% higher than that of the wild type.

[0040] The use of the Aspergillus niger ethyl lactate lipase as described above in the preparation of ester aroma liquid for wine and / or white wine containing ethyl lactate and / or ethyl acetate.

[0041] A recombinant strain of Aspergillus niger lipase comprising the Aspergillus niger lactate ethyl lipase as described above.

[0042] Preferably, the Aspergillus niger lipase recombinant strain is obtained by PEG-mediated transformation;

[0043] Alternatively, the esterification ability of the recombinant strain of Aspergillus niger lipase under pure bran koji conditions is increased by more than 20%;

[0044] Alternatively, the saccharification ability of the recombinant Aspergillus niger lipase strain catalyzing the synthesis of ethyl lactate and ethyl acetate in a pure bran koji system is significantly higher than that of the wild-type Aspergillus niger under the pure bran koji conditions.

[0045] The method for constructing the above-mentioned Aspergillus niger lipase recombinant strain comprises the following steps:

[0046] The Aspergillus niger lactate ethyl lipase repair template was transformed into Aspergillus niger protoplasts through PEG6000 and cultured at 30°C for 3-5 days, and the single clone was verified by PCR strain verification.

[0047] The method for catalyzing the synthesis of ethyl lactate and ethyl acetate using the above-mentioned Aspergillus niger lipase recombinant strain comprises the following steps:

[0048] The recombinant strain of Aspergillus niger lipase is inoculated into a 65% bran medium and cultured at 30°C for 48 hours; the bran with mycelium is placed at 37°C for drying and shaken to be broken up to obtain the first-class seeds; after cooling, the first-class seeds are inoculated into an 85% bran medium, shaken well, and cultured at 30°C for 40 hours, and when the mycelium is fully covered and the bran is connected into a cake shape, the bottle is buckled to separate the bran from the bottom and continue to culture for 24 hours before the bottle is taken out; after the bottle is taken out, it is placed in a cowhide bag, quickly dried at 37-40°C to make its water content less than 12%, and fully broken up to obtain ethyl lactate and ethyl acetate;

[0049] The formula of 65% bran medium per 100 g is: 100 g bran, 65 mL water;

[0050] The formula of 85% bran medium per 100 g is: 100 g bran, 85 mL water.

[0051] Specifically, the relevant preparation and detection are as follows:

[0052] The present invention provides Aspergillus niger lipases, which are respectively defined as Aspergillus niger lipase ANI_1_58064 and Aspergillus niger lipase ANI_1_2344024. The object of the present invention is to provide lipases ANI_1_58064 and ANI_1_2344024, which catalyze the synthesis of ethyl lactate and ethyl acetate by esterification enzyme pathway under pure bran koji conditions, and both are derived from Aspergillus niger.

[0053] The amino acid sequences of the lipases ANI_1_58064 and ANI_1_2344024 of the present invention are shown in SEQ ID No. 1 or SEQ ID No. 2, respectively.

[0054] The gene sequences encoding lipase ANI_1_58064 and ANI_1_2344024 of the present invention are shown as SEQ ID No. 3 or SEQ ID No. 4 respectively.

[0055] The present invention first provides Aspergillus niger lipase, the amino acid sequence of which is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0056] Aspergillus niger lipase ANI_1_58064 amino acid sequence SEQ ID NO.1:

[0057] MYIPSVLLLAASLFHGATALPTPGSTPIPPSQDPWYSAPEGFEEADPGAILRVRPAPGNLTVVVGNASAAYNILYRTTDSQYKPSWAVTTLLVPPVAASAAVNQ SVLLSHQIAYDSFDVNASPSYAMYTSPPSDIILALQRGWFVNVPDYEGPNASFTAGVQSGHATLDSVRSVLASGFGLNEDAQYALWGYSGGALASEWAAELQMQY APELNIAGLAVGGLTPNVTSVMDTVTSTISAGLIPAAALGLSSQHPETYEFILSQLKTTGPYNRTGFLAAKDLTLSEAEVFYAFQNIFDYFVNGSATFQAEVVQK ALNQDGYMGYHGFPQMPVLAYKAIHDEISPIQDTDRVIKRYCGGLLNILYERNTIGGHSAEQVNGNARAWNWLTSIFDGTYAQQYKTEGCTIRNVTLNTTSSVY.

