CN113151201B

CN113151201B - High-thermal-stability and high-activity isoeugenol monooxygenase mutant and application thereof

Info

Publication number: CN113151201B
Application number: CN202110315426.6A
Authority: CN
Inventors: 徐毅; 陆馨怡; 马宝娣; 王倩; 吴小梅; 张磊; 刘胜利
Original assignee: Shanghai Institute of Technology
Current assignee: Sinopharm International Technology Ltd
Priority date: 2021-03-24
Filing date: 2021-03-24
Publication date: 2022-12-16
Anticipated expiration: 2041-03-24
Also published as: CN113151201A

Abstract

The invention discloses an isoeugenol monooxygenase mutant and application thereof, belonging to the field of bioengineering. The isoeugenol monooxygenase gene originated from Pseudomonas nitroreducens (Jin 1) is subjected to site-directed mutagenesis, so that the coded amino acid sequence of the isoeugenol monooxygenase gene is mutated from lysine K to arginine R at the 83 th position, from lysine K to arginine R at the 95 th position and from leucine to phenylalanine F at the 273 th position. The specific activity of the isoeugenol monooxygenase mutant K83R/K95R/L273F is 2.1 times of that before mutation, and the half-life period is respectively improved by 3.0 times, 12.0 times and 24.7 times at 25 ℃,30 ℃ and 35 ℃. The isoeugenol monooxygenase mutant can catalyze the propenyl on the benzene ring of the substrate isoeugenol to be oxidized in a wider temperature range under the action of oxygen so as to generate vanillin, can be widely applied to the industries of food, chemical industry, pharmacy and the like, and has wide prospect.

Description

Isoeugenol monooxygenase mutant with high thermal stability and high activity and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a high-thermostability and high-activity isoeugenol monooxygenase mutant and application thereof.

Background

Isoeugenol Monooxygenase (Isoeugenol Monooxygenase), abbreviated as IEM, generates vanillin by oxidative cleavage of the propenyl group linked to the benzene ring, using Isoeugenol as substrate, in the absence of any coenzymes and cofactors. Vanillin (4-hydroxy-3-methoxybenzaldehyde), also known as vanillin, is widely used in the fields of food, chemical industry, medicine, etc. due to its unique aroma and properties. The vanillin can be used as a flavoring agent in food and added into cakes and ice cream to enhance the fragrance and flavor of the food; the method is commonly used for detecting the existence of resorcinol and tannin in the chemical industry; it is also a raw material for the production and important intermediates of many drugs in the pharmaceutical industry, for example, it can be used for the preparation of dopamine, antiepileptic drugs, etc. (Huangxishun, huang Lijuan, weijian Keke. Vanillin for the observation of efficacy in treating epilepsy [ J ]. J.Utility neurological disease J.2005, 8 (4): 78.).

Most of vanillin in the current market is synthesized by a chemical method, and researches show that the vanillin synthesized by a large-dose chemical method can cause adverse reactions such as headache, vomit, dyspnea and the like, and has great harm to human bodies. Therefore, in recent years, consumers have been calling for natural perfumes more and more, and the demand of natural vanillin has been greatly increased. The natural vanillin is mainly present in vanilla beans and can be obtained by supercritical CO ₂ The extraction technology is used for extraction, but the natural vanillin extracted by the method is very expensive and cannot meet the market demand (Xiehirang, zhoujiang, huangmaoyang, and the like, supercritical CO) ₂ Process study and composition analysis of extracted Vanilla [ J]Food and machinery 2002, (2): 12-14.). With the rapid development of the biocatalysis technology, the method can carry out high-efficiency and rapid reaction in a short time, and has the advantages of environmental protection, strong selectivity and specificity and the like, so that the method is more suitable for commercial production.

Isoeugenol is the best precursor substance for synthesizing vanillin, while IEM is the only key enzyme for biologically converting isoeugenol, and the vanillin synthesized by the method is closest to natural vanillin in aroma, thereby causing wide attention.

The rational design in protein engineering is based on the understanding of the sequence, structure, catalytic mechanism and other enzyme information of target protein, and with the help of various bioinformatics software, the influence of the mutation of different sites on the stability, catalytic performance and other aspects of the target protein is predicted. The whole plasmid PCR method is one of the most widely applied site-directed mutagenesis methods at present, and has the advantages of simple and convenient operation, rapidness, high efficiency and the like. IEM studies on reported microbial sources have found that studies on exogenous expression, enzymatic properties, have largely been centered (Yamada M, okada Y, yoshida Y, et al, vanillin production using Escherichia coli cells over-expressing isoeugenol monoxygenase of Pseudomonas putida [ J ]. Biotechnology letters.2008,30 (4): 665-670.), while studies on molecular engineering have been less. Chinese patent application CN 106754802B discloses an isoeugenol monooxygenase mutant and application thereof, belongs to the field of biotechnology, and aims at the problem of product inhibition, and carries out site-directed mutagenesis on isoeugenol monooxygenase genes to finally obtain a mutant enzyme with catalytic activity at least 117% higher than that of a parent, however, the isoeugenol monooxygenase mutant does not have high thermal stability. Therefore, the invention aims at the problem of poor thermal stability of the enzyme for the first time, carries out site-directed mutagenesis on the enzyme by means of bioinformatics software, and further improves the catalytic activity of the isoeugenol monooxygenase while improving the thermal stability.

The inventor obtains a gene sequence for coding isoeugenol monooxygenase (IEM) from a nitroreduction Pseudomonas (Pseudomonas nitroreducens) Jin1 genome reported in an NCBI library in earlier research, clones the gene sequence into escherichia coli BL21 (DE 3) to successfully realize expression, but the enzyme has certain defects, particularly poor thermal stability, and only has good catalytic effect at a lower temperature, so that the enzyme has certain limitation in industrial application.

Disclosure of Invention

The invention aims to solve the problems and provides an isoeugenol monooxygenase mutant and application thereof.

In order to solve the technical problems, the invention firstly utilizes GetARea software to find total 6 Gly and 14 Lys on the surface of IEM based on the principle that amino acid on the surface of the thermophilic protein has Gly replaced Ala and Lys is replaced Arg, and carries out site-directed mutagenesis on the total 6 Gly and 14 Lys on the surface of the IEM respectively to obtain 2 mutases with improved thermal stability; based on the principle that conserved amino acids in homologous sequences are more stable than non-conserved amino acids, the Consensus Finder software is used for carrying out multi-sequence comparison on the IEM, 9 total amino acids with the threshold value larger than 70% are screened out, and after the amino acids are respectively subjected to site-specific mutagenesis to form corresponding conserved amino acids, 1 mutant enzyme with improved enzyme activity is obtained.

