CN114231507A - Choline oxidase mutant of Arthrobacter bilis and application thereof - Google Patents

Abstract

The invention discloses a choline oxidase mutant of Arthrobacter choledochii and application thereof, belonging to the technical field of biological engineering. The invention overcomes the limitations of substrate specificity and regioselectivity of the prior wild enzyme by modifying choline oxidase derived from the Arthrobacter choline, and finally obtains the best mutant Q5 (AcCOx)^{S101A/H351V/V355T/F357R/T357Q}) Having a molar conversion of 1, 4-cyclohexanedimethanol to 1, 4-cyclohexanedicarboxaldehyde of wild type6.8 times, effectively improves the catalytic efficiency of choline oxidase AcCOx, and lays a foundation for the industrial preparation of 1, 4-cyclohexane dicarbaldehyde.

Description

Choline oxidase mutant of Arthrobacter bilis and application thereof

Technical Field

The invention relates to a choline oxidase mutant of Arthrobacter cholestans and application thereof, belonging to the technical field of biological engineering.

Background

The 1, 4-cyclohexane dicarbaldehyde is a spiral compound containing carbocycle, can be widely applied to the field of optical displays and screen manufacturing as a heating liquid crystal module, and is attracted attention as an initial synthetic raw material. 1, 4-cyclohexanedicarboxaldehyde can be further reductively aminated to form 1, 4-cyclohexanedimethanamine, a diamine of an aliphatic diisocyanate precursor that can be used as a chain extender and as a curing agent for epoxy resins in certain polyurethane systems.

Currently, 1, 4-cyclohexanedimethanol is produced mainly by chemical methods, including metal-catalyzed methods, solid multi-phase catalytic methods, and supported catalytic methods. The methods have the advantages of high reaction yield, simple process, avoidance of use of solvents and strong oxidants, strong operability, safety and environmental protection, but have the problems of high reaction temperature, high pressure and the like, and do not meet the requirements of green production, safe production and sustainable development. In contrast, the method for catalyzing the oxidation of 1, 4-cyclohexanedimethanol to generate 1, 4-cyclohexanedicarboxaldehyde by a one-enzyme two-step method belongs to a biological catalysis method, has the characteristics of stable and safe product quality, mild process conditions, high efficiency, environmental protection and the like, can reduce the environmental and resource pressure, and promotes the development of low carbon and circular economy in China, so that an effective biological method for efficiently preparing 1, 4-cyclohexanedicarboxaldehyde is urgently needed.

The microbiological process for producing 1, 4-cyclohexane dicarbaldehyde relates to a key enzyme choline oxidase (AcCOx, choline-oxyg)en 1-oxidaducedutase, e.c.1.1.3.17), primary or secondary alcohols can be oxidized to the corresponding aldehydes or ketones. 1, 4-Cyclohexanedimethanol (CHDM) is taken as a substrate, one hydroxyl group is oxidized into an aldehyde group under the action of choline oxidase to generate an intermediate product (4-hydroxymethyl) cyclohexyl formaldehyde, and then the other hydroxyl group at the para position is oxidized to generate a final product 1, 4-cyclohexanedicarboxaldehyde. Then using catalase to consume H which is generated in the oxidation process and has toxic effect on cells₂O₂While generating 1/2O₂To promote the reaction to proceed forward. However, the current enzyme suffers from the problems of (1) narrow substrate spectrum, limited range of catalytic substrates, resulting in low activity towards naphthenic compounds; (2) two hydroxyl groups need to be oxidized simultaneously in the reaction process, and the two hydroxyl groups are far away from each other, so that a certain regioselectivity problem exists.

In recent years, protein engineering has become the most effective strategy for improving the properties of enzymes at the molecular level, such as enlarging the substrate range, increasing the activity of enzymes, and changing the regioselectivity of enzymes. Therefore, it is possible to solve the problem of catalytic regioselectivity by protein engineering design of acox. Protein engineering can be mainly classified into four categories: traditional directed evolution (i.e., irrational design), semi-rational design, rational design (based on structural and computer techniques), and the combined use of multiple strategies. At present, some research progress has been made on the engineering of AcCOx by using protein, however, the improvement effect of regioselectivity is still limited, and the actual industrialization requirement is far from being met. Therefore, the further improvement of the biological method for producing 1, 4-cyclohexane dicarbaldehyde is a problem to be solved urgently, and is one of the focuses of scientific researchers in all countries in the world at present.

Disclosure of Invention

The invention provides an AcCOx mutant capable of efficiently preparing 1, 4-cyclohexane dicarbaldehyde and a modification method thereof, the mutant protein is used for catalyzing 1, 4-cyclohexane dimethanol to prepare 1, 4-cyclohexane dicarbaldehyde, and H which has toxic action on cells and is generated in the process of peroxidase consumption reaction is used₂O₂. The strain constructed by the invention has high production strength and high regioselectivity in the preparation of 1, 4-cyclohexane dicarbaldehydeThe bacterial charge in the conversion is reduced, and the industrial production cost is greatly saved.

The invention provides a choline oxidase AcCOx mutant of a binoculus, which takes choline oxidase AcCOx with an amino acid sequence shown as SEQ ID NO.1 as a parent and mutates one or more of the amino acids at the 101 th position, the 351 th position, the 355 th position, the 357 th position and the 376 th position of the parent.

In one embodiment, the nucleic acid sequence of the coding gene of the choline oxidase AcCOx of Arthrobacter cholestae is shown as SEQ ID No. 2.

In one embodiment, the mutant has its amino acid 101 mutated to alanine relative to the acox parent, resulting in mutant S101A.

In one embodiment, the mutant has a mutation of amino acid 351 to valine relative to the acox parent to obtain mutant H351V.

In one embodiment, the mutant has been mutated to alanine and valine at amino acids 101 and 351, respectively, simultaneously relative to the AcCOx parent to obtain mutant S101A/H351V.

In one embodiment, the mutant has mutation of amino acids 101, 351 and 355 simultaneously to alanine, valine and threonine, respectively, relative to the AcCOx parent to obtain mutant S101A/H351V/V355T.

In one embodiment, the mutant is mutated simultaneously to alanine, valine, threonine and arginine at amino acids 101, 351, 355 and 357, respectively, relative to the AcCOx parent to obtain mutant S101A/H351V/V355T/F357R.

In one embodiment, the mutant is mutated simultaneously with respect to the AcCOx parent at amino acids 101, 351, 355, 357 and 376 to alanine, valine, threonine, arginine and glutamine, respectively, to obtain mutant S101A/H351V/V355T/F357R/T376Q.

The invention provides a gene for coding the choline oxidase AcCOx mutant of the binoculus cholla.

In one embodiment, the nucleotide sequences of the genes encoding said mutants S101A, H351V, S101A/H351V, S101A/H351V/V355T, S101A/H351V/V355T/F357R, S101A/H351V/V355T/F357R/T376Q are shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, respectively.

