CN114231507B

CN114231507B - Choline Arthrobacter choline oxidase mutant and application thereof

Info

Publication number: CN114231507B
Application number: CN202111611347.6A
Authority: CN
Inventors: 吴静; 成雅琪; 宋伟; 周怡雯; 陈修来; 高聪; 刘佳
Original assignee: Wuxi Acryl Technology Co ltd; Jiangnan University
Current assignee: Wuxi Acryl Technology Co ltd; Jiangnan University
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2023-11-14
Anticipated expiration: 2041-12-27
Also published as: CN114231507A

Abstract

The invention discloses a choline oxidase mutant of Arthrobacter cholangii and application thereof, belonging to the technical field of bioengineering. The invention overcomes the limitations of the specificity and the regioselectivity of the prior wild type enzyme substrate by modifying the choline oxidase from the Arthrobacter cholangii, and finally obtains the optimal mutant Q5 (AcCOx ^{S101A/H351V/V355T/F357R/T357Q} ) The molar conversion rate of 1, 4-cyclohexanedimethanol converted by the method is 6.8 times of that of a wild type 1, 4-cyclohexanediformaldehyde, so that the catalytic efficiency of choline oxidase AcCOx is effectively improved, and a foundation is laid for the industrialized preparation of 1, 4-cyclohexanediformaldehyde.

Description

Choline Arthrobacter choline oxidase mutant and application thereof

Technical Field

The invention relates to a choline oxidase mutant of Arthrobacter cholangii and application thereof, belonging to the technical field of bioengineering.

Background

1, 4-cyclohexanedicarbaldehyde is a carbocyclic helical compound that is useful as a heating liquid crystal module in the field of optical display and screen manufacturing, and is of great interest as an initial synthetic raw material. The 1, 4-cyclohexanedicarbaldehyde may be further reductively aminated to form 1, 4-cyclohexanedimethylamine, a diamine that is an aliphatic diisocyanate precursor that may be used as a chain extender and as an epoxy resin curing agent in certain polyurethane systems.

At present, 1, 4-cyclohexanedimethylene oxide is mainly produced by chemical methods, including metal catalysis, solid heterogeneous catalysis and supported catalysis. The method has the advantages of higher reaction yield, simple process, strong operability, safety and environmental protection, avoiding the use of solvents and strong oxidants, but having the problems of higher reaction temperature, higher pressure and the like, and being not in line with the requirements of green production, safe production and sustainable development. In contrast, the method for catalyzing 1, 4-cyclohexanedimethanol to generate 1, 4-cyclohexanediformaldehyde by a one-enzyme two-step method belongs to a biocatalysis method, has the characteristics of stable and safe product quality, mild process conditions, high efficiency, environmental protection and the like, can relieve the environmental and resource pressure and promote the development of low carbon and circular economy in China, and therefore, the efficient preparation of 1, 4-cyclohexanediformaldehyde by an effective biological method is urgently needed.

Microbial production of 1, 4-cyclohexanedicarbaldehyde involves a key enzyme, choline-oxidase (E.C.1.1.3.17), which oxidizes primary or secondary alcohols to the corresponding aldehydes or ketones. 1, 4-Cyclohexanedimethanol (CHDM) is used as a substrate, one hydroxyl group is oxidized into 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 the final product 1, 4-cyclohexanediformaldehyde. Then using hydrogen peroxidase to consume H produced in oxidation process and having toxic action on cell ₂ O ₂ Simultaneously generating 1/2O ₂ The reaction is promoted to proceed forward. However, the problem with the current enzyme is (1) a narrow substrate spectrum, limited catalytic substrate range, resulting in lower activity towards naphthenes; (2) The reaction process needs to oxidize two hydroxyl groups simultaneously, 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 expanding the substrate range, increasing the activity of enzymes, and altering the regioselectivity of enzymes. Thus, by engineering AcCOx protein, it is possible to solve the problem of its catalytic regioselectivity. Protein engineering can be largely divided into four categories: traditional directed evolution (i.e., irrational design), semi-rational design, rational design (based on architecture and computer technology), and the combined application of multiple strategies. At present, a certain research progress has been made on the engineering of AcCOx by using protein, however, the improvement effect of regioselectivity is still limited, and the practical industrialization requirement cannot be met. Therefore, further improvement of the biological method for producing the 1, 4-cyclohexanedimethanol is a problem to be solved urgently, and is one of the focuses of current scientific researchers around the world.