After the three amino acids are subjected to combined mutation, a three-site mutant with remarkably improved thermal stability and improved specific activity is obtained, and the problems that the existing IEM is poor in thermal stability and cannot meet industrial production are solved.

A high thermal stability and high activity mutant of isoeugenol monooxygenase, which comprises an amino acid sequence shown as SEQ ID NO.1 and has at least one mutation selected from 83 th position, 95 th position or 273 th position.

Further, the lysine K at position 83 is mutated to arginine R.

Further, lysine K at position 95 is mutated to arginine R.

Further, leucine L at position 273 is mutated to phenylalanine F.

Preferably, the amino acid sequence shown as SEQ ID NO.1 has amino acid sequences with amino acid sequence including 83 th lysine K, 95 th lysine K and 273 rd leucine L mutated into arginine R, arginine R and phenylalanine F.

The isoeugenol monooxygenase mutant with improved specific activity and thermal stability is characterized in that isoeugenol monooxygenase IEM (amino acid sequence of which is shown as NCBI accession number ACP17973.1 and nucleotide sequence of which is shown as NCBI accession number FJ 851547) from parent nitroreduction Pseudomonas (Pseudomonas nitroreducens) Jin1 is subjected to site-specific mutagenesis, mutation is introduced into lysine K at 83, lysine R at 95 and leucine L at 273 of IEM by a whole plasmid PCR (polymerase chain reaction) technology, a vector for expressing the mutant is pET21a, and a recombinant plasmid containing mutated genes is transformed into host Escherichia coli (Escherichia coli) BL21 (DE 3) for expression.

The mutation is to mutate lysine K at the 83 th site into arginine R, lysine K at the 95 th site into arginine R, and leucine L at the 273 th site into phenylalanine F. The iso-eugenol monooxygenase mutant K83R/K95R/L273F is indicated by the nomenclature "original amino acid abbreviation + mutation position + replacement amino acid abbreviation".

Compared with the isoeugenol monooxygenase of parent nitroreduction pseudomonas jina 1 (the specific activity of pure enzyme is 3.4U/mg), the isoeugenol monooxygenase mutants K83R, K95R, L273F, K83R/K95R and K83R/K95R/L273F respectively have the specific activities of 6.1U/mg, 5.3U/mg, 5.5U/mg, 6.0U/mg and 7.0U/mg, wherein the K83R/K95R/L273F shows the optimal specific activity and is improved by 2.1 times compared with the parent.

Compared with the parent pseudomonas nitroreducens Jin1 isoeugenol monooxygenase (half-life t at 25 ℃,30 ℃ and 35 ℃) _1/2 Respectively for 16.1h, 49.8min and 7.8 min), the half-life of the isoeugenol monooxygenase mutants K83R, K95R, L273F, K83R/K95R and K83R/K95R/L273F at 25 ℃ is respectively 1.1 times, 0.6 times, 2.3 times and 3.0 times of that of the parent (t is t _1/2 Respectively 18.2h, 18.5h, 10.0h, 37.1h and 47.5 h), and the half-life period at 30 ℃ is respectively improved by 5.3 times, 4.0 times, 2.7 times, 9.9 times and 12.0 times (t is t) compared with that of a parent _1/2 262.2min, 198.0min, 133.2min, 491.4min and 594.0 min), and the half-life at 35 ℃ is improved by 4.1 times, 3.7 times, 1.6 times, 12.2 times and 24.7 times (t is t) respectively compared with the parent _1/2 31.8min, 28.8min, 12.6min, 94.8min and 192.6min, respectively).

The invention also provides application of the isoeugenol monooxygenase mutant, wherein the isoeugenol monooxygenase mutant is applied to preparation of vanillin by taking isoeugenol as a substrate, the concentration of the substrate is 100mM (cosolvent is 10% DMSO), the reaction temperature is 30 ℃, the pH value is 9.0, the cell amount is 15g/L, after 12 hours of conversion, the conversion rate of vanillin can reach 87%, and the conversion rate of parents under the same conditions is 75%, so that the conversion rate is improved by 1.16 times.

Compared with the prior art, the invention has the beneficial effects that:

by taking the substitution and consensus theory of surface amino acid as a guiding idea, the isoeugenol monooxygenase gene is subjected to site-directed mutagenesis, so that the IEM mutant enzyme K83R/K95R/L273F with improved thermal stability and specific activity is obtained, the half life can reach 12.0 times of that of a wild enzyme at the temperature of 30 ℃, and the specific activity is 2.1 times of that of the wild enzyme. The mutant enzyme can use isoeugenol as a substrate, efficiently catalyze and convert isoeugenol at a higher temperature to synthesize vanillin, and the conversion rates of vanillin at 30 ℃ and 35 ℃ are respectively 16% and 35% higher than those of parent strains.

Drawings

FIG. 1 is an electrophoresis diagram of pure enzyme protein of the mutant and wild type IEM constructed by the present invention;

wherein lane 1 represents the parent, lane 2 represents mutant K83R, lane 3 represents mutant K95R, lane 4 represents mutant L273F, lane 5 represents mutant K83R/K95R, and lane 6 represents mutant K83R/K95R/L273F.

FIG. 2 is a comparison of the specific activities of the mutant and wild type IEM constructed according to the present invention;

FIGS. 3a, 3b, 3c are comparison of the inactivation half-life of the mutant and wild type IEM constructed in the present invention at different temperatures.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

The media formulations referred to in the examples are as follows:

LB liquid medium: 10g/L of tryptone, 5g/L of yeast extract powder, 10g/L of NaCl and 7.0 of pHs.

LB solid Medium: 15g/L agar is added on the basis of the formula of the LB liquid culture medium.

Fermentation medium: na (Na) ₂ HPO ₄ 7g/L，KH ₂ PO ₄ 3g/L, yeast extract powder 10g/L, naCl 0.5g/L, glucose 14g/L, pH7.0.

And (3) measuring the enzyme activity of the isoeugenol monooxygenase:

the activity determination reaction system is (1 mL): mixing 50 μ g pure enzyme with 0.1M Tris-HCl (pH 8.0) buffer solution, preheating with 200mM substrate isoeugenol at 30 deg.C for 3min, immediately adding 50 μ L substrate into reaction system to make its final concentration be 10mM, reacting at 30 deg.C and 1000rpm in a constant temperature mixer for 10min, adding 1mL liquid phase methanol for quenching, mixing in a vortex apparatus, centrifuging at 12000rpm for 2min, sucking supernatant with sterile syringe, and making into oral liquid

And (4) HPLC analysis.

