Sugar alcohol oxidase and use thereof

Abstract

The present invention belongs to the technical field of synthetic biology. Disclosed are a sugar alcohol oxidase and the use thereof. D-glucose is used as a substrate to be docked with sugar alcohol oxidase derived from Streptomyces coelicolor A3, amino acid residues around the active center are selected for saturated mutation, and sugar alcohol oxidase having the activity of oxidizing D-glucose is screened by a chromogenic plate, wherein the amino acid sequence of the sugar alcohol oxidase is shown as SEQ ID NO: 2 or SEQ ID NO: 4. The provided sugar alcohol oxidase has the activity of converting D-glucose into D-gluconic acid and the activity of converting D-glyceraldehyde into D-glyceric acid. By means of the sugar alcohol oxidase, the conversion of D-glucose into pyruvic acid is achieved for the first time by using only three enzymes without relying on any coenzymes, thereby having significant originality and application value.

Description

Sugar alcohol oxidase and its application Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to a sugar alcohol oxidase and an application thereof. Background Art

[0002] Pyruvate, also known as 2-oxoacetic acid, is an important α-oxocarboxylic acid with a colorless to pale yellow color. Pyruvate can be converted between sugars, fats, and amino acids in the body through acetyl-CoA and the tricarboxylic acid cycle, playing a pivotal role in the metabolism of the three major nutrients. As an important organic acid, pyruvate is a precursor for the synthesis of various compounds and has a wide range of applications in the chemical, agricultural, pharmaceutical, and food industries.

[0003] Currently, the main methods for synthesizing pyruvate include microbial fermentation, chemical methods, and multi-enzyme catalysis. Enzymatic methods have attracted considerable attention due to their mild reaction conditions and low byproduct counts. The natural ED (Entner-Doudoroff) pathway is an alternative pathway found in some microorganisms lacking a complete EMP pathway. Its characteristic is that one molecule of glucose undergoes only seven steps to produce two molecules of pyruvate, whereas the EMP pathway requires ten steps. In 2012, Sieber et al. reported an improved ED pathway that utilizes the substrate-hybrid dehydratase DHT to simultaneously catalyze the conversion of D-glycerate to pyruvate and D-glucose to 2-keto-3-deoxyglucose, eliminating both the phosphorylation and dephosphorylation steps of the natural ED pathway and reducing the number of enzymes required. Referring to Figure 1, the improved ED pathway utilizes a multi-enzyme catalysis to synthesize pyruvate using D-glucose as a substrate. The specific reaction scheme is as follows: D-glucose is converted to D-glucose under the catalysis of glucose dehydrogenase GDH, consuming one molecule of NAD. + Then, one molecule of D-gluconic acid is converted into 2-keto-3-deoxygluconic acid by the dehydratase DHT, and then 2-keto-3-deoxygluconic acid is converted into one molecule of D-glyceraldehyde and one molecule of pyruvate by the 2-keto-3-deoxygluconic acid aldolase KDGA, and one molecule of D-glyceraldehyde is converted into one molecule of D-glycerate by the glyceraldehyde dehydrogenase ALDH and consumes one molecule of NAD. + One molecule of D-glycerate is converted into another molecule of pyruvate by the dehydratase DHT. Theoretically, one molecule of glucose can be converted into two molecules of pyruvate and consume two molecules of NAD. +However, this multi-enzyme catalytic system still requires a large number of enzymes to synthesize pyruvate. The high cost of enzyme preparation limits the large-scale application of this system. In addition, the pathway requires the consumption of expensive coenzymes. How to further design new enzyme variants to achieve new catalytic properties in which one enzyme has the functions of two enzymes, thereby reducing the number of enzymes used and eliminating the addition of coenzymes to reduce pathway costs has become a key bottleneck problem that needs to be solved in this field.

[0004] Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a sugar alcohol oxidase that simultaneously oxidizes D-glucose and D-glyceraldehyde, and provides an engineered strain expressing the sugar alcohol oxidase.

