A method for improving the 5-hydroxymethylfurfural oxidation ability of Pseudomonas holmesii

Abstract

The invention discloses a culture method for improving the 5-hydroxymethylfurfural oxidation ability of Pseudomonas hollii. Agar agar hydrolyzate is obtained by acidolysis of agar agar with an organic acid, calcium oxide and sodium hydroxide are sequentially added to adjust the pH of the hydrolyzate, the clear liquid obtained after centrifugation is used as a culture medium carbon source, and a small amount of cheap organic nitrogen source and inorganic salt are added on this basis to obtain agar agar acid hydrolyzate culture medium. The culture medium is low in cost, and Pseudomonas hollii is cultured with the culture medium, and the catalytic ability of the obtained cells to oxidize 5-hydroxymethylfurfural is higher than that of cells cultured with an expensive carbon source and nitrogen source culture medium.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis and conversion, and particularly relates to a cultivation method for improving the 5-hydroxymethylfurfural oxidation ability of Pseudomonas hallii. Background Art

[0002] Compared with traditional chemical catalysis, microbial whole-cell catalysis has the advantages of high catalytic efficiency, high reaction selectivity, green process, mild conditions, and environmental friendliness, and has attracted more and more attention. Microbial whole-cell catalysis is divided into two stages: cell culture stage and substrate catalysis stage. Among them, the culture medium components and culture methods in the cell culture stage not only affect the overall catalytic cost, but are also crucial to the cell catalytic ability in the catalytic stage.

[0003] 5-Hydroxymethylfurfural (HMF) is an important platform compound that can be prepared from biomass. HMF can be oxidized to a variety of furan carboxylic acids, among which its aldehyde-specific oxidation product is 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). HMFCA has diverse functions and is also a promising starting material for the synthesis of polyesters. Microorganisms that can catalyze this reaction include Pseudomonas hallii (Chinese invention patent application number 202110724944.3), Pseudomonas putida (Bioprocess and Biosystems Engineering 2020, 43: 67–73), Gluconobacter oxydans (ACS Sustainable Chemical Engineering 2019, 7: 4406-4413), Comamonas testosteroni (Green Chemistry 2017, 19: 4544-4551), etc.

[0004] When culturing the above-mentioned microorganisms, it has been reported that the culture medium contains commercial carbon sources (glucose, sorbitol, etc.), expensive organic nitrogen sources (yeast powder, peptone), etc. For example, on the website of the China Industrial Microbiological Culture Collection Administration Center, for the cultivation of strains such as Pseudomonas hallii and Pseudomonas putida, it is recommended to use commercial finished culture media (such as LB culture medium, nutrient broth agar) containing nutrients such as peptone, beef extract, and yeast powder, which undoubtedly increases the overall catalytic cost. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a cultivation method for improving the 5-hydroxymethylfurfural oxidation ability of Pseudomonas hallii in view of the deficiencies of the prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for improving the 5-hydroxymethylfurfural oxidation ability of Pseudomonas hallii comprises the following steps:

[0008] (1) crushing agar-agar and mixing it with an organic acid solution, reacting it at high temperature, and separating the solid and liquid after the reaction to obtain an agar-agar acid hydrolyzate;

[0009] (2) adjusting the pH of the agar-agar acid hydrolyzate obtained in step (1) to neutral, using the agar-agar acid hydrolyzate after pH adjustment as a carbon source, compounding with an organic nitrogen source and an inorganic salt, to prepare agar-agar acid hydrolyzate culture medium;

[0010] (3) inoculating Pseudomonas hallii into GMM culture medium, activating and culturing to obtain an activated solution; transferring the activated solution to the agar acid hydrolyzate culture medium prepared in step (2) for further expansion and culturing, collecting the cells and washing them with physiological saline, and obtaining a biocatalyst by solid-liquid separation;

[0011] (4) catalyzing 5-hydroxymethylfurfural with the biocatalyst prepared in step (3); after the catalytic reaction is completed, collecting the supernatant by solid-liquid separation to obtain 5-hydroxymethyl-2-furancarboxylic acid.

[0012] Wherein, in step (1), the organic acid is oxalic acid or citric acid; wherein the organic acid solution is a 1wt% to 10wt% organic acid aqueous solution, and the solid-liquid ratio of agar to organic acid is 1:5 to 1:20 g / mL.

[0013] Wherein, in step (1), the high temperature reaction has the following reaction conditions: reaction temperature is 110°C to 140°C, and reaction time is 15min to 90min.

[0014] Wherein, in step (2), the neutralizing agents used to adjust the pH of the agar-agar acid hydrolysate are calcium oxide and sodium hydroxide, and the two neutralizing agents are added in sequence; first, calcium oxide solid is added at a ratio of 5 to 20 g of calcium oxide per liter of agar-agar acid hydrolysate, and after oscillating the reaction for 0.5 to 2 hours, the precipitate is removed by centrifugation, and then the pH of the supernatant is adjusted to 6.0 to 7.5 with sodium hydroxide solution, and the precipitate is removed by centrifugation again.

[0015] Preferably, firstly, calcium oxide solid is added at a ratio of 10 g calcium oxide per liter of agar-agar acid hydrolyzate, and after oscillating the reaction for 1.5 hours, the precipitate is removed by centrifugation, and then the pH of the supernatant is adjusted to 7.0 with sodium hydroxide solution, and the precipitate is removed by centrifugation again.

[0016] Wherein, in step (2), the organic nitrogen source is any one of corn steep liquor powder, peanut cake powder, soybean cake powder or a combination thereof, and the preferred organic nitrogen source is corn steep liquor powder.

