An acetylacetone cleavage enzyme mutant and a method for producing 2-acetylcyclohexanone by an extracellular enzyme reaction

Abstract

The application discloses an acetylacetone cleavage enzyme mutant and a method for generating 2-acetylcyclohexanone through an extracellular enzyme reaction, and belongs to the technical field of biological engineering. In order to solve the technical problem of how to improve the yield of 2-acetylcyclohexanone generated through an extracellular enzyme reaction, the problem of limited Dke1 substrate types in a whole-cell synthesis system is solved, and the toxicity of 2-acetylcyclohexanone to cells is inhibited. The application provides an acetylacetone cleavage enzyme mutant, wherein the mutant is obtained by mutating the amino acid at the 105th position of the acetylacetone cleavage enzyme with the amino acid sequence shown in SEQ ID NO. 3 to alanine from glycine, and the yield of 2-acetylcyclohexanone generated through an enzyme reaction of the mutant in vitro reaches 14.22 mg / L.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to an acetylacetone lyase mutant and a method for producing 2-acetylcyclohexanone by extracellular enzyme reaction. BACKGROUND

[0002] 2-acetylcyclohexanone is an important intermediate of endothelin converting enzyme inhibitor, and can also be used as an intermediate of insect hormone (Albercla Am et al., 2000). The clinical application of endothelin converting enzyme inhibitor makes it possible to cure a large number of cardiovascular disease patients. According to the research status at home and abroad, it is of great significance to study the synthesis of 2-acetylcyclohexanone, an intermediate of endothelin converting enzyme inhibitor (Jasmin Jf et al., 2004). At present, the chemical synthesis method is mainly used, which can be carried out through the following routes: acylation of cyclohexanone (Zhang Zhugui, 2006); first generating enamine, and then acylating with acetic anhydride (Liu Baodian, 2005); using straight-chain β-diketone to form a ring (Huang Xian, 1983); condensation reaction of cyclohexanone and morpholine with toluene as azeotropic agent and p-toluenesulfonic acid as catalyst to generate enamine, then adding triethylamine as acid binding agent, and dropping acetyl chloride and organic solvent mixture, and then enamine and acetyl chloride undergo nucleophilic addition reaction to generate iminium ion, and finally adding concentrated hydrochloric acid to hydrolyze the iminium ion to generate the research product 2-acetylcyclohexanone (Han Feng, 2009). The chemical synthesis method has the disadvantages of low yield, many by-products, difficult to obtain raw materials, high price, need to use metal organic catalyst in the reaction, and great environmental pressure, which does not meet the needs of the current low-carbon economy. The biological method has the advantages of low energy consumption, low cost, and avoidance of generation of a large amount of pollutants, and has obvious advantages compared with the chemical method.

[0003] The biological synthesis method of 2-acetylcyclohexanone has not attracted widespread attention. Since there are few studies showing that Dke1 can catalyze other substrates, it is very important to expand the substrate spectrum of Dke1 catalysis. At the same time, acetylacetone has toxicity to microbial cells (Water Research et al., 1980), and 2-acetylcyclohexanone has a similar structure to acetylacetone and may have strong toxic effects, resulting in relatively low yield of 2-acetylcyclohexanone. SUMMARY

[0004] The purpose of the present application is to solve the technical problem of how to improve the yield of 2-acetylcyclohexanone produced by extracellular enzyme reaction. The present application solves the problem of limited substrate types of Dke1 in the whole cell synthesis system and the problem of toxicity inhibition of 2-acetylcyclohexanone to cells.

[0005] The present application provides an acetoacetate decarboxylase mutant, which is obtained by mutating the amino acid at position 105 of the acetoacetate decarboxylase with the amino acid sequence shown in SEQ ID NO. 3.

[0006] Further limitation, the glycine at position 105 is mutated to alanine.

[0007] The present application also provides a gene encoding the mutant.

[0008] The present application provides a recombinant vector carrying the gene.

[0009] Further limitation, the starting vector of the recombinant vector is any one of pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series.