[0006] The first object of the present invention is to provide a sugar alcohol oxidase, the amino acid sequence of which is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0007] Compared with the existing technology, the sugar alcohol oxidase provided by the present invention has the activity of converting D-glucose into D-gluconic acid and the activity of converting D-glyceraldehyde into D-glyceric acid. Using this sugar alcohol oxidase, it will be the first time to realize the conversion of D-glucose into pyruvate using only three enzymes, and does not need to rely on any coenzyme, which has great originality and application value.

[0008] The second object of the present invention is to provide a gene encoding the sugar alcohol oxidase.

[0009] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO: 3 or SEQ ID NO: 5.

[0010] The third object of the present invention is to provide a recombinant vector or recombinant strain comprising the sugar alcohol oxidase encoding gene.

[0011] Furthermore, the recombinant vector uses pET28a as an expression vector.

[0012] Furthermore, the recombinant strain uses Escherichia coli BL21 (ED3) as a host.

[0013] The fourth object of the present invention is to provide a method for synthesizing pyruvate, wherein the method uses D-glucose as a substrate and synthesizes pyruvate through a reaction catalyzed by the sugar alcohol oxidase, dehydratase and 2-keto-3-deoxyglucose aldolase.

[0014] Furthermore, the final concentration of glucose in the reaction system is 1-10 mM, the final concentration of the sugar alcohol oxidase is 0.3-0.7 mg / mL, the final concentration of the dehydratase is 0.3-0.5 mg / mL, and the final concentration of the 2-keto-3-deoxyglucose aldolase is 0.1-0.3 mg / mL.

[0015] Furthermore, the reaction system further comprises MgCl2, Tween 20 and manganese dioxide, and the reaction temperature of the reaction system is 38-42°C.

[0016] The fifth object of the present invention is to provide the use of the sugar alcohol oxidase in catalyzing the oxidation of aldose, wherein the aldose is one or more of D-xylose, D-ribose, L-arabinose, D-erythrose, D-mannose and D-galactose. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 is a reaction scheme for synthesizing pyruvic acid using D-glucose as a substrate by a multi-enzyme catalysis method in the prior art.

[0018] FIG2 shows the relative enzymatic activities of the sugar alcohol oxidase of the present invention for oxidizing D-glucose and D-glyceraldehyde.

[0019] FIG3 is a concentration-time curve of the three-enzyme catalytic reaction of the sugar alcohol oxidase of the present invention using D-glucose as a substrate to synthesize pyruvate.

[0020] FIG4 is a liquid phase peak diagram of the three-enzyme catalytic reaction of the sugar alcohol oxidase of the present invention using D-glucose as a substrate to synthesize pyruvate.

[0021] FIG5 is a reaction circuit for synthesizing pyruvic acid using D-glucose as a substrate by the three-enzyme catalysis method of the present invention.

[0022] FIG6 shows the relative enzymatic activities of the sugar alcohol oxidase of the present invention in oxidizing different aldoses. DETAILED DESCRIPTION

[0023] The present invention is further described below with reference to specific examples to enable those skilled in the art to better understand and implement the present invention, but the examples are not intended to limit the present invention. The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the sugar alcohol oxidase. For example, "AxxB" indicates that the amino acid at position xx is replaced by amino acid B from amino acid A in the wild-type enzyme. The position numbers correspond to the amino acid sequence numbering of the wild-type sugar alcohol oxidase in SEQ ID No: 1.

[0024] The whole gene synthesis in the examples of the present invention was completed by Suzhou Genewise Biotechnology Co., Ltd.

[0025] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available.

[0026] The reagents used in the embodiments of the present invention are as follows:

[0027] LB medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, prepared with deionized water, sterilized by high-pressure steam, and kept at room temperature for later use.

[0028] TB medium: 12 g / L peptone, 24 g / L yeast extract, 4 mL / L glycerol, containing potassium phosphate buffer.