Analytical method (HPLC method): the column was reversed phase column C18 (Diamonsil plus,4.6mm 250mm 5 μm); mobile phase (gradient elution): 1-8min methanol: water (pH 2.5) =35, 9-16min methanol: water (pH 2.5) = 65; 17-24min methanol: water (pH 2.5) = 35; the detection wavelength is 280nm; the column temperature was set at 25 ℃; the sample size was 20. Mu.L.

Definition of enzyme activity: the amount of enzyme required to produce 1. Mu. Mol of product vanillin per unit time at 30 ℃ and pH 8.0 is defined as one unit U.

Determination of thermal stability: subpackaging the diluted pure enzyme, respectively placing the pure enzyme in a metal water bath kettle with the temperature of 25 ℃,30 ℃ and 35 ℃ for heat preservation, sampling every interval for a period of time to determine residual enzyme activity, fitting a linear relation graph by taking the heat preservation time as an x axis and taking logarithm of the residual enzyme activity as a y axis, and then fitting the linear relation graph according to t _1/2 ＝ln2/k _d The half-life is obtained.

The full cell catalysis of isoeugenol to synthesize vanillin: taking 15g/L of cells of the isoeugenol monooxygenase and mutants thereof, and reacting with 100mM of substrate isoeugenol (solvent: DMSO) under the condition of pH 9.0, wherein the reaction temperature is 20-35 ℃, and the reaction time is 2, 4, 8 and 12 hours, thereby observing the change of the vanillin conversion rate.

The conversion of the product vanillin was calculated as follows:

conversion α = (0.8823 × (a-A0) + 0.0286) × B/C × 100%;

wherein A represents the integrated peak area of an external standard method, A0 represents the spontaneous oxidation peak area of a substrate, B represents the dilution multiple of the total reaction system, and C represents the concentration of the substrate isoeugenol. The relation y =0.8823x +0.0286 is the standard curve of vanillin by external standard method.

The invention is further illustrated by the following specific examples.

Example 1

Cloning of isoeugenol monooxygenase gene and construction of recombinant engineering bacteria

According to the NCBI library of proteins, a segment of isoeugenol monooxygenase base is obtained from nitroreduction pseudomonas Jin1Therefore (the amino acid sequence is shown as NCBI accession number ACP17973.1, and the nucleotide sequence is shown as NCBI accession number FJ 851547), and is synthesized by Shanghai Jieli bioengineering, inc., the amino acid sequence of the isoeugenol monooxygenase IEM is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 7. By upstream primer 5' -CGCCATATGATGGCGAGACTCAACCGCAAC-3 '(underlined bases are recognition sites for restriction enzyme Nde I) and downstream primer 5' -CCGCTCGAGTTAAGGTCTGGGTACCCCAGCA-3' (underlined bases are recognition sites of restriction enzyme Xho I) is used for amplifying a target gene, and PrimeSTAR Max high fidelity polymerase of Takara China (Beijing) Limited company is used for PCR amplification, wherein the PCR reaction system is as follows: (primer concentration 10. Mu. Mol/L):

the PCR amplification procedure was: pre-denaturation at 98 ℃ for 2min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 60s, and 30 cycles; extending for 7min at 72 ℃; storing at 4 ℃.

After the reaction, the PCR product was detected by 1% agarose gel electrophoresis to obtain a 1.5kb band, which corresponds to the expected target protein length. According to the kit operation, the target fragment is recovered and purified by tapping, the recovered fragment and the pET21a plasmid are subjected to double digestion by using restriction enzymes Nde I and Xho I, ligation Mix is used for Ligation, a Ligation product is transformed into escherichia coli BL21 (DE 3) competent cells, the escherichia coli BL21 (DE 3) competent cells are coated on an LB plate containing ampicillin (100 mu g/mL), a positive clone plasmid is extracted and sent for sequencing, and the result shows that the inserted IEM gene sequence is correct, and the named recombinant bacterium is E.Coli BL21 (DE 3)/pET 21a-IEM.

Example 2

Construction of isoeugenol monooxygenase mutant based on whole plasmid PCR method

The plasmid containing the mutant gene is amplified in vitro by PCR with the recombinant plasmid pET21a-IEM as a template.

Primers used for site-directed mutagenesis were (mutation sites underlined):

K83R primer-F:5’-CATCAGTCGCTGGGTTAGGACCGCTCGATTCACG-3’

K83R primer-R:5’-CGTGAATCGAGCGGTCCTAACCCAGCGACTGATG-3’

K95R primer-F:5’-GAACGACTAGCGCGAAGATCGCTATTTGGCATG-3’

K95R primer-R:5’-CATGCCAAATAGCGATCTTCGCGCTAGTCGTTC-3’

L273F primer-F:5’-GTCAGATTCGCTGGTTCAAGGCACCGGCGCTC-3’

L273F primer-R:5’-GAGCGCCGGTGCCTTGAACCAGCGAATCTGAC-3’

the PCR reaction system is as follows: (primer concentration 10. Mu. Mol/L)

The PCR amplification procedure was: pre-denaturation at 95 ℃ for 10min; denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 75s, and 24 cycles; extending for 5min at 72 ℃; storing at 4 ℃.

And (3) taking 10 mu L of product obtained by PCR, and recovering the reaction solution by using a universal DNA purification recovery kit after the product is verified to be correct by nucleic acid electrophoresis. And digesting the DNA template by utilizing Dpn I endonuclease, wherein the digestion reaction temperature is 37 ℃, the reaction time is 4h, and then transforming the digestion product into escherichia coli BL21 (DE 3) competent cells. Coating an LB resistant solid plate (containing 100 mu g/mL Amp), culturing at 37 ℃ for 12-14h, selecting 1-3 transformants, transferring the transformants into an LB liquid culture medium (containing 100 mu g/mL Amp), culturing for 6h until the liquid is turbid, and taking a bacterial liquid for sequencing.

The strain E.coli BL21 (DE 3)/pET 21a-IEM with improved heat stability compared with the original strain is obtained by screening dominant strains ^K83R (the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO. 8), E.Coli BL21 (DE 3)/pET 21a-IEM ^K95R (the amino acid sequence is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 9), and a strain E.Coli BL21 (DE 3)/pET 21a-IEM with improved enzyme activity compared with the original strain ^L273F (the amino acid sequence is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 10).

