Esterification enzyme mutant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a saccharomyces cerevisiae esterifying enzyme mutant for catalyzing production of ethyl hexanoate as well as a preparation method and application of the saccharomyces cerevisiae esterifying enzyme mutant. In order to improve the yield of ethyl hexanoate produced by the existing esterifying enzyme, the 28th site, the 69th site, the 125th site, the 180th site and the 214th site of the amino acid sequence of the esterifying enzyme derived from saccharomyces cerevisiae are subjected to substitution and / or deletion and / or insertion of at least one amino acid through a genetic engineering technology to obtain various esterifying enzyme mutants, so that the molecular modification is realized, and the yield of ethyl hexanoate produced by the esterifying enzyme is improved. Therefore, the reaction activity of catalyzing the esterification enzyme to produce ethyl hexanoate is improved. According to the method, the biosynthesis efficiency of ethyl hexanoate is improved through enzyme molecule modification, new power is injected for transformation and upgrading of the modern brewing industry, and the traditional brewing industry is promoted to develop towards the intelligent and precise direction.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a Saccharomyces cerevisiae esterase mutant that catalyzes the production of ethyl hexanoate, and a preparation method and application thereof. Background Art

[0002] Ethyl hexanoate is an ester flavor substance with an important aroma contribution. Its fresh and elegant apple aroma plays a key flavor regulation role in various fermented wines. Ethyl hexanoate can enhance the fruity aroma level of the wine body, and can also act synergistically with other flavor substances to enhance the mellow feeling and coordination of the wine body. It is an important component for constructing the flavor system of high-quality wines. In traditional brewing processes, ethyl hexanoate is mainly produced by the esterification reaction of hexanoic acid and ethanol catalyzed by intracellular esterase of Saccharomyces cerevisiae, and its content directly affects the sensory quality and market acceptance of the product. However, there are many limiting factors for the esterase produced by natural Saccharomyces cerevisiae in the actual fermentation process. The catalytic activity of the enzyme is low, resulting in insufficient conversion efficiency of ethyl hexanoate and difficult to meet the industrial production demand in terms of output. Secondly, the environmental adaptability of this enzyme in the fermentation process is poor, and it is easy to inactivate under high-concentration ethanol and acidic conditions, seriously affecting the accumulation of products. Moreover, the substrate specificity of traditional esterase is insufficient, and it is easy to catalyze the formation of other non-target ester substances, which not only reduces the conversion efficiency of the target product, but also leads to the accumulation of fusel alcohols and esters, affecting the purity of the product flavor.

[0003] There are two methods in the prior art to produce ethyl hexanoate: synthesis from hexanoic acid and ethanol catalyzed by lipase or esterase; and synthesis from ethanol and hexanoyl coenzyme A catalyzed by ethanol hexanoyltransferase. Previous studies have shown that ethyl hexanoate is mainly produced in the later stage of liquor brewing. Therefore, in order to increase the content of ethyl hexanoate in liquor, the brewing time of liquor is usually extended. However, this approach not only results in high grain consumption and low production efficiency, but also produces unpleasant flavor compounds. Traditional enzyme modification methods such as random mutation screening may obtain mutants with improved performance, but the screening period is long, the workload is large, and it is difficult to obtain mutants with target performance directionally. In recent years, with the development of protein engineering technology, the strategy of combining semi-rational design and directed evolution to modify enzyme molecules has become an effective way to improve the catalytic performance of enzymes. This method can precisely modify the key amino acid sites of enzyme molecules and specifically enhance their catalytic activity, substrate selectivity and environmental tolerance.

[0004] Therefore, developing esterase mutants with high catalytic activity, high substrate selectivity and good stability has important practical significance for improving the biosynthesis efficiency of ethyl hexanoate and the quality of wine products. Summary of the Invention

[0005] In order to increase the yield of ethyl hexanoate by existing esterifying enzymes, the present invention uses genetic engineering and enzyme engineering means to perform molecular modification on the esterifying enzyme of Saccharomyces cerevisiae, thereby increasing the yield of ethyl hexanoate and creating conditions for expanding its industrial application.

[0006] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0007] In the first aspect, the present invention provides an esterifying enzyme mutant, which is obtained by substituting and / or deleting and / or inserting at least one amino acid at positions 28, 69, 125, 180, and 214 of the amino acid sequence shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] MHLKALTVSLLSYSLLIASLPLTPRNQTPLNSLITAVLEHVPAINGTINAVVGILTDFETLVAGITKAQTTYNEVDDSKPCTEYTLIFARGTTEPGNVGILVGPPLINALIEKVGSDALTVQGVNNYPATIGGYTAGGDPQGSEEMASEIEKVHSTCPDTHLIASGYSQGSQLVHNSIAKLPAATAEWISSVLVFGDPDDNDPIPNVDSSRVFTACHDGDNICQDGDLILPAHLTYAENVRDAAAFAVSAASS.

[0010] Among them, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 is as shown in SEQ ID NO.2.

[0011] SEQ ID NO.2:

[0012] ATGCACCTCAAAGCTCTAACGGTCTCCCTCCTCTCTTACTCCCTCCTCATCGCCTCCCTCCCCCTAACACCCCGGAATCAAACCCCGCTCAATAGCCTAATAACAGCCGTCCTTGAGCACGTCCCCGCCATAAACGGCACCATCAATGCTGTCGTCGGCATCCTAACCGATTTCGAGACACTCGTCGCCGGCATCACCAAGGCGCAGACAACATACAACGAGGTCGACGACTCCAAGCCCTGCACAGAGTACACCCTCATTTTTGCACGGGGGACCACCGAACCTGGCAATGTCGGCATCCTCGTTGGACCCCCGCTGATCAACGCTTTGATTGAGAAGGTGGGGAGCGACGCTTTGACTGTGCAGGGGGTTAATAACTATCCTGCCACTATTGGGGGGTATACGGCTGGCGGAGATCCTCAGGGGAGTGAGGAGATGGCATCTGAAATCGAAAAAGTACACTCCACCTGCCCCGACACCCATCTCATCGCATCAGGATACTCGCAGGGTTCGCAGCTCGTTCATAATTCCATCGCCAAGCTGCCTGCCGCCACGGCAGAGTGGATTAGCAGTGTTCTTGTCTTCGGGGATCCAGATGACAACGACCCAATTCCCAACGTCGATTCGTCCAGGGTCTTCACGGCTTGCCATGATGGCGATAATATCTGCCAGGATGGGGATCTGATCTTGCCTGCACATTTGACATATGCGGAGAATGTGAGGGATGCGGCTGCGTTCGCGGTGTCTGCTGCTTCATCGTGA。