[0029] Isopropyl β-D-Thiogalactoside (IPTG) solution (1 M): Weigh 2.383 g of IPTG and dissolve it in deionized water. After complete dissolution, dilute to 10 mL and dispense into 2 mL seed tubes. Store frozen at -20°C until ready to use.

[0030] Kanamycin solution (50 mg / mL): Weigh 0.5 g of kanamycin sulfate and dissolve it in deionized water. After complete dissolution, dilute to 10 mL and dispense into 2 mL seed tubes. Store frozen at -20°C until ready to use.

[0031] In the following examples, the enzymes used were all expressed by Escherichia coli BL21 (DE3), including alditol oxidase (ALDO), dehydratase (DHT), and 2-keto-3-deoxygluconate aldolase (KDGA).

[0032] In a preferred embodiment of the present invention, the preparation method of the sugar alcohol oxidase of the present invention comprises the following steps: (1) constructing the gene of the corresponding mutation site of the sugar alcohol oxidase into the pET28a expression vector to obtain a recombinant plasmid carrying the target enzyme gene. (2) Transforming the recombinant plasmid into a host bacterial cell, preferably Escherichia coli BL21 (DE3), to obtain the corresponding engineered strain. (3) Inoculating the engineered strain into LB medium, culturing at 37°C overnight, controlling the initial OD value to 0.1, transferring to TB medium, culturing until the OD value reaches above 0.6, then adding 0.1mM-1mM IPTG, and culturing at 12-30°C for 12-20 hours. (4) Collecting the bacteria by centrifugation.

[0033] Example 1: Construction of sugar alcohol oxidase genetically engineered bacteria

[0034] (1) Construction of gene expression vector:

[0035] The wild-type alditol oxidase (ALDO) gene from Streptomyces coelicolor A3 (Gene ID: 1101588) was obtained from NCBI. It contains four mutations: V125M, A244T, V133M, and G399R. Its amino acid sequence is shown in SEQ ID No: 1. The wild-type alditol oxidase sequence was codon-optimized, and the optimized sequence was synthesized and subcloned into the Escherichia coli gene expression vector pET28a to generate the recombinant plasmid pET28a-ScALDO.

[0036] (2) Construction of BL21 (DE3) genetically engineered bacteria for expressing the enzymes in step (1)

[0037] (21) Transformation of recombinant plasmid into competent Escherichia coli

[0038] The recombinant plasmid pET28a-ScALDO constructed in step (1) was transformed into the Escherichia coli expression host BL21 (DE3) using the calcium chloride method.

[0039] In a clean bench, place the BL21 (DE3) competent cell on ice for 2 minutes to thaw it, add the recombinant plasmid pET28a-ScALDO to the BL21 (DE3) competent cell, gently pipette to mix it, let it stand on ice for 5 minutes, then spread it on an LB resistance plate containing 50 mg / L kanamycin and culture it in a 37°C incubator overnight.

[0040] (22) Colony PCR identification

[0041] A positive single colony was picked from the plate in step (21) and preliminarily identified by colony PCR using 2×U Taq PCR MasterMix (Beijing Zhuangmeng International Biogene Technology Co., Ltd., catalog number: ZT201A-1). The colony PCR reaction system is shown in Table 1, and the colony PCR amplification reaction procedure is shown in Table 2. After the colony PCR amplification is completed, the colony PCR product is spotted into a 1% agarose gel electrophoresis gel well (another well is spotted with a DS 5000 DNA Marker as a control), separated in an electrophoresis tank (110V) for 30 minutes, and after separation, the agarose gel electrophoresis gel is immersed in Gelred staining solution for 15 minutes. The approximate size of the colony PCR product band is determined based on the DS 5000 DNA Marker in the gel imaging instrument and compared with the expected band size to determine whether the expression strain construction is complete. In this example, positive transformants were identified by colony PCR to obtain the expression strain BL21 (DE3) / pET28a-ScALDO.