The invention provides a recombinant vector carrying the gene for coding the choline oxidase AcCOx mutant of the binoculus cholla.

In one embodiment, the recombinant vector includes, but is not limited to, pET series, Duet series, pGEX series, pHY300PLK, pPIC3K, or pPIC9K series vectors.

Preferably, the recombinant vector is pET28a (+).

The invention provides a method for obtaining the AcCOx mutant, which comprises the following steps:

(1) determining mutation sites on the basis of an amino acid sequence of choline oxidase AcCOx of the binoculus chohneri; designing a mutation primer of site-directed mutagenesis, and carrying out site-directed mutagenesis by taking a carrier carrying choline oxidase AcCOx gene as a template; constructing a plasmid vector containing the mutant;

(2) transforming the mutant plasmid into a host cell;

(3) and selecting positive clones for fermentation culture, and purifying the choline oxidase mutant AccOx.

In one embodiment, the host cell is a bacterial cell.

In one embodiment, the host cell is E.coli.

The invention provides host cells expressing the mutants or containing the genes.

In one embodiment, the host cell comprises escherichia coli.

Preferably, the host cell is preferably escherichia coli BL 21.

The invention provides a method for preparing 1, 4-cyclohexanedicarboxaldehyde from whole cells, which takes 1, 4-cyclohexanedimethanol as a substrate and utilizes host cells to convert and produce the 1, 4-cyclohexanedicarboxaldehyde.

In one embodiment, the host cell is added into the reaction system, so that the cell concentration is 10-40 g/L, the concentration of the 1, 4-cyclohexanedimethanol in the system is 10-40 g/L, and the reaction is carried out for 10-15 h at the pH of 7.5-8.5 and at the temperature of 25-30 ℃.

Preferably, the cell concentration is 30 g/L.

Preferably, the concentration of the 1, 4-cyclohexanedimethanol in the system is 30g/L, and the reaction is carried out for 12h at the pH of 8.0-8.5 and at the temperature of 25-30 ℃.

In one embodiment, the reaction system further comprises a cosolvent, wherein the cosolvent comprises methanol, ethanol or dimethyl sulfoxide; the cosolvent is preferably dimethyl sulfoxide.

In one embodiment, the co-solvent is present at a concentration of 0% to 5% (v/v).

Preferably, the co-solvent concentration is 5% (v/v).

In one embodiment, the reaction system further comprises peroxidase and a phosphate buffer.

The invention provides an application of the AcCOx mutant, the gene or the recombinant vector in preparation of 1, 4-cyclohexane dicarbaldehyde.

The invention provides application of the host cell in preparation of 1, 4-cyclohexane dicarbaldehyde.

The invention has the beneficial effects that:

(1) the choline oxidase mutant is constructed by mutating choline oxidase derived from Arthrobacter cholecystolicus and is used for catalytically producing 1, 4-cyclohexane dicarbaldehyde;

(2) the choline oxidase mutant obtained by the invention improves the conversion rate (68.3%) of 1, 4-cyclohexanedimethanol to 1, 4-cyclohexanedicarboxaldehyde by about 6.8 times compared with the contrast (WT), improves the production capacity of a unit catalyst, effectively reduces the production cost, only uses water as a catalytic medium in the reaction, has the advantages of mild reaction conditions, simple and convenient operation, high yield and the like, and accelerates the industrial process of producing 1, 4-cyclohexanedicarboxaldehyde by an enzyme conversion method.

Drawings

FIG. 1 is a schematic diagram of a synthesis route of 1, 4-cyclohexanedicarboxaldehyde.

FIG. 2 is a graph showing the relationship between the different mutants and the conversion rates of 1, 4-cyclohexanedicarboxaldehyde and (4-hydroxymethyl) cyclohexylformaldehyde.

FIG. 3 is a SDS-PAGE pattern of AcCOx enzyme-induced expression of the present invention; lanes 1-3 are the sizes of the target protein bands in whole cells, supernatants and precipitates after induction expression at 35 ℃ with 0.2mM IPTG, respectively, and lane 4 is the protein purified by AcCOx enzyme.

FIG. 4 is a graph showing the relationship between the conversion temperature and the conversion rates of 1, 4-cyclohexanedicarboxaldehyde and (4-hydroxymethyl) cyclohexylformaldehyde.

FIG. 5 is a graph showing the relationship between the pH of the conversion buffer and the conversion rates of 1, 4-cyclohexanedicarboxaldehyde and (4-hydroxymethyl) cyclohexylformaldehyde.

Detailed Description

The gene source is as follows: the choline oxidase AcCOx gene of the mycobacterium cholinesterase related to the patent is derived from Arthrobacter chromorphenolichius.

The pET28a (+) plasmid was purchased from Novagen (Madison, WI, u.s.a.).

Restriction enzymes, T4 DNA ligase, primeSTAR, etc., were purchased from TaKaRa (Dalian, China). 1, 4-cyclohexanedimethanol, 1, 4-cyclohexanedicarboxaldehyde and (4-hydroxymethyl) cyclohexylformaldehyde are available from aladdin. Peroxidase is horseradish peroxidase, purchased from Shanghai Michelin Biotechnology, Inc., and has activity: more than 200 u/mg.

The AcCOx mutant is obtained by molecular modification, and other reagents are obtained by market purchase.

Preparing an LB culture medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride, and sterilizing at 121 ℃ for 20 min.

Preparing a fermentation medium: tryptone 12g/L, yeast extract (Angel yeast powder 802)24g/L, glycerin 4mL/L, KH₂PO₄2.31g/L and K₂HPO₄ 12.31g/L。

Preparing a sodium phosphate flushing solution with the pH of 8.0: the specific formula of 0.2mol/L mixed buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate is described in technical handbook for industrial microorganism experiments (published by California Chuugenjian, China).

Example 1: construction and screening of Single-outburst variants

The choline oxidase AcCOx gene (nucleotide sequence is shown as SEQ ID NO. 2) of the segmented choline bacillus derived from Arthrobacter chromophilus is connected to a polyclonal enzyme cutting site of pET28a (+) plasmid to construct a gene AcCOx containing a wild type AcCOx gene^WTThe recombinant plasmid of (1).

(1) Single-knob Q1 construction: design AcCOx^S101APrimers for the mutation sites were constructed by whole plasmid PCR as shown in Table 1.

TABLE 1 Single mutant primer sequences

Constructing a reaction PCR amplification system: PrimSTAR enzyme 0.5. mu.L, 5 XPrimeSTAR Buffer 10. mu. L, dNTP 4. mu.L of each of the two primers for each mutation site, 1. mu.L, template (AcCOx)^WT)4 mul and 32.5 mul of water; the reaction conditions are as follows: firstly, 94 ℃ for 3 min; ② 10s at 98 ℃; ③ 30s at 55 ℃; fourthly, 3min at 72 ℃; fifthly, circulating the three steps from the second step to the fourth step for 29 times; sixthly, the temperature is 72 ℃ for 5 min; keeping the temperature at 12 ℃.