[0013] Among them, the esterase mutant contains at least one of the following mutations:

[0014] The amino acid sequence shown in SEQ ID NO.1 is subjected to:

[0015] (1) The 28th threonine (T) is mutated to alanine (A), arginine (R), asparagine (N), cysteine (C), glutamine (Q), glutamate (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), aspartic acid (D), tryptophan (W), tyrosine (Y), valine (V);

[0016] (2) The 69th glutamine (Q) is mutated to alanine (A), arginine (R), asparagine (N), cysteine (C), threonine (T), glutamate (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), aspartic acid (D), tryptophan (W), tyrosine (Y), valine (V);

[0017] (3) The 125th asparagine (N) is mutated to alanine (A), arginine (R), glutamine (Q), cysteine (C), threonine (T), glutamate (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), aspartic acid (D), tryptophan (W), tyrosine (Y), valine (V);

[0018] (4) The 180th lysine (K) is mutated to alanine (A), arginine (R), asparagine (N), cysteine (C), threonine (T), glutamate (E), glycine (G), histidine (H), isoleucine (I), leucine (L), glutamine (Q), methionine (M), phenylalanine (F), proline (P), serine (S), aspartic acid (D), tryptophan (W), tyrosine (Y), valine (V);

[0019] and / or

[0020] (5) The 214th threonine (T) is mutated to alanine (A), arginine (R), asparagine (N), cysteine (C), glutamine (Q), glutamate (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), aspartic acid (D), tryptophan (W), tyrosine (Y), valine (V).

[0021] Preferably, the esterase mutant is obtained by mutating the 28th threonine (T) in the amino acid sequence shown in SEQ ID NO.1 to alanine (T28A), and the amino acid sequence is as shown in SEQ ID NO.3.

[0022] SEQ ID NO.3:

[0023] MHLKALTVSLLSYSLLIASLPLTPRNQAPLNSLITAVLEHVPAINGTINAVVGILTDFETLVAGITKAQTTYNEVDDSKPCTEYTLIFARGTTEPGNVGILVGPPLINALIEKVGSDALTVQGVNNYPATIGGYTAGGDPQGSEEMASEIEKVHSTCPDTHLIASGYSQGSQLVHNSIAKLPAATAEWISSVLVFGDPDDNDPIPNVDSSRVFTACHDGDNICQDGDLILPAHLTYAENVRDAAAFAVSAASS.

[0024] Preferably, the esterase mutant is obtained by mutating the 69th glutamine (Q) in the amino acid sequence shown in SEQ ID NO.1 to serine (Q69S), and the amino acid sequence is as shown in SEQ ID NO.4.

[0025] SEQ ID NO.4:

[0026] MHLKALTVSLLSYSLLIASLPLTPRNQTPLNSLITAVLEHVPAINGTINAVVGILTDFETLVAGITKASTTYNEVDDSKPCTEYTLIFARGTTEPGNVGILVGPPLINALIEKVGSDALTVQGVNNYPATIGGYTAGGDPQGSEEMASEIEKVHSTCPDTHLIASGYSQGSQLVHNSIAKLPAATAEWISSVLVFGDPDDNDPIPNVDSSRVFTACHDGDNICQDGDLILPAHLTYAENVRDAAAFAVSAASS.

[0027] Preferably, the esterase mutant is obtained by mutating the 125th asparagine (N) in the amino acid sequence shown in SEQ ID NO.1 to proline (N125P), and the amino acid sequence is as shown in SEQ ID NO.5.

[0028] SEQ ID NO.5:

[0029] MHLKALTVSLLSYSLLIASLPLTPRNQTPLNSLITAVLEHVPAINGTINAVVGILTDFETLVAGITKAQTTYNEVDDSKPCTEYTLIFARGTTEPGNVGILVGPPLINALIEKVGSDALTVQGVPNYPATIGGYTAGGDPQGSEEMASEIEKVHSTCPDTHLIASGYSQGSQLVHNSIAKLPAATAEWISSVLVFGDPDDNDPIPNVDSSRVFTACHDGDNICQDGDLILPAHLTYAENVRDAAAFAVSAASS。

[0030] Preferably, the esterase mutant is the amino acid sequence shown in SEQ ID NO.6, in which lysine (K) at position 180 of the amino acid sequence shown in SEQ ID NO.1 is mutated to histidine (K180H).

[0031] SEQ ID NO.6:

[0032] MHLKALTVSLLSYSLLIASLPLTPRNQTPLNSLITAVLEHVPAINGTINAVVGILTDFETLVAGITKAQTTYNEVDDSKPCTEYTLIFARGTTEPGNVGILVGPPLINALIEKVGSDALTVQGVNNYPATIGGYTAGGDPQGSEEMASEIEKVHSTCPDTHLIASGYSQGSQLVHNSIAHLPAATAEWISSVLVFGDPDDNDPIPNVDSSRVFTACHDGDNICQDGDLILPAHLTYAENVRDAAAFAVSAASS。

[0033] Preferably, the esterase mutant is the amino acid sequence shown in SEQ ID NO.7, in which threonine (T) at position 214 of the amino acid sequence shown in SEQ ID NO.1 is mutated to leucine (T214L).

[0034] SEQ ID NO.7:

[0035] MHLKALTVSLLSYSLLIASLPLTPRNQTPLNSLITAVLEHVPAINGTINAVVGILTDFETLVAGITKAQTTYNEVDDSKPCTEYTLIFARGTTEPGNVGILVGPPLINALIEKVGSDALTVQGVNNYPATIGGYTAGGDPQGSEEMASEIEKVHSTCPDTHLIASGYSQGSQLVHNSIAKLPAATAEWISSVLVFGDPDDNDPIPNVDSSRVFLACHDGDNICQDGDLILPAHLTYAENVRDAAAFAVSAASS。

[0036] Most preferably, the esterase mutant is one in which the 28th threonine (T) in the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine (A), the 69th glutamine (Q) is mutated to serine (S), the 125th asparagine (N) is mutated to proline (P), the 180th lysine (K) is mutated to histidine (H), and the 214th threonine (T) is mutated to leucine (L) (T28A / Q69S / N125P / K180H / T214L), and its amino acid sequence is as shown in SEQ ID NO.8.