[0010] The present application provides a recombinant microbial cell carrying the gene or expressing the mutant.

[0011] The present application provides the use of the mutant, the gene, the vector or the recombinant microbial cell in improving the yield of 2-acetylcyclohexanone.

[0012] The present application provides a method for producing 2-acetylcyclohexanone by extracellular enzyme reaction, comprising the following steps:

[0013] 1) connecting the acetoacetate decarboxylase mutant gene to an expression vector to obtain a recombinant vector;

[0014] 2) transforming the recombinant vector obtained in step 1) into a host bacterium to obtain a recombinant bacterium, and fermenting the recombinant bacterium to obtain a culture solution;

[0015] 3) pretreating the culture solution obtained in step 2) to obtain an acetoacetate decarboxylase mutant protease solution;

[0016] 4) mixing the enzyme solution obtained in step 3) with a reaction substrate and reacting to prepare 2-acetylcyclohexanone.

[0017] Further limitation, the gene sequence of the acetoacetate decarboxylase mutant in step 1) is shown in SEQ ID NO. 2.

[0018] Further limitation, the host bacterium in step 2) is Escherichia coli.

[0019] Further limitation, step 1) the fermentation culture is to inoculate the recombinant bacteria into the fermentation medium, cultivate at 37 DEG C, 180 rpm to the bacterial concentration is OD600 0.6~0.8, after adding the final concentration 0.2mM IPTG, cultivate at 30 DEG C for 4~6h, obtain the culture solution.

[0020] Further limitation, the culture solution obtained in step 2) is pretreated, and the bacteria in the culture solution are broken by ultrasonic wave to obtain a broken bacteria solution, the broken bacteria solution is centrifuged to retain the supernatant, and the supernatant is purified by a nickel column to obtain an acetoacetone lyase mutant Dke1G105A protease solution.

[0021] Further limitation, step 2) the pretreatment is to centrifuge the culture solution at 4 DEG C, 10000 rpm for 5 min, discard the supernatant, wash twice with 50mM pH7.4 phosphate buffer solution, and then resuspend with 5mL of the above phosphate buffer; the ultrasonic breaking is treated by ultrasonic breaking instrument at 20KHz, 60W for 20 min; the centrifugal separation is centrifuged at 4 DEG C, 13000 rpm for 15 min.

[0022] Further limitation, the reaction substrate is composed of the following components: MgCl2·6H2O 10mM, KCl 10mM, dithiothreitol 1mM, Tris-HCl buffer 20mM, FeSO4·7H2O 0.5mM, 1,2-cyclohexanedione 10mM, acetic acid 10mM.

[0023] Beneficial effect: the method provided by the application can convert 1,2-cyclohexanedione and acetic acid into 2-acetylcyclohexanone in vitro by enzyme reaction, and the yield reaches 14.22mg / L, which provides a new idea for solving the problem of limited Dke1 substrate types in whole cell synthesis system and the problem of 2-acetylcyclohexanone toxicity inhibition to cells. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 In vitro enzyme reaction product 2-acetylcyclohexanone GC-MS detection result.

[0025] Figure 2 SDS-PAGE detection result for purifying enzyme solution. DETAILED DESCRIPTION

[0026] Acetoacetone lyase gene: Dke1

[0027] Escherichia coli (Escherichia coli): E.coli

[0028] "Overexpression" or "overexpress" refers to the expression of a particular gene in an organism at a level greater than normal (i.e., wild-type expression level), which can be achieved by enhancing endogenous expression or introducing an exogenous gene.

[0029] The present application is further illustrated by the following examples. However, the present application is not limited to the following examples.

[0030] The experimental methods used in the following examples are routine methods unless otherwise specified.

[0031] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0032] Culture medium formula:

[0033] LB liquid medium: 5 g / L yeast powder, 10 g / L NaCl, 10 g / L peptone, pH 7.

