Method for preparing high-yield galactonic acid by combining chemical and biological methods

Abstract

The application discloses a method for preparing high-yield galacturonic acid by combining chemical and biological methods, which comprises the following steps: firstly, mixing gelidium and a maleic acid solution, and performing microwave heating and acidolysis to obtain D-galactose; and then taking engineered Pseudomonas putida as a biological catalyst to efficiently oxidize the D-galactose into galacturonic acid. In the application, the D-galactose obtained in the microwave heating and acidolysis step has high concentration and high yield, and the D-galactose can be completely oxidized into galacturonic acid in the subsequent biological catalysis step. The method does not need strong acid or special catalyst, is green and environment-friendly, has low raw material cost, simple process, high galacturonic acid yield, and realizes high-value development and utilization of the biomass gelidium.

Description

Technical Field

[0001] This invention belongs to the field of biomass degradation and biotransformation, specifically relating to a method for preparing high-yield galactobionic acid by combining chemical and biological methods. Background Technology

[0002] Seaweed resources are abundant, and large seaweeds can be divided into red algae, green algae, and brown algae. Red algae differ significantly in composition from terrestrial plants, with a carbohydrate content exceeding 70%. The most abundant component in red algae is polysaccharides, including cellulose, agar, and carrageenan; it also contains proteins, lipids, and trace amounts of lignin. Based on the differences in carbohydrate composition, red algae can be divided into agar-type red algae and carrageenan-type red algae.

[0003] The most abundant carbohydrate in Gracilaria is agar, thus Gracilaria is an agar-type red algae. Agar, also known as agar, is the main component of the cell wall of Gracilaria red algae, mainly composed of agarose and agar gum. Agarose is a linear chain polysaccharide molecule composed of D-galactose and 3,6-endoether-L-galactose linked by alternating α-1,3- and β-1,4-glycosidic bonds. Chemical or enzymatic hydrolysis of agarose releases D-galactose and 3,6-endoether-L-galactose. The main chain structure of agar gum is similar to that of agarose, but its molecular weight is smaller and its structure is more complex. Galacturonic acid is the oxidation product of D-galactose aldehydes, a novel aldonic acid similar in structure and chemical properties to gluconic acid. It can selectively replace citric acid as a food acidifier and can be used in the development of sweeteners, pharmaceutical intermediates, and dispersants.

[0004] While acid hydrolysis of agar-agar has been reported, the yields are low. No reports have yet documented the successful acid hydrolysis of agar-agar with common organic acids to obtain high yields of D-galactose. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing D-galactose by microwave-assisted catalytic hydrolysis of maleic acid using agar as a raw material, which is abundant in nature, and further oxidizing D-galactose to prepare galacturonic acid using a biocatalyst.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A method for preparing high-yield galactobionic acid using a combination of chemical and biological methods includes the following steps:

[0008] (1) Mix agar with maleic acid solution and heat to hydrolyze;

[0009] (2) Separate the solid and liquid of the acid hydrolysate after the reaction in step (1). The supernatant is the hydrolysate containing D-galactose. Neutralize with alkali to obtain the substrate for the biocatalytic reaction.

[0010] (3) Culture engineered Pseudomonas putida to the logarithmic phase, collect the microbial cells by centrifugation, and obtain the biocatalyst;

[0011] (4) Mix the substrate obtained in step (2) with the biocatalyst obtained in step (3) and add calcium carbonate to react. After the reaction is completed, separate the solid and liquid and collect the supernatant, which is a solution containing galacturonic acid.

[0012] In step (1), the mass concentration of the maleic acid solution is 1.5% to 8%, preferably 2% to 3%; the solid-liquid ratio of the agar to the maleic acid solution is 1:5 to 1:25 g / mL, preferably 1:10 to 1:15 g / mL.

[0013] In step (1), it is preferred to heat the acid hydrolysis in a microwave reactor. The microwave acid hydrolysis reaction temperature is 140℃~165℃, preferably 155℃~160℃, and the reaction time is 20~40min, preferably 25~35min.

[0014] In step (2), the alkali is solid calcium oxide.

[0015] In step (3), the engineered *Pseudomonas fragi* strain was constructed as follows: genomic DNA of *Pseudomonas fragi* NL20W was extracted, the target gene shown in SEQ ID NO.1 was amplified, and a recombinant plasmid was constructed and electroporated into *Pseudomonas fragi* ATCC No. 47054. *Pseudomonas fragi* NL20W has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 17, 2021, with accession number CCTCC NO: M 2021245.