[0058] Aspergillus niger lipase ANI_1_2344024 amino acid sequence SEQ ID NO.2:

[0059] MPFFRAKSSKRQIMFVKWHFLVLGAIPMIPAYPSGAAYKGGFEWDSTKYLIAFGDSYTYVQGTDGLQNYSFIGDLQNFAYDPHTLLTDKIVQNQTATAEGGPNWVEYLTGCGLEEGLTSPFDCDQQLWDFAFAGSDISVEYTPLHHNFTVSLVNQVKQFNTYAQPVLKKTVDQSHAL VAIWIGINDIGDSSKYDVDFPTFYNELMNTLFSSVQTIYSQGYRSYLFMNLPPLDRRPGNLGSADPSPNATQITWYNDALAQHASAFHDRYADTNVMLFDAHSELSYILDNPGDFGIVNITNFCAGYDQPDIAWNYQAYGCPTPLDTYFWYNSGHMTSHVHEILAGAVERKLEEWSD.

[0060] The present invention also provides a gene encoding the Aspergillus niger lipase, which is shown in SEQ ID NO.3 or SEQ ID NO.4 respectively.

[0061] SEQ ID NO.3 of the coding gene of Aspergillus niger lipase ANI_1_58064:

[0062]

[0063] SEQ ID NO.4:

[0064] ATGCCCTTCTTTAGAGCTAAATCAAGCAAACGTCAAATCATGTTCGTAAAATGGCATTTTCTGGTCCTCGGGGCCATTCCGATGATTCCGGCATACCCTTCTGGTGCTGCTTATAAAGGCGGGTTTGAATGGGACTCGACGAAATACTTGTTCGTGCTTGCTCTCATTGATGGCCGCATATGGAGATCCGAGGGCGGTCCCAATTGGGTGGAATACCTGACGGGTTGCGGTCTCGAGGAGGGACTCACCTCACCCTTCGACTGCGACCAACAGCTATGGGACTTCGCCTTTGCAGGATCAGACATCTCAGTTGAATACACCCCACTCCACCACAACTTCACCGTCTCGCTTGTCAACCAAGTTAAGCAATTCAACACCTACGCGCAACCCGTGCTGAAGAAGACCGTAGACCAGTCACACGCACTAGTAGCCATCTGGATCGGGATCAACGACATCGGCGACAGTTCGAAATACGATGTCGACTTCCCGACCTTCTACAACGAGCTCATGAATACACTCTTTTCCTCCGTCCAGACTATCTACTCCCAGGGATACCGTTCCTATCTCTTCATGAACCTTCCCCCGCTGGACCGCAGACCAGGAAACCTAGGCAGCGCTGATCCCAGTCCCAACGCAACACAAATCACCTGGTACAATGATGCGTTGGCTCAGCATGCCAGTGCGTTCCATGATCGCTACGCCGACACCAATGTGATGTTGTTCGACGCGCATAGCGAACTCAGCTATATTCTGGATAACCCGGGTGACTTCGGCATTGTGAATATCACGAACTTCTGTGCGGGATATGATCAGCCGGATATCGCGTGGAATTATCAGGCGTATGGCTGTCCTACGCCGTTGGATACTTATTTCTGGTATAATTCGGGGCATATGACGAGCCATGTTCATGAGATCTTGGCTGGTGCTGTGGAGAGGAAGTTGGAGGAGTGGTCGGATTGA。

[0065] The present invention also provides the use of preparing bran koji from the above-mentioned Aspergillus niger lipase recombinant strain, and catalyzing the synthesis of ethyl lactate and / or ethyl acetate under water phase conditions.

[0066] The present invention also provides the use of the above-mentioned Aspergillus niger lipase recombinant strain in the preparation of ester aroma liquid or white wine containing ethyl lactate and / or ethyl acetate.

[0067] Wherein, in the application, the recombinant Aspergillus niger strain is obtained through PEG-mediated transformation.

[0068] In the application, the synthesis of ethyl lactate and ethyl acetate catalyzed by the recombinant strain of Aspergillus niger lipase is in a pure bran koji system.

[0069] Among them, in the application, the ethyl lactate synthesized by lipase ANI_1_58064 and ANI_1_2344024 is significantly higher than that of the wild type, with an increase of more than 20%.

[0070] In the application, the ethyl acetate synthesized by catalysis of lipase ANI_1_58064 and ANI_1_2344024 increased by more than 70%.

[0071] Among them, in the application, while increasing ethyl lactate, the saccharifying enzyme activity of lipase ANI_1_58064 and ANI_1_2344024 is promoted to a certain extent.

[0072] The present invention also provides a recombinant vector, which contains the coding genes of the above-mentioned lipase ANI_1_58064 and ANI_1_2344024; wherein the recombinant vector is selected from at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, and a yeast expression vector. In a preferred embodiment of the present invention, the recombinant vector is a nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.4 inserted into the multiple cloning site of the basic plasmid pLH454.