[0037] SEQ ID NO.8:

[0038] MHLKALTVSLLSYSLLIASLPLTPRNQAPLNSLITAVLEHVPAINGTINAVVGILTDFETLVAGITKASTTYNEVDDSKPCTEYTLIFARGTTEPGNVGILVGPPLINALIEKVGSDALTVQGVPNYPATIGGYTAGGDPQGSEEMASEIEKVHSTCPDTHLIASGYSQGSQLVHNSIAHLPAATAEWISSVLVFGDPDDNDPIPNVDSSRVFLACHDGDNICQDGDLILPAHLTYAENVRDAAAFAVSAASS。

[0039] In a second aspect, the present invention provides a nucleic acid molecule containing a nucleotide sequence encoding the amino acid sequence of the above-mentioned esterase mutant.

[0040] Wherein, the amino acid sequence of the esterase mutant contains at least one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.8.

[0041] In a third aspect, the present invention provides an expression vector containing the above-mentioned nucleic acid molecule.

[0042] Among them, the expression vector is selected from at least one of p416 GPD, pYES2, pYES2 / NT, pYES2 / CT, pYES3, pYES6, pYCplac22-GFP, pUG6, pSH47, pGADT7, pGBKT7, pYEP13.

[0043] Preferably, the expression vector is the p416 GPD vector.

[0044] Fourthly, the present invention provides a host bacterium containing the above-mentioned expression vector.

[0045] Among them, the host bacterium is selected from at least one of Saccharomyces cerevisiae Y190, Y187, INVSC1, AH109, BY4742, YM4271, FY837.

[0046] Preferably, the host bacterium is Saccharomyces cerevisiae Y190.

[0047] Fifthly, the present invention provides a method for preparing the above-mentioned esterase mutant, which includes the following steps:

[0048] Using the gene encoding the sequence shown in SEQ ID NO.1 as a template, primers shown in SEQ ID NO.9 to SEQ ID NO.108 are used to amplify the gene by PCR technology to obtain a recombinant plasmid containing the encoding genes of different esterase mutants, and then transferred into host cells to obtain a recombinant engineering bacterium; after the recombinant engineering bacterium is fermented and cultured, it is broken and extracted to obtain different esterase mutants.

[0049] Fifthly, the present invention provides the application of the above-mentioned esterase mutant, nucleic acid molecule, expression vector or host bacterium in the catalytic synthesis of ethyl hexanoate.

[0050] Among them, in the above application, the wet cells obtained after the engineering bacterium containing the encoding gene of the above-mentioned esterase mutant is fermented and cultured or the enzyme extracted after the wet cells are broken is used as a biocatalyst to synthesize ethyl hexanoate.

[0051] Preferably, the fermentation culture environment is a YPD medium, and its components include: glucose 15 - 20 g / L, peptone 15 - 20 g / L, yeast extract powder 5 - 10 g / L, natural pH.

[0052] Beneficial effects: Through genetic engineering technology, at least one amino acid substitution and / or deletion and / or insertion is performed on the 28th, 69th, 125th, 180th, and 214th positions of the amino acid sequence of the esterase derived from Saccharomyces cerevisiae, obtaining a variety of esterase mutants, achieving molecular-level transformation, and thus improving the reaction activity of the esterase in catalyzing the production of ethyl hexanoate. Among them, when the constructed esterase mutant T28A / Q69S / N125P / K180H / T214L catalyzes the production of ethyl hexanoate, the yield of ethyl hexanoate is the best, reaching 13.6 g / L. The breakthrough of the technology of the present invention not only helps to solve the problem of directional regulation of flavor substances in the brewing industry, but also provides technical support for the development of new functional brewing strains. By improving the biosynthesis efficiency of ethyl hexanoate through enzyme molecule modification, it injects new impetus into the transformation and upgrading of the modern brewing industry, and promotes the development of the traditional brewing industry towards the direction of intelligence and precision. Brief description of the drawings

[0053] Figure 1 It is a data graph of the production of ethyl hexanoate catalyzed by different mutants obtained by mutating the 28th amino acid of the Saccharomyces cerevisiae esterase in Example 1 of the present invention;

[0054] Figure 2 It is a data graph of the production of ethyl hexanoate catalyzed by different mutants obtained by mutating the 69th amino acid of the Saccharomyces cerevisiae esterase in Example 2 of the present invention;

[0055] Figure 3 It is a data graph of the production of ethyl hexanoate catalyzed by different mutants obtained by mutating the 125th amino acid of the Saccharomyces cerevisiae esterase in Example 3 of the present invention;

[0056] Figure 4 It is a data graph of the production of ethyl hexanoate catalyzed by different mutants obtained by mutating the 180th amino acid of the Saccharomyces cerevisiae esterase in Example 4 of the present invention;

[0057] Figure 5 It is a data graph of the production of ethyl hexanoate catalyzed by different mutants obtained by mutating the 214th amino acid of the Saccharomyces cerevisiae esterase in Example 5 of the present invention. Detailed implementation manners

[0058] In order to make the technical problems to be solved, technical solutions and beneficial effects of the present application clearer, the following further elaborates on the present application in combination with the implementation manners. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0059] The key to the functional differences of esterifying enzymes lies in the differences in microorganisms. Due to the differences in microorganisms, the functional genes carried by the microorganisms and the enzymes encoded by them have obvious differences in amino acid sequences. The sequence determines the structure of the enzyme, and the structure determines the function of the enzyme. It is precisely because the amino acid sequence is different from the sequences related in the existing literature that there are obvious differences in the catalytic characteristics of the enzyme.

[0060] The innovation of the present invention lies in optimizing the amino acid sequence of the existing esterifying enzyme derived from Saccharomyces cerevisiae through genetic engineering technology, so as to increase the content of ethyl hexanoate catalyzed by the enzyme encoded by the gene of this sequence.

