CN110964704B

CN110964704B - Preparation method of hydroxy oxidase CYB5A mutant and ring system product

Info

Publication number: CN110964704B
Application number: CN201911377541.5A
Authority: CN
Inventors: 周始航; 周敏; 唐沁莹
Original assignee: Zhejiang Zetian Fine Chemical Co ltd
Current assignee: Zhejiang Tianjiang Bioengineering Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2021-07-23
Anticipated expiration: 2039-12-27
Also published as: CN110964704A

Abstract

The invention provides a preparation method of a hydroxy oxidase CYB5A mutant and a ring series product. The mutation sites of the hydroxyl oxidase CYB5A mutant comprise: the 83 th amino acid is mutated from lysine into isoleucine, the 137 th amino acid is mutated from proline into arginine, and the 233 th amino acid is mutated from glutamic acid into valine. The hydroxyl oxidase CYB5A mutant has higher conversion rate to the cyclic alcohol compounds, and has good industrial application prospect.

Description

Preparation method of hydroxy oxidase CYB5A mutant and ring system product

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a preparation method of a hydroxyl oxidase CYB5A mutant and a ring series product.

Background

The ring series products such as cyclobutanone and cyclopropanecarboxaldehyde have higher commercial value and better application prospect, however, the prior preparation methods of the ring series products are mostly chemical synthesis methods, the yield is low, and the production process is easy to cause environmental pollution.

Microbial transformation is the structural modification of a complex substrate by a microbial cell, i.e., a catalytic reaction in which a specific site of the substrate is modified by a certain enzyme or a series of enzymes produced during microbial metabolism.

The microbial transformation technology has the following advantages:

1. the reaction condition is mild, the pollution is less, the equipment is simple and the reaction speed is high;

in the reaction of microbial conversion, harsh reaction conditions such as high pressure, intense heat and the like are generally not needed, and the reaction is carried out at normal temperature and in an environment with pH of about 7; the raw materials have no other chemicals except common culture media and substrates, and are generally regarded as less pollution; is provided withThe preparation is simple, the reaction condition is mild, and the production is safe; the microbial conversion reaction is an enzyme-catalyzed reaction, and under the most suitable conditions, the number of molecules of the order of magnitude of 10 which the enzyme can catalyze the conversion of a substrate to a product within one second²～10⁶。

2. The reaction steps are fewer;

with the rapid development of modern biotechnology, genetically engineered bacteria constructed by enzyme synthesis genes can convert intermediates needing multi-step catalytic synthesis in one fermentation.

3. The recovery rate is high and the cost is low;

the microbial reaction can be continuously carried out, the reaction amount is large, the recovery rate is high, the large-scale industrial production can be realized, and the production cost is lower than that of an enzymatic method and a chemical synthesis method in terms of equipment and raw materials.

Disclosure of Invention

The invention aims to provide a mutant of hydroxyl oxidase CYB5A, which has higher conversion rate on cyclic alkanols and has good industrial application prospect.

The invention also aims to provide polydeoxyribonucleotides containing the genetic information of the above-mentioned mutant of the oxyhydroxide enzyme CYB 5A.

The present invention also aims to provide a recombinant vector comprising the above-mentioned polydeoxyribonucleotide.

The invention also aims to provide a recombinant cell comprising the recombinant vector.

The invention also aims to provide a preparation method of the ring series products.

In order to realize the aim, the invention firstly provides a hydroxy oxidase CYB5A mutant, wherein the 83 th amino acid is mutated from lysine to isoleucine, the 137 th amino acid is mutated from proline to arginine, and the 233 th amino acid is mutated from glutamic acid to valine.

In some embodiments of the invention, the mutant of the oxyhydroxide enzyme CYB5A comprises an amino acid sequence shown as sequence 4 in the sequence table.

The invention also provides polydeoxyribonucleotides comprising at least one of the DNA coding sequence of the above-mentioned oxyhydroxide enzyme CYB5A mutant and a DNA sequence complementary to said DNA coding sequence.