[0042] Table 1 Colony PCR reaction system

[0043] Table 2 Colony PCR amplification reaction procedure

[0044] Example 2: Construction and screening of mutant sugar alcohol oxidases

[0045] (1) Construction of a sugar alcohol oxidase mutant library

[0046] Wild-type sugar alcohol oxidase (amino acid sequence: SEQ ID NO: 1) was docked using D-glucose as a substrate, and amino acid residues around the active center were subjected to saturation mutagenesis to construct a saturation mutation library.

[0047] (2) Screening for mutant sugar alcohol oxidases that simultaneously oxidize D-glucose and D-glyceraldehyde

[0048] The saturated mutant library was transformed into competent E. coli BL21(DE3) cells, then plated onto LB plates containing 50 mg / L kanamycin and incubated overnight at 37°C. Monoclonal colonies were screened for activity using a laboratory-developed plate colorimetric strategy. The basic principle is that catalytically active mutant sugar alcohol oxidases release H2O2 during the oxidation of the substrate glucose. Using this H2O2 colorimetric system, monoclonal colonies can be visualized, allowing for rapid identification of active mutations.

[0049] Two mutant strains with D-glucose oxidation activity were screened and obtained. The two mutant strains were cultured separately, and plasmids were extracted. DNA sequencing was performed. The sequencing results showed that the mutation sites that conferred D-glucose oxidation activity to the mutant sugar alcohol oxidase were the Q288G monomer mutation (corresponding to a mutation of glutamine at position 288 of the amino acid sequence of SEQ ID NO: 1 to glycine) and the Q288A monomer mutation (corresponding to a mutation of glutamine at position 288 of the amino acid sequence of SEQ ID NO: 1 to alanine).

[0050] The mutant sugar alcohol oxidase containing the Q288G monomer mutation was named ScALDO-Q288G, its amino acid sequence is SEQ ID NO: 2, its encoding nucleotide sequence is SEQ ID NO: 3, and the corresponding expression strain is BL21(DE3) / pET28a-ScALDO-Q288G; the mutant sugar alcohol oxidase containing the Q288A monomer mutation was named ScALDO-Q288A, its amino acid sequence is SEQ ID NO: 4, its encoding nucleotide sequence is SEQ ID NO: 5, and the corresponding expression strain is BL21(DE3) / pET28a-ScALDO-Q288A.

[0051] Example 3: Construction of genetically engineered bacteria producing dehydratase and 2-keto-3-deoxyglucose aldolase

[0052] The dehydratase (DHT) gene from Paralcaligenes ureilyticus (NCBI Reference Sequence: WP_132585145.1) and the 2-keto-3-deoxygluconate aldolase (KDGA) gene from Picrophilus torridus (NCBI Reference Sequence: NC_005877.1) were obtained from the literature. After codon optimization of the dehydratase and 2-keto-3-deoxygluconate aldolase sequences, the optimized sequences were fully synthesized and subcloned into the Escherichia coli gene expression vector pET28a to obtain the recombinant plasmids pET28a-PuDHT and pET28a-PtKDGA, respectively.

[0053] The recombinant plasmids pET28a-PuDHT and pET28a-PtKDGA were transformed into the Escherichia coli expression host BL21 (DE3) using the calcium chloride method, and the positive transformants were identified to obtain the expression strains BL21 (DE3) / pET28a-PuDHT and BL21 (DE3) / pET28a-PtKDGA.

[0054] Example 4: Enzyme expression and purification

[0055] The wild-type genetically engineered bacteria and mutant genetically engineered bacteria constructed in Examples 1 to 3, namely BL21 (DE3) / pET28a-ScALDO, BL21 (DE3) / pET28a-PuDHT, BL21 (DE3) / pET28a-PtKDGA, BL21 (DE3) / pET28a-ScALDO-Q288G, and BL21 (DE3) / pET28a-ScALDO-Q288A, were inoculated into 10 mL of LB medium containing 50 mg / L kanamycin and cultured overnight at 37 ° C and 250 rpm to obtain seed solution.