The reaction system is incubated at 37 ℃ for 30min to digest the plasmid template (the digestion system is 0.3 mu L of DpnI fast-cutting enzyme, 8.7 mu L of the reaction PCR product and 1 mu L of 10 XT Buffer), and the digestion product obtained after the digestion is introduced into competent cells of Escherichia coli BL21 by a chemical transformation method, wherein the chemical transformation method comprises the following steps:

(1) 10. mu.l of the homologous recombination product was introduced into 100. mu.l of BL21 competent cells;

(2) ice-bath for 15-30 min;

(3) performing water bath heat shock at 42 ℃ for 90s, taking out, rapidly placing into ice, standing, and performing ice bath for 3-5 min;

(4) adding 800 μ l of non-resistant LB culture medium, mixing, culturing at 37 deg.C and 200rpm for 1 h;

(5) centrifuging at 5000rpm for 2min to collect bacteria;

(6) the supernatant was removed, and the remaining 100. mu.l of the supernatant was applied to a plate containing 0.05mg/mL kanamycin resistance by pipetting and incubated at 37 ℃ for about 12 hours.

(7) And (3) selecting the single clone to be cultured in the LB containing 0.05mg/mL kanamycin resistance at the constant temperature of 200rpm and 37 ℃ for 12 hours, sending the single clone to a company for sequencing, and obtaining a positive transformant if the sequencing is correct.

Example 2: construction and screening of double, triple and quadruple mutant

(1) Double-knob Q2 construction: in mutant AcCOx^S101ABased on the above, the construction of the double-process variant AcCOx was carried out by whole-plasmid PCR using mutation primers H351-F and H351-R (Table 2), as shown in step (1) of example 1, and the primers used were H351-F and H351-R^S101A/H351V。

(2) Construction of the Triplex variant Q3: in mutant AcCOx^S101A/H351VBased on the above, site-directed mutagenesis was performed using the mutagenesis primers V355T-F and V355T-R, V355Y-F and V355Y-R, V355R-F and V355R-R, V355Q-F and V355Q-R and V355K-F and V355K-R (Table 2) to construct three mutants by whole plasmid PCR, see example 1 for specific embodiments, to prepare five types of three mutants, AcCOx^{S101A/H351V/V355T}、AcCOx^{S101A/H351V/V355Y}、AcCOx^{S101A/H351V/V355R}、AcCOx^{S101A/H351V/V355Q}、AcCOx^{S101A/H351V/V355K}。

TABLE 2 triple mutant primer sequences

(4) Screening of multiple mutants: inoculating correctly sequenced mutant strain into LB seed culture medium, culturing at 200rpm and 37 deg.C for about 10h, respectively inoculating with 5% (v/v) inoculum size into shake flask fermentation culture medium, culturing at 200rpm and 37 deg.C to OD₆₀₀When the concentration is about 0.8, IPTG is added to the mixture to induce the mixture in a final concentration of 0.5mM, and the induction conditions are 200rpm and 16 ℃ for 18 h. Freezing the bacteria liquid after induction expression in a refrigerator at-80 ℃ for 24h, and repeatedly freezing and thawing for 3 times.

The transformation conditions were: adding final concentration into 100mL conical bottles respectivelyWhole cell, 30 g/L1, 4-cyclohexanedimethanol (C) with a degree of 30g/L mutant protein after induction culture₈H₁₆O₂CHDM), 200mM phosphate buffer (pH 8.0) and 0.01g/L peroxidase, wherein the total reaction system is 10mL, the reaction is carried out at 30 ℃, the pH is 8.0 and the rotation speed is 200rpm for 12 hours.

The conversion solution after the completion of the conversion was extracted with methylene chloride, and the yield of 1, 4-cyclohexanedicarboxaldehyde was measured by the GC method, and the result is shown in FIG. 3, which shows that three mutants AcCOx^{S101A/H351V/V355T}The catalytic efficiency is highest.

TABLE 3 different Tri-mutant molar conversions

(5) Tetrad variant Q4 (in mutant AcCOx)^{S101A/H351V/V355T}Base F357R mutation): to contain AcCOx^{S101A/H351V/V355T}The plasmid of (2) was used as a template, and the whole plasmid PCR was performed using the mutant primers F357R-F and F357R-R, and the PCR product was digested in the same manner as in digestion system and example 1.

(6) Five mutant Q5 (in mutant AcCOx)^{S101A/H351V/V355T/F357R}Mutation based on T376Q): to contain AcCOx^{S101A/H351V/V355T/F357R}The plasmid of (2) was used as a template, and the whole plasmid PCR was carried out using the mutant primers T376Q-F and T367Q-R, and the PCR product was digested in the same manner as in the digestion system and example 1.

Example 3: expression and purification method of mutant enzyme

Positive transformants of the mutant recombinant strains prepared in examples 2 and 3 were inoculated into LB medium and cultured at 37 ℃ to OD₆₀₀And when the concentration is 0.6-1.0, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5mM to induce the expression of the enzyme, wherein the induction temperature is 16 ℃, and the induction time is 18 hours to obtain the fermentation liquor. The fermentation broth was centrifuged at 6000rpm for 10min at 4 ℃ to obtain the cells. Adding 10mL of binding solution A (20mM sodium phosphate, 0.5mM NaCl, 20mM imidazole, 1% (v/v) glycerol, adjusting pH to 8.0 with HCl), fully suspending the thallus, placing the centrifuge tube in an ice bath, placing in an ultrasonic cell disruptor, and ultrasonically disrupting stripsThe parts are as follows: working time 4s, interval time 4s, 10min in total. And centrifuging the obtained crushed solution at low temperature and high speed for 30min at 4 ℃ and 8000rpm to obtain a crude enzyme solution. Filtering with 0.22 μm microporous membrane.

Preparing a nickel ion affinity chromatography column, firstly pumping ultrapure water into the column by using a constant flow pump at the temperature of 4 ℃ to flush the column (about 6-12 times of the volume of the column), and then balancing the environment of the column by using 10mL of the binding solution A. When the effluent at the lower end of the column and the low salt concentration buffer pumped into the column have the same pH value (about 5 column volumes of buffer), the resulting membrane-passed crude enzyme solution is added to the column. The heteroproteins are first washed with binding solution A to baseline equilibrium and then eluted with eluent B (20mM sodium phosphate, 0.5mM NaCl, 500mM imidazole). Collecting the eluent of the absorption peak, and measuring the enzyme activity to obtain the target protein reaching the electrophoretic purity.

Example 4: determination of the Whole-cell catalytic efficiency of the mutant

The positive transformant of the mutant recombinant strain prepared in example 2 was inoculated into LB medium and cultured at 37 ℃ to OD₆₀₀And when the concentration is 0.6-1.0, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5mM to induce the expression of the enzyme, wherein the induction temperature is 16 ℃, and the induction time is 18 hours to obtain the fermentation liquor. The fermentation broth was centrifuged at 6000rpm for 10min at 4 ℃ to obtain the cells. Freezing the bacteria liquid after induction expression in a refrigerator at minus 80 ℃ for 24h, and repeatedly freezing and thawing for 3 times for transformation.