The second round with pET21a-IEM ^K83R The plasmid is used as a template, K95R-F and K95R-R are used as primers, and after full plasmid PCR, transformation and plate coating, the dominant strain E.Coli BL21 (DE 3)/pET 21a-IEM with further improved thermal stability is obtained ^K83R-K95R (the amino acid sequence is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 11). The third round was pET21a-IEM ^K83R-K95R Plasmid is taken as a template, L273F-F and L273F-R are taken as primers, and after full plasmid PCR, transformation and plate coating, the dominant strain E.Coli BL21 (DE 3)/pET 21a-IEM with improved enzyme activity and thermal stability compared with the original strain is obtained ^{K83R-K95R-L273F} (the amino acid sequence is shown as SEQ ID NO.6, and the nucleotide sequence is shown as SEQ ID NO. 12).

Example 3

Inducible expression of parent and mutant strains

Activating a flat plate: the glycerol pipefish liquid is dipped by an inoculating loop, streaked on an LB resistant solid plate (containing 100 mu g/mL Amp) four areas, and inverted cultured for 12h at 37 ℃ in an incubator.

Seed culture: single colonies were picked from the solid plate and inoculated into LB liquid medium (containing 100. Mu.g/mL Amp), the liquid content was 50mL/250mL, the shake culture was performed at 37 ℃ for 12 hours on a shaker at 200 rpm.

And (3) shaking flask fermentation: inoculating the seed solution cultured for 12h into fermentation medium (containing 100 μ g/mL Amp) at an inoculum size of 1%, loading 50mL/250mL, culturing at 37 deg.C and 200rpm to OD ₆₀₀ And (4) cooling the liquid to room temperature, adding 0.1mM IPTG (isopropyl thiogalactoside) for induction, and performing induction expression at 25 ℃ for 12h. After fermentation, all the cells were collected by centrifugation at 10000rpm for 10min in a 4 ℃ centrifuge, and the cells were washed twice with 0.8% physiological saline.

Example 4

Purification of parent and mutant of isoeugenol monooxygenase

Adding a corresponding buffer solution into the collected thalli to resuspend the cells until the thallus concentration is 10g/L, placing the thalli in a beaker containing ice water, and carrying out cell disruption by using an ultrasonic disruptor under the conditions as follows: ultrasonic treatment for 2s, intermittent treatment for 4s, power for 38%, time for 10min, crushing twiceAfter that, the mixture was centrifuged at 12000rpm for 30min, and the supernatant was obtained as a crude enzyme solution. Purifying parent and positive mutant K83R, K95R, L273F, K83R/K95R and K83R/K95R/L273F of isoeugenol monooxygenase by using Suzhou beaver His-tag protein purification magnetic beads, and purifying Ni on the surface of the magnetic beads ²⁺ Can form a coordinate bond with histidine in a protein His label, bind a target protein to magnetic beads, remove adsorbed foreign protein by 100mM imidazole, elute the target protein by 500mM imidazole, and concentrate and replace buffer solution by putting the target protein in a Millipore ultrafiltration centrifugal tube. After purification, samples were taken at the same protein amount and same volume and verified by SDS-PAGE as shown in FIG. 1, wherein lane 1 represents the parent of isoeugenol monooxygenase, lane 2 represents mutant K83R, lane 3 represents mutant K95R, lane 4 represents mutant L273F, lane 5 represents mutant K83R/K95R, and lane 6 represents mutant K83R/K95R/L273F.

Example 5

Comparison of the Activity of different mutants with the parent enzyme

The concentrated pure enzyme was diluted, the enzyme activity was measured as described above, and the protein concentration was measured by the Bradford method. As shown in figure 2, the specific activity of the parent IEM is 3.4 +/-0.15U/mg, and the specific activities of the mutants K83R, K95R, L273F, K83R/K95R and K83R/K95R/L273F are respectively 6.1 +/-0.04U/mg, 5.3 +/-0.02U/mg, 5.5 +/-0.09U/mg, 6.0 +/-0.14U/mg and 7.0 +/-0.04U/mg, which are respectively improved by 1.8, 1.6, 1.8 and 2.1 times compared with the parent.

Example 6

Comparison of thermostability of different mutants with the parent enzyme

Comparison of thermostability of different mutants with the parent enzyme was mainly determined by measuring half-lives at different temperatures. As shown in FIG. 3a, the half-life of the parent at 25 ℃ is 16.1h, while the half-lives of the mutants K83R, K95R, L273F, K83R/K95R and K83R/K95R/L273F are 18.2h, 18.5h, 10.0h, 37.1h and 47.5h, which are 1.1 times, 0.6 times, 2.3 times and 3.0 times the parent, respectively. As shown in FIG. 3b, the half-life of the parent at 30 ℃ is 49.8min, while the half-life of the mutants K83R, K95R, L273F, K83R/K95R and K83R/K95R/L273F is 262.2min, 198.0min, 133.2min, 491.4min and 594.0min, which are respectively improved by 5.3 times, 4.0 times, 2.7 times, 9.9 times and 12.0 times compared with the parent. As shown in FIG. 3c, the half-life of the parent at 35 ℃ is 7.8min, while the half-life of the mutants K83R, K95R, L273F, K83R/K95R and K83R/K95R/L273F is 31.8min, 28.8min, 12.6min, 94.8min and 192.6min, which are respectively improved by 4.1 times, 3.7 times, 1.6 times, 12.2 times and 24.7 times compared with the parent. Therefore, the three-site mutant K83R/K95R/L273F shows the optimal thermal stability at both low temperature and high temperature, and the combination mutation can further improve the thermal stability of the enzyme.

Example 7

Comparison of conversion rates of the mutant IEMK83R-K95R-L273F and the parent enzyme in the synthesis of vanillin at different temperatures

In a 50mL centrifuge tube, 9mL of Gly-NaOH buffer solution (100 mM pH 9.0), 9mL of the substrate isoeugenol (100 mM cosolvent: 10% DMSO), 15g/L of the cell mass (parent and mutant enzymes), and 1mL of the substrate isoeugenol were added, and the mixture was transformed at 20, 25, 30, 35 ℃ and 180rpm for 12 hours, respectively. After the reaction, an equal volume of liquid phase methanol was added to quench the reaction, and after mixing, the supernatant was centrifuged and extracted for HPLC analysis. As a result, the conversion rates of vanillin of the mutant enzyme and the parent at 20 ℃ and 25 ℃ are close to 100%, while the mutant enzyme shows better catalytic efficiency than the parent at 30 ℃ and 35 ℃, wherein the conversion rates of vanillin are respectively 87% and 70%, and are respectively 16% and 35% (30 ℃ and 75%;35 ℃ and 52%) higher than the conversion rates of the parent.