[0056] Inoculate the seed solution in 100 mL of TB medium containing 50 mg / L kanamycin to control the initial OD value of the fermentation medium. 600 The fermentation medium was placed at 37°C and 220 rpm for cultivation. 600When the pH value was 0.6 or above, 50 μL of 1 M IPTG was added for induction, so that the final concentration of IPTG in the fermentation medium was 0.5 mM. The fermentation medium was placed at 16° C. and induced at 180 rpm for 16 h.

[0057] After induction, transfer the bacterial suspension to a 100 mL centrifuge tube and centrifuge at 6000 rpm for 5 minutes at room temperature. Discard the supernatant, resuspend in sterile water, and centrifuge at 6000 rpm for 5 minutes at room temperature. Discard the supernatant. Add a certain volume of HEPES buffer to each of the centrifuged bacterial pellets. Place the pellet in a Toshiba ultrasonic disruptor and sonicate at 35% power for 3 seconds, followed by a 3-second interval, for a total of 40 minutes. Then, centrifuge at 10,000 rpm for 30 minutes at 4°C to obtain a crude enzyme solution.

[0058] The target protein was purified using an AKTA purifier and a nickel ion affinity chromatography column with an imidazole concentration of 0.3M-0.5M to obtain the target protein; the imidazole in the target protein was then removed using a desalting column.

[0059] Example 5: Enzyme activity determination of wild-type and mutant sugar alcohol oxidase

[0060] The relative activities of wild-type and mutant sugar alcohol oxidases on D-glucose were assessed in a system containing 10 mM D-glucose, 1 mM 4-AAP, 1 mM DHBS, 4 μL of 0.3 g / L horseradish peroxidase, and 0.1 mg / mL of wild-type or mutant sugar alcohol oxidase. The enzyme activities of ScALDO, ScALDO-Q288G, and ScALDO-Q288A on D-glucose were calculated by measuring the change in absorbance over 5 minutes. The relative activities were calculated with the highest activity as 100%.

[0061] The relative activities of wild-type and mutant sugar alcohol oxidases towards D-glyceraldehyde were assessed in a system containing 10 mM D-glyceraldehyde, 1 mM 4-AAP, 1 mM DHBS, 4 μL of 0.3 g / L horseradish peroxidase, and 0.1 mg / mL of wild-type or mutant sugar alcohol oxidase. The enzyme activities of ScALDO, ScALDO-Q288G, and ScALDO-Q288A towards D-glyceraldehyde were calculated by measuring the change in absorbance over 5 minutes. The relative activities were calculated with the maximum enzyme activity as 100%.

[0062] Refer to Figure 2, which shows the relative enzymatic activities of the sugar alcohol oxidases of the present invention for oxidizing D-glucose and D-glyceraldehyde. The wild-type sugar alcohol oxidase ScALDO lacks activity in oxidizing D-glucose to D-gluconic acid and exhibits high substrate specificity. However, the mutant sugar alcohol oxidases ScALDO-Q288G and ScALDO-Q288A both exhibit significant activity in oxidizing D-glucose to D-gluconic acid and retain some activity in oxidizing D-glyceraldehyde to D-glycerate. Both ScALDO-Q288G and ScALDO-Q288A achieve high substrate promiscuity. Clearly, the Q288G and Q288A site-directed mutations confer upon the wild-type sugar alcohol oxidase ScALDO the novel property of simultaneously oxidizing D-glucose and D-glyceraldehyde. Theoretically, the sugar alcohol oxidases (ScALDO-Q288G and ScALDO-Q288A) of the present invention can replace glucose dehydrogenase GDH and glyceraldehyde dehydrogenase ALDH in the pyruvate synthesis pathway from glucose, reducing the number of enzymes required to synthesize pyruvate from D-glucose from 4 to 3.

