Uronic acid dehydrogenase and method for synthesizing glucaric acid

Abstract

According to the present invention, the glucaric acid is catalyzed and synthesized by using the biological method, such that the feeding amount of the reaction substrate is high, the solid waste produced by the reaction is less, the environmental protection is provided, the heavy metal ions introduced during the chemical method metal catalyst catalysis preparation of the glucaric acid are avoided, the reaction selectivity is high, and the high yield glucaric acid can be obtained.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an aldehyde dehydrogenase and a method for synthesizing glucaric acid. Background Art

[0002] Glucaric acid is a naturally occurring organic acid that is widely used in food, health products, pharmaceuticals and chemicals. It is known as "one of the most valuable biorefining products" because of its high application value in the fields of health care and chemical industry. FDCA is a high value-added chemical that can be used as a substitute for terephthalic acid, a petrochemical derivative, and has very broad prospects in the fields of pharmaceuticals, chemicals, plastics, etc. Glucaric acid can be used as a precursor for the synthesis of FDCA and has very broad market prospects.

[0003] The method currently used for synthesizing glucaric acid is mainly microbial fermentation and chemical oxidation. Chemical oxidation usually requires very harsh reaction conditions, such as strong oxidants such as nitric acid, high temperature and high pressure reaction conditions. At the same time, the reaction selectivity of chemical oxidation is low, more by-products are produced, and the separation and purification of compounds is very challenging. In order to obtain higher quality products, the enzyme catalytic reaction to prepare glucaric acid technology has gradually emerged, such as enzymatic synthesis of glucaric acid with sugar or uronic acid as substrate, in patent CN113337555A, Beijing University of Chemical Technology uses flavoprotein oxidase HMFO to catalyze glucose and glucosyl derivatives to prepare glucaric acid, which is 1.96g / L gluconic acid, 10% (V / V) HMFO enzyme solution, 1% (V / V) 1mg / ml catalase solution. After reacting for 24 hours, the reaction yield is 84%, and the time-space yield of the reaction is low.

[0004] There are many defects in the current biocatalytic preparation of glucaric acid. For example, the fermentation method for preparing glucaric acid is acidic, which causes the pH of the fermentation system to decrease during the fermentation process, limiting the yield of glucaric acid. The existing enzymatic preparation of glucaric acid has a low substrate input, which limits the space-time yield of the enzyme catalysis. Summary of the invention

[0005] In order to overcome the problems existing in the prior art, the technical solution adopted in this application is as follows:

[0006] L-guluronic acid, D-glucuronic acid or galacturonic acid are used as substrates to generate glucaric acid through urate dehydrogenase (UDH). The reaction can be carried out through the coenzyme cycle of NADH oxidase (NOx), or the catalytic activity of ketones by UDH itself or ketoreductase (KRED) can be used to carry out the coenzyme NAD + cycle.

[0007] The reaction mechanism is as follows

[0008] Using L-guluronic acid as substrate, the enzyme in the NOx coenzyme cycle system catalyzes the synthesis of glucaric acid:

[0009]

[0010] Using L-guluronic acid as substrate, the enzymes of the UDH or ketoreductase (KRED) coenzyme cycle system catalyze the synthesis of glucaric acid:

[0011]

[0012] Using D-glucuronic acid as substrate, the enzyme in the NOx coenzyme cycle system catalyzes the synthesis of glucaric acid:

[0013]

[0014] Using D-glucuronic acid as substrate, the enzymes of the UDH or ketoreductase (KRED) coenzyme cycle system catalyze the synthesis of glucaric acid:

[0015]

[0016] In order to obtain a highly active and highly regioselective aldehyde dehydrogenase (UDH) capable of catalyzing the above reaction, the inventors screened and obtained an aldehyde dehydrogenase with catalytic activity for L-guluronic acid and D-glucuronic acid. The aldehyde dehydrogenase is derived from Fusarium longipes. However, under high substrate concentrations, the catalytic activity of the aldehyde dehydrogenase is very low. Therefore, the inventors mutated it to obtain an aldehyde dehydrogenase mutant SEQ ID NO: 2 with improved activity. The mutation of the mutant compared to the wild-type enzyme is A43I. The enzyme catalyzed reaction uses the NOx cycle coenzyme NAD + The NOx SEQ ID NO: 4 used in the reaction is derived from Streptococcus agalactiae. The mutant was tested to have a conversion rate of more than 95% in the presence of 200 g / L D-glucuronic acid, 10 g / L UDH wet bacteria, and a NOx coenzyme circulation system for 24 hours.

