Sugar alcohol oxidase and use thereof

Abstract

The application belongs to the technical field of synthetic biology, and discloses a sugar alcohol oxidase and application thereof. The application docks a sugar alcohol oxidase derived from Streptomyces coelicolor A3 on D-glucose as a substrate, and selects amino acid residues around an active center for saturated mutation, and through plate color development screening, a sugar alcohol oxidase with oxidation D-glucose activity is screened, and the amino acid sequence of the sugar alcohol oxidase is shown as SEQ ID NO: 2 or SEQ ID NO: 4. The sugar alcohol oxidase provided by the application has the activity of converting D-glucose into D-gluconic acid and the activity of converting D-glyceraldehyde into D-glyceric acid, and by using the sugar alcohol oxidase, the conversion of D-glucose into pyruvic acid is completed by only using three enzymes for the first time, and does not need to depend on any coenzyme, and has great originality and application value.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and in particular to a sugar alcohol oxidase and its applications. Background Technology

[0002] Pyruvate, also known as 2-oxoacetic acid, is an important α-oxocarboxylic acid, colorless to pale yellow in color. Pyruvate plays a crucial pivotal role in the metabolism of the three major nutrients by facilitating the interconversion of carbohydrates, fats, and amino acids within the body through acetyl-CoA and the tricarboxylic acid cycle. As an important organic acid, pyruvate is a precursor for the synthesis of many compounds and has wide applications in chemical, agricultural, pharmaceutical, and food industries.

[0003] Currently, the main methods for synthesizing pyruvate include microbial fermentation, chemical methods, and multi-enzyme catalysis. Among these, enzymatic methods have attracted considerable attention from researchers due to their mild reaction conditions and low byproduct production. The natural ED (Entner-Doudoroff) pathway is an alternative pathway found in some microorganisms lacking the complete EMP pathway. Its characteristic is that one molecule of glucose can be converted into two molecules of pyruvate in just seven steps, 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-glyceroate to pyruvate and D-gluconic acid to 2-keto-3-deoxygluconic acid, eliminating the phosphorylation and dephosphorylation steps in the natural ED pathway, thereby reducing the amount of enzyme required. (See also...) Figure 1 The improved ED pathway employs a multi-enzyme catalytic method to synthesize pyruvate using D-glucose as a substrate. The specific reaction route is as follows: D-glucose is converted to D-gluconic acid by glucose dehydrogenase GDH, consuming one molecule of NAD. + Subsequently, one molecule of D-gluconic acid is converted to 2-keto-3-deoxygluconic acid by the dehydratase DHT. Then, 2-keto-3-deoxygluconic acid is converted to one molecule of D-glyceraldehyde and one molecule of pyruvate by the 2-keto-3-deoxygluconic acid aldolase KDGA. Finally, one molecule of D-glyceraldehyde is converted to D-glyceric acid by the glyceraldehyde dehydrogenase ALDH, consuming one molecule of NAD+. + One molecule of D-glyceric acid is converted into another molecule of pyruvate by the dehydrating enzyme DHT. Theoretically, one molecule of glucose can be converted into two molecules of pyruvate and consume two molecules of NAD. +However, the synthesis of pyruvate using this multi-enzyme catalytic system still requires a relatively large number of enzymes, and the high cost of enzyme preparation limits the large-scale application of this system. In addition, the pathway requires expensive coenzymes. How to further design new enzyme variants to achieve new catalytic properties that combine the functions of two enzymes, thereby reducing the number of enzymes used and eliminating the need for coenzyme addition to reduce pathway costs, has become a key bottleneck problem that urgently needs to be solved in this field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a sugar alcohol oxidase that simultaneously oxidizes D-glucose and D-glyceraldehyde, and provides an engineered strain expressing this sugar alcohol oxidase.

[0005] The first object of the present invention is to provide a sugar alcohol oxidase having an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:4.

[0006] Compared with the prior art, the sugar alcohol oxidase provided by the present invention has the activity of converting D-glucose to D-gluconic acid and D-glyceraldehyde to D-glyceric acid. Using this sugar alcohol oxidase, the conversion of D-glucose to pyruvate can be completed for the first time using only three enzymes without relying on any coenzymes, which has significant originality and application value.