Sequence listing

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85 90 95

Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys

100 105 110

Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly

115 120 125

Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro

130 135 140

Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr

145 150 155 160

Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp

165 170 175

Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met

180 185 190

Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp

195 200 205

Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg

210 215 220

Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg

225 230 235 240

Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser

245 250 255

Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp

260 265 270

Leu Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu

275 280 285

Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro

290 295 300

Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala

305 310 315 320

Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp

325 330 335

Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile

340 345 350

Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly

355 360 365

Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile

370 375 380

Phe Ala Tyr Asn Ser Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr

385 390 395 400

Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe

405 410 415

Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr

420 425 430

Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp

435 440 445

Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe

450 455 460

Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro

465 470 475

<210> 3

<211> 478

<212> PRT

<213> Artificial sequence (Isoeugenol Monooxygene)

<400> 3

Met Ala Arg Leu Asn Arg Asn Asp Pro Gln Leu Val Gly Thr Leu Leu

1 5 10 15

Pro Thr Arg Ile Glu Ala Asp Leu Phe Asp Leu Glu Val Asp Gly Glu

20 25 30

Ile Pro Lys Ser Ile Asn Gly Thr Phe Tyr Arg Asn Thr Pro Glu Pro

35 40 45

Gln Val Thr Pro Gln Lys Phe His Thr Phe Ile Asp Gly Asp Gly Met

50 55 60

Ala Ser Ala Phe His Phe Glu Asp Gly His Val Asp Phe Ile Ser Arg

65 70 75 80

Trp Val Lys Thr Ala Arg Phe Thr Ala Glu Arg Leu Ala Arg Arg Ser

85 90 95

Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys

100 105 110

Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly

115 120 125

Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro

130 135 140

Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr

145 150 155 160

Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp

165 170 175

Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met

180 185 190

Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp

195 200 205

Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg

210 215 220

Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg

225 230 235 240

Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser

245 250 255

Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp

260 265 270

Leu Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu

275 280 285

Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro

290 295 300

Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala

305 310 315 320

Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp

325 330 335

Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile

340 345 350

Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly

355 360 365

Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile

370 375 380

Phe Ala Tyr Asn Ser Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr

385 390 395 400

Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe

405 410 415

Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr

420 425 430

Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp

435 440 445

Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe

450 455 460

Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro

465 470 475

<210> 4

<211> 478

<212> PRT

<213> Artificial sequence (Isoeugenol Monooxgene)

<400> 4

Met Ala Arg Leu Asn Arg Asn Asp Pro Gln Leu Val Gly Thr Leu Leu

1 5 10 15

Pro Thr Arg Ile Glu Ala Asp Leu Phe Asp Leu Glu Val Asp Gly Glu

20 25 30

Ile Pro Lys Ser Ile Asn Gly Thr Phe Tyr Arg Asn Thr Pro Glu Pro

35 40 45

Gln Val Thr Pro Gln Lys Phe His Thr Phe Ile Asp Gly Asp Gly Met

50 55 60

Ala Ser Ala Phe His Phe Glu Asp Gly His Val Asp Phe Ile Ser Arg

65 70 75 80

Trp Val Lys Thr Ala Arg Phe Thr Ala Glu Arg Leu Ala Arg Arg Ser

85 90 95

Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys

100 105 110

Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly

115 120 125

Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro

130 135 140

Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr

145 150 155 160

Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp

165 170 175

Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met

180 185 190

Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp

195 200 205

Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg

210 215 220

Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg

225 230 235 240

Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser

245 250 255

Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp

260 265 270

Phe Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu

275 280 285

Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro

290 295 300

Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala

305 310 315 320

Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp

325 330 335

Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile

340 345 350

Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly

355 360 365

Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile

370 375 380

Phe Ala Tyr Asn Ser Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr

385 390 395 400

Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe

405 410 415

Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr

420 425 430

Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp

435 440 445

Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe

450 455 460

Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro

465 470 475

<210> 5

<211> 478

<212> PRT

<213> Artificial sequence (Isoeugenol Monooxgene)

<400> 5

Met Ala Arg Leu Asn Arg Asn Asp Pro Gln Leu Val Gly Thr Leu Leu

1 5 10 15

Pro Thr Arg Ile Glu Ala Asp Leu Phe Asp Leu Glu Val Asp Gly Glu

20 25 30

Ile Pro Lys Ser Ile Asn Gly Thr Phe Tyr Arg Asn Thr Pro Glu Pro

35 40 45

Gln Val Thr Pro Gln Lys Phe His Thr Phe Ile Asp Gly Asp Gly Met

50 55 60

Ala Ser Ala Phe His Phe Glu Asp Gly His Val Asp Phe Ile Ser Arg

65 70 75 80

Trp Val Arg Thr Ala Arg Phe Thr Ala Glu Arg Leu Ala Arg Arg Ser

85 90 95

Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys

100 105 110

Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly

115 120 125

Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro

130 135 140

Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr

145 150 155 160

Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp

165 170 175

Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met

180 185 190

Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp

195 200 205

Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg

210 215 220

Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg

225 230 235 240

Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser

245 250 255

Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp

260 265 270

Leu Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu

275 280 285

Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro

290 295 300

Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala

305 310 315 320

Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp

325 330 335

Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile

340 345 350

Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly

355 360 365

Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile

370 375 380

Phe Ala Tyr Asn Ser Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr

385 390 395 400

Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe

405 410 415

Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr

420 425 430

Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp

435 440 445

Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe

450 455 460

Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro

465 470 475

<210> 6

<211> 478

<212> PRT

<213> Artificial sequence (Isoeugenol Monooxgene)

<400> 6

Met Ala Arg Leu Asn Arg Asn Asp Pro Gln Leu Val Gly Thr Leu Leu

1 5 10 15

Pro Thr Arg Ile Glu Ala Asp Leu Phe Asp Leu Glu Val Asp Gly Glu

20 25 30

Ile Pro Lys Ser Ile Asn Gly Thr Phe Tyr Arg Asn Thr Pro Glu Pro

35 40 45

Gln Val Thr Pro Gln Lys Phe His Thr Phe Ile Asp Gly Asp Gly Met

50 55 60

Ala Ser Ala Phe His Phe Glu Asp Gly His Val Asp Phe Ile Ser Arg

65 70 75 80

Trp Val Arg Thr Ala Arg Phe Thr Ala Glu Arg Leu Ala Arg Arg Ser

85 90 95

Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys

100 105 110

Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly

115 120 125

Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro

130 135 140

Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr

145 150 155 160

Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp

165 170 175

Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met

180 185 190

Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp

195 200 205

Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg

210 215 220

Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg

225 230 235 240

Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser

245 250 255

Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp

260 265 270

Phe Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu

275 280 285

Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro

290 295 300

Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala

305 310 315 320

Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp

325 330 335

Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile

340 345 350

Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly

355 360 365

Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile

370 375 380

Phe Ala Tyr Asn Ser Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr

385 390 395 400

Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe

405 410 415

Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr

420 425 430

Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp

435 440 445

Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe

450 455 460

Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro

465 470 475

<210> 7

<211> 1437

<212> DNA

<213> Artificial sequence (Isoeugenol Monooxygene)