Disclosure of Invention

The invention provides an AcCOx mutant capable of efficiently preparing 1, 4-cyclohexanedimethanol and a transformation method thereof, and the mutant protein is utilized to catalyze 1, 4-cyclohexanedimethanol to prepare 1, 4-cyclohexanediformaldehyde, and H which is generated in the process of peroxidase consumption reaction and has toxic action on cells is utilized ₂ O ₂ . The strain constructed by the invention has high production strength and high regioselectivity in the preparation of 1, 4-cyclohexanediformaldehyde, reduces the bacterial dosage in the conversion and greatly saves the industrialized production cost.

The invention provides a choline oxidase Accox mutant of Arthrobacter cholangii, 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 101 st, 351 st, 355 th, 357 th and 376 th amino acids of the parent.

In one embodiment, the nucleic acid sequence of the coding gene of the choline oxidase Accox of the Arthrobacter cholate is shown as SEQ ID NO. 2.

In one embodiment, the mutant is mutated to alanine at amino acid 101 relative to the AcCOx parent to obtain mutant S101A.

In one embodiment, the mutant is mutated from valine at amino acid 351 relative to the AcCOx parent to obtain mutant H351V.

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

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

The invention provides a gene for encoding the Arthrobacter cholate choline oxidase AcCOx mutant.

In one embodiment, the nucleotide sequences of the genes encoding the mutants S101A, H351V, S A/H351V, S A/H351V/V355T, S A/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 encoding the segmented choline oxidase AcCOx mutant of the Arthrobacter cholate.

In one embodiment, the recombinant vector includes, but is not limited to, pET series, duret 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 the amino acid sequence of the choline oxidase AcCOx of the Arthrobacter cholate; designing a mutation primer of site-directed mutagenesis, and carrying out site-directed mutagenesis by taking a carrier carrying a choline oxidase AcCOx gene as a template; constructing a plasmid vector containing the mutant;

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

(3) Positive clones were selected for fermentation culture and choline oxidase mutant AccOx was purified.

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

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

The invention provides a host cell expressing the mutant, or containing the gene.

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

Preferably, the host cell is preferably E.coli BL21.

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

In one embodiment, the host cells are added to the reaction system such that the cell concentration is 10 to 40g/L and the 1, 4-cyclohexanedimethanol concentration in the system is 10 to 40g/L and reacted at pH7.5 to 8.5 at 25 to 30℃for 10 to 15 hours.

Preferably, the cell concentration is 30g/L.

Preferably, the concentration of 1, 4-cyclohexanedimethanol in the system is 30g/L, and the reaction is carried out for 12 hours at the pH of 8.0-8.5 and 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 concentration of the co-solvent is 0% to 5% (v/v).

Preferably, the cosolvent concentration is 5% (v/v).

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

The invention provides an application of the Accox mutant, the gene or the recombinant vector in preparing 1, 4-cyclohexanediformaldehyde.

The invention provides application of the host cell in preparing 1, 4-cyclohexanediformaldehyde.

The invention has the beneficial effects that:

(1) The invention constructs a choline oxidase mutant by mutating choline oxidase from Arthrobacter cholangii, which is used for catalyzing and producing 1, 4-cyclohexane dicarboxaldehyde;

(2) The conversion rate (68.3%) of the choline oxidase mutant obtained by the invention for converting 1, 4-cyclohexanedimethanol into 1, 4-cyclohexanediformaldehyde is improved by about 6.8 times compared with that of a control (WT), the production capacity of a unit catalyst is improved, the production cost is effectively reduced, and only water is used as a catalytic medium in the reaction, so that the choline oxidase mutant has the advantages of mild reaction condition, simplicity and convenience in operation, high yield and the like, and the industrial process of producing 1, 4-cyclohexanediformaldehyde by an enzyme conversion method is accelerated.