[0073] The present invention also provides a recombinant cell containing the above-mentioned recombinant vector. The recombinant cell is at least one of an insect cell, a mammalian cell, Escherichia coli, and a yeast. In a preferred embodiment of the present invention, the present invention provides a recombinant bacterium containing the recombinant cell, which is obtained by introducing the above-mentioned recombinant vector into a host cell, wherein the host cell is Aspergillus niger.

[0074] The present invention also provides the use of the lipase ANI_1_58064 and ANI_1_2344024 recombinant strains in catalyzing the synthesis of ethyl lactate and ethyl acetate by using an esterification enzyme pathway.

[0075] The method for synthesizing ethyl lactate and ethyl acetate by catalyzing the lipase recombinant strain comprises the following steps:

[0076] 1) Using the coding gene corresponding to lipase as a template, PCR amplification is performed using a primer pair to obtain a target gene fragment;

[0077] 2) inserting the target gene fragment into a plasmid expression vector to obtain a recombinant vector;

[0078] 3) PEG-mediated transformation of the recombinant vector into Aspergillus niger protoplasts to obtain a recombinant strain;

[0079] 4) preparing the recombinant strain successfully expressed in Aspergillus niger into pure bran koji;

[0080] 5) Mixing pure bran koji with acid and alcohol, esterifying at constant temperature for 7 days, and analyzing by gas phase after distillation;

[0081] In one embodiment of the present invention, the total volume of the optimal reaction system for synthesizing ethyl lactate by the lipase involved in pure bran koji is 100 mL, including 5 g of absolute dry bran koji, 20 mL of ethanol, and 1 mL of lactic acid or acetic acid.

[0082] The present invention provides three other Aspergillus niger control groups, which are Aspergillus niger lipase ANI_1_1202034, ANI_1_2588074 and ANI_1_582104, and their amino acid sequences are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.

[0083] Aspergillus niger lipase ANI_1_1202034 amino acid sequence SEQ ID NO.5:

[0084] MKGLRGHNKKSFWGNTRLSDLLWPVTLLPGLISAYQPVYLGSRQSSPFLPPQIPLGDSPPLSDTHEFTLRHIFHRGTYQDPDLHRRLDITPDTRLRAVSDAGIESDVVTPNTPLVASSQPLIIERLADRRLSVIEEHLVTARSSGSAVALSPSDWVMDTLPGPNVTEKKTVVALAMMTANDYIEVPGTGDWQDIHGRFNHSGSFGWQGDGLRGHVYADKTNSTVVISLKGTSPALFDGEGTTTNDKINDNLFFSCCCGQGGSYLWRQVCDCQTTLYNANLTCIVEAMLDENRYYRASLDLYSNITEMYPNANVWLTGHSLGGAVSSLLGLTFGVPVVTFEAVPEALPAARLGLPSPPGHDSRYPQSRRNTGSYHFGHTADPVYMGTCNGVSSVCTWGGYAMESACHTGQMCVYDTVEDKGWRVGLSKHRINYVIANVLAGYDDVPPCAPEEECYDCELWKFFRSNGSEITTTTSATSTSTSTSTTTSRTSTCKTPGWWGCLDQTTTTETTSTSTSTSTCKTPGWFGCKDPTTTSDITSVPTITTTEPPMTSTTTCKTPGWFGCKDETTTQVIAPAATLTITEPPVTSTTTGKHLGRF

[0085] Amino acid sequence of Aspergillus niger lipase ANI_1_2588074: SEQ ID NO.6

[0086] MHWEYSLVILLGACAVSVDGSPQVDLGYARYRGVRLPAGVDEYLGMRYAAPPLGQQRFRAPGDPSSTSSIQDAFEFGPTCIGVGEEISPLVGEDCLFINVFTPSHATTLSRLPVWVHIQGGGYASNANANFNGTNVIQHSGYDLVFVNFNYRVGALGFLASEEIRRDGDLNVGLLDQRKALAWVKQHISQFGGDPDHIIAHGDSAGAGSVTHHMTAYGGRAEGLFAGAVLESPFWPTLRTVAEMEFQYTRFVQRVGCSDASSALACLQSADLAAIQQGNVVDPFPDAAVDPPPLWYFLPVIDGAVVQDQLSRLFDQGKTVKIPVLVGDDTNEGSTFAYNASDASDMSRFLNANYPGLSSASLAAIGDAYPRMRPVADHAAYFPSASAAYGDAAFTCPGNRVAASMADHLPSGRVWSYRYNVRAPELVDEGLGVPHIFELSAIFGVGFAGNSEITSYNGINANAVATVMDYWISFVKALDPNPRRRSQAPRWEAWKPGLGQRLKIQTNTTAMEPIPPQQADRCSLWSALAPEMEI