<400> 7

atggcgagac tcaaccgcaa cgacccgcaa ttagtaggaa cacttctccc cacccgtatc 60

gaggcagact tgttcgatct agaggttgac ggcgaaatcc caaaatcaat aaatggaacg 120

ttctaccgta atacgccaga acctcaagtt accccgcaaa aattccacac cttcatagat 180

ggagatggaa tggcctctgc cttccacttc gaagatggtc atgtcgactt catcagtcgc 240

tgggttaaaa ccgctcgatt cacggccgaa cgactagcgc gaaaatcgct atttggcatg 300

tacagaaacc cctataccga cgacaccagt gtaaaaggac tagaccgcac cgttgccaat 360

acaagcatca ttagccatca cggcaaggtg ctggcggtga aggaagacgg cctaccgtac 420

gaactggatc ctcgtacact tgaaactcgc ggacgcttcg actacgacgg ccaagttacc 480

agccaaaccc acaccgccca tccaaaatat gacccggaaa cgggtgactt gttgttcttc 540

ggttcggcag ctaagggcga agcaactcca gacatggcct attacattgt cgacaagcac 600

ggcaaggtga cacatgaaac ttggtttgag cagccctatg gcgcattcat gcacgacttt 660

gccattaccc gaaattggtc cattttccca attatgccgg ccaccaacag cctgtcccgc 720

ctcaaggcga aacagccaat ttatatgtgg gagccggaac tgggcagcta cattggcgta 780

ctgccgcgcc gcggccaggg cagtcagatt cgctggctca aggcaccggc gctctgggta 840

tttcatgttg tgaatgcttg ggaagtcgga accaagattt atatcgacct tatggaaagt 900

gaaatcctgc cgttcccctt ccccaactca caaaaccaac ccttcgcccc tgagaaagcc 960

gtaccacgcc tgactcgttg ggaaattgac ctcgatagca gcagcgacga gatcaagcga 1020

acccggctac acgatttctt tgcggaaatg ccaatcatgg attttcgctt cgccctgcaa 1080

tgcaaccgct atggctttat gggggtggac gatccacgca aaccacttgc gcatcagcag 1140

gccgagaaga tatttgcgta caactcactc ggcatctggg acaaccaccg aggtgactac 1200

gacctctggt actccggaga agcctcggcg gcccaggagc cggccttcgt ccctagaagt 1260

ccgaccgccg ccgaaggtga tgggtacttg ctgaccgtgg ttggtcgtct cgatgaaaat 1320

cgcagtgatc tggtaattct cgacactcaa gacatccagt ctggtcccgt ggcaaccatc 1380

aagctgccat tccggttaag ggccgctctc catggctgct gggtacccag accttaa 1437

<210> 8

<211> 1437

<212> DNA

<213> Artificial sequence (Isoeugenol Monooxgene)

<400> 8

atggcgagac tcaaccgcaa cgacccgcaa ttagtaggaa cacttctccc cacccgtatc 60

gaggcagact tgttcgatct agaggttgac ggcgaaatcc caaaatcaat aaatggaacg 120

ttctaccgta atacgccaga acctcaagtt accccgcaaa aattccacac cttcatagat 180

ggagatggaa tggcctctgc cttccacttc gaagatggtc atgtcgactt catcagtcgc 240

tgggttagga ccgctcgatt cacggccgaa cgactagcgc gaaaatcgct atttggcatg 300

tacagaaacc cctataccga cgacaccagt gtaaaaggac tagaccgcac cgttgccaat 360

acaagcatca ttagccatca cggcaaggtg ctggcggtga aggaagacgg cctaccgtac 420

gaactggatc ctcgtacact tgaaactcgc ggacgcttcg actacgacgg ccaagttacc 480

agccaaaccc acaccgccca tccaaaatat gacccggaaa cgggtgactt gttgttcttc 540

ggttcggcag ctaagggcga agcaactcca gacatggcct attacattgt cgacaagcac 600

ggcaaggtga cacatgaaac ttggtttgag cagccctatg gcgcattcat gcacgacttt 660

gccattaccc gaaattggtc cattttccca attatgccgg ccaccaacag cctgtcccgc 720

ctcaaggcga aacagccaat ttatatgtgg gagccggaac tgggcagcta cattggcgta 780

ctgccgcgcc gcggccaggg cagtcagatt cgctggctca aggcaccggc gctctgggta 840

tttcatgttg tgaatgcttg ggaagtcgga accaagattt atatcgacct tatggaaagt 900

gaaatcctgc cgttcccctt ccccaactca caaaaccaac ccttcgcccc tgagaaagcc 960

gtaccacgcc tgactcgttg ggaaattgac ctcgatagca gcagcgacga gatcaagcga 1020

acccggctac acgatttctt tgcggaaatg ccaatcatgg attttcgctt cgccctgcaa 1080

tgcaaccgct atggctttat gggggtggac gatccacgca aaccacttgc gcatcagcag 1140

gccgagaaga tatttgcgta caactcactc ggcatctggg acaaccaccg aggtgactac 1200

gacctctggt actccggaga agcctcggcg gcccaggagc cggccttcgt ccctagaagt 1260

ccgaccgccg ccgaaggtga tgggtacttg ctgaccgtgg ttggtcgtct cgatgaaaat 1320

cgcagtgatc tggtaattct cgacactcaa gacatccagt ctggtcccgt ggcaaccatc 1380

aagctgccat tccggttaag ggccgctctc catggctgct gggtacccag accttaa 1437

<210> 9

<211> 1437

<212> DNA

<213> Artificial sequence (Isoeugenol Monooxgene)