[0034] LB solid medium: 5 g / L yeast powder, 10 g / L NaCl, 10 g / L peptone, 15 g / L agar, pH 7.

[0035] Shaking flask fermentation medium: 20 g / L glucose, 5 g / L yeast powder, 10 g / L NaCl, 10 g / L peptone, pH 7.

[0036] During actual cultivation, a certain concentration of antibiotic can be added to the above-mentioned culture medium to maintain the stability of the plasmid, such as 100 μg·mL -1 Ampicillin.

[0037] For the amino acid sequence of acetoacetone cleavage enzyme derived from Acinetobacter johnsonii (SEQ ID NO. 3). Acinetobacter johnsonii ) (SEQ ID NO. 3).

[0038] Example 1. Construction of recombinant plasmid

[0039] For the amino acid sequence of acetoacetone cleavage enzyme derived from Acinetobacter johnsonii Acinetobacter johnsonii The gene sequence of acetoacetone cleavage enzyme derived from Acinetobacter johnsonii (SEQ ID NO. 6) was codon-optimized, and the gene (SEQ ID NO. 4) was synthesized by Suzhou Jinweizhi Company. The synthesized gene was used as a template, and primers were designed using site-directed mutagenesis.

[0040] The upstream fragment (primers F: GATGCGTCCGGCGTAGAGG (SEQ ID NO. 7) and R: GAAGGTTTTAGCGTGCAGAGCACCAGAAGATTCG, SEQ ID NO. 8) and the downstream fragment (primers F: CGAATCTTCTGGTGCTCTGCACGCTAAAACCTTC (SEQ ID NO. 9) and R: GCTAGTTATTGCTCAGCGG, SEQ ID NO. 10) of the dke1 gene were obtained by PCR amplification, and the target fragments were recovered by a recovery kit. The obtained upstream and downstream fragments were used as substrates to obtain the acetoacetate decarboxylase mutant gene sequence Dke1 in which the glycine at position 105 was changed to alanine by bridge PCR (primers F: CGGGATCCGATGGACTACTGCAACA (SEQ ID NO. 11) and R: CGGAATTCTTAAGCAGCTTCGTTTTTGGTA, SEQ ID NO. 12), and the sequence is shown in SEQ ID NO. 2. The PCR conditions were 95°C for 3 min, 35 cycles of 95°C for 30 s, 53°C for 30 s, 72°C for 1 min, 72°C for 5 min, and 16°C for incubation. The PCR amplification system was 1 μL of template, 1 μL of upstream and downstream primers, 25 μL of 2x Prime STAR max, and 22 μL of ddH2O. G105A

[0041] The obtained Dke1 G105A gene fragment and the plasmid pETDuet-1 were digested with BamHI and EcoRI, and the digestion products were recovered. The recovered vector and the Dke1 G105A gene fragment were connected at a molar ratio of 1:5 at 16°C for more than 6 h. The connection product was recovered to obtain an expression vector of the acetoacetate decarboxylase mutant gene for improving the synthesis efficiency of 2-acetylcyclohexanone, which was named pETDuet-Dke1 G105A .

[0042] Example 2. Construction of recombinant bacteria

[0043] The obtained vector pETDuet-Dke1 G105A was introduced into E. coli ​BL21(DE3) competent cells were coated on LB solid plates containing 100 μg·mL-1 ampicillin; the coated plates were placed in a 37 °C incubator and incubated until single colonies grew. The single colonies were streaked on solid LB plates and incubated at 37 °C in an incubator until single colonies grew again. The single colonies were used as templates to verify the correct expression of the mutant acetone cleavage enzyme by colony PCR (primers F: GATGCGTCCGGCGTAGAGG and R: GCTAGTTATTGCTCAGCGG). After verification, the E. coli containing the mutant acetone cleavage enzyme was obtained E. coli BL21 (pETDuet-Dke1 G105A ).

[0044] The mutant acetone cleavage enzyme gene Dke1 G105A The amino acid sequence of the expressed mutant acetone cleavage enzyme is shown in SEQ ID NO. 1.