[0016] In step (4), the reaction temperature is 20℃~40℃, preferably 25℃~35℃; the reaction pH is 5.0~8.0, preferably 5.3~5.8; and the reaction speed is 0~500rpm, preferably 250~350rpm.

[0017] In step (4), the ratio of substrate to biocatalyst is: 3g substrate / g dry weight cells to 8g substrate / g dry weight cells.

[0018] Step (4) is carried out in a biocatalytic reaction vessel, which is a wide-mouth type and sealed with double gauze. The reaction time range is 3 to 15 hours.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) Compared with terrestrial lignocellulosic biomass resources, agar-agar grows rapidly, is rich in resources, has low lignin content, is easy to hydrolyze, and the hydrolysate is rich in D-galactose. (2) When hydrolyzing biomass, inorganic acids, such as dilute sulfuric acid, are usually used. Free sugars obtained by hydrolysis with inorganic acids are easily further degraded during high-temperature treatment. In addition, inorganic acids have a strong corrosive effect. Maleic acid used in the present invention is a common organic acid that is inexpensive. Under microwave-assisted conditions, a low acid concentration can be used to obtain high concentration and high yield of D-galactose. The acid hydrolysis process is green and environmentally friendly and the process is simple. (3) The engineered bacteria of Pseudomonas putida in the present invention oxidize D-galactose faster and without the production of by-products. (4) Based on the galactan content in agar-agar, the galactose acid yield prepared by the technical solution described in the present invention is high. Detailed Implementation

[0020] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0021] Method for determining the moisture content of agar-agar: Weigh 0.5g of crushed agar-agar and determine the moisture content using an Infrared Moisture Determination Balance FD-720 instrument.

[0022] Method for determining the galactomannan content in agar-agar: After pulverizing the agar-agar, remove the water, weigh 0.3 g of the completely dry material and add it to a hydrolysis flask. Add 3 mL of 72% sulfuric acid and stir until the raw materials are fully mixed. Place the mixture in a 30℃ constant temperature shaking water bath and keep it at that temperature for 1 h, shaking it with a vortex mixer every 5-10 min. Then, add 84 g of water to the reaction solution, mix well, and acid hydrolyze it at 120℃ for 1 h. After cooling, filter it through G3 glass sand, dilute the filtrate appropriately, filter the filtrate through a 0.22 μm filter membrane, and analyze the D-galactose content by high performance liquid chromatography (HPLC).

[0023] Galactan content (%) = (Cgal × 0.9 × V / W) × 100%

[0024] (Where, Cgal is the concentration of D-galactose, g / L; 0.9 is the conversion coefficient between C6 monosaccharides and polysaccharides; V is the total volume of the filtrate, L; W is the oven-dry weight of the sample, g)

[0025] In this embodiment of the invention, the moisture content of the pulverized agar-agar is 9.21%. The galactomannan content in the oven-dried material is 49.16%.

[0026] Based on 1 gram of crushed agar, the content of galactomannan is 0.446g.

[0027] The determination method for D-galactose and galacturonic acid was as follows: High-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Chromatograph: Agilent 1260 HPLC system; Column: Coregel ION 300; Injection volume: 10 μL; Mobile phase: 0.5 mM sulfuric acid; Flow rate: 0.4 mL / min; Column temperature: 75℃; Detector: Differential refractive index detector.

[0028] In the microwave acid hydrolysis step, the yield of D-galactose is calculated according to the following formula (1):

[0029]

[0030] In the biocatalytic step, D-galactose or a microwave acid hydrolysate rich in D-galactose is used as the substrate, and the galacturonic acid yield is calculated according to the following formula (2):

[0031]

[0032] Using agar as a substrate, and combining chemical catalysis and biocatalysis, the yield of galacturonic acid was calculated according to the following formula (3):

[0033] Galacturonic acid yield (%) = (1) × (2) × 100 (3)

[0034] Example 1

[0035] Weigh 1 gram of crushed agar and mix it evenly with 10 mL of 2% maleic acid solution. Add the mixture to a microwave digestion tube, place it in a microwave reactor, and seal it. Heat to 160℃ and maintain for 30 min. After the reaction is complete, wait for the temperature to drop below 80℃, remove the reaction solution, and separate the solid and liquid phases to obtain a supernatant containing D-galactose. The D-galactose concentration was determined by HPLC to be 46.3 g / L. The D-galactose yield was calculated to be 93.4% according to formula (1).