[0061] In some specific embodiments of the present invention, through genetic engineering technology, the 28th amino acid of the amino acid sequence of the Saccharomyces cerevisiae esterifying enzyme is mutated into T28A, T28R, T28N, T28C, T28Q, T28E, T28G, T28H, T28I, T28L, T28K, T28M, T28F, T28P, T28S, T28D, T28W, T28Y, T28V respectively, and recombinant plasmids containing different Saccharomyces cerevisiae esterifying enzyme mutant genes are obtained. Then the constructed recombinant plasmids are respectively transferred into host bacteria to obtain recombinant engineering bacteria. The obtained recombinant engineering bacteria are fermented and cultured, and can catalyze the synthesis of ethyl hexanoate with different yields.

[0062] In some specific embodiments of the present invention, through genetic engineering technology, the 69th amino acid of the amino acid sequence of the Saccharomyces cerevisiae esterifying enzyme is mutated into Q69A, Q69R, Q69N, Q69C, Q69T, Q69E, Q69G, Q69H, Q69I, Q69L, Q69K, Q69M, Q69F, Q69P, Q69S, Q69D, Q69W, Q69Y, Q69V respectively, and recombinant plasmids containing different Saccharomyces cerevisiae esterifying enzyme mutant genes are obtained. Then the constructed recombinant plasmids are respectively transferred into host bacteria to obtain recombinant engineering bacteria. The obtained recombinant engineering bacteria are fermented and cultured, and can catalyze the synthesis of ethyl hexanoate with different yields.

[0063] In some specific embodiments of the present invention, through genetic engineering technology, the 125th amino acid of the amino acid sequence of the Saccharomyces cerevisiae esterifying enzyme is mutated into N125A, N125R, N125Q, N125C, N125T, N125E, N125G, N125H, N125I, N125L, N125K, N125M, N125F, N125P, N125S, N125D, N125W, N125Y, N125V respectively, and recombinant plasmids containing different Saccharomyces cerevisiae esterifying enzyme mutant genes are obtained. Then the constructed recombinant plasmids are respectively transferred into host bacteria to obtain recombinant engineering bacteria. The obtained recombinant engineering bacteria are fermented and cultured, and can catalyze the synthesis of ethyl hexanoate with different yields.

[0064] In some specific embodiments of the present invention, through genetic engineering techniques, the 180th amino acid of the amino acid sequence of Saccharomyces cerevisiae esterase is mutated to K180A, K180R, K180N, K180C, K180T, K180E, K180G, K180H, K180I, K180L, K180Q, K180M, K180F, K180P, K180S, K180D, K180W, K180Y, K180V, respectively, to obtain recombinant plasmids containing different Saccharomyces cerevisiae esterase mutant genes, and then the constructed recombinant plasmids are respectively transferred into host bacteria to obtain recombinant engineering bacteria. The obtained recombinant engineering bacteria are fermented and cultured, and can catalyze the synthesis of ethyl hexanoate with different yields.

[0065] In some specific embodiments of the present invention, through genetic engineering techniques, the 214th amino acid of the amino acid sequence of Saccharomyces cerevisiae esterase is mutated to T214A, T214R, T214N, T214C, T214Q, T214E, T214G, T214H, T214I, T214L, T214K, T214M, T214F, T214P, T214S, T214D, T214W, T214Y, T214V, respectively, to obtain recombinant plasmids containing different Saccharomyces cerevisiae esterase mutant genes, and then the constructed recombinant plasmids are respectively transferred into host bacteria to obtain recombinant engineering bacteria. The obtained recombinant engineering bacteria are fermented and cultured, and can catalyze the synthesis of ethyl hexanoate with different yields.

[0066] In a preferred embodiment of the present invention, when the esterase mutant T28A / Q69S / N125P / K180H / T214L is constructed to catalyze the production of ethyl hexanoate, the yield of ethyl hexanoate is the best, reaching 13.6 g / L.

[0067] In a more specific embodiment of the present invention, the construction of the recombinant engineering bacteria includes the following steps:

[0068] Using wild-type Saccharomyces cerevisiae esterase as a template, different esterase mutant genes are amplified by PCR technology using different primers. The Saccharomyces cerevisiae esterase mutant genes are inserted into the XbaI and SmaI sites of the p416 GPD vector to construct a recombinant plasmid containing the Saccharomyces cerevisiae esterase mutant gene. The constructed recombinant plasmid is transferred into the host bacterium Saccharomyces cerevisiae Y190 to obtain recombinant engineering bacteria.

[0069] Specific embodiments will be listed below to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those without specific techniques or conditions noted in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. Reagents or instruments without the manufacturer noted are all conventional products that can be obtained through commercial purchase.

[0070] The YPD medium used in the embodiment has a formulation of 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, natural pH, and is sterilized at 121 °C for 20 minutes.

[0071] Saccharomyces cerevisiae Y190: Purchased from SHMCC D25047.

[0072] The preparation method of the sorghum enzymatic hydrolysate used in the embodiment is as follows: Sorghum is pulverized and distilled water is added according to distilled water: sorghum = 4:1 (v / w). It is boiled for 30 minutes for gelatinization. The gelatinized solution is cooled to 90 °C, and 0.2% of the high-temperature α-amylase based on the dry weight of sorghum is added and stirred at a constant temperature for 1 hour for liquefaction. After the liquefied solution is cooled, 0.2% of the glucoamylase based on the dry weight of sorghum is added for saccharification for 2 hours. After saccharification, it is filtered and adjusted to 8 Brix and then aliquoted into Erlenmeyer flasks, and sterilized at 115 °C for 20 minutes to obtain the sorghum enzymatic hydrolysate.