Whole cells of mutant Q5 protein at a final concentration of 30g/L after induction culture, 30 g/L1, 4-cyclohexanedimethanol (C) were added to 100mL Erlenmeyer flasks, respectively₈H₁₆O₂CHDM), 200mM phosphate buffer (pH 8.5) and 0.1g/L peroxidase at 25 ℃ for 48h, extracting with dichloromethane, centrifuging at 12000r/min for 10min, sucking the upper organic phase, drying over anhydrous magnesium sulfate, passing through a 0.22-micron organic membrane, and performing gas chromatography.

The specific gas chromatographic analysis method comprises the following steps: the sample analysis adopts a gas chromatograph Agilent GC-7890B and a chiral gas chromatographic column DB-5; the temperature of a sample inlet is 200 ℃; the initial column temperature is 80 ℃, the retention time is 2min, the temperature is raised to 200 ℃ at the speed of 10 ℃/min, and the retention time is 5 min; the carrier gas is nitrogen, the flow rate is 1.0mL/min, and the flow is not divided. Under the detection conditions, the retention time of cis-and trans-1, 4-cyclohexanedicarboxaldehyde, cis-and trans- (4-hydroxymethyl) cyclohexylformaldehyde, cis-and trans-1, 4-cyclohexanedimethanol is 12.736 and 12.851min, 13.899min and 13.925min, 14.796min and 15.069min respectively.

(1, 4-cyclohexanedicarboxaldehyde) molar yield ═ P/S₀)×100％；

Wherein: p represents the final molar concentration of 1, 4-cyclohexanedicarboxaldehyde, S₀Represents the initial molar concentration of 1, 4-cyclohexanedimethanol.

The results showed that the enzymatic effect of whole cells was 56.3% molar yield. From this, it was found that the mutant Q5 had good transformation efficiency.

Example 5: mutant whole cell reaction condition optimization

(1) Reaction temperature optimization

See example 2 for a difference that the yield of 1, 4-cyclohexanedicarboxaldehyde after 48h of conversion of the mutant Q5 at different temperatures (20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃) was determined in the buffer pH8.5, and the yield of 1, 4-cyclohexanedicarboxaldehyde was determined according to the above-described assay and the molar conversion was calculated. As shown in FIG. 4, the catalytic activity of mutant Q5 increased with increasing temperature between 20 ℃ and 30 ℃ and peaked at about 30 ℃ with a molar conversion of 57.8%, and then decreased with further increasing temperature. This indicates that higher temperature affects the activity of the cells and decreases the catalytic activity, and the optimal temperature for the reaction is 30 ℃.

TABLE 1 conversion of the product at different temperatures

(2) Reaction pH optimization

See example 2 for a difference in that the yield of 1, 4-cyclohexanedicarboxaldehyde after 48h conversion of mutant Q5 at a temperature of 30 ℃ in different buffers pH (6.5, 7.0, 7.5, 8.0, 8.5, 9.0) was determined and the yield of 1, 4-cyclohexanedicarboxaldehyde was determined according to the above described assay and the molar conversion was calculated. The results show that the catalytic activity of the mutant Q5 increased with increasing pH in the range of 6.5-8.0 ℃ and peaked at around 8.0 with a molar conversion of 58.2%, and subsequently decreased with further increasing pH. This indicates that higher pH (alkaline environment) is more favorable for mutant Q5 to catalyze the reaction, and whole cells have better catalytic activity at pH 8.0.

TABLE 2 product conversion at different pH

(3) Co-solvent type optimization

Referring to example 2, except that the yield of 1, 4-cyclohexanedicarboxaldehyde after 48h of conversion of the mutant Q5 in different cosolvents (the cosolvents were methanol, ethanol, isopropanol, ethyl acetate, dichloromethane, DMSO, respectively, and added in an amount of 5% of the reaction volume) was measured at 30 ℃ in a buffer pH of 8.5, the yield of 1, 4-cyclohexanedicarboxaldehyde was measured according to the above-described assay method, and the molar conversion was calculated. The result shows that the transformation efficiency of the mutant Q5 is the highest when DMSO is used as a cosolvent, and is 59.3%.

TABLE 3 conversion of the products with different cosolvents

(4) Flux concentration optimization

See example 2 for a difference in that the yield of 1, 4-cyclohexanedicarboxaldehyde after 48h of the conversion of mutant Q5 with different concentrations of cosolvent (0%, 1%, 2%, 3%, 4%, 5% by volume) was determined at pH8.5 in buffer at 30 ℃ in DMSO as cosolvent, and the yield of 1, 4-cyclohexanedicarboxaldehyde was determined according to the above-described assay and the molar conversion was calculated. The results show that the mutant Q5 has the highest transformation efficiency of 68.3% when 1% DMSO is used as a cosolvent.

TABLE 4 conversion of the product in different concentrations of DMSO

Comparative example 1

See example 4 for a specific embodiment, except that the mutant Q5 strain was replaced with the wild-type Q0 strain to perform the transformation experiment. After the conversion was completed, a part of the converted solution was extracted with methylene chloride, centrifuged at 12,000 Xg for 15min, and the supernatant was collected and filtered through a 0.22 μm organic filter and subjected to GC analysis. The GC chromatogram results show that: the molar conversion of 1, 4-cyclohexanedicarboxaldehyde production was 10.5%.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Sn-free Ackir science and technology Co., Ltd