[0017] The substrate that can be catalyzed by aldehyde dehydrogenase is S1, where the R group can be a carbonyl group or a hydroxyl group, specifically L-guluronic acid, D-glucuronic acid, D-galacturonic acid, etc.; the corresponding product generated is P1:

[0018]

[0019] L-guluronic acid, one of the catalytic substrates of aldehyde dehydrogenase, can be obtained by oxidase-catalyzed gluconic acid. The oxidase used is an engineered galactose oxidase mutant, and its sequence is SEQ ID NO: 6; the sequence of catalase is SEQ ID NO: 8.

[0020]

[0021] The currently known galactose oxidase (GOase) does not have the activity of catalyzing the oxidation of gluconic acid. The inventors carried out engineering modification based on theoretical research and bioinformatics analysis, taking a wild-type galactose oxidase as the starting point. The wild-type galactose oxidase is derived from Fusarium longipes, and it was found that it could not oxidize the hydroxyl group at position 6 of glucose. After directed evolution modification, the inventors obtained a galactose oxidase mutant SEQ ID NO: 6 with oxidation activity for the hydroxyl group at position 6 of gluconic acid. Compared with the wild-type enzyme, the mutations included are A53P; W333F; R373K; Q449T; V537A; N578D. According to the inventors' detection, the mutant galactose oxidase has the catalytic activity of catalyzing gluconic acid to produce L-guluronic acid. It reacts for 24 hours under 50g / L gluconic acid feeding, and the reaction yield can reach more than 90%.

[0022] The active site of galactose oxidase (GOase) is composed of a single copper ion coordinated with two histidine residues, a tyrosine residue and a cross-linked cysteine-tyrosine unit, so 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. The present application uses horseradish peroxidase (HRP) as an activator of galactose oxidase, and galactose oxidase in a semi-reduced form is oxidized by 1e-oxidation to make it catalytically active. The hydrogen peroxide produced in the reaction is decomposed by catalase to avoid the inhibition of hydrogen peroxide on the enzyme. The galactose oxidase activator is not limited to horseradish peroxidase, and small molecule chemical oxidants such as manganese (III) fluoride, manganese (III) acetate, sodium persulfate, ammonium persulfate, etc. can all be used as activators of galactose oxidase.

[0023] The aldehyde dehydrogenase, NOx, galactose oxidase and catalase are products expressed by Escherichia coli, and the host cell is E. Coli, BL21 (DE3).

[0024] The coenzyme cycle system used in the aldehyde dehydrogenase reaction is not limited to the NOx coenzyme cycle system, and may be a ketoreductase (alcohol dehydrogenase) coenzyme cycle system.

[0025] The coenzyme of the aldehyde dehydrogenase system is NAD + (nicotinamide adenine dinucleotide) or NADP + (nicotinamide adenine dinucleotide phosphate), preferably NAD + .

[0026] The hydrogen acceptor in the ketoreductase (alcohol dehydrogenase) coenzyme cycle system is not limited to acetone, and may also be other ketones, such as 2-butanone, pyruvic acid, etc.

[0027] The preparation forms of aldehyde dehydrogenase, NOx, galactose oxidase and catalase used in the reaction system can be wet Escherichia coli cells, cell disruption supernatant, enzyme powder or immobilized enzyme.

[0028] The reaction system can be a phosphate buffer system, a borate buffer system, a Tris-HCl buffer system, etc.

[0029] The pH range is 6-9, preferably pH9.

[0030] The reaction temperature used in the reaction is 20-60°C, preferably 30°C.