[0087] Amino acid sequence of Aspergillus niger lipase ANI_1_582104, SEQ ID NO.7:

[0088] MSNTVAAAEEEGAYTPYDQFFLFGDSITEYSNAQDMGFGFSAALQNAYARRLDVVNRGLAGYNTLHAIKAFPKCFPTPERANVRLMTIWFGANDASLPGFEQHVPIETYKQNLRNIIQHPLTKAQNPRIMIITPPPINEYQLAGFDAMKGNPHPTRTNAHARKYGQAAREVAAEFGLPVADVWSAFMSTVGWKEGQPLVGSRDLPEDGKFASLFTDGLHLAANGYRIVFEEVMKTIQENWPDQVPEKLPYVFPSWALAPK

[0089] The present invention also provides Aspergillus niger lipase control groups ANI_1_1202034, ANI_1_2588074, and ANI_1_582104, and the encoding genes thereof are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0090] Aspergillus niger lipase ANI_1_1202034 amino acid sequence SEQ ID NO.8:

[0091]

[0092] Aspergillus niger lipase ANI_1_2588074 amino acid sequence SEQ ID NO.9:

[0093]

[0094] Aspergillus niger lipase ANI_1_582104 amino acid sequence SEQ ID NO.10:

[0095] ATGTCCAACACCGTTGCAGCAGCCGAGGAAGAAGGCGCTTACACGCCTTACGACCAATTCTTCCTCTTCGGCGACTCGATCACCGAGTACTCCAATGCCCAGGACATGGGATTCGGGTTCTCCGCAGCATTGCAGAATGGTAGGCAATCATTCCCTTATCCCCTTTTTGTACAGACATACACTGATATCTTGTAGCCTACGCCCGCCGCCTGGACGTTGTGAACCGTGGATTAGCGTGCGTCCCTCTATACCAAAACTGAAGCAACAAATAAAAGACAACAACCTCTGACTCTTTCCACAGCGGATACAATACTCTCCACGCCATCAAAGCCTTCCCCAAATGCTTCCCTACCCCAGAGCGCGCCAATGTCCGACTTATGGTAAGCACTCTTACCACACACCCTTACCAACACCCTCATAACTAACGAGCACCACCAGACCATCTGGTTCGGCGCCAACGACGCCAGTCTCCCAGGTTTCGAGCAACACGTCCCCATCGAAACCTACAAGCAAAACCTTAGAAACATCATCCAGCACCCCTTAACCAAGGCCCAGAACCCCCGCATCATGATCATCACTCCTCCCCCCATCAACGAGTACCAATTGGCCGGGTTCGACGCCATGAAGGGGAACCCCCATCCGACTCGGACCAACGCGCATGCCAGGAAGTACGGCCAGGCTGCGCGCGAAGTTGCTGCGGAGTTCGGTCTGCCTGTTGCGGATGTCTGGAGCGCGTTCATGTCGACGGTTGGGTGGAAAGAGGGTCAGCCGCTGGTTGGGTCGAGGGATTTGCCTGAGGATGGCAAGTTTGCTAGTCTGTTTACGGACGGCTTGCATTTGGCTGCTAATGGGTATCGCATTGTGTTTGAGGAGGTTATGAAGACTATTCAGGAGAACTGGCCGGATCAGGTGCCGGAGAAGCTTCCTTATGTGTTTCCTTCTTGGGCGTTGGCGCCGAAATAG

[0096] The primer synthesis and sequencing work used in the following examples were completed by a sequencing company. The expression vector used was pLH454. The expression host used was Aspergillus niger ATCC 1015.

[0097] Example 1 Construction of recombinant plasmid

[0098] Taking Aspergillus niger lipase ANI_1_58064 as an example, the construction process of recombinant strains such as Aspergillus niger lipase ANI_1_2344024 and ANI_1_1202034 is consistent with it. Using the DNA of Aspergillus niger lipase ANI_1_58064 as a template, according to the amino acid sequence of fungal lipase reported in GenBank, the online software Block Maker was used to search for conserved regions, and then the online primer design software CODEHOP (http: / / blocks.fhcrc.org / codehop.html) was used to design primers as follows:

[0099] The primer pair used to amplify ANI_1_58064 is forward primer SEQ ID No.11:

[0100] 5'-CATAGACACATCTAAACAATGGAATTCATGTATATCCCCTCGGTGCT-3';

[0101] Reverse primer SEQ ID No.12:

[0102] 5'-GATGATTTCAGTAACGTTAAGTGGATCCCTAATAAACGGAGGAAGTCGTGTTC-3'

[0103] The primer pair used to amplify ANI_1_2344024 is forward primer SEQ ID No.13:

[0104] 5'-CGCCACATAGACACATCTAAACAATGGAATTCATGCCCTTCTTTAGAGCTAAATC-3';

[0105] Reverse primer SEQ ID No.14:

[0106] 5'-GATGATTTCAGTAACGTTAAGTGGATCCTCAATCCGACCACTCCTCC-3'

[0107] The primer pair used to amplify ANI_1_1202034 is forward primer SEQ ID No.15:

[0108] 5'-CATAGACACATCTAAACAATGGAATTCATGAAAGGCCTTCGAGGG-3'

[0109] Reverse primer SEQ ID No.16:

[0110] 5'-GATGATTTCAGTAACGTTAAGTGGATCCTCAAAACCTGCCCAAGTGC-3'

[0111] The primer pair used to amplify ANI_1_2588074 is forward primer SEQ ID No.17:

[0112] 5'-CATAGACACATCTAAACAATGGAATTCATGCATTGGGAATATTCTCTGG-3'

[0113] Reverse primer SEQ ID No.18:

[0114] 5'-GATGATTTCAGTAACGTTAAGTGGATCCCTAAATCTCCATTTCCGGCG-3'

[0115] The primer pair used to amplify ANI_1_582104 is forward primer SEQ ID No.19:

[0116] 5'-CATAGACACATCTAAACAATGGAATTCATGTCCAACACCGTTGCAG-3'

[0117] Reverse primer SEQ ID No.20:

[0118] 5'-GATGATTTCAGTAACGTTAAGTGGATCCCTATTTCGGCGCCAACGC-3'

[0119] PCR reaction: Aspergillus niger ATCC 1015 genomic DNA was used as template, SEQ ID No. 11 and SEQ ID No. 12 were used as primers, and 2× Phanta Flash Master Mix (Cat. No. P520) was used. The PCR reaction conditions were: 98°C pre-denaturation for 30s, 98°C denaturation for 10s, 55°C annealing for 5s, 72°C extension for 30s, and 72°C final extension for 10min. After the PCR amplification was completed, the product was detected by 1% agarose gel electrophoresis. The results are shown in Figure 1 As shown, from Figure 1 It can be seen that a band of 1257 bp in length can be detected by gel electrophoresis, which is consistent with the theoretical value.

[0120] After the detection is correct, the product is purified and recovered using a DNA purification / agarose gel recovery kit (Cat. No.: AG21003).

[0121] The above-mentioned purified product and pLH454 (the applicant's previously authorized patent, patent number: ZL201810985901.9) were used as starting plasmids, and double-digested with EcoRⅠ and BamHⅠ, respectively. After digestion, they were connected and transformed into E. coli DH5α, and positive transformants were screened on LB plates containing G418 antibiotics (100 mg / mL). The obtained plasmids were sequenced and aligned to be correct, which were the correct recombinant plasmids pLH454-ANI_1_58064, pLH454-ANI_1_2344024, pLH454-ANI_1_1202034, pLH454-ANI_1_2588074 and pLH454-ANI_1_582104.

[0122] The composition of LB plate culture medium is: 10g / L sodium chloride, 10g / L peptone, 5g / L yeast extract powder, 100μg / mL G418, and 20g / L agar.

[0123] Example 2 Preparation of Aspergillus niger protoplasts and PEG-mediated transformation

[0124] The primers used to amplify the recombinant plasmid were designed as follows:

[0125] SEQ ID No.9: 5'-CACATGAGCGAAACCCTATAGG-3'

[0126] SEQ ID No.10: 5'-GATCGGTGCGGGCCTCTTCG-3'

[0127] The primer pair used to verify the recombinant strain ANI_1_58064 is the forward primer SEQ ID No.21:

[0128] 5'-AGAGGTTCCCTCCCCTAC-3'

[0129] Reverse primer SEQ ID No.22:

[0130] 5'-CTAGGTTGCAGTCAATGCATTAATG-3'

[0131] The primer pair used to verify the recombinant strain ANI_1_2344024 is the forward primer SEQ ID No.23:

[0132] 5'-CCTACCCTTTTTCTACTTCCCC-3'

[0133] Reverse primer SEQ ID No.24:

[0134] 5'-CATTAATGCATTGCAGATGAGC-3'