Jiangnan University

<120> Choline arthrobacterium choline oxidase mutant and application thereof

<130> BAA211717A

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 546

<212> PRT

<213> Artificial sequence

<400> 1

Met His Ile Asp Asn Ile Glu Asn Leu Ser Asp Arg Gly Phe Asp Tyr

1 5 10 15

Val Val Ile Gly Gly Gly Ser Ala Gly Ala Ala Val Ala Ala Arg Leu

20 25 30

Ser Glu Asp Pro Asp Val Ser Val Ala Leu Val Glu Ala Gly Pro Asp

35 40 45

Asp Arg Asn Ile Pro Glu Ile Leu Gln Leu Asp Arg Trp Met Glu Leu

50 55 60

Leu Glu Ser Gly Tyr Asp Trp Asp Tyr Pro Ile Glu Pro Gln Glu Asn

65 70 75 80

Gly Asn Ser Phe Met Arg His Ala Arg Ala Lys Val Met Gly Gly Cys

85 90 95

Ser Ser His Asn Ser Cys Ile Ala Phe Trp Ala Pro Arg Glu Asp Leu

100 105 110

Asp Glu Trp Glu Ser Lys Tyr Gly Ala Thr Gly Trp Asn Ala Ala Asn

115 120 125

Ala Trp Pro Leu Tyr Lys Arg Leu Glu Thr Asn Gln Asp Ala Gly Pro

130 135 140

Asp Ala Pro His His Gly Asp Ser Gly Pro Val His Leu Met Asn Val

145 150 155 160

Pro Pro Ala Asp Pro Ser Gly Val Ala Leu Leu Asp Ala Cys Glu Glu

165 170 175

Ala Gly Ile Pro Arg Ala Arg Phe Asn Thr Gly Thr Thr Val Val Asn

180 185 190

Gly Ala Asn Phe Phe Gln Ile Asn Arg Arg Gly Asp Gly Thr Arg Ser

195 200 205

Ser Ser Ser Val Ser Tyr Ile His Pro Ile Ile Glu Arg Asp Asn Phe

210 215 220

Thr Leu Leu Thr Gly Leu Arg Ala Arg Gln Leu Val Phe Asp Ala Asp

225 230 235 240

Lys Arg Cys Thr Gly Val Glu Val Val Asp Gly Ala Phe Gly Arg Thr

245 250 255

His Arg Leu Thr Ala Arg His Glu Val Ile Leu Ser Thr Gly Ala Ile

260 265 270

Asp Ser Pro Lys Leu Leu Met Leu Ser Gly Ile Gly Pro Ala Glu His

275 280 285

Leu Ala Gln His Gly Ile Glu Val Leu Val Asp Ser Pro Gly Val Gly

290 295 300

Glu Asn Leu Gln Asp His Pro Glu Gly Val Val Gln Phe Glu Ala Lys

305 310 315 320

Gln Pro Met Val Gln Thr Ser Thr Gln Trp Trp Glu Ile Gly Ile Phe

325 330 335

Thr Pro Thr Glu Asp Gly Leu Asp Arg Pro Asp Leu Met Met His Tyr

340 345 350

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

355 360 365

Thr Glu Asn Gly Phe Ser Leu Thr Pro Asn Val Thr His Ala Arg Ser

370 375 380

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

385 390 395 400

Val Asp Pro Arg Tyr Phe Thr Asp Pro Glu Gly His Asp Met Arg Val

405 410 415

Met Val Ala Gly Ile Arg Lys Ala Arg Glu Ile Ala Ala Gln Pro Ala

420 425 430

Met Ser Ala Trp Thr Gly Arg Glu Leu Ser Pro Gly Val Gly Ala Gln

435 440 445

Thr Asp Glu Glu Leu Gln Asp Tyr Ile Arg Lys Thr His Asn Thr Val

450 455 460

Tyr His Pro Val Gly Thr Val Arg Met Gly Ala Asp Asp Asp Gly Met

465 470 475 480

Ser Pro Leu Asp Ala Arg Leu Arg Val Lys Gly Val Thr Gly Leu Arg

485 490 495

Val Ala Asp Ala Ser Val Met Pro Glu His Val Thr Val Asn Pro Asn

500 505 510

Ile Thr Val Met Met Ile Gly Glu Arg Cys Ala Asp Leu Ile Lys Ala

515 520 525

Asp Tyr Ala Gly Ala Asp Ala Leu Glu Glu Lys Glu Leu Thr Thr Ser

530 535 540

Phe Ala

545

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<213> Artificial sequence

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atgcatatcg ataacatcga aaatttgtca gatcgcggtt ttgattacgt tgtgattggc 60

gggggctcgg cgggtgctgc ggtagccgcg cgtttgtcgg aggacccgga cgtaagtgta 120

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ggcaattcct tcatgcgcca tgctcgtgct aaggttatgg gtggttgctc cagtcacaat 300