[0031] Beneficial effects:

[0032] This application uses biological catalytic synthesis of glucaric acid (such as Figure 1 As shown), the reaction substrate feed amount is high, the substrate feed amount can reach more than 200g / L, and at the same time, the reaction produces less solid waste, which is green and environmentally friendly, avoiding the introduction of heavy metal ions in the preparation of glucaric acid by chemical metal catalysts. The reaction has high selectivity and can obtain a higher yield of glucaric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :Catalytic synthesis of glucaric acid using gluconic acid as raw material by all-biological method Specific implementation plan:

[0034] Example 1

[0035] 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 IPTG was added to induce protein expression, and the culture was shaken at 30°C for 18 hours. The bacterial cells were collected by centrifugation at 8000 rpm and stored at -20°C for later use.

[0036] Example 2

[0037] 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. The cells were centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant of the homogenate was collected and freeze-dried at -20°C to obtain aldehyde dehydrogenase or NOx enzyme powder, which was stored at -20°C.

[0038] Example 3

[0039] The reaction samples were detected by the following LC-MS method.

[0040]

[0041]

[0042] Example 4

[0043] In a 30 mL reaction bottle, add 0.25 g sodium D-glucuronic acid, 0.05 g wet bacteria expressing UDH shown in SEQ ID No: 2, 50 mM Tris-HCl (pH = 9), 0.5 mL 1 g / L NAD + Mother liquor, 0.5 mL acetone (final concentration 10% (v / v)), stirring, final reaction volume 5 mL, reaction at 30°C for 24 h, sampling LC-MS detection, 24 h reaction yield is 65%. Under the same conditions, the reaction yield of wild-type enzyme is about 20%.

[0044] Example 5

[0045] In a 30 mL reaction bottle, add 0.25 g sodium D-glucuronic acid, 0.05 g wet bacteria expressing UDH shown in SEQ ID No: 2, 0.05 g wet bacteria expressing KRED shown in SEQ ID No: 10, 50 mM Tris-HCl (pH = 9), 0.5 mL 1 g / L NAD +Mother liquor, 0.5 mL acetone (final concentration 10% (v / v)), stirring was started, the final reaction volume was 5 mL, reaction was carried out at 30°C for 24 h, sampling was performed for LC-MS detection, and the 24 h reaction yield was 71%.

[0046] Example 6

[0047] In 4 reaction bottles with a volume of 100 mL, 2.5 g, 5 g, 7.5 g, 10 g, and 15 g of sodium D-glucuronic acid, 0.5 g of wet bacteria expressing UDH shown in SEQ ID No: 2, 0.5 g of wet bacteria expressing NOx shown in SEQ ID No: 4, 50 mM Tris-HCl (pH = 9), and 5 mL of 1 g / L NAD were added respectively. + The mother liquor, the final reaction volume is 50mL, during the reaction process, the pH is adjusted to pH9 in real time with 10M sodium hydroxide, the reaction is carried out at 30°C for 24h, and the sample is taken for LC-MS detection. The reaction results are as follows:

[0048] Substrate concentration Reaction yield 50g / L 99％ 100g / L 99％ 150g / L 97％ 200g / L 95％ 300g / L 72％

[0049] For the reaction system with a substrate concentration of 200 g / L, reaction tests were carried out at different temperatures and pH values. Under the reaction condition of real-time adjustment of pH to maintain pH 9, the reaction yields were 64%, 95%, 90%, 76%, and 53% at temperatures of 20°C, 30°C, 40°C, 50°C, and 60°C, respectively. Under the temperature condition of 30°C, when the pH value of the reaction system was maintained at 6, 7, 8, and 9, respectively, the reaction yields were 43%, 65%, 95%, and 79%, respectively.

[0050] Under the reaction condition of substrate concentration of 300 g / L and other reaction conditions being the same as above, the catalytic activity of wild-type UDH was inhibited and the catalytic reaction could hardly proceed.