[0007] A second object of the present invention is to provide a gene encoding the sugar alcohol oxidase.

[0008] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO:3 or SEQ ID NO:5.

[0009] A third objective of this invention is to provide a recombinant vector or recombinant strain containing the sugar alcohol oxidase encoding gene.

[0010] Furthermore, the recombinant vector uses pET28a as the expression vector.

[0011] Furthermore, the recombinant strain uses Escherichia coli BL21(ED3) as its host.

[0012] The fourth objective of this 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 sugar alcohol oxidase, dehydratase and 2-keto-3-deoxyglucuronide aldolase.

[0013] Furthermore, the final concentration of glucose in the reaction system is 1~10mM, the final concentration of sugar alcohol oxidase is 0.3~0.7mg / mL, the final concentration of dehydrating enzyme is 0.3~0.5mg / mL, and the final concentration of 2-keto-3-deoxyglucuronide aldolase is 0.1~0.3mg / mL.

[0014] Furthermore, the reaction system also includes MgCl2, Tween 20 and manganese dioxide, and the reaction temperature of the reaction system is 38~42℃.

[0015] A fifth object of the present invention is to provide the application of the sugar alcohol oxidase in catalyzing the oxidation of aldoses, wherein the aldose is one or more selected from D-xylose, D-ribose, L-arabinose, D-erythrose, D-mannose and D-galactose. Attached Figure Description

[0016] Figure 1 This is the reaction route for synthesizing pyruvate using D-glucose as a substrate in the existing multi-enzyme catalytic method.

[0017] Figure 2 The relative enzyme activities of sugar alcohol oxidase in the oxidation of D-glucose and D-glyceraldehyde are given in this invention.

[0018] Figure 3 This is a concentration-time curve of the three-enzyme catalytic reaction of the sugar alcohol oxidase of the present invention synthesizing pyruvate using D-glucose as a substrate.

[0019] Figure 4 This is a liquid phase peak diagram of the three-enzyme catalytic reaction of sugar alcohol oxidase in this invention, which synthesizes pyruvate using D-glucose as a substrate.

[0020] Figure 5 This is the reaction circuit for the synthesis of pyruvate using D-glucose as a substrate in the three-enzyme catalytic method of this invention.

[0021] Figure 6 The relative enzyme activity of sugar alcohol oxidase for oxidizing different aldoses is presented in this invention. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention. In this context, "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the sugar alcohol oxidase, such as "AxxB" indicating that the amino acid at position xx is replaced by amino acid B in the wild-type enzyme, and the position number corresponds to the amino acid sequence number of the wild-type sugar alcohol oxidase in SEQ ID No:1.

[0023] All whole-genome synthesis in the embodiments of this invention was completed by Suzhou Genewiz Biotechnology Co., Ltd.

[0024] Unless otherwise specified, the materials and reagents used in the embodiments of this invention are commercially available.

[0025] The reagents used in the embodiments of this invention are as follows: LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, prepared with deionized water, autoclaved, and kept at room temperature for later use.

[0026] TB medium: 12 g / L peptone, 24 g / L yeast extract, 4 mL / L glycerol, containing potassium phosphate buffer.

[0027] Isopropyl β-D-Thiogalactoside solution (IPTG) (1M): Weigh 2.383g of IPTG, dissolve it in deionized water, and after complete dissolution, bring the volume to 10mL. Aliquot the solution into 2mL seed tubes and store at -20℃ for later use.

[0028] Kanamycin solution (50mg / mL): Weigh 0.5g of kanamycin sulfate, dissolve it in deionized water, and after complete dissolution, bring the volume to 10mL. Aliquot the solution into 2mL preservation tubes and store at -20℃ for later use.

[0029] 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).

[0030] In a preferred embodiment of the present invention, the method for preparing the sugar alcohol oxidase of the present invention includes the following steps: (1) The gene of the corresponding mutation site of sugar alcohol oxidase is constructed into the pET28a expression vector to obtain a recombinant plasmid carrying the target enzyme gene. (2) The recombinant plasmid is transferred into a host bacterial cell, preferably Escherichia coli BL21(DE3), to obtain the corresponding engineered strain. (3) The engineered strain is inoculated into LB medium and cultured overnight at 37°C. The initial OD is controlled at 0.1 and transferred to TB medium. The culture is carried out until the OD value reaches 0.6 or higher. Then, 0.1mM-1mM IPTG is added and cultured at 12-30°C for 12-20 hours. (4) The bacterial cells are collected by centrifugation.