tcctgtattg cgttctgggc gccccgtgaa gatttagacg agtgggagtc gaagtatggg 360

gcaaccggtt ggaatgctgc taacgcctgg cccttatata agcgcctgga gacaaatcaa 420

gacgccggcc cggacgcacc acaccatgga gactcgggtc ccgttcactt gatgaacgta 480

ccaccggcag atccatctgg cgtggccctg ctggacgctt gcgaggaagc tggcatccct 540

cgcgctcgtt tcaatactgg gacgacggtc gtcaatggag ctaacttctt ccaaatcaat 600

cgccgcggag atggtacgcg ttcgtccagc tccgttagct acatccaccc gatcattgaa 660

cgtgacaatt tcacgttgct tacagggctg cgcgctcgtc aacttgtatt tgacgcggac 720

aaacgttgta cgggagttga ggtagtggac ggggctttcg gtcgcacaca ccgtttgaca 780

gcccgtcatg aggtcatctt gagcacaggg gccattgact ccccgaagct gttaatgctg 840

tccgggatcg gcccggcaga acatcttgcg cagcatggca ttgaagtttt agtagacagt 900

cccggtgtgg gcgaaaatct gcaagatcat ccagaaggag tcgtacaatt cgaagcaaaa 960

caacctatgg tccaaacaag cacgcaatgg tgggaaatcg gaatttttac acctacagag 1020

gatggacttg accgccccga cttgatgatg cattatggct cagtaccatt tgatatgaac 1080

actcttcgcc acggatatcc tacaacggag aatggattta gcttgacccc taacgttaca 1140

cacgcacgtt cgcgcggtac cgttcgtctt cgctcacgcg attttcgtga caaaccgatg 1200

gtcgatcctc gttacttcac tgatccagag ggccacgata tgcgcgtgat ggtagcgggc 1260

attcgtaaag cccgcgaaat tgcggctcag cctgctatgt cggcctggac ggggcgcgaa 1320

ttgagccccg gagtcggtgc gcaaacagat gaggagctgc aagactacat ccgtaagacg 1380

cataataccg tctaccatcc tgtaggcacg gtccgcatgg gagcagatga tgatgggatg 1440

tcgcccttag acgcacgttt gcgcgttaag ggcgttacgg ggctgcgcgt agccgatgcg 1500

tccgttatgc ctgaacacgt gactgttaat ccaaacatca ctgtgatgat gattggggag 1560

cgttgcgcag atttaattaa ggccgactat gcgggagccg acgcgcttga agaaaaggag 1620

ttgaccacat cattcgctta a 1641

<210> 3

<211> 1641

<212> DNA

<213> Artificial sequence

<400> 3

atgcatatcg ataacatcga aaatttgtca gatcgcggtt ttgattacgt tgtgattggc 60

gggggctcgg cgggtgctgc ggtagccgcg cgtttgtcgg aggacccgga cgtaagtgta 120

gccctggtgg aagcagggcc cgatgatcgt aatatccctg aaattctgca gttagatcgc 180

tggatggaat tacttgagtc cgggtatgac tgggactatc caatcgagcc gcaagagaac 240

ggcaattcct tcatgcgcca tgctcgtgct aaggttatgg gtggttgctc cagtcacaat 300

gcttgtattg cgttctgggc gccccgtgaa gatttagacg agtgggagtc gaagtatggg 360

gcaaccggtt ggaatgctgc taacgcctgg cccttatata agcgcctgga gacaaatcaa 420

gacgccggcc cggacgcacc acaccatgga gactcgggtc ccgttcactt gatgaacgta 480

ccaccggcag atccatctgg cgtggccctg ctggacgctt gcgaggaagc tggcatccct 540

cgcgctcgtt tcaatactgg gacgacggtc gtcaatggag ctaacttctt ccaaatcaat 600

cgccgcggag atggtacgcg ttcgtccagc tccgttagct acatccaccc gatcattgaa 660

cgtgacaatt tcacgttgct tacagggctg cgcgctcgtc aacttgtatt tgacgcggac 720

aaacgttgta cgggagttga ggtagtggac ggggctttcg gtcgcacaca ccgtttgaca 780

gcccgtcatg aggtcatctt gagcacaggg gccattgact ccccgaagct gttaatgctg 840

tccgggatcg gcccggcaga acatcttgcg cagcatggca ttgaagtttt agtagacagt 900

cccggtgtgg gcgaaaatct gcaagatcat ccagaaggag tcgtacaatt cgaagcaaaa 960

caacctatgg tccaaacaag cacgcaatgg tgggaaatcg gaatttttac acctacagag 1020

gatggacttg accgccccga cttgatgatg cattatggct cagtaccatt tgatatgaac 1080

actcttcgcc acggatatcc tacaacggag aatggattta gcttgacccc taacgttaca 1140

cacgcacgtt cgcgcggtac cgttcgtctt cgctcacgcg attttcgtga caaaccgatg 1200

gtcgatcctc gttacttcac tgatccagag ggccacgata tgcgcgtgat ggtagcgggc 1260

attcgtaaag cccgcgaaat tgcggctcag cctgctatgt cggcctggac ggggcgcgaa 1320

ttgagccccg gagtcggtgc gcaaacagat gaggagctgc aagactacat ccgtaagacg 1380

cataataccg tctaccatcc tgtaggcacg gtccgcatgg gagcagatga tgatgggatg 1440

tcgcccttag acgcacgttt gcgcgttaag ggcgttacgg ggctgcgcgt agccgatgcg 1500

tccgttatgc ctgaacacgt gactgttaat ccaaacatca ctgtgatgat gattggggag 1560

cgttgcgcag atttaattaa ggccgactat gcgggagccg acgcgcttga agaaaaggag 1620

ttgaccacat cattcgctta a 1641

<210> 4

<211> 1641

<212> DNA

<213> Artificial sequence

<400> 4

atgcatatcg ataacatcga aaatttgtca gatcgcggtt ttgattacgt tgtgattggc 60

gggggctcgg cgggtgctgc ggtagccgcg cgtttgtcgg aggacccgga cgtaagtgta 120

gccctggtgg aagcagggcc cgatgatcgt aatatccctg aaattctgca gttagatcgc 180

tggatggaat tacttgagtc cgggtatgac tgggactatc caatcgagcc gcaagagaac 240

ggcaattcct tcatgcgcca tgctcgtgct aaggttatgg gtggttgctc cagtcacaat 300

tcctgtattg cgttctgggc gccccgtgaa gatttagacg agtgggagtc gaagtatggg 360

gcaaccggtt ggaatgctgc taacgcctgg cccttatata agcgcctgga gacaaatcaa 420

gacgccggcc cggacgcacc acaccatgga gactcgggtc ccgttcactt gatgaacgta 480

ccaccggcag atccatctgg cgtggccctg ctggacgctt gcgaggaagc tggcatccct 540

cgcgctcgtt tcaatactgg gacgacggtc gtcaatggag ctaacttctt ccaaatcaat 600

cgccgcggag atggtacgcg ttcgtccagc tccgttagct acatccaccc gatcattgaa 660

cgtgacaatt tcacgttgct tacagggctg cgcgctcgtc aacttgtatt tgacgcggac 720

aaacgttgta cgggagttga ggtagtggac ggggctttcg gtcgcacaca ccgtttgaca 780

gcccgtcatg aggtcatctt gagcacaggg gccattgact ccccgaagct gttaatgctg 840

tccgggatcg gcccggcaga acatcttgcg cagcatggca ttgaagtttt agtagacagt 900

cccggtgtgg gcgaaaatct gcaagatcat ccagaaggag tcgtacaatt cgaagcaaaa 960

caacctatgg tccaaacaag cacgcaatgg tgggaaatcg gaatttttac acctacagag 1020

gatggacttg accgccccga cttgatgatg gtatatggct cagtaccatt tgatatgaac 1080

actcttcgcc acggatatcc tacaacggag aatggattta gcttgacccc taacgttaca 1140

cacgcacgtt cgcgcggtac cgttcgtctt cgctcacgcg attttcgtga caaaccgatg 1200

gtcgatcctc gttacttcac tgatccagag ggccacgata tgcgcgtgat ggtagcgggc 1260

attcgtaaag cccgcgaaat tgcggctcag cctgctatgt cggcctggac ggggcgcgaa 1320

ttgagccccg gagtcggtgc gcaaacagat gaggagctgc aagactacat ccgtaagacg 1380