In some embodiments of the invention, the DNA coding sequence comprises a DNA sequence as shown in sequence 3 of the sequence listing.

The invention also provides a recombinant vector comprising the polydeoxyribonucleotide.

The invention also provides a recombinant cell, which comprises a host cell and the recombinant vector positioned in the host cell.

In some embodiments of the invention, the host cell is escherichia coli.

Preferably, the escherichia coli is e.coli BL21(DE 3).

The invention also provides a preparation method of the ring series products, which comprises the steps of converting the ring series alcohol compounds by using the hydroxy oxidase CYB5A mutant to obtain the ring series products; the ring series alcohol compound is cyclobutanol or cyclopropyl methanol, and the ring series product is cyclobutanone or cyclopropane formaldehyde.

In some embodiments of the invention, the process for preparing the ring system product comprises: preparing a recombinant vector of the encoding gene of the hydroxyl oxidase CYB5A mutant, transferring the recombinant vector into a host cell to obtain a recombinant cell, and transforming the cyclic alcohol compounds by using the recombinant cell.

In some embodiments of the invention, the DNA coding sequence is a DNA sequence shown as sequence 2 in the sequence table, the recombinant vector is a pET-30a plasmid containing the gene coding for the mutant of the oxyhydroxide enzyme CYB5A, and the host cell is e.coli BL21(DE 3).

In some embodiments of the present invention, when the cyclic alcohol compound is cyclobutanol, the temperature condition of the conversion is 20 to 50 ℃, and the reaction system of the conversion includes: cyclobutanol, recombinant cells containing a hydroxy oxidase CYB5A mutant and a buffer solution with the pH value of 5.5-10.5.

Specifically, the initial concentration of cyclobutanol in the reaction system is 10-800 mmol/L.

Specifically, the amount of the recombinant cells in the reaction system is 100-300 g/L based on the wet weight of the cells.

Optionally, the reaction system further comprises an organic solvent, such as ethanol, isopropanol, and the like, to improve the solubility of the conversion product.

Preferably, after the ring series alcohol compounds are transformed by the recombinant cells, the reaction solution is extracted by using ethyl acetate with a proper volume, the organic layer is a crude product containing a target product (aldehyde compound or ketone compound), and the crude product is purified to obtain the target product.

The method for transforming the cyclic alcohol compounds by using the recombinant cell has the following advantages: the method has the advantages of mild reaction conditions, high substrate adaptability, environmental friendliness, capability of efficiently converting the high-concentration ring system alcohol compound in a reaction system without adding any coenzyme, high reaction efficiency, greenness, environmental friendliness, no generation of solid waste and waste gas, and good industrial application prospect.

The invention has the beneficial effects that: the invention provides a hydroxyl oxidase CYB5A mutant, the mutation site of which comprises: the 83 th amino acid is mutated from lysine into isoleucine, the 137 th amino acid is mutated from proline into arginine, and the 233 th amino acid is mutated from glutamic acid into valine. The hydroxyl oxidase CYB5A mutant has higher conversion rate to the cyclic alcohol compounds, and has good industrial application prospect.

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of the present invention.

FIG. 1 is the SDS-PAGE electrophoresis of the purified mutant of the oxyhydroxide enzyme CYB5A expressed by the genetically engineered bacterium K83I of the oxyhydroxide enzyme CYB5A in example 2;

FIG. 2 is an HPLC chromatogram of the product of transformation of cyclobutanol with the mutant genetically engineered bacterium K83I of oxyhydroxide CYB5A in example 4;

FIG. 3 is the NMR spectrum of the product of transforming cyclobutanol with the mutant genetically engineered bacterium K83I of oxyhydroxide CYB5A in example 4;

FIG. 4 is an HPLC chromatogram of the product of the transformation of cyclopropylmethanol by the mutant genetically engineered bacterium K83I of the oxyhydroxide enzyme CYB5A in example 5.

Detailed Description

The terms as used herein:

"prepared from … …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of … …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 ~ 5" is disclosed, the ranges described should be construed to include the ranges "1 ~ 4", "1 ~ 3", "1 ~ 2 and 4 ~ 5", "1 ~ 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

In these examples, the parts and percentages are by mass unless otherwise indicated.