[0135] The primer pair used to verify the recombinant strain ANI_1_1202034 is the forward primer SEQ ID No.25:

[0136] 5'-CTCTCTCTTCCTCTTCATCCATC-3'

[0137] Reverse primer SEQ ID No.26:

[0138] 5'-ATTGCAGATGAGCTGTATCTGG-3'

[0139] The primer pair used to verify the recombinant strain ANI_1_2588074 is the forward primer SEQ ID No.27:

[0140] 5'-GTCAGTCCAGAGGTTCCCTC-3'

[0141] Reverse primer SEQ ID No.28:

[0142] 5'-CTGTATCTGGAAGAGGTAAACCCG-3'

[0143] The primer pair used to verify the recombinant strain ANI_1_582104 is the forward primer SEQ ID No.29:

[0144] 5'-CGCCGCTCAACTTTTCTTTC-3'

[0145] Reverse primer SEQ ID No.30:

[0146] 5'-AAGAGGTAAACCCGAAAACG-3'

[0147] Aspergillus niger liquid culture medium: glucose 20g / L, yeast extract 10g / L, magnesium sulfate 1g / L, potassium dihydrogen phosphate 1g / L.

[0148] Enzyme solution: Lysing Enzymes 5mg / mL, Yatalase 4mg / mL, β-glucuronidase 460U / mL.

[0149] STC solution: sorbitol 218.6 g / L, calcium chloride 7.35 g / L, Tris 1.21 g / L.

[0150] PEG6000 solution: PEG6000 570g / L, calcium chloride 7.35g / L.

[0151] Upper culture medium: malt extract 20 g / L, tryptone 5 g / L, sorbitol 218.6 g / L, agar 10 g / L, hygromycin 250 μg / mL.

[0152] Lower culture medium: malt extract 20 g / L, tryptone 5 g / L, sorbitol 218.6 g / L, agar 20 g / L, hygromycin 250 μg / mL.

[0153] 1.Fix template acquisition

[0154] After fragment amplification of the expression cassettes of the recombinant plasmids pLH454-ANI_1_58064, pLH454-ANI_1_2344024, pLH454-ANI_1_1202034, pLH454-ANI_1_2588074 and pLH454-ANI_1_582104 using primers SEQ ID No.9 / SEQ ID No.10, the DNA fragments of the reaction products were recovered and frozen for storage.

[0155] 2. Preparation of Protoplasts

[0156] (1) Use a sterile pipette tip to pick up spores from the Aspergillus niger plate, spot-spot the spores onto a PDA plate, and culture in a 30°C incubator in the dark for 3-5 days.

[0157] (2) Use the flat end of a sterile weighing spoon to scrape the spores from the Aspergillus niger plate and transfer them to a centrifuge tube containing 5 mL of sterile water.

[0158] (3) Filter the mycelium with sterile filter paper and filter the spores into another sterile centrifuge tube. Use a hemocytometer to determine the spore concentration. 8 Inoculate 50 mL of Aspergillus niger liquid culture medium with a CFU / mL inoculation amount, culture at 30°C, 200 rpm, shaker for 14-16 h, take 10 μL to make slices, and observe the spore germination. Ideally, the head should not be bifurcated.

[0159] (4) Filter the germinated spores and wash them repeatedly with precooled sterile water to remove the residual culture medium.

[0160] (5) Use a sterilized weighing spoon to pick up an appropriate amount of germinated spores and add them to a 50 mL Erlenmeyer flask containing 10 mL of enzymatic solution. Incubate at 120 rpm and 37°C for 3.5 h. Observe under a microscope to obtain the best protoplasts. Place on ice immediately after lysis.

[0161] (6) Add 10 mL of pre-cooled STC solution to the lysis solution and gently resuspend the protoplast pellet. Centrifuge several times and resuspend the protoplasts to obtain the final protoplast cells.

[0162] 3. PEG-mediated transformation of Aspergillus niger protoplasts

[0163] (1) Add 200 μL of Aspergillus niger protoplasts, 50 μg of repair template, and 680 μL of PEG 6000 solution to a sterile centrifuge tube and place on ice for 20 min;

[0164] (2) Add 2 mL of PEG 6000 solution and leave at room temperature for 10 min;

[0165] (3) Add 4 mL of STC solution and 4 mL of upper culture medium and plate on the lower culture medium;

[0166] (4) The plate is placed in an incubator and incubated for about 3-5 days to produce colonies.