cataataccg tctaccatcc tgtaggcacg gtccgcatgg gagcagatga tgatgggatg 1440

tcgcccttag acgcacgttt gcgcgttaag ggcgttacgg ggctgcgcgt agccgatgcg 1500

tccgttatgc ctgaacacgt gactgttaat ccaaacatca ctgtgatgat gattggggag 1560

cgttgcgcag atttaattaa ggccgactat gcgggagccg acgcgcttga agaaaaggag 1620

ttgaccacat cattcgctta a 1641

<210> 5

<211> 1641

<212> DNA

<213> Artificial sequence

<400> 5

atgcatatcg ataacatcga aaatttgtca gatcgcggtt ttgattacgt tgtgattggc 60

gggggctcgg cgggtgctgc ggtagccgcg cgtttgtcgg aggacccgga cgtaagtgta 120

gccctggtgg aagcagggcc cgatgatcgt aatatccctg aaattctgca gttagatcgc 180

tggatggaat tacttgagtc cgggtatgac tgggactatc caatcgagcc gcaagagaac 240

ggcaattcct tcatgcgcca tgctcgtgct aaggttatgg gtggttgctc cagtcacaat 300

gcttgtattg cgttctgggc gccccgtgaa gatttagacg agtgggagtc gaagtatggg 360

gcaaccggtt ggaatgctgc taacgcctgg cccttatata agcgcctgga gacaaatcaa 420

gacgccggcc cggacgcacc acaccatgga gactcgggtc ccgttcactt gatgaacgta 480

ccaccggcag atccatctgg cgtggccctg ctggacgctt gcgaggaagc tggcatccct 540

cgcgctcgtt tcaatactgg gacgacggtc gtcaatggag ctaacttctt ccaaatcaat 600

cgccgcggag atggtacgcg ttcgtccagc tccgttagct acatccaccc gatcattgaa 660

cgtgacaatt tcacgttgct tacagggctg cgcgctcgtc aacttgtatt tgacgcggac 720

aaacgttgta cgggagttga ggtagtggac ggggctttcg gtcgcacaca ccgtttgaca 780

gcccgtcatg aggtcatctt gagcacaggg gccattgact ccccgaagct gttaatgctg 840

tccgggatcg gcccggcaga acatcttgcg cagcatggca ttgaagtttt agtagacagt 900

cccggtgtgg gcgaaaatct gcaagatcat ccagaaggag tcgtacaatt cgaagcaaaa 960

caacctatgg tccaaacaag cacgcaatgg tgggaaatcg gaatttttac acctacagag 1020

gatggacttg accgccccga cttgatgatg gtatatggct cagtaccatt tgatatgaac 1080

actcttcgcc acggatatcc tacaacggag aatggattta gcttgacccc taacgttaca 1140

cacgcacgtt cgcgcggtac cgttcgtctt cgctcacgcg attttcgtga caaaccgatg 1200

gtcgatcctc gttacttcac tgatccagag ggccacgata tgcgcgtgat ggtagcgggc 1260

attcgtaaag cccgcgaaat tgcggctcag cctgctatgt cggcctggac ggggcgcgaa 1320

ttgagccccg gagtcggtgc gcaaacagat gaggagctgc aagactacat ccgtaagacg 1380

cataataccg tctaccatcc tgtaggcacg gtccgcatgg gagcagatga tgatgggatg 1440

tcgcccttag acgcacgttt gcgcgttaag ggcgttacgg ggctgcgcgt agccgatgcg 1500

tccgttatgc ctgaacacgt gactgttaat ccaaacatca ctgtgatgat gattggggag 1560

cgttgcgcag atttaattaa ggccgactat gcgggagccg acgcgcttga agaaaaggag 1620

ttgaccacat cattcgctta a 1641

<210> 6

<211> 1641

<212> DNA

<213> Artificial sequence

<400> 6

atgcatatcg ataacatcga aaatttgtca gatcgcggtt ttgattacgt tgtgattggc 60

gggggctcgg cgggtgctgc ggtagccgcg cgtttgtcgg aggacccgga cgtaagtgta 120

gccctggtgg aagcagggcc cgatgatcgt aatatccctg aaattctgca gttagatcgc 180

tggatggaat tacttgagtc cgggtatgac tgggactatc caatcgagcc gcaagagaac 240

ggcaattcct tcatgcgcca tgctcgtgct aaggttatgg gtggttgctc cagtcacaat 300

gcttgtattg cgttctgggc gccccgtgaa gatttagacg agtgggagtc gaagtatggg 360

gcaaccggtt ggaatgctgc taacgcctgg cccttatata agcgcctgga gacaaatcaa 420

gacgccggcc cggacgcacc acaccatgga gactcgggtc ccgttcactt gatgaacgta 480

ccaccggcag atccatctgg cgtggccctg ctggacgctt gcgaggaagc tggcatccct 540

cgcgctcgtt tcaatactgg gacgacggtc gtcaatggag ctaacttctt ccaaatcaat 600

cgccgcggag atggtacgcg ttcgtccagc tccgttagct acatccaccc gatcattgaa 660

cgtgacaatt tcacgttgct tacagggctg cgcgctcgtc aacttgtatt tgacgcggac 720

aaacgttgta cgggagttga ggtagtggac ggggctttcg gtcgcacaca ccgtttgaca 780

gcccgtcatg aggtcatctt gagcacaggg gccattgact ccccgaagct gttaatgctg 840

tccgggatcg gcccggcaga acatcttgcg cagcatggca ttgaagtttt agtagacagt 900

cccggtgtgg gcgaaaatct gcaagatcat ccagaaggag tcgtacaatt cgaagcaaaa 960

caacctatgg tccaaacaag cacgcaatgg tgggaaatcg gaatttttac acctacagag 1020

gatggacttg accgccccga cttgatgatg gtatatggct caacaccatt tgatatgaac 1080

actcttcgcc acggatatcc tacaacggag aatggattta gcttgacccc taacgttaca 1140

cacgcacgtt cgcgcggtac cgttcgtctt cgctcacgcg attttcgtga caaaccgatg 1200

gtcgatcctc gttacttcac tgatccagag ggccacgata tgcgcgtgat ggtagcgggc 1260

attcgtaaag cccgcgaaat tgcggctcag cctgctatgt cggcctggac ggggcgcgaa 1320

ttgagccccg gagtcggtgc gcaaacagat gaggagctgc aagactacat ccgtaagacg 1380

cataataccg tctaccatcc tgtaggcacg gtccgcatgg gagcagatga tgatgggatg 1440

tcgcccttag acgcacgttt gcgcgttaag ggcgttacgg ggctgcgcgt agccgatgcg 1500

tccgttatgc ctgaacacgt gactgttaat ccaaacatca ctgtgatgat gattggggag 1560

cgttgcgcag atttaattaa ggccgactat gcgggagccg acgcgcttga agaaaaggag 1620

ttgaccacat cattcgctta a 1641

<210> 7

<211> 1641

<212> DNA

<213> Artificial sequence

<400> 7

atgcatatcg ataacatcga aaatttgtca gatcgcggtt ttgattacgt tgtgattggc 60

gggggctcgg cgggtgctgc ggtagccgcg cgtttgtcgg aggacccgga cgtaagtgta 120

gccctggtgg aagcagggcc cgatgatcgt aatatccctg aaattctgca gttagatcgc 180

tggatggaat tacttgagtc cgggtatgac tgggactatc caatcgagcc gcaagagaac 240

ggcaattcct tcatgcgcca tgctcgtgct aaggttatgg gtggttgctc cagtcacaat 300

gcttgtattg cgttctgggc gccccgtgaa gatttagacg agtgggagtc gaagtatggg 360

gcaaccggtt ggaatgctgc taacgcctgg cccttatata agcgcctgga gacaaatcaa 420

gacgccggcc cggacgcacc acaccatgga gactcgggtc ccgttcactt gatgaacgta 480

ccaccggcag atccatctgg cgtggccctg ctggacgctt gcgaggaagc tggcatccct 540

cgcgctcgtt tcaatactgg gacgacggtc gtcaatggag ctaacttctt ccaaatcaat 600

cgccgcggag atggtacgcg ttcgtccagc tccgttagct acatccaccc gatcattgaa 660

cgtgacaatt tcacgttgct tacagggctg cgcgctcgtc aacttgtatt tgacgcggac 720

aaacgttgta cgggagttga ggtagtggac ggggctttcg gtcgcacaca ccgtttgaca 780