[0036] Example 2

[0037] Weigh 1 gram of crushed agar and mix it evenly with 10 mL of 6% maleic acid solution. Add the mixture to a microwave digestion tube, place it in a microwave reactor, and seal it. Heat to 160℃ and maintain for 30 min. After the reaction is complete, wait for the temperature to drop below 80℃, remove the reaction solution, separate the solid and liquid, and obtain a supernatant containing D-galactose. The D-galactose concentration was determined by HPLC to be 45.2 g / L. The D-galactose yield was calculated to be 91.2% according to formula (1).

[0038] Example 3

[0039] Weigh 1 gram of pulverized agar and mix it thoroughly with 10 mL of 2% maleic acid solution. Add the mixture to a microwave digestion tube, place it in a microwave reactor, and seal it. Heat to 155℃ and maintain for 25 min. After the reaction is complete, wait for the temperature to drop below 80℃, remove the reaction solution, and separate the solid and liquid phases to obtain a supernatant containing D-galactose. HPLC analysis showed that the D-galactose concentration was 46.0 g / L. The D-galactose yield was calculated to be 92.8% according to formula (1).

[0040] Example 4

[0041] Weigh 1 gram of crushed agar and mix it evenly with 15 mL of 2% maleic acid solution. Add the mixture to a microwave digestion tube, place it in a microwave reactor, and seal it. Heat to 155℃ and maintain for 33 min. After the reaction is complete, wait for the temperature to drop below 80℃, remove the reaction solution, separate the solid and liquid, and obtain a supernatant containing D-galactose. The D-galactose concentration was determined by HPLC to be 31.1 g / L. The D-galactose yield was calculated to be 94.1% according to formula (1).

[0042] Example 5

[0043] 1. Screening and preservation of Pseudomonas berries.

[0044] Soil samples were collected from various habitats in Zijin Mountain. 1g of the mixed soil sample was thoroughly mixed with 9mL of sterile physiological saline, and 500µL of the mixture was inoculated into 50mL of LB medium. The mixture was incubated at 30℃ and 200rpm for 12 hours. The culture medium was then... -8 10 -9 10 -10 Three dilutions were plated on solid LB medium containing 50 g / L lactose and 25 g / L light calcium carbonate, and incubated at 30°C until microbial colonies appeared. Based on the size of the clear zone around the colony, a sufficient number of single colonies with large clear zones were selected and inoculated into 50 mL of liquid LB medium containing 50 g / L lactose and 25 g / L calcium carbonate. The culture time was 12 hours, with samples taken every 2 hours to measure lactobionic acid production. The strain with the best lactobionic acid production and yield was selected and stored for later use.

[0045] Alignment of its 16S rDNA sequence with sequences of other strains in the NCBI database revealed that this strain is most closely related to *Pseudomonas fragi*, exhibiting a 16S rDNA sequence similarity greater than 99% with multiple *Pseudomonas fragi* strains in the database. Phylogenetic analysis also indicated that this strain belongs to the same evolutionary branch as other *Pseudomonas fragi* strains. This strain is classified and named *Pseudomonas fragi* NL20W. It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 17, 2021, with accession number CCTCCNO:M 2021245. Its 16S rDNA sequence is as follows: (SEQ ID NO. 6)

[0046] GAACTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAA GTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAATACCTAGGAATCTGCCTGATAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGA GAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTG AGGTAATGGCTCACCAAGGCTACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAA AGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTGGTGTCTTGACGTTACCGACAGAATAAGCACCGGCTAA CTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAA AGCGCGCGTAGGTGGTTTGTTAAGTTGAATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGT AGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGTGC GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGTCTTGAACTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGA GATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCAT GGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCG AGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACA CACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGA CGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTA.

[0047] 2. Genomic DNA of *Pseudomonas berries* NL20W was prepared using conventional methods. The procedure can be found in the small-scale preparation method of bacterial genomes in *A Concise Guide to Molecular Biology* published by Science Press. The target gene was amplified from the genomic DNA using synthetic primers, and its nucleotide sequence is shown in SEQ ID NO.1.

[0048] Following conventional molecular biology methods, using -Use the Uni Seamless Cloning and Assembly Kit (purchased from TransGen) to clone the target gene obtained from PCR amplification into the pBBR1MCS-2 plasmid (purchased from Addgene). The specific steps are as follows:

[0049] 1) Design two pairs of primers, one for amplifying the target gene and the other for amplifying the plasmid.