[0045] Example 3. Expression and purification of mutant acetone cleavage enzyme

[0046] The E. coli containing the mutant acetone cleavage enzyme obtained in Example 2 was inoculated in fermentation medium and incubated at 37 °C and 180 rpm until the cell concentration reached OD600 of 0.6-0.8. After adding IPTG at a final concentration of 0.2 mM, the culture was incubated at 30 °C for 4-6 h to obtain the culture solution. E. coli BL21 (pETDuet-Dke1 G105A ).

[0047] The obtained culture solution was centrifuged at 1000 rpm at 4 °C for 5 min, washed twice with 50 mM phosphate buffer solution at pH 7.4, and then resuspended with 5 mL of phosphate buffer solution. The ultrasonic disrupter was used at 20 KHz and 60 W for 20 min. The supernatant was obtained by centrifugation at 13000 rpm at 4 °C for 15 min. The obtained supernatant was purified by nickel column to obtain the pure enzyme solution. The nickel column purification conditions were as follows: the supernatant after cell disruption and centrifugation was poured into the nickel column treated with Binding buffer at pH 7.5. After the liquid completely flowed out, Elution buffer was added to wash out the protein and collect the enzyme solution containing the protein. Figure 2 The SDS-PAGE detection results of the purified enzyme solution.

[0048] Example 4. Application of mutant acetone cleavage enzyme

[0049] The acetylacetone cleavage enzyme mutant enzyme solution obtained in Example 3 was mixed with the reaction substrate, and the final concentration of the added pure enzyme solution was 0.1 mM, and a reaction system containing the following components was formed: MgCl2·6H2O 10 mM, KCl 10 mM, dithiothreitol 1 mM, Tris-HCl buffer 20 mM, FeSO4·7H2O 0.5 mM, 1,2-cyclohexanedione 10 mM, acetic acid 10 mM. The reaction system was sealed in a 1.5 ml centrifuge tube and placed in a 37°C, 180 rpm shaker for 12 h.

[0050] Comparative Example 1.

[0051] According to the same strategy as in Examples 1-4, the sequence shown in SEQ ID NO. 4 was used to replace the acetylacetone cleavage enzyme mutant gene sequence Dke1 G105A The experiment was carried out, and the acetylacetone cleavage enzyme (amino acid sequence as shown in SEQ ID NO. 3) expressed by the in vitro enzyme reaction produced 2-acetylcyclohexanone. Only qualitative 2-acetylcyclohexanone could be achieved, and quantitative analysis could not be achieved.

[0052] Comparative Example 2.

[0053] According to the same strategy as in Examples 1-4, the amino acid at position 105 of the acetylacetone cleavage enzyme was mutated from glycine to lysine, and the upstream fragment amplification primer was F: GATGCGTCCGGCGTAGAGG and R: GAAGAAGGTTTTTTTGTGCAGAGCAC (SEQ ID NO. 13). The downstream fragment amplification primer was F: GTGCTCTGCACAAAAAAACCTTCTTC (SEQ ID NO. 14) and R: GCTAGTTATTGCTCAGCGG. The bridging PCR primer was F: CGGGATCCGATGGACTACTGCAACA and R: CGGAATTCTTAAGCAGCTTCGTTTTTGGTA. The acetylacetone cleavage enzyme mutant gene Dke1 G105K .

[0054] The following experiments were used to verify the experimental effect:

[0055] 2-acetylcyclohexanone detection:

[0056] The reaction solution obtained from Example 2 was subjected to product analysis by GC-MS injection of 1 μL. The 2-acetylcyclohexanone product was detected. The gas chromatography column of the GC-MS was HP-5MS 5% Phenyl Methyl Silox (30 m x 0.25 mm, 0.25 μm), and the temperature program was 50 °C for 5 min, then increased to 240 °C at a rate of 15 °C / min, and maintained for 5 min. The yield of 2-acetylcyclohexanone was measured to be 14.22 mg / L. Figure 1 GC-MS detection results of 2-acetylcyclohexanone produced by in vitro enzyme reaction.