"part by mass" means a basic unit of measure indicating a mass ratio of a plurality of components, and 1 part may represent any unit mass, for example, 1g or 2.689 g. If we say that the part by mass of the component A is a part by mass and the part by mass of the component B is B part by mass, the ratio of the part by mass of the component A to the part by mass of the component B is a: b. alternatively, the mass of the A component is aK and the mass of the B component is bK (K is an arbitrary number, and represents a multiple factor). It is unmistakable that, unlike the parts by mass, the sum of the parts by mass of all the components is not limited to 100 parts.

"and/or" is used to indicate that one or both of the illustrated conditions may occur, e.g., a and/or B includes (a and B) and (a or B).

Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the invention aims to provide a hydroxyl oxidase CYB5A mutant, wherein the amino acid sequence of the mutant has three sites to realize site-directed mutagenesis, and the three site-directed mutagenesis respectively are as follows: the 83 th amino acid is mutated into isoleucine (I) from lysine (K), the 137 th amino acid is mutated into arginine (R) from proline (P), and the 233 th amino acid is mutated into valine (V) from glutamic acid (E). The invention performs multi-site-specific mutagenesis on the amino acid sequence of the hydroxyl oxidase CYB5A to obtain the hydroxyl oxidase CYB5A mutant which has higher conversion rate on the cyclic canol compounds and has good industrial application prospect.

The encoding gene of the hydroxyl oxidase CYB5A mutant is transferred into a host cell to obtain a recombinant cell, and the recombinant cell can convert a cyclic terpineol compound to obtain an aldehyde compound or a ketone compound with high conversion rate, so that the recombinant cell has a good industrial application prospect. The method for transforming the cyclic alcohol compounds by using the recombinant cells has the following advantages: the method has the advantages of mild reaction conditions, high substrate adaptability and environmental friendliness, and can efficiently catalyze the oxidation reaction of the high-concentration ring system alcohols (such as cyclobutanol or cyclopropyl methanol) in a reaction system without adding any coenzyme to generate high-purity aldehyde compounds or ketone compounds.

Conservative substitution forms of other amino acid sites than the mutation site of the hydroxyl oxidase CYB5A mutant, addition or deletion forms of one or more amino acids, amino-terminal truncation forms, and carboxyl-terminal truncation forms are also included in the scope of the present invention.

Example 1: construction of the Hydroxyoxidase CYB5A mutant

PCR amplification was carried out according to the QuikChange Site-directed Mutagenesis (Stratagene, LaJolla, Calif.) method using a nucleotide sequence containing a mutation Site as a mutation primer (Table 1) and a pET-30a recombinant vector containing the hydroxyoxidase CYB5A gene as a template.

As shown in table 1, the mutant primers include: K83I-F1 and K83I-R1, K83I-F2 and K83I-R2, K83I-F3 and K83I-R3.

Specifically, the PCR amplification is carried out for 3 times, the first amplification is to carry out PCR amplification on a pET-30a recombinant vector containing a hydroxy oxidase CYB5A gene by using primers K83I-F1 and K83I-R1, the second amplification is to carry out PCR amplification on a product obtained by the first amplification by using primers K83I-F2 and K83I-R2, and the third amplification is to carry out PCR amplification on a product obtained by the second amplification by using primers K35 83I-F3 and K83I-R3.

TABLE 1 mutant primer sequences

In the primer sequences of Table 1 above, the underlined positions indicate the mutation sites.

And (3) PCR reaction system:

5×PrimerSTAR buffer(Mg²⁺plus)，5μL；

dNTPs (2.5 mM each), 2.0. mu.L;

forward primer (10. mu.M), 1.0. mu.L;

downstream primer (10. mu.M), 1.0. mu.L;

template, 15 ng;

Primer STAR^TM HS DNA Polymerase(2.5U/μL)，0.5μL；

add ddH₂O to a total volume of 25. mu.L.