[0051] Example 7

[0052] In a reaction bottle with a volume of 100 mL, 2.5 g of sodium gluconate, 0.5 g of galactose oxidase mutant enzyme powder of SEQ ID No: 6, 5 mL of 1 g / L SEQ ID NO: 8 catalase mother solution, 5 mL of copper sulfate mother solution (5 mM), 50 mM Tris-HCl (pH = 8), 5 mL of 1 g / L horseradish peroxidase (HRP, purchased from Sigma, product number P8125) mother solution were added, stirring was started, the final reaction volume was 50 mL, the reaction was carried out at room temperature for 24 h, and sampling was performed for LC-MS detection. The reaction yield of L-guluronic acid was 90.6%.

[0053] Example 8

[0054] In a reaction bottle with a volume of 100 mL, 25 mL of the reaction solution prepared in Example 7 was added, the reaction solution contained about 45 g / L L-guluronic acid, the final concentration of L-guluronic acid in the reaction system was 22.5 g / L, 0.5 g of wet bacteria expressing UDH shown in SEQ ID No: 2, 0.5 g of wet bacteria expressing NOx shown in SEQ ID No: 4, 50 mM Tris-HCl (pH = 9), 5 mL 1 g / L NAD + The mother liquor had a final reaction volume of 50 mL. During the reaction, 10 M sodium hydroxide was used to adjust the pH to pH 9 in real time. The reaction was carried out at 30° C. for 24 h. Samples were taken for LC-MS detection. 91% of the L-guluronic acid in the reaction solution was converted into glucaric acid.

[0055] Example 9

[0056] In a 30 mL reaction bottle, add 0.25 g D-galacturonic acid, 0.05 g wet bacteria expressing UDH shown in SEQ ID No: 2, 0.05 g wet bacteria expressing NOx shown in SEQ ID No: 4, 50 mM Tris-HCl (pH = 9), 0.5 mL 1 g / L NAD + The mother liquor was used, and the final reaction volume was 5 mL. The reaction was carried out at 30° C. for 24 h. Samples were taken for LC-MS detection. The yield of galactaric acid was 93.7%.

[0057] 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.

[0058]

[0059]

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[0065]

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Claims

1. A aldehyde dehydrogenase, which can catalyze uronic acid to produce saccharic acid, and its sequence is shown in SEQ ID No:

2.

2. The aldehyde dehydrogenase according to claim 1, wherein Aldehyde dehydrogenase uses a coenzyme cycle system during the reaction process, wherein the coenzyme cycle system is a NOx coenzyme cycle system or a ketoreductase (alcohol dehydrogenase) coenzyme cycle system, and the coenzyme is NAD + or NADP + .

3. The aldehyde dehydrogenase according to claim 2, wherein The NOx used by aldehyde dehydrogenase in the reaction process is the sequence shown in SEQ ID NO: 4, and the hydrogen acceptor in the coenzyme circulation system of ketoreductase (alcohol dehydrogenase) is selected from acetone, 2-butanone and pyruvate.

4. A method for preparing a compound having a structure of P1, using an aldehyde dehydrogenase 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.

5. The method according to claim 4, wherein the substrate is L-guluronic acid, D-glucuronic acid or D-galacturonic acid.

6. The method according to claim 4-5, wherein a coenzyme circulation system is used in the reaction process, the coenzyme circulation system is selected from the NOx coenzyme circulation system or the ketoreductase (alcohol dehydrogenase) coenzyme circulation system, and the coenzyme is NAD + or NADP + .

7. The method according to claims 4-6, wherein the reaction pH ranges from 6 to 9, preferably pH 9, and the reaction temperature is 20 to 60°C, preferably 30°C.

8. The method according to claim 5-7, wherein L-guluronic acid can be obtained by catalyzing gluconic acid with galactose oxidase, and the reaction mechanism is as follows, wherein: The sequence of galactose oxidase is shown in SEQ ID NO:6, and the sequence of catalase is shown in SEQ ID NO:

8.

9. A method for preparing glucaric acid by enzymatic reaction throughout the process, wherein glucaric acid is prepared by two-step enzymatic oxidation using gluconic acid as a raw material; in the first step, galactose oxidase (GOase) is used to catalyze the oxidation of glucuronic acid to obtain L-guluronic acid, and in the second step, uronate dehydrogenase (UDH) is used to catalyze the oxidation of L-guluronic acid to obtain glucaric acid.

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