Drawings

FIG. 1 is a schematic diagram of the synthetic route of 1, 4-cyclohexanedicarbaldehyde.

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

FIG. 3 is a SDS-PAGE diagram of the Accox enzyme-induced expression of the present invention; lanes 1 to 3 are the sizes of the target protein bands in whole cells, supernatant and pellet after induction of expression at 35℃at 0.2mM IPTG concentration, respectively, and lane 4 is the protein purified by Accox enzyme.

FIG. 4 is a graph of conversion temperature versus conversion of 1, 4-cyclohexanediformaldehyde and (4-hydroxymethyl) cyclohexylformaldehyde.

FIG. 5 is a graph of conversion buffer pH versus conversion of 1, 4-cyclohexanediformaldehyde and (4-hydroxymethyl) cyclohexylformaldehyde.

Detailed Description

Gene source: the AcCOx gene of the choline oxidase of the Arthrobacter cholate is derived from Arthrobacter cholorphenolicus.

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

Restriction enzymes, T4 DNA ligase, primestaR, etc. are available from TaKaRa (Dalia, china). 1, 4-cyclohexanedimethanol, 1, 4-cyclohexanediformaldehyde and (4-hydroxymethyl) cyclohexylformaldehyde were purchased from aladin. Peroxidase is horseradish peroxidase, available from Shanghai Meilin Biochemical technologies Co., ltd., activity: greater than 200u/mg.

The AcCOx mutants are obtained by molecular modification, and the rest reagents are obtained by market purchase.

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

Preparing a fermentation medium: tryptone 12g/L, yeast extract (Angel Yeast powder 802) 24g/L, glycerol 4mL/L, KH ₂ PO ₄ 2.31g/L and K ₂ HPO ₄ 12.31g/L。

Preparing a phosphate sodium flushing solution with the pH value of 8.0: the specific formula of the 0.2mol/L disodium hydrogen phosphate and sodium dihydrogen phosphate mixed buffer solution is shown in industrial microbiological experiment technical Manual (China light industry Press, zhu Gejian, main edition).

Example 1: construction and screening of Single mutation variants

Accox gene (nucleotide sequence shown in SEQ ID NO. 2) of choline oxidase of Arthrobacter cholangii from Arthrobacter cholorphenolicus is connected to a polyclonal enzyme cutting site of pET28a (+) plasmid, and is constructed to obtain Accox gene containing wild type Accox ^WT Is a recombinant plasmid of (a).

(1) Single mutation variant Q1 construction:design of AcCOx ^S101A The primers 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 two primers per mutation site 1. Mu.L each, template (AcCOx ^WT ) 4 mu L of water 32.5 mu L; the reaction conditions are as follows: (1) 94 ℃ for 3min; (2) 98 ℃ for 10s; (3) 30s at 55 ℃; (4) 72 ℃ for 3min; (5) cycling the steps (2) - (4) 29 times; (6) 72 ℃ for 5min; (7) preserving heat at 12 ℃.

The reaction system is incubated for 30min at 37 ℃ to digest a plasmid template (the digestion system is that DpnI fast-cutting enzyme is 0.3 mu L, the reaction PCR product is 8.7 mu L and 10 xT Buffer is 1 mu L), and the digestion product obtained after the digestion is finished is introduced into competent cells of escherichia coli BL21 by a chemical conversion method, wherein the specific steps of the chemical conversion method are as follows:

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

(2) Ice bath for 15-30min;

(3) Heat shock in a water bath at 42 ℃ for 90s, taking out, rapidly putting into ice, and standing for ice bath for 3-5min;

(4) Adding 800 μl of non-resistant LB culture medium, mixing, and culturing at 37deg.C and 200rpm for 1 hr;

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

(6) The supernatant was removed, and the remaining 100-200. Mu.l was applied to a kanamycin-resistant plate containing 0.05mg/mL by pipetting, and incubated at 37℃for about 12 hours.