PCR procedure:

(1)98℃，1min；

(2)98℃，10s；

(3)55℃，10s；

(4)72℃，7min。

after the steps (2) - (4) were circulated 20 times, the temperature was 72 ℃ for 10min, and then the temperature was cooled to 4 ℃.

And washing a product obtained by the third PCR amplification, and digesting by using a restriction enzyme Dpn I for specifically recognizing a methylation site to degrade a template plasmid.

An enzyme digestion reaction system: mu.L of the washed PCR product, 2.0. mu.L of 10 Xbuffer, 1.0. mu.L of restriction enzyme Dpn I. Wherein, the buffer liquid system is: 50mM potassium acetate, 20mM Tris acetate, 10mM magnesium acetate, 100. mu.g/mL bovine serum albumin, pH 7.9.

The enzyme digestion reaction conditions are as follows: incubate at 37 ℃ for 1 h.

The PCR product after enzyme digestion treatment is transformed into E.coli BL21(DE3) to obtain corresponding recombinant escherichia coli, the recombinant escherichia coli is coated on a flat plate containing kanamycin and cultured overnight at 37 ℃, a single clone is randomly selected to carry out colony PCR identification and sequencing verification, and the result shows that the recombinant vector containing the encoding gene of the oxyhydroxide CYB5A mutant is successfully transformed into an expression host E.coli BL21(DE3), and finally the oxyhydroxide CYB5A mutant gene engineering bacterium K83I is obtained. Sequencing the gene sequence of the hydroxyl oxidase CYB5A mutant in the genetically engineered bacteria K83I, wherein the result shows that after mutation, the gene sequence of the hydroxyl oxidase CYB5A mutant is shown as a sequence 3 in a sequence table, and the amino acid sequence of the hydroxyl oxidase CYB5A mutant is shown as a sequence 4 in the sequence table.

Before mutation, the gene sequence of the hydroxyl oxidase CYB5A is shown as a sequence 1 in a sequence table, and the amino acid sequence of the hydroxyl oxidase CYB5A is shown as a sequence 2 in the sequence table.

Specifically, by comparing the gene sequence of the oxyhydroxide enzyme CYB5A shown as the sequence 1 in the sequence table with the gene sequence of the oxyhydroxide enzyme CYB5A mutant shown as the sequence 3 in the sequence table, it can be seen that the 248 th base is mutated from A to T, the 249 th base is mutated from A to T, the 250 th base is mutated from A to C, the 410 th base is mutated from C to G, the 411 th base is mutated from G to C, the 697 th base is mutated from A to T, and the 698 th base is mutated from A to T.

Comparing the amino acid sequence of the hydroxyl oxidase CYB5A shown in the sequence 2 in the sequence table with the amino acid sequence of the hydroxyl oxidase CYB5A mutant shown in the sequence 4 in the sequence table, the 83 th amino acid is mutated from lysine (K) to isoleucine (I), the 137 th amino acid is mutated from proline (P) to arginine (R), and the 233 th amino acid is mutated from glutamic acid (E) to valine (V).

Example 2: induction expression of hydroxyl oxidase CYB5A mutant gene engineering bacteria K83I

The mutant gene engineering bacteria K83I of the hydroxyl oxidase CYB5A are inoculated into LB culture medium of 50 mu g/mL kanamycin, cultured overnight at 37 ℃ at 200rpm, then inoculated into LB culture medium containing 50 mu g/mL kanamycin in a 1% inoculum size (v/v), cultured at 37 ℃ at 200rpm until the thallus concentration OD600 is about 0.6, added with IPTG with the final concentration of 0.1mM, induced and cultured for 6h at 26 ℃, centrifuged at 4 ℃ at 8000rpm for 10min, and stored at 80 ℃ for later use.

FIG. 1 is a SDS-PAGE electrophoresis picture of a purified oxyhydroxide enzyme CYB5A mutant expressed by genetically engineered bacteria K83I, and the calculation of an amino acid sequence shown as a sequence 4 in a sequence table shows that the theoretical molecular weight of the oxyhydroxide CYB5A mutant is 38.79kDa, and as can be seen from FIG. 1, the molecular weight of the purified oxyhydroxide CYB5A mutant is between 35 and 50 and is consistent with the theoretical value.

