Galactose oxidase mutant and application thereof

Abstract

The invention discloses a galactose oxidase mutant which has the activity of catalytically oxidizing hydroxyl at the sixth position of gluconic acid and can be used for synthesizing L-guluronic acid. The method has the advantages of high reaction selectivity, less solid waste generated in the reaction, environmental protection, high safety, avoidance of dangerous chemicals used in the chemical method, safety and reliability, and can be used for enzyme catalysis reaction under the condition of high substrate.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an oxidase for synthesizing L-guluronic acid and a preparation method thereof. Background Art

[0002] L-guluronic acid can be used as a precursor for synthesizing a variety of compounds, such as glucaric acid, etc., but most sugars and uronic acid compounds in nature are D-configuration, and it is difficult to obtain L-guluronic acid through natural enzyme-catalyzed reactions. The use of chemical methods to synthesize L-configuration guluronic acid faces great difficulties, such as poor reaction selectivity, so L-guluronic acid has not been widely used. In the article "Short and Sweet: d-Glucose to l-Glucose and l-Glucuronic Acid", R. Fernando Martnez et al. described a method for preparing L-guluronic acid by chemical method. The reaction uses D-glucose as the starting material and obtains L-guluronic acid through 5 steps of reaction. The reaction process uses highly toxic sodium cyanide, which has certain safety issues. Summary of the invention

[0003] In order to solve the problems of poor selectivity and low safety of synthesizing L-guluronic acid in the prior art, the present application has developed a biosynthetic route for synthesizing L-guluronic acid by enzyme catalysis. However, since the galactose oxidase (GOase) in the prior art does not have the activity of catalyzing the oxidation of gluconic acid, the inventors of the present application have modified it to obtain catalytic activity. The technical solution adopted is as follows:

[0004] Using gluconic acid as a substrate, 6-galactose oxidase is modified to have oxidase activity for oxidizing the 6-hydroxyl group of glucose. The active site of galactose oxidase consists of a single copper ion coordinated with two histidine residues, a tyrosine residue and a cross-linked cysteine-tyrosine unit. Therefore, the reaction requires the participation of copper ions to form catalytic activity. During the reaction, some copper ions in the active center of the enzyme will exist in a semi-reduced state, causing the enzyme to lose activity. This application uses horseradish peroxidase (Horseradish Peroxidase, HRP) as an activator of galactose oxidase. - The galactose oxidase in its semi-reduced form is oxidized to make it catalytically active, and the hydrogen peroxide produced in the reaction is decomposed by catalase to avoid the inhibition of the enzyme by hydrogen peroxide.

[0005] The reaction mechanism is as follows

[0006]

[0007] In order to obtain an oxidase with high activity and high regioselectivity that can catalyze the above reaction, the inventor first screened 10 wild-type galactose oxidases, among which none of the above galactose oxidases had the activity of catalyzing the oxidation of gluconic acid. The inventor selected a wild-type galactose oxidase from them through theoretical research and bioinformatics analysis for modification. The wild-type galactose oxidase originated from Fusarium longipes. It was detected that it could not oxidize the 6th hydroxyl group of glucose. The inventor obtained a galactose oxidase mutant SEQ ID NO: 2 that was active in oxidizing the 6th hydroxyl group of gluconic acid through directed evolution modification. Compared with the wild-type enzyme, the mutations included were A53P; W333F; R373K; Q449T; V537A; N578D. The inventor detected that the mutated galactose oxidase had the catalytic activity of catalyzing gluconic acid to produce L-guluronic acid. The reaction yield could reach more than 90% when reacted for 24 hours under 50g / L gluconic acid feeding.

[0008] The galactose oxidase and catalase are products expressed by E. coli, and the host cell is E. coli, BL21 (DE3).

[0009] The amino acid sequence of the catalase is shown in SEQ ID NO:4.

[0010] The galactose oxidase activator is not limited to horseradish peroxidase (HRP). Small molecule chemical oxidants, such as manganese (III) fluoride, manganese (III) acetate, sodium persulfate, ammonium persulfate, etc., can all be used as galactose oxidase activators.

[0011] The HRP was purchased from Sigma with the product number P8125.

[0012] The substrate structure that the oxidase can catalyze is S1, where R 1-4 It can be a carbonyl or a hydroxyl group, R 5 It can be a hydroxyl group, an aldehyde group or a carboxyl group. The catalytic substrate is not limited to gluconic acid, but can also be galactose, glucose, L-sorbose, L-sorbone, etc. The structure of the corresponding oxidation product is such as P1.

[0013]

[0014] The galactose oxidase mutant can directly catalyze sorbose to produce 2-keto-L-gulonic acid. The reaction equation is as follows:

[0015]

[0016] pH range is 5-9, preferably pH 7;

[0017] The copper ion used can be in the form of copper chloride, copper sulfate, etc., preferably copper sulfate;

[0018] The copper ion concentration is 0.05mM-20mM, preferably 0.5mM;

[0019] The reaction temperature used in the reaction is 20-60°C, preferably 30°C.