[0050] 2) The primers for amplifying the target gene are:

[0051] Upstream primer: 5'-ttactcagccagtttgaacg-3' (SEQ ID NO.2)

[0052] Downstream primer: 5'-atgagcactgaaggtgcttt-3' (SEQ ID NO.3)

[0053] 3) The primers for amplifying the plasmid are:

[0054] Upstream primer:

[0055] 5'-aaagcaccttcagtgctcatagctgtttcctgtgtgaaat-3'(SEQ ID NO.4)

[0056] Downstream primer:

[0057] 5'-cgttcaaactggctgagtaagcgttaatattttgttaaaa-3'(SEQ ID NO.5)

[0058] 4) The PCR product was purified using a purification kit, and then processed according to... The Uni Seamless Cloning and Assembly Kit instruction manual explains how to connect the target fragment and plasmid.

[0059] 5) The obtained recombinant plasmid was electroporated into competent *Pseudomonas putida* cells (ATCC No. 47054). The methods for preparing competent cells and electroporation are detailed in patent CN113073072A. After electroporation, the bacterial culture in the electroporation vessel was transferred to centrifuge tubes and cultured on a shaker at 30°C for 1 hour to resuscitate the cells. After resuscitation, the cells were screened on LB agar plates containing kanamycin resistance. The resulting colonies were inoculated into LB liquid medium containing kanamycin resistance and cultured at 30°C until mid-log phase. The bacterial cells were then collected and stored in a cryogenic freezer for later use. This strain is the engineered *Pseudomonas putida* strain.

[0060] Example 6

[0061] A loopful of *Pseudomonas putida* engineered bacteria was inoculated onto an agar slant medium (components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L kanamycin, 18 g / L agar powder) and inoculated onto a liquid medium (components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L kanamycin) and cultured at 30°C and 200 rpm for 12 h to activate the strain. The activated bacterial solution was then inoculated onto the same medium at 1% by volume for further culture. After culturing at 30°C and 200 rpm for 10 h, the bacterial cells were collected by centrifugation. The cells were washed twice with physiological saline, and the intact microbial cells obtained by centrifugation were used as the biocatalyst.

[0062] The cells were resuspended in phosphate buffer (200 mM, pH 7.0) and mixed with D-galactose (purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.). The concentrations of each substance in the reaction system were adjusted so that the concentration of the biocatalyst was 8 g dry weight / L, D-galactose was 50 g / L, and calcium carbonate was added to make the concentration 28 g / L. The reaction was carried out at 30℃ and 200 rpm for 6 hours, and samples were taken every 2 hours to detect the changes in the concentrations of D-galactose and galacturonic acid. The yield of galacturonic acid was calculated according to formula (2). The data showed that the yield of galacturonic acid was 87% after 4 hours of reaction and 99% after 6 hours of reaction.

[0063] Example 7

[0064] Biocatalysts were prepared by culturing engineered Pseudomonas putida according to the method described in Example 6.

[0065] The pH of the acid hydrolysate obtained in Example 1 was adjusted to 5.5 with calcium oxide, and then mixed with the cells to adjust the cell concentration in the reaction system so that the concentration of the biocatalyst was 8 g dry weight / L. Calcium carbonate was added to make its concentration 25 g / L. The reaction was carried out at 35°C and 300 rpm, and samples were taken every 2 hours to detect the changes in the concentrations of D-galactose and galacturonic acid. The yield of galacturonic acid was calculated according to formula (2). The data showed that the yield of galacturonic acid was 95% after 6 hours of reaction and 100% after 8 hours of reaction. Therefore, using agar as a substrate, combined with chemical catalysis and biocatalysis, according to formula (3), the overall yield of galacturonic acid was 93.4%.

[0066] Example 8

[0067] Biocatalysts were prepared by culturing engineered Pseudomonas putida according to the method described in Example 6.

[0068] The pH of the acid hydrolysate obtained in Example 4 was adjusted to 6.0 with calcium oxide, and then mixed with the cells described above. The cell concentration in the reaction system was adjusted to make the concentration of the biocatalyst 8 g dry weight / L, and calcium carbonate was added to make its concentration 17 g / L. The reaction was carried out at 30°C and 350 rpm, and samples were taken every 2 hours to detect the changes in the concentrations of D-galactose and galacturonic acid. The yield of galacturonic acid was calculated according to formula (2). The data showed that the yield of galacturonic acid was 100% after 4 hours of reaction. Therefore, using agar as a substrate, combined with chemical catalysis and biocatalysis, according to formula (3), the overall yield of galacturonic acid was 94.1%.