[0031] Example 1: Construction of genetically engineered bacteria for sugar alcohol oxidase (1) Constructing gene expression vectors: Sky blue Streptomyces A3 was obtained through NCBI. Streptomyces coelicolorThe wild-type alditol oxidase (ALDO) gene (Gene ID: 1101588) from gene A3 contains mutations at four sites: V125M, A244T, V133M, and G399R. Its amino acid sequence is shown in SEQ ID No: 1. After codon optimization of the wild-type alditol oxidase sequence, the optimized sequence was synthesized and subcloned into the *E. coli* gene expression vector pET28a to obtain the recombinant plasmid pET28a-ScALDO.

[0032] (2) Construct BL21(DE3) genetically engineered bacteria for expressing the enzymes related to step (1). (21) Transformation of E. coli competent cells with recombinant plasmid The recombinant plasmid pET28a-ScALDO constructed in step (1) was transformed into the Escherichia coli expression host BL21(DE3) using the calcium chloride method.

[0033] In a clean bench, BL21(DE3) competent cells were placed on ice for 2 minutes to thaw. The recombinant plasmid pET28a-ScALDO was added to the BL21(DE3) competent cells and gently piped to mix. The cells were then placed on ice for 5 minutes and then spread on LB resistant plates containing 50 mg / L kanamycin. The plates were incubated overnight at 37°C.

[0034] (22) Colony PCR identification Positive single colonies were picked from the plates in step (21) and preliminarily identified by colony PCR using 2×Utaq PCR MasterMix (Beijing Zhuangmeng International Biotechnology 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 colony PCR amplification, the colony PCR products were spotted into the wells of a 1% agarose gel electrophoresis gel (another well was spotted with a DS 5000 DNA Marker as a control). The gel was separated in the electrophoresis tank (110V) for 30 min. After separation, the agarose gel was immersed in Gelred staining solution for 15 min. The approximate size of the colony PCR product band was determined by the DS5000 DNA Marker in the gel electrophoresis apparatus and compared with the expected band size to determine whether the expression strain construction was completed. In this embodiment, positive transformants were identified by colony PCR to obtain the expression strain BL21(DE3) / pET28a-ScALDO.

[0035] Table 1 Colony PCR Reaction System

[0036] Table 2 Colony PCR Amplification Reaction Procedure

[0037] Example 2: Construction and screening of mutant sugar alcohol oxidase (1) Construction of a sugar alcohol oxidase mutant library Using D-glucose as a substrate, wild-type sugar alcohol oxidase (amino acid sequence SEQ ID NO:1) was docked, and amino acid residues around the active site were selected for saturation mutation to construct a saturation mutant library; (2) Screening for mutant sugar alcohol oxidases that simultaneously oxidize D-glucose and D-glyceraldehyde The saturated mutant library was transformed into *E. coli* BL21(DE3) competent cells, then plated on LB agar plates containing 50 mg / L kanamycin and incubated overnight at 37°C. A laboratory-developed plate staining strategy was used to screen single colonies for activity. The basic principle is that mutant sugar alcohol oxidases with catalytic activity release H2O2 during the oxidation of glucose, and the H2O2 staining system allows for the staining of single colonies, thus rapidly identifying active mutants.

[0038] Two mutant strains with D-glucose oxidizing activity were screened. These two mutant strains were cultured and plasmids were extracted. DNA sequencing was performed. The sequencing results showed that the mutation sites that enable the mutant sugar alcohol oxidase to have D-glucose oxidizing activity were the Q288G monomer mutation (corresponding to the mutation of glutamine to glycine at amino acid position 288 of the SEQ ID NO:1 amino acid sequence) and the Q288A monomer mutation (corresponding to the mutation of glutamine to alanine at amino acid position 288 of the SEQ ID NO:1 amino acid sequence).