<400> 9

atggcgagac tcaaccgcaa cgacccgcaa ttagtaggaa cacttctccc cacccgtatc 60

gaggcagact tgttcgatct agaggttgac ggcgaaatcc caaaatcaat aaatggaacg 120

ttctaccgta atacgccaga acctcaagtt accccgcaaa aattccacac cttcatagat 180

ggagatggaa tggcctctgc cttccacttc gaagatggtc atgtcgactt catcagtcgc 240

tgggttaaaa ccgctcgatt cacggccgaa cgactagcgc gaagatcgct atttggcatg 300

tacagaaacc cctataccga cgacaccagt gtaaaaggac tagaccgcac cgttgccaat 360

acaagcatca ttagccatca cggcaaggtg ctggcggtga aggaagacgg cctaccgtac 420

gaactggatc ctcgtacact tgaaactcgc ggacgcttcg actacgacgg ccaagttacc 480

agccaaaccc acaccgccca tccaaaatat gacccggaaa cgggtgactt gttgttcttc 540

ggttcggcag ctaagggcga agcaactcca gacatggcct attacattgt cgacaagcac 600

ggcaaggtga cacatgaaac ttggtttgag cagccctatg gcgcattcat gcacgacttt 660

gccattaccc gaaattggtc cattttccca attatgccgg ccaccaacag cctgtcccgc 720

ctcaaggcga aacagccaat ttatatgtgg gagccggaac tgggcagcta cattggcgta 780

ctgccgcgcc gcggccaggg cagtcagatt cgctggctca aggcaccggc gctctgggta 840

tttcatgttg tgaatgcttg ggaagtcgga accaagattt atatcgacct tatggaaagt 900

gaaatcctgc cgttcccctt ccccaactca caaaaccaac ccttcgcccc tgagaaagcc 960

gtaccacgcc tgactcgttg ggaaattgac ctcgatagca gcagcgacga gatcaagcga 1020

acccggctac acgatttctt tgcggaaatg ccaatcatgg attttcgctt cgccctgcaa 1080

tgcaaccgct atggctttat gggggtggac gatccacgca aaccacttgc gcatcagcag 1140

gccgagaaga tatttgcgta caactcactc ggcatctggg acaaccaccg aggtgactac 1200

gacctctggt actccggaga agcctcggcg gcccaggagc cggccttcgt ccctagaagt 1260

ccgaccgccg ccgaaggtga tgggtacttg ctgaccgtgg ttggtcgtct cgatgaaaat 1320

cgcagtgatc tggtaattct cgacactcaa gacatccagt ctggtcccgt ggcaaccatc 1380

aagctgccat tccggttaag ggccgctctc catggctgct gggtacccag accttaa 1437

<210> 10

<211> 1437

<212> DNA

<213> Artificial sequence (Isoeugenol Monooxgene)

<400> 10

atggcgagac tcaaccgcaa cgacccgcaa ttagtaggaa cacttctccc cacccgtatc 60

gaggcagact tgttcgatct agaggttgac ggcgaaatcc caaaatcaat aaatggaacg 120

ttctaccgta atacgccaga acctcaagtt accccgcaaa aattccacac cttcatagat 180

ggagatggaa tggcctctgc cttccacttc gaagatggtc atgtcgactt catcagtcgc 240

tgggttaaaa ccgctcgatt cacggccgaa cgactagcgc gaaaatcgct atttggcatg 300

tacagaaacc cctataccga cgacaccagt gtaaaaggac tagaccgcac cgttgccaat 360

acaagcatca ttagccatca cggcaaggtg ctggcggtga aggaagacgg cctaccgtac 420

gaactggatc ctcgtacact tgaaactcgc ggacgcttcg actacgacgg ccaagttacc 480

agccaaaccc acaccgccca tccaaaatat gacccggaaa cgggtgactt gttgttcttc 540

ggttcggcag ctaagggcga agcaactcca gacatggcct attacattgt cgacaagcac 600

ggcaaggtga cacatgaaac ttggtttgag cagccctatg gcgcattcat gcacgacttt 660

gccattaccc gaaattggtc cattttccca attatgccgg ccaccaacag cctgtcccgc 720

ctcaaggcga aacagccaat ttatatgtgg gagccggaac tgggcagcta cattggcgta 780

ctgccgcgcc gcggccaggg cagtcagatt cgctggttca aggcaccggc gctctgggta 840

tttcatgttg tgaatgcttg ggaagtcgga accaagattt atatcgacct tatggaaagt 900

gaaatcctgc cgttcccctt ccccaactca caaaaccaac ccttcgcccc tgagaaagcc 960

gtaccacgcc tgactcgttg ggaaattgac ctcgatagca gcagcgacga gatcaagcga 1020

acccggctac acgatttctt tgcggaaatg ccaatcatgg attttcgctt cgccctgcaa 1080

tgcaaccgct atggctttat gggggtggac gatccacgca aaccacttgc gcatcagcag 1140

gccgagaaga tatttgcgta caactcactc ggcatctggg acaaccaccg aggtgactac 1200

gacctctggt actccggaga agcctcggcg gcccaggagc cggccttcgt ccctagaagt 1260

ccgaccgccg ccgaaggtga tgggtacttg ctgaccgtgg ttggtcgtct cgatgaaaat 1320

cgcagtgatc tggtaattct cgacactcaa gacatccagt ctggtcccgt ggcaaccatc 1380

aagctgccat tccggttaag ggccgctctc catggctgct gggtacccag accttaa 1437

<210> 11

<211> 1437

<212> DNA

<213> Artificial sequence (Isoeugenol Monooxgene)