[0069] Comparative Example 1

[0070] Weigh 1 gram of crushed agar and mix it evenly with 10 mL of 2% maleic acid solution. Add the mixture to a pressure-resistant tube, seal it, and incubate it in an oil bath at 160℃ for 30 min. After the reaction is complete, remove the pressure-resistant tube and cool it to room temperature. Pour out the reaction solution, separate the solid and liquid, and obtain a supernatant containing D-galactose. The concentration of D-galactose was determined by HPLC to be 39.8 g / L. The yield of D-galactose was calculated to be 80.3% according to formula (1).

[0071] Comparative Example 2

[0072] Weigh 1 gram of pulverized agar and mix it thoroughly with 10 mL of 5% acetic acid solution. Add the mixture to a microwave digestion tube, place it in a microwave reactor, and seal it. Heat to 160℃ and maintain for 30 min. After the reaction is complete, wait for the temperature to drop below 80℃, remove the reaction solution, and separate the solid and liquid phases to obtain a supernatant containing D-galactose. HPLC analysis showed that the D-galactose concentration was 39.6 g / L. The D-galactose yield was calculated to be 79.9% according to formula (1).

[0073] Comparative Example 3

[0074] Pseudomonas putida (ATCC No. 47054) was used as a biocatalyst.

[0075] The biocatalyst was prepared using the same method as in Example 6, but the culture medium did not contain kanamycin.