[0020] Beneficial effects:

[0021] The present application adopts biological method to catalyze and synthesize L-guluronic acid, and transforms the galactose oxidase which is inactive to gluconic acid through enzyme directed evolution to make it obtain the activity of catalyzing and oxidizing the 6th hydroxyl group of gluconic acid, and the oxidation reaction can obtain a higher yield, and the yield can reach more than 90% after 24 hours of reaction under the condition of 50g / L gluconic acid as substrate. At the same time, enzyme catalysis can also be carried out under high substrate conditions, and the feeding amount of substrate gluconic acid can be increased to 200g / L. The reaction selectivity is higher than that of the chemical method, and the reaction produces less solid waste, is green and environmentally friendly, and has high safety, avoiding the hazardous chemicals used in the chemical method, and is safe and reliable. Specific implementation plan:

[0022] Example 1

[0023] The recombinant E. coli was inoculated into LB solid medium containing chloramphenicol resistance and cultured at 37°C for 20 hours. A single colony was picked and inoculated into 50 mL LB liquid medium containing chloramphenicol resistance and cultured with shaking for 20 hours. After the culture was completed, the bacterial solution was transferred to 250 mL TB liquid medium. After culturing for 2.5 hours, the bacterial solution was diluted and the OD value was 0.7. 0.1 mM was added to induce protein expression, cultured with shaking at 25°C for 18 hours, and the bacterial cells were collected by centrifugation at 8000 rpm and stored at -20°C for later use.

[0024] Example 2

[0025] The wet E. coli cells prepared in Example 1 were dissolved in a ratio of pure water: cells = 5:1, and homogenized and crushed twice using a homogenizer at 800 bar. Centrifuged at 8000 rpm for 10 min at 4°C and collected the supernatant of the homogenate to obtain an enzyme solution; the enzyme solution was freeze-dried at -20°C to obtain an enzyme powder of oxidase or catalase. The enzyme solution or enzyme powder can be stored at -20°C for use.

[0026] Example 3

[0027] The reaction samples were detected by the following LC-MS method.

[0028]

[0029]

[0030] Example 4

[0031] In a reaction bottle with a volume of 30 mL, add 0.25 g of sodium gluconate, 0.05 g of galactose oxidase mutant enzyme powder of SEQ ID No: 2, 0.5 mL of 1 g / L catalase enzyme solution of SEQ ID NO: 4, 0.5 mL of copper sulfate mother solution (5 mM), 50 mM Tris-HCl (pH = 7.5), and 0.5 mL of 1 g / L horseradish peroxidase (HRP) mother solution. Start stirring. The final reaction volume is 5 mL. React at room temperature for 24 hours. Sample LC-MS detection. The reaction yield is 93.7%.

[0032] Example 5

[0033] In 4 reaction bottles with a volume of 30 mL, 0.125 g, 0.25 g, 0.5 g, and 1 g of sodium gluconate, 0.05 g of galactose oxidase mutant enzyme powder of SEQ ID No: 2, 0.5 mL of 1 g / L catalase enzyme solution of SEQ ID NO: 4, 0.5 mL of copper sulfate mother solution (5 mM), 50 mM Tris-HCl (pH = 7), and 0.5 mL of 1 g / L horseradish peroxidase (HRP) mother solution were added respectively, stirring was started, the final reaction volume was 5 mL, the reaction was carried out at room temperature for 24 h, and samples were taken for LC-MS detection. The reaction results are shown in the following table.

[0034] Final concentration of sodium gluconate Reaction yield of L-guluronic acid 25g / L 96.1％ 50g / L 93.7％ 100g / L 35.1％ 200g / L 18.6％

[0035] The reaction system with a final sodium gluconate concentration of 200 g / L was tested at different temperatures and pH values. Under the reaction condition of pH 7, the reaction yields were 18.6%, 21.4%, 17.3%, 11.2%, and 5.3% at temperatures of 20°C, 30°C, 40°C, 50°C, and 60°C, respectively. At room temperature of 25°C, the reaction yields were 7.1%, 14.5%, 20.6%, 20.2%, and 17.3%, respectively, at pH values ​​of 5, 6, 7, 8, and 9, respectively.

[0036] Example 6

[0037] In 5 reaction bottles with a volume of 30 mL, 0.5 mL of 1 g / L horseradish peroxidase (HRP) mother solution, 6 mg of manganese (III) fluoride, 14 mg of manganese (III) acetate dihydrate, 12 mg of sodium persulfate, 11 mg of ammonium persulfate, 0.25 g of sodium gluconate, 0.05 g of galactose oxidase mutant enzyme powder of SEQ ID No: 2, 0.5 mL of 1 g / L catalase enzyme solution of SEQ ID NO: 4, 0.5 mL of copper sulfate mother solution (5 mM), and 50 mM Tris-HCl (pH = 8) were added respectively, stirring was started, the final reaction volume was 5 mL, the reaction was carried out at room temperature for 24 h, and samples were taken for LC-MS detection. The reaction results are shown in the following table.