(7) The monoclonal is selected and cultured in LB containing 0.05mg/mL kanamycin resistance for 12 hours at a constant temperature of 200rpm and 37 ℃, and then sent to a company for sequencing, and the positive transformant is obtained after the sequencing is correct.

Example 2: construction and screening of double, triple and tetramutant variants

(1) Double mutation variant Q2 construction: in mutant AcCOx ^S101A Is based on (a)Above, the double mutant variant AcCOx was prepared by whole plasmid PCR using mutant primers H351-F and H351-R (Table 2), and the specific embodiment was described in step (1) of example 1, using primers H351-F and H351-R ^S101A/H351V 。

(2) Construction of the Tri-mutant variant Q3: in mutant AcCOx ^S101A/H351V Based on the above, site-directed mutagenesis was performed, and three mutants were constructed by whole-plasmid PCR using 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), as described in example 1, to prepare five three mutant AcCOx variants ^{S101A/H351V/V355T} 、AcCOx ^{S101A/H351V/V355Y} 、AcCOx ^{S101A/H351V/V355R} 、AcCOx ^{S101A/H351V/V355Q} 、AcCOx ^{S101A/H351V/V355K} 。

TABLE 2 three mutant primer sequences

(4) Screening of multiple mutants: inoculating mutant strain with correct sequence into LB seed culture medium, culturing at 200rpm and 37deg.C for about 10 hr, inoculating into shake flask fermentation culture medium with 5% (v/v) inoculum size, and culturing at 200rpm and 37deg.C to OD ₆₀₀ About=0.8, IPTG was added at a final concentration of 0.5mM for induction at 200rpm at 16 ℃ for 18h. Freezing the induced and expressed bacterial liquid for 24 hours at the temperature of-80 ℃ in a refrigerator, and repeatedly freezing and thawing for 3 times.

The conversion conditions are as follows: whole cells of the mutant protein after induction culture at a final concentration of 30g/L, 30 g/L1, 4-cyclohexanedimethanol (C) were added to a 100mL Erlenmeyer flask, respectively ₈ H ₁₆ O ₂ CHDM), 200mM phosphate buffer (pH 8.0) and 0.01g/L peroxidase, the total reaction system was 10mL and reacted at 30℃and pH8.0 at a rotation speed of 200rpm for 12 hours.

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

TABLE 3 molar conversion of the different triple mutants

(5) Tetramutant variant Q4 (in mutant AcCOx ^{S101A/H351V/V355T} F357R mutations on the basis): to contain AcCOx ^{S101A/H351V/V355T} The whole plasmid PCR was performed using the mutant primers F357R-F and F357R-R as templates, and the PCR product was digested, and the PCR system was the same as that of the digestion system and example 1.

(6) Five mutant Q5 (in mutant AcCOx) ^{S101A/H351V/V355T/F357R} Mutation of T376Q on the basis): to contain AcCOx ^{S101A/H351V/V355T/F357R} The whole plasmid PCR was performed using the mutant primers T376Q-F and T367Q-R as templates, and the PCR product was digested, and the PCR system was the same as that of the digestion system and example 1.

Example 3: expression purification method of mutant enzyme

The mutant recombinant strain positive transformants prepared in examples 2 and 3 were inoculated into LB medium and cultured at 37℃to OD ₆₀₀ And adding 0.5mM IPTG to induce enzyme expression at the final concentration of 0.6-1.0, wherein the induction temperature is 16 ℃, and the induction time is 18 hours, so as to obtain fermentation liquor. Centrifuging the fermentation broth at 4deg.C and 6000rpm for 10min, and collecting thallus. 10mL of the binding solution A (20 mM sodium phosphate, 0.5mM NaCl, 20mM imidazole, 1% (v/v) glycerol, pH-adjusted to 8.0 with HCl) was added to the cells to fully resuspend the cells, and then the centrifuge tube was placed in an ice bath and placed in an ultrasonic cell disruption apparatus under the following conditions: the working time is 4s, the interval time is 4s, and the total time is 10min. And (3) centrifuging the obtained crushed liquid at a low temperature and a high speed for 30min at a temperature of 4 ℃ and at a speed of 8000rpm to obtain crude enzyme liquid. Filtering with 0.22 μm microporous membrane for use.