[0076] The cells were resuspended in phosphate buffer (200 mM, pH 7.0) and mixed with D-galactose (purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.). The concentrations of each substance in the reaction system were adjusted so that the concentration of the biocatalyst was 8 g dry weight / L, D-galactose was 50 g / L, and calcium carbonate was added to make the concentration 28 g / L. The reaction was carried out at 30℃ and 200 rpm for 6 hours, and samples were taken every 2 hours to detect the changes in the concentrations of D-galactose and galacturonic acid. The yield of galacturonic acid was calculated according to formula (2). The data showed that the yield of galacturonic acid was 69% after 4 hours of reaction and 89% after 6 hours of reaction. sequence list <110> Nanjing Forestry University <120> A method for preparing high-yield galactobionic acid by combining chemical and biological methods <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2418 <212> DNA <213> Pseudomonas fragi <400> 1 atgagcactg aaggtgcttt cagtcgaagc cgcctgctac cgagccttct cggtatcttg 60 ctgctgctaa tgggcctggc catgttggcc gggggtatca aactggtcac gctgggcggg 120 tcgtggtact acctgctggc cgggatcggt tttggcttgt cgggcgcact gctgattgcc 180 gggcgccgcg ctgcactggc tctatacgcg ctgacgctgt tcgccagcac cgtatgggca 240 ctgatggaag tgggtctgga ctggtggcaa ctggtgccgc gcctggccat gtggttcgcc 300 atcggtatcg ttctgctgct gccatggttc cgtcgtccgg ttctgcgcgg tcagtcggca 360 cctttggcta ccggcgcact gagcgttgcc gtggttctgg caggtgctgc tgcactggcc 420 agccagttca ccagcccggg cgaaatcaaa ggccaactgg atcgtgatgc cgtacccggc 480 atgaccaacg ccgcaccggc catgcccgat ggcgactggc agtcctacgg ccgcaccgct 540 tttggtgacc gttactcgcc gctgaaagaa atcacccctg agaatgccca caagctggtt 600 ccagcctgga cattccgcac cggtgacatg ccaggtgaag gcgatcccgg cgaaacaacc 660 gccgagaaca ccccgctgaa agtcaacggc atgctgtatg tgtgtacccc acacagccag 720 gtaattgccc ttgacccgga caccggcaag gaaatctggc gttacgatcc gaagatcagc 780 acgcagaacg ctgagaactt caaaggctgg gcacacatga cctgccgcgg cgtgacttat 840 cacgacgaaa atgcctacgc caaagccagc actgaacaaa gcgctgccga gcctgctgct 900 gccacatcca gcaactcgtg cccgcgtcgc ctgttcctgc cgactgccga cacccgtctg 960 atcgccttga acgccgacac cggcaaacct tgtgaagact tcggtgacca cggttcggta 1020 gacctgcgtc acaacatcgg cagctttgct ccaggtggtt actactccac ttcgccacct 1080 gccgtgacca aagacttggt agtgattggc ggccacgtga ccgacaacat ctccaacgac 1140 gagccgtcgg gcgtgatccg tgcgtacgac gtacgtaccg gcaagctggt ctggaactgg 1200 gacagcggca acccggagaa aaccactccg attgctgaag gcgaaaccta cacccgtaac 1260 tcgccaaaca tgtggtcgat gttcgctgtc gacgaagacc tcggcatgct gtacctgccg 1320 atgggcaacc agacccctga ccaatttggc ggcgatcgta ccgaagattc cgagcgttat 1380 gccgctggca tcaccgccct ggacatcaac actggtaaag tccgctggta ccgtccgctt 1440 actcaccatg acctgtggga catggacgta ggtggtcaac caaccctgat ggacctgaaa 1500 accgccgatg gcgtgaaacc ggccctgctg gcttccacca aacaaggcag catctacgtc 1560 atggaccgtc gcactggcga agccattgtg ccgatcaccg agatccctgc accgggcggc 1620 gctgtagaag gtgaccacac tgcaccgaca cagcctcgtt cggacctgaa catgatcccg 1680 ccggtgctga ccgaacgtga catgtggggc gtgacgccat tcgaccagat gctgtgccgg 1740 atcaacttca aatccctgcg ttatgacggc atgtacaccc cgccatcgct gcaaggttcg 1800 atcgtttatc caggcaactt cggcgtgttc gactggggcg gcatctcggt tgacccggtt 1860 cgccagattg ccttcctgaa cccgagctac atggcgttca cctccaagct ggttccgcag 1920 gccgacgtgg ctgcaatggg cccgcgcaaa ggcgaaacct caggcgttca accgaacaaa 1980 ggcgcacctt acggcgtgat tctggagcca ctgttgtcgc cactgggcct gccttgccag 2040 gcaccggcgt ggggttatgt tgctgcagtc gacctgacca acaacgaagt gatctggaaa 2100 cacaaaaacg gtaccgtgcg tgacagctcg ccggttccga tcccgttgtc catgggtgtt 2160 ccaagcctgg gcgggacctt caccaccgca ggtggtgtgg ccttcctgag cggtacactt 2220 gaccagtacc tgcgtgctta cgacgtaagc aacggtaaag tactgtggga aggtcgcctg 2280 cctgctggcg gccagaccac cccgatgacc tacaccggca aggacggcac tcaatatgtg 2340 ctggtcatgg cgggcggtca cggcggcctg ggcaccaaaa aaggtgacta tgtcatggcg 2400 ttcaaactgg ctgagtaa 2418 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ttactcagcc agtttgaacg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 atgagcactg aaggtgcttt 20 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <400> 4 aaagcacctt cagtgctcat agctgtttcc tgtgtgaaat 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <400> 5 cgttcaaact ggctgagtaa gcgttaatattttgttaaaa 40 <210> 6 <211> 1537 <212> DNA <213> Artificial Sequence <400> 6 gaactgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggccta acacatgcaa 60 gtcgagcggt agagagaagc ttgcttctct tgagagcggc ggacgggtga gtaataccta 120 ggaatctgcc tgatagtggg ggataacgtt cggaaacgga cgctaatacc gcatacgtcc 180 tacgggagaa agcaggggac cttcgggcct tgcgctatca gatgagccta ggtcggatta 240 gctagttggt gaggtaatgg ctcaccaagg ctacgatccg taactggtct gaggatga 300 tcagtcacac tggaactgag acacggtcca gactcctacg ggaggcagca gtgggggaata 360 ttggacaatg ggcgaaagcc tgatccagcc atgccgcgtg tgtgaagaag gtttcggat 420 tgtaaagcac tttaagttgg gaggaagggc attaacctaa tacgttggtg tcttgacgtt 480 accgacagaa taagcaccgg ctaactctgt gccagcagcc gcggtaatac agagggtgca 540 agcgttaatc ggaattactg ggcgtaaagc gcgcgtaggt ggtttgttaa gttgaatgtg 600 aaatccccgg gctcaacctg ggaactgcat ccaaaactgg caagctagag tatggtagag 660 ggtagtggaa tttcctgtgt agcggtgaaa tgcgtagata taggaaggaa caccagtggc 720 gaaggcgact acctggactg atactgacac tgaggtgcga aagcgtgggg agcaaacagg 780 attagatacc ctggtagtcc acgccgtaaa cgatgtcaac tagccgttgg gagtcttgaa 840 ctcttagtgg cgcagctaac gcattaagtt gaccgcctgg ggagtacggc cgcaaggtta 900 aaactcaaat gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 960 caacgcgaag aaccttacca ggccttgaca tccaatgaac tttccagaga tggattggtg 1020 ccttcgggaa cattgagaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1080 gggttaagtc ccgtaacgag cgcaaccctt gtccttagtt accagcacgt aatggtgggc 1140 actctaagga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcatcat 1200 ggcccttacg gcctgggcta cacacgtgct acaatggtcg gtacaaaggg ttgccaagcc 1260 gcgaggtgga gctaatccca taaaaccgat cgtagtccgg atcgcagtct gcaactcgac 1320 tgcgtgaagt cggaatcgct agtaatcgtg aatcagaatg tcacggtgaa tacgttcccg 1380 ggccttgtac acaccgcccg tcacaccatg ggagtgggtt gcaccagaag tagctagtct 1440 aaccttcggg aggacggtta ccacggtgtg attcatgact ggggtgaagt cgtaacaagg 1500 tagccgtagg ggaacctgcg gctggatcac ctcctta 1537