<400> 11

atggcgagac tcaaccgcaa cgacccgcaa ttagtaggaa cacttctccc cacccgtatc 60

gaggcagact tgttcgatct agaggttgac ggcgaaatcc caaaatcaat aaatggaacg 120

ttctaccgta atacgccaga acctcaagtt accccgcaaa aattccacac cttcatagat 180

ggagatggaa tggcctctgc cttccacttc gaagatggtc atgtcgactt catcagtcgc 240

tgggttagga ccgctcgatt cacggccgaa cgactagcgc gaagatcgct atttggcatg 300

tacagaaacc cctataccga cgacaccagt gtaaaaggac tagaccgcac cgttgccaat 360

acaagcatca ttagccatca cggcaaggtg ctggcggtga aggaagacgg cctaccgtac 420

gaactggatc ctcgtacact tgaaactcgc ggacgcttcg actacgacgg ccaagttacc 480

agccaaaccc acaccgccca tccaaaatat gacccggaaa cgggtgactt gttgttcttc 540

ggttcggcag ctaagggcga agcaactcca gacatggcct attacattgt cgacaagcac 600

ggcaaggtga cacatgaaac ttggtttgag cagccctatg gcgcattcat gcacgacttt 660

gccattaccc gaaattggtc cattttccca attatgccgg ccaccaacag cctgtcccgc 720

ctcaaggcga aacagccaat ttatatgtgg gagccggaac tgggcagcta cattggcgta 780

ctgccgcgcc gcggccaggg cagtcagatt cgctggctca aggcaccggc gctctgggta 840

tttcatgttg tgaatgcttg ggaagtcgga accaagattt atatcgacct tatggaaagt 900

gaaatcctgc cgttcccctt ccccaactca caaaaccaac ccttcgcccc tgagaaagcc 960

gtaccacgcc tgactcgttg ggaaattgac ctcgatagca gcagcgacga gatcaagcga 1020

acccggctac acgatttctt tgcggaaatg ccaatcatgg attttcgctt cgccctgcaa 1080

tgcaaccgct atggctttat gggggtggac gatccacgca aaccacttgc gcatcagcag 1140

gccgagaaga tatttgcgta caactcactc ggcatctggg acaaccaccg aggtgactac 1200

gacctctggt actccggaga agcctcggcg gcccaggagc cggccttcgt ccctagaagt 1260

ccgaccgccg ccgaaggtga tgggtacttg ctgaccgtgg ttggtcgtct cgatgaaaat 1320

cgcagtgatc tggtaattct cgacactcaa gacatccagt ctggtcccgt ggcaaccatc 1380

aagctgccat tccggttaag ggccgctctc catggctgct gggtacccag accttaa 1437

<210> 12

<211> 1437

<212> DNA

<213> Artificial sequence (Isoeugenol Monooxgene)

<400> 12

atggcgagac tcaaccgcaa cgacccgcaa ttagtaggaa cacttctccc cacccgtatc 60

gaggcagact tgttcgatct agaggttgac ggcgaaatcc caaaatcaat aaatggaacg 120

ttctaccgta atacgccaga acctcaagtt accccgcaaa aattccacac cttcatagat 180

ggagatggaa tggcctctgc cttccacttc gaagatggtc atgtcgactt catcagtcgc 240

tgggttagga ccgctcgatt cacggccgaa cgactagcgc gaagatcgct atttggcatg 300

tacagaaacc cctataccga cgacaccagt gtaaaaggac tagaccgcac cgttgccaat 360

acaagcatca ttagccatca cggcaaggtg ctggcggtga aggaagacgg cctaccgtac 420

gaactggatc ctcgtacact tgaaactcgc ggacgcttcg actacgacgg ccaagttacc 480

agccaaaccc acaccgccca tccaaaatat gacccggaaa cgggtgactt gttgttcttc 540

ggttcggcag ctaagggcga agcaactcca gacatggcct attacattgt cgacaagcac 600

ggcaaggtga cacatgaaac ttggtttgag cagccctatg gcgcattcat gcacgacttt 660

gccattaccc gaaattggtc cattttccca attatgccgg ccaccaacag cctgtcccgc 720

ctcaaggcga aacagccaat ttatatgtgg gagccggaac tgggcagcta cattggcgta 780

ctgccgcgcc gcggccaggg cagtcagatt cgctggttca aggcaccggc gctctgggta 840

tttcatgttg tgaatgcttg ggaagtcgga accaagattt atatcgacct tatggaaagt 900

gaaatcctgc cgttcccctt ccccaactca caaaaccaac ccttcgcccc tgagaaagcc 960

gtaccacgcc tgactcgttg ggaaattgac ctcgatagca gcagcgacga gatcaagcga 1020

acccggctac acgatttctt tgcggaaatg ccaatcatgg attttcgctt cgccctgcaa 1080

tgcaaccgct atggctttat gggggtggac gatccacgca aaccacttgc gcatcagcag 1140

gccgagaaga tatttgcgta caactcactc ggcatctggg acaaccaccg aggtgactac 1200

gacctctggt actccggaga agcctcggcg gcccaggagc cggccttcgt ccctagaagt 1260

ccgaccgccg ccgaaggtga tgggtacttg ctgaccgtgg ttggtcgtct cgatgaaaat 1320

cgcagtgatc tggtaattct cgacactcaa gacatccagt ctggtcccgt ggcaaccatc 1380

aagctgccat tccggttaag ggccgctctc catggctgct gggtacccag accttaa 1437

Claims

1. A mutant of isoeugenol monooxygenase with high thermal stability and high activity is characterized in that on the basis of the amino acid sequence shown in SEQ ID NO.1, the mutant has at least one mutation selected from 83 th position, 95 th position or 273 th position; when the position 83 is mutated, the lysine K at the position 83 is mutated into arginine R, when the position 95 is mutated, the lysine K at the position 95 is mutated into arginine R, and when the position 273 is mutated, the leucine L at the position 273 is mutated into phenylalanine F.

2. The iso-eugenol monooxygenase mutant with high thermal stability and activity as claimed in claim 1, wherein lysine K at position 83, lysine K at position 95 and leucine L at position 273 in the amino acid sequence shown in SEQ ID No.1 are respectively mutated into arginine R, arginine R and phenylalanine F.

3. A gene encoding a thermostable and highly active mutant of isoeugenol monooxygenase according to any one of claims 1-2.

4. The method for obtaining the isoeugenol monooxygenase mutant with high thermal stability and high activity as claimed in claim 1, wherein plasmid pET21a-IEM is used as a template, primers are designed, genes and plasmids for coding the mutant are obtained through PCR, and Escherichia coli is used as a host for expressing the isoeugenol monooxygenase mutant.

5. The method of claim 4, wherein specifically, plasmid pET21a-IEM is used as a template, a primer is designed, a recombinant plasmid for coding the mutant is obtained through PCR, escherichia coli seed solution containing the recombinant plasmid is inoculated into a fermentation medium, and the fermentation time is 12h under the conditions of 25 ℃,200 rpm; the fermentation medium comprises the following components: na (Na) ₂ HPO ₄ 7g/L，KH ₂ PO ₄ 3g/L, 10g/L yeast extract powder, 0.5g/L NaCl, 14g/L glucose and 7.0 pH; the IPTG induction concentration was 0.1mM.

6. The method of claim 4 or 5, wherein the isoeugenol monooxygenase mutant is expressed using E.coli as a host and pET21a as a vector.

7. Use of a mutant encoding the thermostable and highly active isoeugenol monooxygenase according to any of claims 1-2, wherein vanillin is produced using isoeugenol as a substrate.