gcccgtcatg aggtcatctt gagcacaggg gccattgact ccccgaagct gttaatgctg 840

tccgggatcg gcccggcaga acatcttgcg cagcatggca ttgaagtttt agtagacagt 900

cccggtgtgg gcgaaaatct gcaagatcat ccagaaggag tcgtacaatt cgaagcaaaa 960

caacctatgg tccaaacaag cacgcaatgg tgggaaatcg gaatttttac acctacagag 1020

gatggacttg accgccccga cttgatgatg gtatatggct caacaccacg cgatatgaac 1080

actcttcgcc acggatatcc tacaacggag aatggattta gcttgacccc taacgttaca 1140

cacgcacgtt cgcgcggtac cgttcgtctt cgctcacgcg attttcgtga caaaccgatg 1200

gtcgatcctc gttacttcac tgatccagag ggccacgata tgcgcgtgat ggtagcgggc 1260

attcgtaaag cccgcgaaat tgcggctcag cctgctatgt cggcctggac ggggcgcgaa 1320

ttgagccccg gagtcggtgc gcaaacagat gaggagctgc aagactacat ccgtaagacg 1380

cataataccg tctaccatcc tgtaggcacg gtccgcatgg gagcagatga tgatgggatg 1440

tcgcccttag acgcacgttt gcgcgttaag ggcgttacgg ggctgcgcgt agccgatgcg 1500

tccgttatgc ctgaacacgt gactgttaat ccaaacatca ctgtgatgat gattggggag 1560

cgttgcgcag atttaattaa ggccgactat gcgggagccg acgcgcttga agaaaaggag 1620

ttgaccacat cattcgctta a 1641

<210> 8

<211> 1641

<212> DNA

<213> Artificial sequence

<400> 8

atgcatatcg ataacatcga aaatttgtca gatcgcggtt ttgattacgt tgtgattggc 60

gggggctcgg cgggtgctgc ggtagccgcg cgtttgtcgg aggacccgga cgtaagtgta 120

gccctggtgg aagcagggcc cgatgatcgt aatatccctg aaattctgca gttagatcgc 180

tggatggaat tacttgagtc cgggtatgac tgggactatc caatcgagcc gcaagagaac 240

ggcaattcct tcatgcgcca tgctcgtgct aaggttatgg gtggttgctc cagtcacaat 300

gcttgtattg cgttctgggc gccccgtgaa gatttagacg agtgggagtc gaagtatggg 360

gcaaccggtt ggaatgctgc taacgcctgg cccttatata agcgcctgga gacaaatcaa 420

gacgccggcc cggacgcacc acaccatgga gactcgggtc ccgttcactt gatgaacgta 480

ccaccggcag atccatctgg cgtggccctg ctggacgctt gcgaggaagc tggcatccct 540

cgcgctcgtt tcaatactgg gacgacggtc gtcaatggag ctaacttctt ccaaatcaat 600

cgccgcggag atggtacgcg ttcgtccagc tccgttagct acatccaccc gatcattgaa 660

cgtgacaatt tcacgttgct tacagggctg cgcgctcgtc aacttgtatt tgacgcggac 720

aaacgttgta cgggagttga ggtagtggac ggggctttcg gtcgcacaca ccgtttgaca 780

gcccgtcatg aggtcatctt gagcacaggg gccattgact ccccgaagct gttaatgctg 840

tccgggatcg gcccggcaga acatcttgcg cagcatggca ttgaagtttt agtagacagt 900

cccggtgtgg gcgaaaatct gcaagatcat ccagaaggag tcgtacaatt cgaagcaaaa 960

caacctatgg tccaaacaag cacgcaatgg tgggaaatcg gaatttttac acctacagag 1020

gatggacttg accgccccga cttgatgatg gtatatggct caacaccacg cgatatgaac 1080

actcttcgcc acggatatcc tacaacggag aatggattta gcttgcagcc taacgttaca 1140

cacgcacgtt cgcgcggtac cgttcgtctt cgctcacgcg attttcgtga caaaccgatg 1200

gtcgatcctc gttacttcac tgatccagag ggccacgata tgcgcgtgat ggtagcgggc 1260

attcgtaaag cccgcgaaat tgcggctcag cctgctatgt cggcctggac ggggcgcgaa 1320

ttgagccccg gagtcggtgc gcaaacagat gaggagctgc aagactacat ccgtaagacg 1380

cataataccg tctaccatcc tgtaggcacg gtccgcatgg gagcagatga tgatgggatg 1440

tcgcccttag acgcacgttt gcgcgttaag ggcgttacgg ggctgcgcgt agccgatgcg 1500

tccgttatgc ctgaacacgt gactgttaat ccaaacatca ctgtgatgat gattggggag 1560

cgttgcgcag atttaattaa ggccgactat gcgggagccg acgcgcttga agaaaaggag 1620

ttgaccacat cattcgctta a 1641

Claims (10)

1. The choline oxidase AcCOx mutant takes the choline oxidase AcCOx with an amino acid sequence shown as SEQ ID NO.1 as a parent, and one or more of the amino acids at the 101 th position, the 351 th position, the 355 th position, the 357 th position and the 376 th position of the parent are mutated.

2. The mutant according to claim 1, wherein the mutant is one of the following (a) to (f):

(a) mutating the 101 th site of the parent into alanine;

(b) mutation of 351 th position of parent into valine;

(c) simultaneously mutating the 101 th site and the 351 th site of the parent into alanine and valine respectively;

(d) simultaneously mutating the 101 th site, the 351 th site and the 355 th site of the parent into alanine, valine and valine respectively;

(e) simultaneously mutating the 101 th, 351 th, 355 th and 357 th positions of the parents into alanine, valine and arginine;

(f) the 101 th, 351 th, 355 th, 357 th and 376 th positions of the parents are mutated into alanine, valine, arginine and glutamine at the same time.

3. A gene encoding the mutant of claim 1 or 2.

4. A recombinant vector carrying the gene of claim 3.

5. A host cell expressing the mutant of claim 1 or 2, or carrying the gene of claim 3.

6. A method for preparing 1, 4-cyclohexanedicarboxaldehyde from whole cells, which is characterized in that 1, 4-cyclohexanedimethanol is used as a substrate, and the host cell of claim 5 is used for transforming and producing the 1, 4-cyclohexanedicarboxaldehyde.

7. The method according to claim 6, wherein the host cell according to claim 5 is added to the reaction system so that the cell concentration is 10 to 40g/L, the 1, 4-cyclohexanedimethanol concentration in the system is 10 to 40g/L, and the reaction is carried out at pH7.5 to 8.5 at 25 to 30 ℃ for 10 to 15 hours.

8. The method of claim 7, wherein the reaction system further comprises a cosolvent, and the cosolvent comprises methanol, ethanol or dimethyl sulfoxide.

9. The method of claim 8, wherein the co-solvent is present at a concentration of 0% to 5% (v/v).

10. Use of the mutant according to any one of claims 1 to 2, or the gene according to claim 3, or the recombinant vector according to claim 4, or the host cell according to claim 5 for the production of 1, 4-cyclohexanedicarboxaldehyde.

Images