[0167] The above repair template was transformed into Aspergillus niger protoplasts, and transformants were selected from the plate and transferred to a new strain PDA plate. After a sufficient amount of Aspergillus niger spores were grown, the genome was verified. Figure 2 As shown, after PCR amplification, gel electrophoresis detection can produce a band of 1793 bp in length, which is consistent with the positive result. The transformants with consistent length verification are genome-verified positive transformants, which can prove that the ANI_1_58064 gene expression work has been completed based on the Aspergillus niger strain.

[0168] Example 3 Preparation of pure bran koji

[0169] 65% bran medium (100 g): 100 g bran, 65 mL water.

[0170] 85% bran medium (100 g): 100 g bran, 85 mL water.

[0171] The recombinant strains ANI_1_58064, ANI_1_2344024, ANI_1_1202034, ANI_1_2588074 and ANI_1_582104 were inoculated into 65% bran medium and cultured at 30°C for 48 hours. The bran with mycelium was placed at 37°C for drying and shaken to break up to obtain the first-level seeds. After cooling, the first-level seeds were inoculated into 85% bran medium, shaken well, and cultured at 30°C for 40 hours. When the mycelium was full and the bran was connected into a cake shape, the bottle was buckled to separate the bran from the bottom and continue to culture for 24 hours before the bottle was taken out. After taking out the bottle, it was placed in a cowhide bag, dried quickly at 37-40°C (water content <12%), and then fully broken up and stored.

[0172] Example 4: Synthesis of ethyl lactate and ethyl acetate catalyzed by lipase ANI_1_58064, ANI_1_2344024, ANI_1_1202034, ANI_1_2588074 and ANI_1_582104 recombinant strains under bran conditions

[0173] Esterification power determination method: 1mL lactic acid and 20mL anhydrous ethanol are diluted to 100mL with distilled water, mixed and transferred to a 250mL conical flask, 5g of absolutely dry pure bran koji is added, and esterification is carried out at 30℃ for 7 days. Then 100mL of distilled water is added and heated and distilled in a 1000mL distillation flask, and 100mL of distillate is collected. The esterified distillate is taken for gas chromatography analysis.

[0174] The chromatographic detection conditions are as follows: Agilent CP-WAX57 chromatographic column (50m×250μm×0.25μm); the carrier gas is high-purity nitrogen (N2) (purity>99.999%); the column flow rate is 1mL / min; the detector temperature is 260℃; the injection port temperature is 250℃; the programmed temperature is: the starting temperature is 35℃, maintained for 1min, increased to 70℃ at 3℃ / min, maintained for 0min, and then increased to 190℃ at 3.5℃ / min, maintained for 5min; the injection volume is 1μL; split injection, the split ratio is 30:1.

[0175] The results are as follows Figure 3As shown, under the condition of pure bran koji, through the determination of esterification power, it can be seen that the wild-type Aspergillus niger synthesized 1220 mg / L of ethyl lactate. Compared with the wild-type Aspergillus niger, the five recombinant strains ANI_1_582104 successfully constructed in Aspergillus niger synthesized 1189.38 mg / L of ethyl lactate, which was 2.51% lower than the wild-type Aspergillus niger. The ethyl lactate synthesized by the recombinant strains ANI_1_1202034 and ANI_1_2588074 were 1304.91 mg / L and 1321.38 mg / L, respectively, which were not significantly improved compared with the wild-type Aspergillus niger. Only the recombinant strains ANI_1_58064 and ANI_1_2344024 significantly increased ethyl lactate, among which the recombinant strain ANI_1_58064 synthesized ethyl lactate to 1830 mg / L, an increase of 50%. The synthesis of ethyl lactate by the recombinant strain ANI_1_2344024 was 1519.39 mg / L, which was 20% higher. It can be seen that not all lipases can make an important contribution to the synthesis of ethyl lactate. Combined with the determination of the synthesis of ethyl acetate by the recombinant strain ANI_1_58064 and the recombinant strain ANI_1_2344024, it was found that the synthesis of ethyl acetate by the recombinant strain ANI_1_58064 was 416.3 mg / L, which was 80.73% higher than that of the wild type, and the synthesis of ethyl acetate by the recombinant strain ANI_1_2344024 was 424.9 mg / L, which was 84.74% higher than that of the wild type. This shows that under the conditions of bran koji, these two lipase recombinant strains can efficiently synthesize ethyl acetate while efficiently synthesizing ethyl lactate.