[0039] The mutant sugar alcohol oxidase containing the Q288G monomer mutation is named ScALDO-Q288G, with its amino acid sequence being SEQ ID NO:2 and its encoding nucleotide sequence being SEQ ID NO:3. The corresponding expression strain is BL21(DE3) / pET28a-ScALDO-Q288G. The mutant sugar alcohol oxidase containing the Q288A monomer mutation is named ScALDO-Q288A, with its amino acid sequence being SEQ ID NO:4 and its encoding nucleotide sequence being SEQ ID NO:5. The corresponding expression strain is BL21(DE3) / pET28a-ScALDO-Q288A.

[0040] Example 3: Construction of genetically engineered bacteria containing dehydrating enzymes and 2-keto-3-deoxyglucuronide aldolase Ureaplasma urealyticum (a byproduct of alkalogenesis) was obtained from the literature. Paralcaligenes ureilyticusThe dehydratase (DHT) gene (NCBI Reference Sequence: WP_132585145.1) from which the heat-loving acidophilic archaea were obtained. Picrophilus torridus The 2-keto-3-deoxygluconate aldolase (KDGA) gene (NCBI Reference Sequence: NC_005877.1) is derived from this gene. ) After codon optimization of the dehydrase and 2-keto-3-deoxyglucuronide aldolase sequences, the optimized sequences were synthesized from the whole genome and subcloned into the E. coli gene expression vector pET28a, resulting in recombinant plasmids pET28a-PuDHT and pET28a-PtKDGA, respectively.

[0041] The recombinant plasmids pET28a-PuDHT and pET28a-PtKDGA were transformed into the expression host BL21(DE3) of Escherichia coli using the calcium chloride method. Positive transformants were identified, and the expression strains BL21(DE3) / pET28a-PuDHT and BL21(DE3) / pET28a-PtKDGA were obtained.

[0042] Example 4: Enzyme Induction, Expression, and Purification The wild-type and mutant genetically engineered bacteria constructed in Examples 1-3, 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 culture.

[0043] Seed culture was inoculated into 100 mL of TB medium containing 50 mg / L kanamycin, and the initial OD of the fermentation medium was controlled. 600 The concentration was 0.1, and the fermentation medium was incubated at 37°C and 220 rpm. When the OD reached... 600 When the concentration is above 0.6, add 50 μL of 1M IPTG for induction, so that the final concentration of IPTG in the fermentation medium is 0.5 mM. Place the fermentation medium at 16℃ and 180 rpm for 16 h.

[0044] After induction, the bacterial culture was transferred to a 100 mL centrifuge tube and centrifuged at 6000 rpm for 5 min at room temperature. The supernatant was discarded, and the culture was resuspended in sterile water and centrifuged at 6000 rpm for 5 min at room temperature. The supernatant was then discarded. A certain volume of HEPES buffer was added to the centrifuged bacterial pellet, and the mixture was then placed in a Toshiba ultrasonic homogenizer and sonicated at 35% power for 3 seconds, with a 3-second interval, for a total of 40 min. Finally, the mixture was centrifuged at 10000 rpm at 4°C for 30 min to obtain the crude enzyme solution.

[0045] The target protein was purified using an AKTA purification instrument and a nickel ion affinity chromatography column with an imidazole concentration of 0.3M-0.5M. The target protein was then purified using a desalting column to remove the imidazole from the target protein.

[0046] Example 5: Enzyme activity assay of wild-type and mutant sugar alcohol oxidases The relative activities of wild-type and mutant sugar alcohol oxidases to D-glucose were tested: The system contained 10 mM D-glucose, 1 mM 4-AAP, 1 mM DHBS, 4 μL of horseradish peroxidase at a concentration of 0.3 g / L, and 0.1 mg / mL of wild-type or mutant sugar alcohol oxidase. The enzyme activities of ScALDO, ScALDO-Q288G, and ScALDO-Q288A to D-glucose were calculated by measuring the change in absorbance of the system over 5 minutes. The relative enzyme activity was calculated with the highest enzyme activity as 100%.

[0047] The relative activities of wild-type and mutant sugar alcohol oxidases to D-glyceraldehyde were tested: The system contained 10 mM D-glyceraldehyde, 1 mM 4-AAP, 1 mM DHBS, 4 μL of horseradish peroxidase at a concentration of 0.3 g / L, and 0.1 mg / mL of wild-type or mutant sugar alcohol oxidase. The enzyme activities of ScALDO, ScALDO-Q288G, and ScALDO-Q288A to D-glyceraldehyde were calculated by measuring the change in absorbance of the system over 5 minutes. The relative enzyme activities were calculated with the highest enzyme activity as 100%.