Example 3: hydroxyoxidase CYB5A mutant genetically engineered bacterium K83I fermentation tank culture

The genetically engineered bacterium K83I was inoculated into LB medium containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃ at 200rpm, further inoculated into LB medium containing 50. mu.g/mL kanamycin at 2% inoculum size (v/v), cultured at 37 ℃ at 200rpm, inoculated into a fermenter containing 15L of fermentation medium containing 50. mu.g/mL kanamycin at 10% inoculum size (v/v) in the middle logarithmic phase, cultured at 37 ℃ for about 14 hours (middle and late logarithmic phase), induced with lactose for 20 hours, and then centrifuged by a tubular centrifuge to collect the cells for use. The fermentation medium may be a medium known in the art, such as LB medium, which enables the genetically engineered bacteria to grow and express.

Example 4: experiment for converting cyclobutanol into hydroxy oxidase CYB5A mutant genetically engineered bacteria K83I

Reaction system (10.0 mL): 2g of wet cells of the genetically engineered bacterium K83I collected in example 3, 100mM cyclobutanol, 5.0mL of Na₂HPO₄-NaH₂PO₄Buffer (100mM, pH 7.0).

Reaction conditions are as follows: the reaction was carried out at 37 ℃ and 200rpm for 6 h.

Extracting the reaction liquid with ethyl acetate to obtain a crude product containing cyclobutanone, purifying the crude product, and detecting the purified product by using a High Performance Liquid Chromatography (HPLC) and a nuclear magnetic resonance method to obtain an HPLC (high performance liquid chromatography) spectrum shown in figure 2 and a nuclear magnetic resonance spectrum shown in figure 3, wherein the results of figures 2 and 3 can confirm that the main product of cyclobutanol converted by the genetically engineered bacterium K83I in example 4 is cyclobutanone.

Example 5: experiment for converting hydroxy oxidase CYB5A mutant genetically engineered bacterium K83I into cyclopropylmethanol

Reaction system (10.0 mL): 2g of wet cells of the genetically engineered bacterium K83I collected in example 3, 100mM cyclopropylmethanol, 5.0mL of Na₂HPO₄-NaH₂PO₄Buffer (100mM, pH 7.0).

Extracting the reaction liquid with ethyl acetate to obtain a crude product containing cyclobutanone, purifying the crude product, and detecting the purified product by adopting a high performance liquid chromatography and a nuclear magnetic resonance method to obtain an HPLC (high performance liquid chromatography) spectrum shown in figure 4, wherein the HPLC spectrum can be confirmed by figure 4, and the main product of the cyclopropyl methanol converted by the genetically engineered bacterium K83I in the example 5 is cyclopropane formaldehyde.

Example 6: detection of catalytic activity of genetic engineering bacteria K83I of oxyhydroxide enzyme CYB5A mutant

Experimental group A

Reaction conditions are as follows: the reaction was carried out at 37 ℃ and 200 rpm.

Experimental group B

Reaction system (10.0 mL): 2g of non-mutated oxyhydroxide enzyme CYB5A genetically engineered bacteria wet cells, 100mM cyclobutanol, 5.0mL of Na2HPO4-NaH2PO4 buffer (100mM, pH 7.0).

Specifically, the non-mutated hydroxy oxidase CYB5A genetically engineered bacterium refers to a genetically engineered bacterium containing a non-mutated hydroxy oxidase CYB5A encoding gene (shown as a sequence in a sequence 1 in a sequence table).

The results show that the conversion rate of cyclobutanol in experimental group A is only 2% and the conversion rate of cyclobutanol in experimental group B reaches more than 99.9% when the reaction is carried out for 3 hours. That is, when the reaction is carried out for 3 hours, the conversion rate of the non-mutated hydroxy oxidase CYB5A genetically engineered bacteria to the cyclobutanol is only 2%, and the conversion rate of the hydroxy oxidase CYB5A mutant genetically engineered bacteria K83I to the cyclobutanol reaches more than 99.9%.