Claims

1. A method for preparing high-yield galactobionic acid using a combination of chemical and biological methods, characterized in that, Includes the following steps: (1) Mix agar with maleic acid solution and heat in a microwave reactor for acid hydrolysis; (2) Separate the solid and liquid of the acid hydrolysate after the reaction in step (1). The supernatant is the hydrolysate containing D-galactose. Neutralize with alkali to obtain the substrate for the biocatalytic reaction. (3) Culture engineered Pseudomonas putida to the logarithmic phase, centrifuge to collect microbial cells, and use them as a biocatalyst; (4) Mix the substrate obtained in step (2) with the biocatalyst obtained in step (3) and add calcium carbonate to react. After the reaction is completed, separate the solid and liquid and collect the supernatant, which is a solution containing galacturonic acid. In step (3), the engineered strain of *Pseudomonas putida* was constructed as follows: genomic DNA of *Pseudomonas berries* NL20W was extracted, the target gene shown in SEQ ID NO.1 was amplified, and a recombinant plasmid was constructed and electroporated into *Pseudomonas putida* ATCC No. 47054.

2. The method for preparing high-yield galactobionic acid using a combination of chemical and biological methods according to claim 1, characterized in that, In step (1), the mass concentration of the maleic acid solution is 1.5%~8%; the solid-liquid ratio of the agar to the maleic acid solution is 1:5~1:25 g / mL.

3. The method for preparing high-yield galactobionic acid using a combination of chemical and biological methods according to claim 1, characterized in that, In step (1), the mass concentration of the maleic acid solution is 2%~3%; the solid-liquid ratio of the agar to the maleic acid solution is 1:10~1:15 g / mL.

4. The method for preparing high-yield galactobionic acid by combining chemical and biological methods according to claim 1, characterized in that, In step (1), the acid hydrolysis reaction temperature is 140℃~165℃ and the reaction time is 20~40 min.

5. The method for preparing high-yield galactobionic acid by combining chemical and biological methods according to claim 1, characterized in that, In step (1), the acid hydrolysis reaction temperature is 155℃~160℃ and the reaction time is 25~35 min.

6. The method for preparing high-yield galactobionic acid by combining chemical and biological methods according to claim 1, characterized in that, In step (2), the alkali is solid calcium oxide.

7. The method for preparing high-yield galactobionic acid by combining chemical and biological methods according to claim 1, characterized in that, In step (4), the reaction temperature is 20℃~40℃; the reaction pH is 5.0~8.0; and the reaction speed is 0~500 rpm.

8. The method for preparing high-yield galactobionic acid by combining chemical and biological methods according to claim 1, characterized in that, In step (4), the reaction temperature is 25℃~35℃; the reaction pH is 5.3~5.8; and the reaction speed is 250~350 rpm.

9. The method for preparing high-yield galactobionic acid by combining chemical and biological methods according to claim 1, characterized in that, In step (4), each gram of dry cell biocatalyst corresponds to 3g~8g of substrate.

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