[0038] Reaction yield of L-guluronic acid HRP 89.2％ Manganese(III) fluoride 68.3％ Manganese(III) acetate dihydrate 71.7％ Sodium Persulfate 56.9％ Ammonium Persulfate 55.1％

[0039] Example 7

[0040] In a reaction bottle with a volume of 100 mL, add 2.5 g of sodium gluconate, 0.5 g of galactose oxidase mutant enzyme powder of SEQ ID No: 2, 5 mL of 1 g / L catalase enzyme solution of SEQ ID NO: 4, 5 mL of copper sulfate mother solution (5 mM), 50 mM Tris-HCl (pH = 8), and 5 mL of 1 g / L horseradish peroxidase (HRP) mother solution. Start stirring. The final reaction volume is 50 mL. React at room temperature for 24 hours. Sample LC-MS detection, the reaction yield of L-guluronic acid is 90.6%.

[0041] Example 8

[0042] In a reaction bottle with a volume of 30 mL, 0.25 g of sorbitol, 0.05 g of galactose oxidase mutant enzyme powder of SEQ ID No: 2, 0.5 mL of 1 g / L catalase enzyme solution of SEQ ID NO: 4, 0.5 mL of 5 mM copper sulfate mother solution, 50 mM Tris-HCl (pH = 7.5), and 0.5 mL of 1 g / L horseradish peroxidase (HRP) mother solution were added, stirring was started, the final reaction volume was 5 mL, the reaction was carried out at room temperature for 24 hours, and sampling was performed for LC-MS detection. The yield of L-sorbitone was 13.5%, and the reaction yield of 2-keto-L-gulonic acid was 1.3%.

[0043] Example 9

[0044] In a reaction bottle with a volume of 30 mL, add 0.25 g of glucose, 0.05 g of galactose oxidase mutant enzyme powder of SEQ ID No: 2, 0.5 mL of 1 g / L catalase mother solution of SEQ ID NO: 4, 0.5 mL of copper sulfate mother solution (5 mM), 50 mM Tris-HCl (pH = 7.5), and 0.5 mL of 1 g / L horseradish peroxidase (HRP) mother solution. Start stirring. The final reaction volume is 5 mL. React at room temperature for 24 hours. Sample LC-MS detection. The reaction yield is 90.4%.

[0045] Example 10

[0046] In a reaction bottle with a volume of 30 mL, add 0.25 g galactose, 0.05 g galactose oxidase mutant enzyme powder of SEQ ID No: 2, 0.5 mL 1 g / L catalase enzyme solution of SEQ ID NO: 4, 0.5 mL copper sulfate mother solution (5 mM), 50 mM Tris-HCl (pH = 7.5), 0.5 mL 1 g / L horseradish peroxidase (HRP) mother solution, start stirring, the final reaction volume is 5 mL, react at room temperature for 24 hours, take samples for LC-MS detection, the reaction yield is 96.8%.

[0047] It should be understood that after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

Claims

1. A galactose oxidase, which can oxidize the 6-hydroxyl group of glucose, and the sequence is shown in SEQ ID NO:

2.

2. The galactose oxidase according to claim 1, wherein the reaction for oxidizing the 6-hydroxyl group of glucose is as follows: Copper ions are combined with galactose oxidase, and a galactose oxidase activator is used to make the galactose oxidase catalytically active by 1e-oxidation of the semi-reduced form of galactose oxidase, and the hydrogen peroxide produced in the reaction is decomposed by catalase.

3. The galactose oxidase according to claim 2, wherein the galactose oxidase activator used in the reaction is horseradish peroxidase, manganese (III) fluoride, manganese (III) acetate, sodium persulfate, or ammonium persulfate.

4. The galactose oxidase according to claim 2-3, wherein the catalase sequence used in the reaction is shown in SEQ ID NO:

4.

5. The galactose oxidase according to claim 2-4, wherein the copper ion used in the reaction is in the form of copper chloride or copper sulfate.

6. A method for preparing a compound having a structure of P1, using an oxidase having a sequence as shown in SEQ ID NO: 2 to catalyze a substrate having a structure of S1, wherein R 1-4 It can be a carbonyl or a hydroxyl group, R 5 It can be a hydroxyl group, an aldehyde group or a carboxyl group.

7. The method of claim 6, wherein the substrate is selected from the group consisting of gluconic acid, galactose, glucose, L-sorbose, and L-sorbone.

8. The method according to claims 6-7, wherein the pH range is 5-9, preferably pH 7; and the reaction temperature is 20-60°C, preferably 30°C.

9. The method according to claims 6-8, wherein during the reaction, copper ions are combined with galactose oxidase, and an activator of galactose oxidase is used to make the galactose oxidase catalytically active by 1e-oxidizing the semi-reduced form of galactose oxidase, and the hydrogen peroxide produced in the reaction is decomposed by catalase.

10. The method of claim 9, wherein the copper ion used is in the form of copper chloride or copper sulfate, and the activator of galactose oxidase is selected from horseradish peroxidase, manganese (III) fluoride, manganese (III) acetate, sodium persulfate, and ammonium persulfate.

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