[0073] Example 1 Construction of a mutant with the 28th amino acid mutation of Saccharomyces cerevisiae esterase

[0074] In order to perform 19 mutations other than the wild type on the 28th amino acid codon of the Saccharomyces cerevisiae esterase gene, forward primers T28A, T28R, T28N, T28C, T28Q, T28E,, T28G, T28H, T28I, T28L, T28K, T28M, T28F, T28P, T28S, T28D, T28W, T28Y, T28V, and a common reverse primer T28-R were designed. The primer sequences are as follows:

[0075] Forward primers:

[0076] SEQ ID NO.9, T28A: 5’-GCGCCGCTCAATAGCCTAATAACAG-3’

[0077] SEQ ID NO.10, T28R: 5’-CGCCCGCTCAATAGCCTAATAACAG-3’

[0078] SEQ ID NO.11, T28N: 5’-AACCCGCTCAATAGCCTAATAACAG-3’

[0079] SEQ ID NO.12, T28C: 5’-TGCCCGCTCAATAGCCTAATAACAG-3’

[0080] SEQ ID NO.13, T28Q: 5’-CAGCCGCTCAATAGCCTAATAACAG-3’

[0081] SEQ ID NO.14, T28E: 5’-GAGCCGCTCAATAGCCTAATAACAG-3’

[0082] SEQ ID NO.15, T28G: 5’-GGACCGCTCAATAGCCTAATAACAG-3’

[0083] SEQ ID NO.16, T28H: 5’-CATCCGCTCAATAGCCTAATAACAG-3’

[0084] SEQ ID NO.17, T28I: 5’-ATCCCGCTCAATAGCCTAATAACAG-3’

[0085] SEQ ID NO.18, T28L: 5’-CTGCCGCTCAATAGCCTAATAACAG-3’

[0086] SEQ ID NO.19, T28K: 5’-AAGCCGCTCAATAGCCTAATAACAG-3’

[0087] SEQ ID NO.20, T28M: 5’-ATGCCGCTCAATAGCCTAATAACAG-3’

[0088] SEQ ID NO.21, T28F: 5’-TTCCCGCTCAATAGCCTAATAACAG-3’

[0089] SEQ ID NO.22, T28P: 5’-CCTCCGCTCAATAGCCTAATAACAG-3’

[0090] SEQ ID NO.23, T28S: 5’-TCGCCGCTCAATAGCCTAATAACAG-3’

[0091] SEQ ID NO.24, T28D: 5’-GACCCGCTCAATAGCCTAATAACAG-3’

[0092] SEQ ID NO.25, T28W: 5’-TGGCCGCTCAATAGCCTAATAACAG-3’

[0093] SEQ ID NO.26, T28Y: 5’-TACCCGCTCAATAGCCTAATAACAG-3’

[0094] SEQ ID NO.27, T28V: 5’-GTCCCGCTCAATAGCCTAATAACAG-3’

[0095] Reverse primer:

[0096] SEQ ID NO.28, T28-R: 5’-CCTCCCCCTAACACCCCGGAATCAA-3’.

[0097] The esterase (ASPNIDRAFT, NCBI, GenBank accession number: EHA26219.1) gene (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) was inserted into the XbaI and SmaI sites of the p416 GPD vector to construct the recombinant plasmid p416 GPD-ASPNIDRAFT. Using the recombinant plasmid p416 GPD-ASPNIDRAFT as a template, whole plasmid PCR amplification was performed using the above primers. The amplification conditions were as follows:

[0098] PCR reagents: 5 μL of 10×Ex Taq Buffer (Mg 2+ Plus) (20 mM), 4 μL of dNTP Mixture (2.5 mM each), 2 μL of primer (10 μM), 4 μL of template, 0.5 μL of Taq DNA polymerase (5 U / μL), and made up to 50 μL with ddH 2 O.

[0099] The reaction conditions were as follows: pre-denaturation at 90°C for 5 min, denaturation at 94°C for 30 s, annealing at 56.5°C for 30 s, extension at 72°C for 1 min, complete extension at 72°C for 10 min, and 35 cycles were carried out.

[0100] Digest the PCR product with Dpn I enzyme at 37 °C for 1 hour to remove the template plasmid. Ligate the digested PCR product with T4 DNA ligase at 16 °C for 4 hours. Transform the ligation product into E. coli DH5α competent cells, spread on an Amp-resistant plate, and culture at 37 °C for 16 hours. Pick single colonies to obtain recombinant plasmids containing the esterase mutant gene of Saccharomyces cerevisiae. Inoculate Saccharomyces cerevisiae Y190 into YPD liquid medium and culture at 28 °C. Centrifuge to collect cells and prepare competent cells by the PEG / LiAc method. Mix the constructed recombinant plasmid with the competent cells, add the PEG / LiAc solution, incubate at 30 °C for 30 min, add DMSO, and perform heat shock at 42 °C for 15 min, then ice bath for 5 min. After centrifugation, resuspend the cells, spread on an SC-Ura plate, and culture at 28 °C for 2 - 3 days to obtain recombinant engineering bacteria.

[0101] First, activate and culture the recombinant engineering bacteria containing different esterase mutants in YPD liquid medium at 28 °C for 24 hours. Then, inoculate them into the sorghum enzymatic hydrolysate medium at an inoculation amount of 3.5%, and culture at 28 °C and 180 r / min for 24 hours to complete the cell accumulation stage. Subsequently, add 2% ethanol (v / v) and 0.059% caproic acid (v / v) and continue culturing to complete the ethyl caproate synthesis stage. Use a 7820A gas chromatograph-mass spectrometer (GC-MS, Agilent Technologies, USA) to detect the content of ethyl caproate in the fermentation broth.

[0102] The detection method is as follows: Chromatographic column: J&W 122 - 7032 (30 m × 250 μm × 0.25 μm); Carrier gas (high-purity nitrogen) flow rate: 0.8 mL / min; Split ratio: 60∶1; Tail gas blow 25 mL / min; H 2 Flow rate 30 mL / min; Air flow rate 400 mL / min; Injection volume: 0.1 μL; Detector temperature: 220 °C; Injection port temperature 220 °C; Column oven temperature programming conditions are: Equilibrate at 50 °C for 5 min, and program the temperature to 200 °C at a rate of 8 °C / min and hold for 5 min. Treatment method of the esterification liquid: Take 5 mL of the esterification liquid, add 5 mL of anhydrous ethanol, mix well, filter through a 0.22 μm organic filter membrane, and then use gas chromatography for detection. The final ethyl caproate yield is as Figure 1 shown.