[0057] Using the acetoacetate cleavage enzyme mutant gene Dke1 G105K Substituting the acetoacetate cleavage enzyme mutant gene sequence Dke1 G105A The experiment was performed to express the acetoacetate cleavage enzyme mutant Dke1 G105K (acetoacetate cleavage enzyme mutant Dke1 (amino acid sequence shown as SEQ ID NO. 5) to produce 2-acetylcyclohexanone by in vitro enzyme reaction, and the yield of 2-acetylcyclohexanone was 8.51 mg / L, which was 40.15% lower than that of using the acetoacetate cleavage enzyme mutant Dke1 G105A (acetoacetate cleavage enzyme mutant Dke1 (amino acid sequence shown as SEQ ID NO. 5) to produce 2-acetylcyclohexanone by in vitro enzyme reaction, and the yield of 2-acetylcyclohexanone was 8.51 mg / L, which was 40.15% lower than that of using the acetoacetate cleavage enzyme mutant Dke1

[0058] SEQ ID NO. 1

[0059] MDYCNKKHTAEEYVKISDNNYVPFPEAFSDGGITWQLLHSSPETSSWTAIFNCPAGSSFASHIHAGPGEYFLTKGKMEVRGGEQEGGSTAYAPSYGFESSGALHAKTFFPVESQFYMTFLGPLNFIDDNGKVIASIGWAEAQGAWLATKNEAA

[0060] SEQ ID NO. 2

[0061] ATGGACTACTGCAACAAAAAACACACCGCTGAAGAATACGTTAAAATCTCTGACAACAACTACGTTCCGTTCCCGGAAGCGTTCTCTGACGGTGGTATCACCTGGCAGCTGCTGCACTCTTCTCCGGAAACCTCTTCTTGGACCGCTATCTTCAACTGCCCGGCTGGTTCTTCTTTCGCTTCTCACATCCACGCTGGTCCGGGTGAATACTTCCTGACCAAAGGTAAAATGGAAGTTCGTGGTGGTGAACAGGAAGGTGGTTCTACCGCTTACGCTCCGTCTTACGGTTTCGAATCTTCTGGTGCTCTGCACGCTAAAACCTTCTTCCCGGTTGAATCTCAGTTCTACATGACCTTCCTGGGTCCGCTGAACTTCATCGACGACAACGGTAAAGTTATCGCTTCTATCGGTTGGGCTGAAGCTCAGGGTGCTTGGCTGGCTACCAAAAACGAAGCTGCTTAA

[0062] SEQ ID NO. 3

[0063] MDYCNKKHTAEEYVKISDNNYVPFPEAFSDGGITWQLLHSSPETSSWTAIFNCPAGSSFASHIHAGPGEYFLTKGKMEVRGGEQEGGSTAYAPSYGFESSGALHGKTFFPVESQFYMTFLGPLNFIDDNGKVIASIGWAEAQGAWLATKNEAA

[0064] SEQ ID NO. 4

[0065] ATGGACTACTGCAACAAAAAACACACCGCTGAAGAATACGTTAAAATCTCTGACAACAACTACGTTCCGTTCCCGGAAGCGTTCTCTGACGGTGGTATCACCTGGCAGCTGCTGCACTCTTCTCCGGAAACCTCTTCTTGGACCGCTATCTTCAACTGCCCGGCTGGTTCTTCTTTCGCTTCTCACATCCACGCTGGTCCGGGTGAATACTTCCTGACCAAAGGTAAAATGGAAGTTCGTGGTGGTGAACAGGAAGGTGGTTCTACCGCTTACGCTCCGTCTTACGGTTTCGAATCTTCTGGTGCTCTGCACGGTAAAACCTTCTTCCCGGTTGAATCTCAGTTCTACATGACCTTCCTGGGTCCGCTGAACTTCATCGACGACAACGGTAAAGTTATCGCTTCTATCGGTTGGGCTGAAGCTCAGGGTGCTTGGCTGGCTACCAAAAACGAAGCTGCTTAA