[0176] Example 5 Determination of saccharification capacity of lipase ANI_1_58064 and ANI_1_2344024 recombinant strains

[0177] Method for determining saccharification power: 5g of absolutely dry bran koji is placed in a beaker, 10mL of acetic acid-sodium acetate is added, and the volume is made up to 100mL with water. It is placed in a 35℃ constant temperature water bath for 1h, filtered, and the filtrate is collected as the bran koji sample solution. 5mL is taken as a blank solution and titrated directly with a glucose standard solution, and another 5mL is taken and saccharified in a 35℃ constant temperature water bath for 1h and then titrated with a glucose standard solution.

[0178] Aspergillus niger is the main source of saccharifying enzymes in liquor and plays an important role in saccharifying starch. In order to improve the activity of esterification enzymes, it is necessary to ensure that the saccharifying enzymes can play a normal saccharifying role. Therefore, the saccharifying ability of the recombinant strains was determined. Figure 4As shown, the saccharification capacity of the wild-type Aspergillus niger is 1083 mg / L, the saccharification capacity of the recombinant strain ANI_1_58064 is 1331.01 mg / L, an increase of 22.9%; the saccharification capacity of the recombinant strain ANI_1_2344024 is 1379.74 mg / L, an increase of 27.4%. The saccharification capacity of the two recombinant strains that can efficiently synthesize ethyl lactate is significantly higher than that of the wild-type strain, indicating that the activity of the saccharifying enzyme is not inhibited and even improved to a certain extent while the content of ethyl lactate is increased, that is, ethyl lactate is successfully synthesized efficiently through the esterification enzyme pathway, providing an effective method to increase the yield of ethyl lactate in liquor, which is different from the chemical synthesis in previous studies.

[0179] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. An Aspergillus niger ethyl lactate lipase, characterized in that: The amino acid sequence of the Aspergillus niger lactate ethyl lipase is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A gene encoding the Aspergillus niger lactate ethyl lipase as claimed in claim 1.

3. The coding gene according to claim 2, characterized in that: The sequence of the coding gene is shown as SEQ ID NO.3 or SEQ ID NO.

4.

4. Use of the Aspergillus niger ethyl lactate lipase as claimed in claim 1 in catalyzing the synthesis of ethyl lactate and / or ethyl acetate.

5. The use according to claim 4, characterized in that: The ethyl lactate synthesized by the Aspergillus niger lactate ethyl lipase is significantly higher than that of the wild type, with an increase of more than 20%; the ethyl acetate synthesized by the Aspergillus niger lactate ethyl lipase is more than 70% higher than that of the wild type.

6. Use of the Aspergillus niger lactate ethyl lipase as claimed in claim 1 in the preparation of ester aroma liquid for wine and / or white wine containing ethyl lactate and / or ethyl acetate.

7. A recombinant Aspergillus niger lipase strain containing the Aspergillus niger lactate ethyl lipase according to claim 1.

8. The Aspergillus niger lipase recombinant strain according to claim 7, characterized in that: The Aspergillus niger lipase recombinant strain is obtained by PEG-mediated transformation; Alternatively, the esterification ability of the recombinant strain of Aspergillus niger lipase under pure bran koji conditions is increased by more than 20%; Alternatively, the saccharification ability of the recombinant Aspergillus niger lipase strain catalyzing the synthesis of ethyl lactate and ethyl acetate in a pure bran koji system is significantly higher than that of the wild-type Aspergillus niger under the pure bran koji conditions.

9. The method for constructing a recombinant strain of Aspergillus niger lipase according to claim 7 or 8, characterized in that: The steps include: The Aspergillus niger lactate ethyl lipase repair template was transformed into Aspergillus niger protoplasts through PEG6000 and cultured at 30°C for 3-5 days, and the single clone was verified by PCR strain verification.

10. A method for synthesizing ethyl lactate and ethyl acetate by catalyzing the Aspergillus niger lipase recombinant strain as claimed in claim 7, characterized in that: The steps include: The recombinant strain of Aspergillus niger lipase is inoculated into a 65% bran medium and cultured at 30°C for 48 hours; the bran with mycelium is placed at 37°C for drying and shaken to be broken up to obtain the first-class seeds; after cooling, the first-class seeds are inoculated into an 85% bran medium, shaken well, and cultured at 30°C for 40 hours, and when the mycelium is fully covered and the bran is connected into a cake shape, the bottle is buckled to separate the bran from the bottom and continue to culture for 24 hours before the bottle is taken out; after the bottle is taken out, it is placed in a cowhide bag, quickly dried at 37-40°C to make its water content less than 12%, and fully broken up to obtain ethyl lactate and ethyl acetate; The formula of 65% bran medium per 100 g is: 100 g bran, 65 mL water; The formula of 85% bran medium per 100 g is: 100 g bran, 85 mL water.

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