[0103] Example 2 Construction of a mutant with the 69th amino acid mutation of the esterase in Saccharomyces cerevisiae

[0104] To introduce 19 mutations other than the wild type at the 69th amino acid codon of the Saccharomyces cerevisiae esterase gene, forward primers Q69A, Q69R, Q69N, Q69C, Q69T, Q69E, Q69G, Q69H, Q69I, Q69L, Q69K, Q69M, Q69F, Q69P, Q69S, Q69D, Q69W, Q69Y, Q69V and a common reverse primer Q69-R were designed. The primer sequences are shown below:

[0105] Forward primers:

[0106] SEQ ID NO.29, Q69A: 5’-GCGACAACATACAACGAGGTCGAC-3’

[0107] SEQ ID NO.30, Q69R: 5’-CGAACAACATACAACGAGGTCGAC-3’

[0108] SEQ ID NO.31, Q69N: 5’-AACACAACATACAACGAGGTCGAC-3’

[0109] SEQ ID NO.32, Q69C: 5’-TGCACAACATACAACGAGGTCGAC-3’

[0110] SEQ ID NO.33, Q69T: 5’-ACTACAACATACAACGAGGTCGAC-3’

[0111] SEQ ID NO.34, Q69E: 5’-GAGACAACATACAACGAGGTCGAC-3’

[0112] SEQ ID NO.35, Q69G: 5’-GGTACAACATACAACGAGGTCGAC-3’

[0113] SEQ ID NO.36, Q69H: 5’-CATACAACATACAACGAGGTCGAC-3’

[0114] SEQ ID NO.37, Q69I: 5’-ATCACAACATACAACGAGGTCGAC-3’

[0115] SEQ ID NO.38, Q69L: 5’-CTGACAACATACAACGAGGTCGAC-3’

[0116] SEQ ID NO.39, Q69K: 5’-AAGACAACATACAACGAGGTCGAC-3’

[0117] SEQ ID NO.40, Q69M: 5’-ATGACAACATACAACGAGGTCGAC-3’

[0118] SEQ ID NO.41, Q69F: 5’-TTCACAACATACAACGAGGTCGAC-3’

[0119] SEQ ID NO.42, Q69P: 5’-CCTACAACATACAACGAGGTCGAC-3’

[0120] SEQ ID NO.43, Q69S: 5’-TCGACAACATACAACGAGGTCGAC-3’

[0121] SEQ ID NO.44, Q69D: 5’-GACACAACATACAACGAGGTCGAC-3’

[0122] SEQ ID NO.45, Q69W: 5’-TGGACAACATACAACGAGGTCGAC-3’

[0123] SEQ ID NO.46, Q69Y: 5’-TACACAACATACAACGAGGTCGAC-3’

[0124] SEQ ID NO.47, Q69V: 5’-GTCACAACATACAACGAGGTCGAC-3’

[0125] Reverse primer:

[0126] SEQ ID NO.48, Q69-R: 5’-CGCCTTGGTGATGCCGGCGACGAGT-3’

[0127] Perform whole plasmid PCR amplification using the above forward and reverse primers, and other operations are the same as in Example 1.

[0128] After performing single-site site-directed saturation mutagenesis on the 69th amino acid of the Saccharomyces cerevisiae esterase, different mutants are obtained, and finally the yield of ethyl hexanoate catalyzed is as Figure 2 shown.

[0129] Example 3 Construction of mutants with the 125th amino acid mutation of Saccharomyces cerevisiae esterase

[0130] To introduce 19 mutations other than the wild-type at the 125th amino acid codon of the Saccharomyces cerevisiae esterase gene, forward primers N125A, N125R, N125Q, N125C, N125T, N125E, N125G, N125H, N125I, N125L, N125K, N125M, N125F, N125P, N125S, N125D, N125W, N125Y, N125V, and a common reverse primer N125-R were designed. The primer sequences are as follows:

[0131] Forward primers:

[0132] SEQ ID NO.49, N125A: 5’-GCTAACTATCCTGCCACTATTGGGG-3’

[0133] SEQ ID NO.50, N125R: 5’-CGAAACTATCCTGCCACTATTGGGG-3’

[0134] SEQ ID NO.51, N125Q: 5’-CAGAACTATCCTGCCACTATTGGGG-3’

[0135] SEQ ID NO.52, N125C: 5’-TGCAACTATCCTGCCACTATTGGGG-3’

[0136] SEQ ID NO.53, N125T: 5’-ACTAACTATCCTGCCACTATTGGGG-3’

[0137] SEQ ID NO.54, N125E: 5’-GAGAACTATCCTGCCACTATTGGGG-3’

[0138] SEQ ID NO.55, N125G: 5’-GGGAACTATCCTGCCACTATTGGGG-3’

[0139] SEQ ID NO.56, N125H: 5’-CATAACTATCCTGCCACTATTGGGG-3’

[0140] SEQ ID NO.57, N125I: 5’-ATCAACTATCCTGCCACTATTGGGG-3’

[0141] SEQ ID NO.58, N125L: 5’-CTGAACTATCCTGCCACTATTGGGG-3’

[0142] SEQ ID NO.59, N125K: 5’-AAGAACTATCCTGCCACTATTGGGG-3’

[0143] SEQ ID NO.60, N125M: 5’-ATGAACTATCCTGCCACTATTGGGG-3’

[0144] SEQ ID NO.61, N125F: 5’-TTCAACTATCCTGCCACTATTGGGG-3’

[0145] SEQ ID NO.62, N125P: 5’-CCTAACTATCCTGCCACTATTGGGG-3’

[0146] SEQ ID NO.63, N125S: 5’-TCGAACTATCCTGCCACTATTGGGG-3’

[0147] SEQ ID NO.64, N125D: 5’-GACAACTATCCTGCCACTATTGGGG-3’

[0148] SEQ ID NO.65, N125W: 5’-TGGAACTATCCTGCCACTATTGGGG-3’

[0149] SEQ ID NO.66, N125Y: 5’-TACAACTATCCTGCCACTATTGGGG-3’

[0150] SEQ ID NO.67, N125V: 5’-GTCAACTATCCTGCCACTATTGGGG-3’

[0151] Reverse primer:

[0152] SEQ ID NO.68, N125-R: 5’-AACCCCCTGCACAGTCAAAGCGTCG-3’

[0153] Perform whole plasmid PCR amplification using the above forward and reverse primers, and other operations are the same as in Example 1.

[0154] After performing single-site site-directed saturation mutagenesis on the 125th amino acid of the Saccharomyces cerevisiae esterase, different mutants were obtained, and finally the yields of ethyl hexanoate catalyzed are as Figure 3 shown.