[0066] SEQ ID NO. 5

[0067] MDYCNKKHTAEEYVKISDNNYVPFPEAFSDGGITWQLLHSSPETSSWTAIFNCPAGSSFASHIHAGPGEYFLTKGKMEVRGGEQEGGSTAYAPSYGFESSGALHKKTFFPVESQFYMTFLGPLNFIDDNGKVIASIGWAEAQGAWLATKNEAA

[0068] SEQ ID NO. 6

[0069] ATGGACTACTGCAACAAAAAACACACCGCTGAAGAATACGTTAAAATCTCTGACAACAACTACGTTCCGTTCCCGGAAGCGTTCTCTGACGGTGGTATCACCTGGCAGCTGCTGCACTCTTCTCCGGAAACCTCTTCTTGGACCGCTATCTTCAACTGCCCGGCTGGTTCTTCTTTCGCTTCTCACATCCACGCTGGTCCGGGTGAATACTTCCTGACCAAAGGTAAAATGGAAGTTCGTGGTGGTGAACAGGAAGGTGGTTCTACCGCTTACGCTCCGTCTTACGGTTTCGAATCTTCTGGTGCTCTGCACGGTAAAACCTTCTTCCCGGTTGAATCTCAGTTCTACATGACCTTCCTGGGTCCGCTGAACTTCATCGACGACAACGGTAAAGTTATCGCTTCTATCGGTTGGGCTGAAGCTCAGGGTGCTTGGCTGGCTACCAAAAACGAAGCTGCTTAA. SEQUENCE LISTING <110> Qingdao Institute of Bioenergy and Process Research, Chinese Academy of Sciences <120> A mutant of acetoacetate decarboxylase and a method for producing 2-acetylcyclohexanone by extracellular enzyme reaction <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 153 <212> PRT <213> Artificial synthesis <400> 1 Met Asp Tyr Cys Asn Lys Lys His Thr Ala Glu Glu Tyr Val Lys Ile 1 5 10 15 Ser Asp Asn Asn Tyr Val Pro Phe Pro Glu Ala Phe Ser Asp Gly Gly 20 25 30 Ile Thr Trp Gln Leu Leu His Ser Ser Pro Glu Thr Ser Ser Trp Thr 35 40 45 Ala Ile Phe Asn Cys Pro Ala Gly Ser Ser Phe Ala Ser His Ile His 50 55 60 Ala Gly Pro Gly Glu Tyr Phe Leu Thr Lys Gly Lys Met Glu Val Arg 65 70 75 80 Gly Gly Glu Gln Glu Gly Gly Ser Thr Ala Tyr Ala Pro Ser Tyr Gly 85 90 95 Phe Glu Ser Ser Gly Ala Leu His Ala Lys Thr Phe Phe Pro Val Glu 100 105 110 Ser Gln Phe Tyr Met Thr Phe Leu Gly Pro Leu Asn Phe Ile Asp Asp 115 120 125 Asn Gly Lys Val Ile Ala Ser Ile Gly Trp Ala Glu Ala Gln Gly Ala 130 135 140 Trp Leu Ala Thr Lys Asn Glu Ala Ala 145 150 <210> 2 <211> 462 <212> DNA <213> Synthetic <400> 2 atggactact gcaacaaaaa acacaccgct gaagaatacg ttaaaatctc tgacaacaac ​tacgttccgt tcccggaagc gttctctgac ggtggtatca cctggcagct gctgcactct 120 tctccggaaa cctcttcttg gaccgctatc ttcaactgcc cggctggttc ttctttcgct 180 tctcacatcc acgctggtcc gggtgaatac ttcctgacca aaggtaaaat ggaagttcgt 240 ggtggtgaac aggaaggtgg ttctaccgct tacgctccgt cttacggttt cgaatcttct 300 ggtgctctgc acgctaaaac cttcttcccg gttgaatctc agttctacat gaccttcctg 360 ggtccgctga acttcatcga cgacaacggt aaagttatcg cttctatcgg ttgggctgaa 420 gctcagggtg cttggctggc taccaaaaac gaagctgctt aa 462 <210> 3 <211> 153 <212> PRT <213> Acinetobacter johnsonii <400> 3 Met Asp Tyr Cys Asn Lys Lys His Thr Ala Glu Glu Tyr Val Lys Ile 1 5 10 15 Ser Asp Asn Asn Tyr Val Pro Phe Pro Glu Ala Phe Ser Asp Gly Gly 20 25 30 Ile Thr Trp Gln Leu Leu His Ser Ser Pro Glu Thr Ser Ser Trp Thr 35 40 45 Ala lie Phe Asn Cys Pro Ala Gly Ser Ser Phe Ala Ser His lie His 50 55 60 Ala Gly Pro Gly Glu Tyr Phe Leu Thr Lys Gly Lys Met Glu Val Arg 65 70 75 80 Gly Gly Glu Gin Glu Gly Gly Ser Thr Ala Tyr Ala Pro Ser Tyr Gly 85 90 95 Phe Glu Ser Ser Gly Ala Leu His Gly Lys Thr Phe Phe Pro