[0048] See Figure 2 , Figure 2This invention relates to the relative enzymatic activities of the sugar alcohol oxidases 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. In contrast, the mutant sugar alcohol oxidases ScALDO-Q288G and ScALDO-Q288A both demonstrate significant activity in oxidizing D-glucose to D-gluconic acid and retain some activity in oxidizing D-glyceraldehyde to D-glycerol acid. Both ScALDO-Q288G and ScALDO-Q288A achieve high substrate heterogeneity. Clearly, the site-directed mutations in Q288G and Q288A endow the wild-type sugar alcohol oxidase ScALDO with the novel characteristic of simultaneously oxidizing D-glucose and D-glyceraldehyde. In theory, the sugar alcohol oxidases (ScALDO-Q288G and ScALDO-Q288A) of the present invention can replace glucose dehydrogenase GDH and glyceraldehyde dehydrogenase ALDH in the glucose-to-pyruvate pathway, reducing the number of enzymes required for D-glucose-to-pyruvate synthesis from 4 to 3.

[0049] Example 6: Three-enzyme catalytic reaction for the synthesis of pyruvate from glucose as a substrate. The reaction system was supplemented with 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-deoxyglucuronide 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℃, the pH was 8.0, the buffer was 100 mM HEPES-NaOH buffer, and the reaction volume was 100 μL. The sugar alcohol oxidase ScALDO-Q288G was replaced with an equal amount of wild-type sugar alcohol oxidase ScALDO, while the concentrations of the other substances remained unchanged. The reaction was carried out under the same conditions for 24 hours as a control group.

[0050] The concentration of pyruvate in the three-enzyme reaction system of sugar alcohol oxidase ScALDO-Q288G, dehydratase PuDHT and 2-keto-3-deoxyglucuronide aldolase PtKDGA was determined at different reaction times, and the concentration of pyruvate in the control reaction system was also determined.

[0051] See Figure 3 , Figure 3 This is a concentration-time curve of the three-enzyme catalytic reaction for the synthesis of pyruvate using D-glucose as a substrate. The three-enzyme reaction system of sugar alcohol oxidase ScALDO-Q288G, dehydratase PuDHT, and 2-keto-3-deoxyglucuronide aldolase PtKDGA can convert glucose into pyruvate, and the maximum conversion rate can reach 75.8%.

[0052] See Figure 4 , Figure 4 The figure shows the liquid phase peaks of the three-enzyme catalytic reaction for the synthesis of pyruvate using D-glucose as a substrate. No pyruvate was produced in the control group reaction system, which further illustrates that the sugar alcohol oxidase of the present invention has unique substrate mixing characteristics.

[0053] See Figure 1 and Figure 5 In all reports, the oxidation of D-glucose to D-gluconic acid and the oxidation of D-glyceraldehyde to D-glycerol are catalyzed by glucose dehydrogenase (GDH) and glyceraldehyde dehydrogenase (ALDH), respectively. However, in this embodiment, the reaction pathway for the three-enzyme catalytic reaction of D-glucose synthesis using the sugar alcohol oxidase described in this invention is as follows: one molecule of D-glucose is converted to one molecule of D-gluconic acid by sugar alcohol oxidase; subsequently, one molecule of D-gluconic acid is converted to 2-keto-3-deoxygluconic acid by the dehydratase PuDHT; then, 2-keto-3-deoxygluconic acid is converted to one molecule of D-glyceraldehyde and one molecule of pyruvate by the 2-keto-3-deoxyglucuronide aldolase PtKDGA; one molecule of D-glyceraldehyde is converted to one molecule of D-glycerol by sugar alcohol oxidase; and one molecule of D-glycerol is converted to another molecule of pyruvate by the dehydratase PuDHT. Theoretically, one molecule of glucose can be converted to 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 in this invention, reducing the number of enzymes required for the synthesis of pyruvate from D-glucose from four to three, and eliminating the need for the cofactor NAD. + Dependence.