Example 7: amplification test for converting cyclobutanol into hydroxy oxidase CYB5A mutant genetically engineered bacteria K83I

Reaction system (10.0 mL): 2g of wet cells of the genetically engineered bacterium K83I of example 3, cyclobutanol (10mM/L, 100mM/L, 300mM/L, 500mM/L, 800mM/L) at various concentrations, and 5.0mL of Na₂HPO₄-NaH₂PO₄Buffer (100mM, pH 7.0).

The result shows that the hydroxy oxidase CYB5A mutant genetically engineered bacterium K83I can still realize the conversion rate of the cyclobutanol to more than 99% when reacting for 6h under the condition that the concentration of the cyclobutanol is 800 mM/L.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Sequence listing

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145 150 155 160

Asn Thr Asn Cys Val Gly Trp Asn Arg Met Asn Cys Phe Thr Glu Phe

165 170 175

Trp Leu Met Met Met Ala Glu Gln Ser Asp Lys Ala Val Lys Tyr Tyr

180 185 190

Thr Leu Glu Glu Ile Gln Lys His Asn Asn Ser Lys Ser Thr Trp Leu

195 200 205

Ile Leu His His Lys Val Tyr Asp Leu Thr Lys Phe Leu Glu Glu His

210 215 220

Pro Gly Gly Glu Glu Val Leu Arg Val Gln Ala Gly Gly Asp Ala Thr

225 230 235 240

Glu Asp Ser Met Glu Lys Gly Leu Asn Thr Val Cys Leu Lys Phe Gly

245 250 255

Phe Ala Arg Met Thr Asn Pro Ile Ile Val Lys Lys Ile Leu Glu Cys

260 265 270

Arg Pro Asn Asn Ile Val Gly Ala Ile Met Leu Glu Glu Arg Trp His

275 280 285

Cys Ile Ile Asn Ile Gly Phe Arg Asn Ile Trp Tyr Arg Cys Thr Gln

290 295 300

Gly Leu Lys Phe Ile Gly Leu Trp Ile Cys Cys Ile Val Glu Glu Ser

305 310 315 320

Glu Cys Trp

Claims

1. A hydroxy oxidase CYB5A mutant is characterized in that the 83 th amino acid is mutated from lysine to isoleucine, the 137 th amino acid is mutated from proline to arginine, and the 233 th amino acid is mutated from glutamic acid to valine;

before mutation, the gene sequence of the hydroxyl oxidase CYB5A is shown as a sequence 1 in a sequence table.

2. A polydeoxyribonucleotide comprising at least one of the DNA coding sequence of the hydroxyoxidase CYB5A mutant according to claim 1 and a DNA sequence complementary to said DNA coding sequence.

3. The polydeoxyribonucleotide according to claim 2, wherein said DNA coding sequence comprises the DNA sequence shown as sequence 3 in the sequence Listing.

4. A recombinant vector comprising the polydeoxyribonucleotide according to any one of claims 2 to 3.

5. A recombinant cell comprising a host cell and the recombinant vector of claim 4 located within the host cell.

6. The recombinant cell of claim 5, wherein the host cell is E.coli.

7. The recombinant cell of claim 6, wherein the E.coli is E.coli BL21(DE 3).

8. A method for preparing ring series products, which is characterized in that the hydroxy oxidase CYB5A mutant of claim 1 is used for converting ring series alcohol compounds to obtain ring series products; the ring series alcohol compound is cyclobutanol or cyclopropyl methanol, and the ring series product is cyclobutanone or cyclopropane formaldehyde.

9. A process for the preparation of a ring system product according to claim 8, comprising: preparing a recombinant vector containing the encoding gene of the mutant of the hydroxyl oxidase CYB5A, transferring the recombinant vector into a host cell to obtain a recombinant cell, and transforming the cyclic alcohol compounds by using the recombinant cell.