Val Glu 100 105 110 Ser Gin Phe Tyr Met Thr Phe Leu Gly Pro Leu Asn Phe lie Asp Asp 115 120 125 Asn Gly Lys Val lie Ala Ser lie Gly Trp Ala Glu Ala Gin Gly Ala 130 135 140 Trp Leu Ala Thr Lys Asn Glu Ala Ala 145 150 <210> 4 <211> 462 <212> DNA <213> Artificially synthesized <400> 4 atggactact gcaacaaaaa acacaccgct gaagaatacg ttaaaatctc tgacaacaac 60 tacgttccgt tcccggaagc gttctctgac ggtggtatca cctggcagct gctgcactct 120 tctccggaaa cctcttcttg gaccgctatc ttcaactgcc cggctggttc ttctttcgct 180 tctcacatcc acgctggtcc gggtgaatac ttcctgacca aaggtaaaat ggaagttcgt 240 ggtggtgaac aggaaggtgg ttctaccgct tacgctccgt cttacggttt cgaatcttct 300 ggtgctctgc acggtaaaac cttcttcccg gttgaatctc agttctacat gaccttcctg 360 ggtccgctga acttcatcga cgacaacggt aaagttatcg cttctatcgg ttgggctgaa 420 gctcagggtg cttggctggc taccaaaaac gaagctgctt aa 462 <210> 5 <211> 153 <212> PRT <213> Artificial <400> 5 Met Asp Tyr Cys Asn Lys Lys His Thr Ala Glu Glu Tyr Val Lys Ile 1 5 10 15 Ser Asp Asn Asn Tyr Val Pro Phe Pro Glu Ala Phe Ser Asp Gly Gly 20 25 30 Ile Thr Trp Gln Leu Leu His Ser Ser Pro Glu Thr Ser Ser Trp Thr 35 40 45 Ala Ile Phe Asn Cys Pro Ala Gly Ser Ser Phe Ala Ser His Ile His 50 55 60 Ala Gly Pro Gly Glu Tyr Phe Leu Thr Lys Gly Lys Met Glu Val Arg 65 70 75 80 Gly Gly Glu Gin Glu Gly Gly Ser Thr Ala Tyr Ala Pro Ser Tyr Gly 85 90 95 Phe Glu Ser Ser Gly Ala Leu His Lys Lys Thr Phe Phe Pro Val Glu 100 105 110 Ser Gin Phe Tyr Met Thr Phe Leu Gly Pro Leu Asn Phe Ile Asp Asp 115 120 125 Asn Gly Lys Val Ile Ala Ser Ile Gly Trp Ala Glu Ala Gin Gly Ala 130 135 140 Trp Leu Ala Thr Lys Asn Glu Ala Ala 145 150 <210> 6 <211> 462 <212> DNA <213> Acinetobacter johnsonii <400> 6 atggactact gcaacaaaaa acacaccgct gaagaatacg ttaaaatctc tgacaacaac 60 tacgttccgt tcccggaagc gttctctgac ggtggtatca cctggcagct gctgcactct 120 tctccggaaa cctcttcttg gaccgctatc ttcaactgcc cggctggttc ttctttcgct 180 tctcacatcc acgctggtcc gggtgaatac ttcctgacca aaggtaaaat ggaagttcgt 240 ggtggtgaac aggaaggtgg ttctaccgct tacgctccgt cttacggttt cgaatcttct 300 ggtgctctgc acggtaaaac cttcttcccg gttgaatctc agttctacat gaccttcctg 360 ggtccgctga acttcatcga cgacaacggt aaagttatcg cttctatcgg ttgggctgaa 420 gctcagggtg cttggctggc taccaaaaac gaagctgctt aa 462 <210> 7 <211> 19 <212> DNA <213> Synthetic <400> 7 gatgcgtccg gcgtagagg 19 <210> 8 <211> 34 <212> DNA <213> Synthetic <400> 8 gaaggtttta gcgtgcagag caccagaaga ttcg 34 <210> 9 <211> 34 <212> DNA <213> Synthetic <400> 9 cgaatcttct ggtgctctgc acgctaaaac cttc 34 <210> 10 <211> 19 <212> DNA <213> Synthetic <400> 10 gctagttatt gctcagcgg 19 <210> 11 <211> 25 <212> DNA <213> Synthetic <400> 11 cgggatccga tggactactg caaca 25 <210> 12 <211> 30 <212> DNA <213> Artificial <400> 12 cggaattctt aagcagcttc gtttttggta 30 <210> 13 <211> 26 <212> DNA <213> Artificial <400> 13 gaagaaggtt tttttgtgca gagcac 26 <210> 14 <211> 26 <212> DNA <213> Artificial <400> 14 gtgctctgca caaaaaaacc ttcttc 26