[0063] Example 6: Tri-enzyme catalytic reaction for synthesizing pyruvate using glucose as substrate

[0064] The reaction system contained 5 mM glucose, 5 mM MgCl2, 0.1% Tween 20, 1 g / L manganese dioxide, 0.5 mg / mL sugar alcohol oxidase ScALDO-Q288G, 0.4 mg / mL dehydratase PuDHT, and 0.2 mg / mL 2-keto-3-deoxygluconic acid aldolase PtKDGA. The total reaction time was 24 hours, with 0.5 g / L manganese dioxide added at 2 and 4 hours. The reaction temperature was 40°C, the pH was 8.0, and the buffer was 100 mM HEPES-NaOH buffer. The reaction volume was 100 μL. The sugar alcohol oxidase ScALDO-Q288G in the reaction system was replaced with an equal amount of wild-type sugar alcohol oxidase ScALDO. The concentrations of the other substances remained unchanged and the reaction was continued under the same conditions for 24 hours to serve as a control.

[0065] The concentration of pyruvate in the three-enzyme reaction system of sugar alcohol oxidase ScALDO-Q288G, dehydratase PuDHT and 2-keto-3-deoxyglucose aldolase PtKDGA was determined at different reaction times, and the concentration of pyruvate in the control reaction system was also determined.

[0066] Referring to Figure 3 , which shows a concentration-time curve for the three-enzyme catalytic reaction for synthesizing pyruvate using D-glucose as a substrate, the three-enzyme reaction system comprising the sugar alcohol oxidase ScALDO-Q288G, the dehydratase PuDHT, and the 2-keto-3-deoxyglucose aldolase PtKDGA can convert glucose into pyruvate with a maximum conversion rate of 75.8%.

[0067] Referring to FIG4 , FIG4 is a liquid phase peak diagram of the three-enzyme catalytic reaction for synthesizing pyruvate using D-glucose as a substrate. No pyruvate was produced in the control reaction system, further illustrating that the sugar alcohol oxidase of the present invention has a unique substrate promiscuity characteristic.

[0068] Referring to Figures 1 and 5 , in all reports, the two catalytic reactions of D-glucose oxidation to D-gluconic acid and D-glyceraldehyde oxidation to D-glyceric acid are respectively performed by glucose dehydrogenase GDH and glyceraldehyde dehydrogenase ALDH. In this embodiment, the reaction circuit for the three-enzyme catalytic reaction of synthesizing pyruvate using D-glucose as a substrate using the sugar alcohol oxidase of the present invention is as follows: one molecule of D-glucose is converted into one molecule of D-gluconic acid by the sugar alcohol oxidase, followed by conversion of one molecule of D-gluconic acid into 2-keto-3-deoxygluconic acid by the dehydratase PuDHT, followed by conversion of 2-keto-3-deoxygluconic acid into one molecule of D-glyceraldehyde and one molecule of pyruvate by the 2-keto-3-deoxygluconic acid aldolase PtKDGA, followed by conversion of one molecule of D-glyceraldehyde into one molecule of D-glyceric acid by the sugar alcohol oxidase, followed by conversion of one molecule of D-glyceric acid into another molecule of pyruvate by the dehydratase PuDHT. In theory, one molecule of glucose can be converted into two molecules of pyruvate. Compared with other studies, the glucose dehydrogenase GDH and glyceraldehyde dehydrogenase ALDH required for the synthesis of pyruvate from D-glucose are replaced by the sugar alcohol oxidase provided by the present invention, the number of enzymes required for the synthesis of pyruvate from D-glucose is reduced from four to three, and the dependence on the cofactor NAD+ is eliminated.

[0069] Therefore, the present invention provides sugar alcohol oxidases ScALDO-Q288G and ScALDO-Q288A, which have the novel property of simultaneously oxidizing D-glucose and D-glyceraldehyde. This can reduce the number of enzymes required for synthesizing pyruvate from D-glucose from 4 to 3 for the first time, and does not rely on any coenzymes, thus having significant originality and application value.