A nickel ion affinity chromatography column was prepared, first, the column was flushed with ultra-pure water (about 6-12 column volumes) by pumping the column with a constant flow pump at 4℃and then the column environment was equilibrated with 10mL of binding solution A. When the effluent at the lower end of the column is consistent with the pH value of the low salt concentration buffer solution pumped into the column (about 5 column volumes of buffer solution are needed), the obtained membrane-passing crude enzyme solution is added into the column. The hybrid protein was washed with binding solution A to baseline equilibrium and eluted with eluent B (20 mM sodium phosphate, 0.5mM NaCl, 500mM imidazole). Collecting the eluent of absorption peak, and measuring enzyme activity to obtain the target protein reaching electrophoresis purity.

Example 4: mutant whole cell catalytic efficiency assay

Inoculating the positive transformant of the mutant recombinant strain prepared in the example 2 into LB culture medium, and culturing at 37 ℃ until OD ₆₀₀ And adding 0.5mM IPTG to induce enzyme expression at the final concentration of 0.6-1.0, wherein the induction temperature is 16 ℃, and the induction time is 18 hours, so as to obtain fermentation liquor. Centrifuging the fermentation broth at 4deg.C and 6000rpm for 10min, and collecting thallus. Freezing the bacterial liquid after induced expression in a refrigerator at-80 ℃ for 24 hours, and repeatedly freezing and thawing for 3 times to be used for transformation.

Whole cells of mutant Q5 protein after induction culture at a final concentration of 30g/L, 30 g/L1, 4-cyclohexanedimethanol (C) were added to a 100mL Erlenmeyer flask, respectively ₈ H ₁₆ O ₂ CHDM), 200mM phosphate buffer (pH 8.5) and 0.1g/L peroxidase at 25℃for 48 hours, and after methylene chloride extraction, 12000r/min was centrifuged for 10 minutes, and the upper organic phase was extracted, dried over anhydrous magnesium sulfate, and subjected to gas chromatography through a 0.22 μm organic film.

The specific gas chromatography method comprises the following steps: sample analysis adopts a gas chromatograph Agilent GC-7890B and a chiral gas chromatographic column DB-5; the temperature of the sample inlet is 200 ℃; the initial column temperature is 80 ℃, the retention time is 2min, the temperature is increased to 200 ℃ at 10 ℃/min, and the retention time is 5min; the carrier gas is nitrogen with the flow rate of 1.0mL/min without split flow. Under this detection condition, the retention times of cis-and trans-1, 4-cyclohexanedicarbaldehyde, cis-and trans- (4-hydroxymethyl) cyclohexylcarbaldehyde, cis-and trans-1, 4-cyclohexanedimethanol were 12.736 and 12.851min, 13.899min and 13.925min and 14.796min and 15.069min, respectively.

Molar yield = (P/S) of (1, 4-cyclohexanediformaldehyde ₀ )×100％；

Wherein: p represents the final molar concentration of 1, 4-cyclohexanediformaldehyde, S ₀ Represents the initial mole of 1, 4-cyclohexanedimethanolConcentration.

The results showed that the whole cell enzymolysis effect was 56.3% molar yield. From this, mutant Q5 has good transformation efficiency.

Example 5: mutant whole cell reaction condition optimization

(1) Reaction temperature optimization

The specific embodiment is described in example 2, except that the molar conversion of mutant Q5 was calculated by measuring the 1, 4-cyclohexanediformaldehyde yield after 48h conversion at various temperatures (20 ℃,25 ℃, 30 ℃, 35 ℃, 40 ℃) in the presence of buffer pH8.5, and measuring the 1, 4-cyclohexanediformaldehyde yield according to the above-described assay. As a result, as shown in FIG. 4, the catalytic activity of mutant Q5 increased with an increase in temperature at 20℃to 30℃and reached a peak at about 30℃with a molar conversion of 57.8%, followed by a decrease in catalytic activity with a further increase in temperature. This suggests that higher temperatures affect the activity of the cells, decreasing the catalytic activity, and that the optimum temperature for the reaction is 30 ℃.