Claims

1. An acetylacetate cleavage enzyme mutant, characterized in that, The mutant is obtained by mutating the amino acid at position 105 of the acetoacetate decarboxylase with amino acid sequence shown as SEQ ID NO. 3 as the starting sequence; mutating the glycine at position 105 to alanine.

2. A gene encoding the acetoacetate decarboxylase mutant of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector carries the encoding gene of claim 2.

4. The recombinant vector of claim 3, wherein, The starting vector of the recombinant vector is any one of the pET series, the Duet series, the pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series.

5. A recombinant microbial cell, characterized in that, A microbial cell carrying the gene of claim 2 or expressing the mutant of claim 1.

6. Use of the mutant of claim 1, the gene of claim 2, the vector of claim 3 or 4 or the recombinant microbial cell of claim 5 in increasing the production of 2-acetylcyclohexanone.

7. A method for the production of 2-acetylcyclohexanone by an extracellular enzyme reaction, characterized by, Comprising the following steps: 1) connecting the gene of the acetoacetate decarboxylase mutant of claim 2 to an expression vector to obtain a recombinant vector; 2) transforming the recombinant vector obtained in step 1) into a host cell to obtain a recombinant cell, and culturing the recombinant cell to obtain a culture solution; 3) treating the culture solution obtained in step 2) to obtain an acetoacetate decarboxylase mutant enzyme solution; 4) mixing the enzyme solution obtained in step 3) with a reaction substrate and reacting to obtain 2-acetylcyclohexanone.

8. The method of claim 7, wherein: The gene sequence of the acetoacetate decarboxylase mutant in step 1) is shown as SEQ ID NO.

2.

9. The method of claim 7, wherein, The reaction substrate in step 4) is composed of MgCl2·6H2O, KCl, dithiothreitol, Tris-HCl buffer, FeSO4·7H2O, 1,2-cyclohexanedione and acetic acid.

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