[0070] Example 7: Application of mutant sugar alcohol oxidase in oxidation of various aldoses

[0071] This example tested the oxidative activity of the wild-type and mutant sugar alcohol oxidases (ScALDO, ScALDO-Q288G, and ScALDO-Q288G) provided by the present invention toward various aldoses, including D-xylose, D-ribose, L-arabinose, D-erythrose, D-mannose, and D-galactose. During the oxidation of aldoses, the sugar alcohol oxidase releases H₂O₂. This H₂O₂ reacts with 4-AAP and DHBS under the catalysis of horseradish peroxidase to form a chromogenic substance. The oxidative activity of the wild-type and mutant sugar alcohol oxidases toward aldoses was determined by measuring the formation of the chromogenic substance at a wavelength of 515 nm. Relative enzyme activity was calculated with the highest enzyme activity as 100%.

[0072] See Figure 6, which shows the relative enzymatic activities of sugar alcohol oxidases (ScALDO-Q288G and ScALDO-Q288A) for the oxidation of different aldoses. Using D-xylose as the substrate, the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO: 2) provided by the present invention was increased by 59-fold, and the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO: 4) was increased by 4.4-fold compared to the wild-type sugar alcohol oxidase ScALDO.

[0073] When D-ribose is used as a substrate, the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO: 2) provided by the present invention is increased by 226.3 times, and the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO: 4) is increased by 38.3 times compared with the wild-type sugar alcohol oxidase ScALDO.

[0074] When L-arabinose was used as a substrate, the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO: 2) provided by the present invention was increased by 3.61 times, and the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO: 4) was increased by 1.98 times compared with the wild-type sugar alcohol oxidase ScALDO.

[0075] When D-erythrose is used as a substrate, the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO: 2) provided by the present invention is increased by 1.9 times, and the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO: 4) is increased by 1.9 times compared with the wild-type sugar alcohol oxidase ScALDO.

[0076] When D-mannose is used as a substrate, the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO: 2) provided by the present invention is increased by 3.2 times, and the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO: 4) is increased by 2.9 times compared with the wild-type sugar alcohol oxidase ScALDO.

[0077] When D-galactose is used as a substrate, the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO: 2) provided by the present invention is increased by 5.1 times, and the enzymatic activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO: 4) is increased by 3.7 times compared with the wild-type sugar alcohol oxidase ScALDO.

[0078] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A sugar alcohol oxidase, characterized in that Its amino acid sequence is shown in SEQ ID NO:2 or SEQ ID NO:

4.

2. A gene encoding the sugar alcohol oxidase according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO:3 or SEQ ID NO:

5.

4. A recombinant vector or recombinant strain comprising the gene as claimed in claim 2.

5. The recombinant vector or recombinant strain according to claim 4, characterized in that: The recombinant vector uses pET28a as an expression vector.

6. The recombinant vector or recombinant strain according to claim 4, characterized in that: The recombinant strain uses Escherichia coli BL21 (ED3) as a host.

7. A method for synthesizing pyruvic acid, characterized in that: The method uses D-glucose as a substrate and synthesizes pyruvate through a reaction catalyzed by sugar alcohol oxidase, dehydratase and 2-keto-3-deoxyglucose aldolase. The sugar alcohol oxidase is the sugar alcohol oxidase according to claim 1.

8. The method according to claim 7, characterized in that: The final concentration of the sugar alcohol oxidase in the reaction system is 0.3-0.7 mg / mL, the final concentration of the dehydratase is 0.3-0.5 mg / mL, and the final concentration of the 2-keto-3-deoxyglucose aldolase is 0.1-0.3 mg / mL.

9. The method according to claim 8, characterized in that The reaction system also includes MgCl2, Tween 20 and manganese dioxide, and the reaction temperature of the reaction system is 38-42°C.

10. The use of the sugar alcohol oxidase in catalyzing aldose oxidation according to claim 1, characterized in that: The aldose is one or more of D-xylose, D-ribose, L-arabinose, D-erythrose, D-mannose and D-galactose.

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