TABLE 1 conversion of products at different temperatures

(2) Reaction pH optimization

Specific embodiment see example 2, except that the 1, 4-cyclohexanediformaldehyde yield after 48h conversion of mutant Q5 (6.5, 7.0, 7.5, 8.0, 8.5, 9.0) at different buffer pH's was measured at 30℃and the molar conversion was calculated by measuring the 1, 4-cyclohexanediformaldehyde yield according to the above-described assay. The results showed that mutant Q5 increased in catalytic activity with increasing pH at 6.5-8.0℃and peaked at around 8.0 with a molar conversion of 58.2% followed by a decrease in catalytic activity with further pH increase. This suggests that a higher pH (alkaline environment) is more favorable for the mutant Q5 catalytic reaction and that whole cells have better catalytic activity at pH 8.0.

TABLE 2 conversion of products at different pH' s

(3) Cosolvent type optimization

The specific embodiment is described in example 2, with the difference that the 1, 4-cyclohexanediformaldehyde yield after 48h conversion of mutant Q5 in different co-solvents (co-solvents methanol, ethanol, isopropanol, ethyl acetate, dichloromethane, DMSO, added in an amount of 5% by volume of the reaction) is measured at pH8.5 at 30℃and the molar conversion is calculated by measuring the 1, 4-cyclohexanediformaldehyde yield according to the above-described assay method. The results showed that mutant Q5 had the highest conversion efficiency of 59.3% with DMSO as a cosolvent.

TABLE 3 conversion of products with different cosolvents

(4) Cosolvent concentration optimization

Specific embodiment referring to example 2, the difference is that the 1, 4-cyclohexanediformaldehyde yield after 48h conversion of mutant Q5 (0%, 1%, 2%, 3%, 4%, 5% by volume) in different concentrations of the cosolvent (in the presence of DMSO at pH8.5 in the presence of 30℃in the buffer, was measured and the molar conversion was calculated by measuring the 1, 4-cyclohexanediformaldehyde yield according to the above-described assay. The results showed that mutant Q5 had the highest conversion efficiency of 68.3% with 1% DMSO as a cosolvent.

TABLE 4 conversion of products at different concentrations of DMSO

Comparative example 1

For specific embodiments, see example 4, except that the mutant Q5 strain was replaced with a wild type Q0 strain for transformation experiments. After the conversion was completed, a part of the conversion solution was extracted with methylene chloride and centrifuged at 12,000Xg for 15min, and the supernatant was collected and filtered through a 0.22 μm organic filter membrane for GC analysis. GC chromatogram results showed: the molar conversion of 1, 4-cyclohexanediformaldehyde produced was 10.5%.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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> tin-free Alkovic technologies Co., ltd

Jiangnan University

<120> a choline oxidase mutant of Arthrobacter cholatus and application thereof

<130> BAA211717A

<160> 8

<170> PatentIn version 3.3

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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 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

1. The choline oxidase Accox mutant takes choline oxidase Accox with an amino acid sequence shown as SEQ ID NO.1 as a parent, and amino acids at 101 th, 351 th, 355 th, 357 th and 376 th of the parent are simultaneously mutated to be respectively mutated into alanine, valine, threonine, arginine and glutamine.

2. A gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. A host cell expressing the mutant of claim 1.

5. A host cell carrying the gene of claim 2.

6. A method for preparing 1, 4-cyclohexanediformaldehyde by whole cells, which is characterized in that 1, 4-cyclohexanedimethanol is used as a substrate, and the host cells of claim 4 or 5 are used for transformation to produce 1, 4-cyclohexanediformaldehyde.

7. The method according to claim 6, wherein the host cell according to claim 4 or 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 and 25 to 30 ℃ for 10 to 15 hours.

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

9. The method according to claim 8, wherein the cosolvent is added in a volume ratio of 1% -5%.

10. Use of a mutant according to claim 1, or a gene according to claim 2, or a recombinant vector according to claim 3, or a host cell according to claim 4 or 5 for the preparation of 1, 4-cyclohexanediformaldehyde.