[0054] Therefore, the present invention provides a sugar alcohol oxidase ScALDO-Q288G and ScALDO-Q288A, which have the novel property of simultaneously oxidizing D-glucose and D-glyceraldehyde. This reduces the number of enzymes required for the synthesis of pyruvate from D-glucose from 4 to 3 for the first time, and does not require any coenzyme. This has significant originality and application value.

[0055] Example 7: Application of mutant sugar alcohol oxidase in the oxidation of various aldoses This embodiment tests the oxidation activities of wild-type and mutant sugar alcohol oxidases (ScALDO, ScALDO-Q288G, and ScALDO-Q288G) provided by this invention on various aldoses, including D-xylose, D-ribose, L-arabinose, D-erythrose, D-mannose, and D-galactose. During the oxidation of aldoses, sugar alcohol oxidases release H2O2. The released H2O2 reacts with 4-AAP and DHBS to generate a chromogenic substance under the catalysis of horseradish peroxidase. The oxidation activity of wild-type or mutant sugar alcohol oxidases on aldoses is tested by detecting the generation of the chromogenic substance at a wavelength of 515 nm. The relative enzyme activity is calculated with the highest enzyme activity as 100%.

[0056] See Figure 6 , Figure 6 The relative enzyme activities of sugar alcohol oxidases (ScALDO-Q288G and ScALDO-Q288G) for oxidizing different aldoses were determined. When D-xylose was used as a substrate, the enzyme activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO:2) provided by this invention was increased by 59 times compared to the wild-type sugar alcohol oxidase ScALDO, and the enzyme activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO:4) was increased by 4.4 times.

[0057] When D-ribose is used as a substrate, the enzyme activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO:2) provided by this invention is increased by 226.3 times compared with that of wild-type sugar alcohol oxidase ScALDO, and the enzyme activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO:4) is increased by 38.3 times.

[0058] When L-arabinose is used as a substrate, the enzyme activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO:2) provided by this invention is increased by 3.61 times compared with that of wild-type sugar alcohol oxidase ScALDO, and the enzyme activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO:4) is increased by 1.98 times.

[0059] When D-erythrose is used as a substrate, the enzyme activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO:2) provided by this invention is increased by 1.9 times compared with the wild-type sugar alcohol oxidase ScALDO, and the enzyme activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO:4) is increased by 1.9 times.

[0060] When D-mannose is used as a substrate, the enzyme activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO:2) provided by this invention is increased by 3.2 times compared with that of wild-type sugar alcohol oxidase ScALDO, and the enzyme activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO:4) is increased by 2.9 times.

[0061] When D-galactose is used as a substrate, the enzyme activity of the sugar alcohol oxidase ScALDO-Q288G (SEQ ID NO:2) provided by this invention is increased by 5.1 times compared with that of wild-type sugar alcohol oxidase ScALDO, and the enzyme activity of the sugar alcohol oxidase ScALDO-Q288A (SEQ ID NO:4) is increased by 3.7 times.

[0062] The above-described embodiments are merely preferred embodiments provided to fully illustrate 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 all within the scope of protection of the present invention. The scope of protection of the present invention is defined by 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. The gene encoding the sugar alcohol oxidase of claim 1.

3. The gene as described in 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 comprising the gene as described in claim 2.

5. The recombinant vector as described in claim 4, characterized in that, The recombinant vector used is pET28a as the expression vector.

6. A recombinant strain comprising the gene as described in claim 2.

7. The recombinant strain according to claim 6, characterized in that, The recombinant strain used Escherichia coli BL21(ED3) as its host.

8. A method for synthesizing pyruvic acid, characterized in that, The method uses D-glucose as a substrate to synthesize pyruvate via a reaction catalyzed by sugar alcohol oxidase, dehydratase and 2-keto-3-deoxyglucuronide aldolase, wherein the sugar alcohol oxidase is the sugar alcohol oxidase described in claim 1.

9. The method as described in claim 8, 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 dehydrating enzyme is 0.3~0.5 mg / mL, and the final concentration of the 2-keto-3-deoxyglucuronide aldolase is 0.1~0.3 mg / mL.

10. The method as described in claim 9, 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℃.

11. The application of the sugar alcohol oxidase as described in claim 1 in the catalytic oxidation of aldoses, 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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