[0155] Example 4 Construction of mutants with mutations at the 180th amino acid of the Saccharomyces cerevisiae esterase

[0156] To introduce 19 mutations other than the wild-type at the 180th amino acid codon of the Saccharomyces cerevisiae esterase gene, forward primers K180A, K180R, K180N, K180C, K180T, K180E, K180G, K180H, K180I, K180L, K180Q, K180M, K180F, K180P, K180S, K180D, K180W, K180Y, K180V, and the reverse universal primer K180-R were designed. The primer sequences are as follows:

[0157] Forward primers:

[0158] SEQ ID NO.69, K180A: 5’-GCTCTGCCTGCCGCCACGGCAGAGT-3’

[0159] SEQ ID NO.70, K180R: 5’-CGACTGCCTGCCGCCACGGCAGAGT-3’

[0160] SEQ ID NO.71, K180N: 5’-AACCTGCCTGCCGCCACGGCAGAGT-3’

[0161] SEQ ID NO.72, K180C: 5’-TGCCTGCCTGCCGCCACGGCAGAGT-3’

[0162] SEQ ID NO.73, K180T: 5’-ACTCTGCCTGCCGCCACGGCAGAGT-3’

[0163] SEQ ID NO.74, K180E: 5’-GAGCTGCCTGCCGCCACGGCAGAGT-3’

[0164] SEQ ID NO.75, K180G: 5’-GGGCTGCCTGCCGCCACGGCAGAGT-3’

[0165] SEQ ID NO.76, K180H: 5’-CATCTGCCTGCCGCCACGGCAGAGT-3’

[0166] SEQ ID NO.77, K180I: 5’-ATCCTGCCTGCCGCCACGGCAGAGT-3’

[0167] SEQ ID NO.78, K180L: 5’-CTGCTGCCTGCCGCCACGGCAGAGT-3’

[0168] SEQ ID NO.79, K180Q: 5’-CAGCTGCCTGCCGCCACGGCAGAGT-3’

[0169] SEQ ID NO.80, K180M: 5’-ATGCTGCCTGCCGCCACGGCAGAGT-3’

[0170] SEQ ID NO.81, K180F: 5’-TTCCTGCCTGCCGCCACGGCAGAGT-3’

[0171] SEQ ID NO.82, K180P: 5’-CCTCTGCCTGCCGCCACGGCAGAGT-3’

[0172] SEQ ID NO.83, K180S: 5’-TCGCTGCCTGCCGCCACGGCAGAGT-3’

[0173] SEQ ID NO.84, K180D: 5’-GACCTGCCTGCCGCCACGGCAGAGT-3’

[0174] SEQ ID NO.85, K180W: 5’-TGGCTGCCTGCCGCCACGGCAGAGT-3’

[0175] SEQ ID NO.86, K180Y: 5’-TACCTGCCTGCCGCCACGGCAGAGT-3’

[0176] SEQ ID NO.87, K180V: 5’-GTCCTGCCTGCCGCCACGGCAGAGT-3’

[0177] Reverse primer:

[0178] SEQ ID NO.88, K180-R: 5’-GGCGATGGAATTATGAACGAGCTGC-3’

[0179] Perform whole plasmid PCR amplification using the above forward and reverse primers, and other operations are the same as in Example 1.

[0180] After performing single-site site-directed saturation mutagenesis on the 180th amino acid of the Saccharomyces cerevisiae esterase, different mutants were obtained, and finally the yields of ethyl hexanoate catalyzed are as Figure 4 shown.

[0181] Example 5 Construction of mutants with mutations at the 214th amino acid of Saccharomyces cerevisiae esterase

[0182] To introduce 19 mutations other than the wild - type at the 214th amino acid codon of the Saccharomyces cerevisiae esterase gene, forward primers T214A, T214R, T214N, T214C, T214Q, T214E, T214G, T214H, T214I, T214L, T214K, T214M, T214F, T214P, T214S, T214D, T214W, T214Y, T214V, and a reverse universal primer T214 - R were designed. The primer sequences are as follows:

[0183] Forward primers:

[0184] SEQ ID NO.89, T214A: 5’ - GCTGCTTGCCATGATGGCGATAATA - 3’

[0185] SEQ ID NO.90, T214R: 5’ - CGAGCTTGCCATGATGGCGATAATA - 3’

[0186] SEQ ID NO.91, T214N: 5’ - AACGCTTGCCATGATGGCGATAATA - 3’

[0187] SEQ ID NO.92, T214C: 5’ - TGCGCTTGCCATGATGGCGATAATA - 3’

[0188] SEQ ID NO.93, T214Q: 5’ - CAGGCTTGCCATGATGGCGATAATA - 3’

[0189] SEQ ID NO.94, T214E: 5’ - GAGGCTTGCCATGATGGCGATAATA - 3’

[0190] SEQ ID NO.95, T214G: 5’ - GGGGCTTGCCATGATGGCGATAATA - 3’

[0191] SEQ ID NO.96, T214H: 5’ - CATGCTTGCCATGATGGCGATAATA - 3’

[0192] SEQ ID NO.97, T214I: 5’ - ATCGCTTGCCATGATGGCGATAATA - 3’

[0193] SEQ ID NO.98, T214L: 5’ - CTGGCTTGCCATGATGGCGATAATA - 3’

[0194] SEQ ID NO.99, T214K: 5’-AAGGCTTGCCATGATGGCGATAATA-3’

[0195] SEQ ID NO.100, T214M: 5’-ATGGCTTGCCATGATGGCGATAATA-3’

[0196] SEQ ID NO.101, T214F: 5’-TTCGCTTGCCATGATGGCGATAATA-3’

[0197] SEQ ID NO.102, T214P: 5’-CCTGCTTGCCATGATGGCGATAATA-3’

[0198] SEQ ID NO.103, T214S: 5’-TCGGCTTGCCATGATGGCGATAATA-3’

[0199] SEQ ID NO.104, T214D: 5’-GACGCTTGCCATGATGGCGATAATA-3’

[0200] SEQ ID NO.105, T214W: 5’-TGGGCTTGCCATGATGGCGATAATA-3’

[0201] SEQ ID NO.106, T214Y: 5’-TACGCTTGCCATGATGGCGATAATA-3’

[0202] SEQ ID NO.107, T214V: 5’-GTCGCTTGCCATGATGGCGATAATA-3’

[0203] Reverse primer:

[0204] SEQ ID NO.108, T214-R: 5’-GAACACCCTGGACGAATCGACGTTG-3’

[0205] Perform whole plasmid PCR amplification using the above forward and reverse primers, and other operations are the same as in Example 1.

[0206] After performing single-site site-directed saturation mutagenesis on the 214th amino acid of Saccharomyces cerevisiae esterase, different mutants are obtained, and finally the yields of ethyl hexanoate catalyzed are as Figure 5 shown.

[0207] Example 6 Construction of Saccharomyces cerevisiae Esterase Mutant T28A / Q69S / N125P / K180H / T214L

[0208] Forward primer:

[0209] SEQ ID NO.9, T28A: 5’-GCGCCGCTCAATAGCCTAATAACAG-3’

[0210] SEQ ID NO.43, Q69S: 5’-TCGACAACATACAACGAGGTCGAC-3’

[0211] SEQ ID NO.62, N125P: 5’-CCTAACTATCCTGCCACTATTGGGG-3’

[0212] SEQ ID NO.76, K180H: 5’-CATCTGCCTGCCGCCACGGCAGAGT-3’

[0213] SEQ ID NO.98, T214L: 5’-CTGGCTTGCCATGATGGCGATAATA-3’

[0214] Reverse primer:

[0215] SEQ ID NO.28, T28-R: 5’-CCTCCCCCTAACACCCCGGAATCAA-3’.

[0216] SEQ ID NO.48, Q69-R: 5’-CGCCTTGGTGATGCCGGCGACGAGT-3’

[0217] SEQ ID NO.68, N125-R: 5’-AACCCCCTGCACAGTCAAAGCGTCG-3’

[0218] SEQ ID NO.88, K180-R: 5’-GGCGATGGAATTATGAACGAGCTGC-3’

[0219] SEQ ID NO.108, T214-R: 5’-GAACACCCTGGACGAATCGACGTTG-3’

[0220] Perform whole plasmid PCR amplification using the above forward and reverse primers, and other operations are the same as in Example 1.

[0221] By performing site-directed saturation mutagenesis at five sites simultaneously at positions 28, 69, 125, 180, and 214 of the esterase of Saccharomyces cerevisiae, the amino acid sequence of the mutant is as shown in SEQ ID NO.8, and the mutant T28A / Q69S / N125P / K180H / T214L is obtained. Finally, the ethyl hexanoate production reaches 13.6 g / L.

Claims

1. An esterase mutant, characterized in that: The amino acid sequence is obtained by substituting and / or deleting and / or inserting at least one amino acid at positions 28, 69, 125, 180 and 214 of the amino acid sequence shown in SEQ ID NO.

1.

2. The esterification enzyme mutant according to claim 1, characterized in that The esterase mutant comprises at least one of the following mutations: The amino acid sequence shown in SEQ ID NO.1 is: (1) Threonine at position 28 is mutated to alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, aspartic acid, tryptophan, tyrosine, or valine; (2) Glutamine at position 69 mutated to alanine, arginine, asparagine, cysteine, threonine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, aspartic acid, tryptophan, tyrosine, or valine; (3) Asparagine at position 125 mutated to alanine, arginine, glutamine, cysteine, threonine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, aspartic acid, tryptophan, tyrosine, or valine; (4) Lysine 180 is mutated to alanine, arginine, asparagine, cysteine, threonine, glutamic acid, glycine, histidine, isoleucine, leucine, glutamine, methionine, phenylalanine, proline, serine, aspartic acid, tryptophan, tyrosine, or valine; and / or (5) The threonine at position 214 is mutated to alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, aspartic acid, tryptophan, tyrosine, or valine.

3. The esterification enzyme mutant according to claim 2, characterized in that: The esterification enzyme mutant is a mutant in which the threonine at position 28 of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine, and the amino acid sequence is shown in SEQ ID NO.3; and / or The esterification enzyme mutant is a mutant in which the glutamine at position 69 of the amino acid sequence shown in SEQ ID NO.1 is mutated to serine, and the amino acid sequence is as shown in SEQ ID NO.4; and / or The esterification enzyme mutant is a mutant in which the asparagine at position 125 of the amino acid sequence shown in SEQ ID NO.1 is mutated to proline, and the amino acid sequence is shown in SEQ ID NO.5; and / or The esterification enzyme mutant is a mutant in which the 180th lysine in the amino acid sequence shown in SEQ ID NO.1 is mutated to a histidine amino acid sequence as shown in SEQ ID NO.6; and / or The esterase mutant is obtained by mutating the 214th threonine in the amino acid sequence shown in SEQ ID NO.1 to leucine, and the amino acid sequence is shown in SEQ ID NO.

7.

4. The esterification enzyme mutant according to any one of claims 1 to 3, characterized in that: The esterase mutant is a mutant in which the threonine at position 28 of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine, the glutamine at position 69 is mutated to serine, the asparagine at position 125 is mutated to proline, the lysine at position 180 is mutated to histidine, and the threonine at position 214 is mutated to leucine. The amino acid sequence is shown in SEQ ID NO.

8.

5. A nucleic acid molecule, characterized in that: A nucleotide sequence comprising the amino acid sequence encoding the esterification enzyme mutant according to any one of claims 1 to 4.

6. An expression vector, characterized in that: Containing the nucleic acid molecule according to claim 5.

7. A host bacterium, characterized in that: Containing the expression vector according to claim 6.

8. The method for preparing the esterification enzyme mutant according to any one of claims 1 to 4, characterized in that: The following steps are involved: The gene shown in SEQ ID NO.1 is used as a template, and the primers shown in SEQ ID NO.9 to SEQ ID NO.108 are used to amplify the gene by PCR technology to obtain a recombinant plasmid containing genes encoding different esterase mutants, which are then transferred into host cells to obtain recombinant engineered bacteria; the recombinant engineered bacteria are then fermented and cultured, and then crushed and extracted to obtain different esterase mutants.

9. Use of the esterification enzyme mutant according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6 or the host bacteria according to claim 7 in catalyzing the synthesis of ethyl hexanoate.

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