[0017] Wherein, in step (2), the agar-agar acid hydrolyzate culture medium has a formula of: 1L of distilled water contains: 50-200mL of agar-agar acid hydrolyzate after pH adjustment, 2-5g of organic nitrogen source, 0.5-3g of Na2HPO4, 0.2-1.5g of KH2PO4, 0-0.5g of NH4Cl, 0-0.25g of NaCl, 0-0.2g of MgSO4, and 0-2mL of trace element solution, the solvent is water, and the pH is 6.0-7.5. The formula of the trace element solution is as follows: 1L of distilled water contains: H3BO3 0.1-0.5g, ZnCl2 0.01-0.1g, MnCl2·4H2O 0.01-0.05g, CoCl2 0.1-0.5g, CuCl2·2H2O 0.01-0.5g, NiCl2·6H2O 0.01-0.1g, and NaMoO4·2H2O 0.01-0.05g.

[0018] Preferably, the formula of the agar-agar acid hydrolyzate culture medium is as follows: 100 mL of pH-adjusted agar-agar acid hydrolyzate, 5 g of corn steep liquor powder, 0.5 g of Na2HPO4, and 0.2 g of KH2PO4 in 1 L of distilled water, the solvent is water, and the pH value is 7.0.

[0019] Wherein, in step (3), the Pseudomonas hallii is Pseudomonas hallii NL2019, whose preservation number is CCTCC NO: M 2021356, which has been recorded in patent CN113388565A.

[0020] Wherein, in step (3), the formula of the GMM culture medium is: 1L of distilled water contains: 2-10g of galactose, 1-5g of Na2HPO4, 1-3g of KH2PO4, 0.1-1g of NH4Cl, 0.1-1g of NaCl, 0.1-0.4g of MgSO4, and 2-3mL of trace element solution, the solvent is water, and the pH is 6.0-7.5. The formula of the trace element solution is as follows: 1L of distilled water contains: H3BO3 0.1-0.5g, ZnCl2 0.01-0.1g, MnCl2·4H2O 0.01-0.05g, CoCl2 0.1-0.5g, CuCl2·2H2O 0.01-0.5g, NiCl2·6H2O 0.01-0.1g, and NaMoO4·2H2O 0.01-0.05g.

[0021] Preferably, the formula of GMM culture medium is: 5g galactose, 3.5g Na2HPO4, 1.5g KH2PO4, 0.5g NH4Cl, 0.25g NaCl, 0.24g MgSO4, 2.5mL trace element solution in 1L distilled water, the solvent is water, pH 7.0. Wherein, the formula of trace element solution is: 0.3g H3BO3, 0.05g ZnCl2, 0.03g MnCl2·4H2O, 0.2g CoCl2, 0.01g CuCl2·2H2O, 0.02g NiCl2·6H2O, 0.03g NaMoO4·2H2O in 1L distilled water.

[0022] Wherein, in step (3), when the activation solution is transferred to the agar-agar acid hydrolyzate culture medium for further expansion of the culture, the transfer amount is 1% to 10% by volume.

[0023] The preferred transfer amount is 2% by volume.

[0024] Wherein, in step (3), the activation culture and expansion culture are carried out under the following culture conditions: temperature 25-35°C, culture time 8-14 hours.

[0025] The preferred activation culture has the following culture conditions: a temperature of 30° C. and a culture time of 10 hours; the preferred expansion culture has the following culture conditions: a temperature of 30° C. and a culture time of 12 hours.

[0026] Wherein, in step (4), 5-hydroxymethylfurfural is catalyzed by the biocatalyst prepared in step (3), the dry weight concentration of the biocatalyst is 5 to 10 g / L, and the concentration of 5-hydroxymethylfurfural is 2 to 40 g / L.

[0027] Wherein, in step (4), the catalytic reaction has the following reaction conditions: reaction temperature is 25°C to 45°C, reaction pH is 5.5 to 8.0, and reaction time is 0.5 to 10 hours.

[0028] The above method is used to catalyze the conversion of 5-hydroxymethylfurfural to prepare 5-hydroxymethyl-2-furancarboxylic acid, wherein the conversion rate of the 5-hydroxymethylfurfural is above 98%; the yield of the 5-hydroxymethyl-2-furancarboxylic acid is above 92%. It is found that the catalytic ability of the cells obtained by the present invention to oxidize 5-hydroxymethylfurfural is much higher than that of cells cultured in a medium using expensive carbon sources (glucose, sorbitol, etc.) and expensive organic nitrogen sources (yeast powder, peptone, etc.).

[0029] Beneficial effects: Compared with the prior art, the present invention uses agar-agar acid hydrolysate as a carbon source, and compounded with a small amount of cheap organic nitrogen source and inorganic salt as a culture medium to culture Pseudomonas hallii. Compared with the commercial finished product culture medium, the culture medium of the present invention does not contain expensive carbon sources (glucose, sorbitol, etc.) and expensive organic nitrogen sources (yeast powder, peptone, etc.), and the culture medium cost is low. At the same time, in the subsequent biocatalytic process, the ability of the obtained cells to oxidize 5-hydroxymethylfurfural is also stronger than that of cells cultured with expensive carbon and nitrogen source culture media. While ensuring the conversion rate of 5-hydroxymethylfurfural and the yield of 5-hydroxymethyl-2-furancarboxylic acid, the overall catalytic cost is greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the growth curve of the cells in Example 2 when they were expanded and cultured in the acid hydrolyzate medium of Gelidium amansii.

[0031] Figure 2 This is the process curve of Pseudomonas hallii oxidizing 5-hydroxymethylfurfural in Example 3.

[0032] Figure 3 This is the process curve of Pseudomonas hallii oxidizing 5-hydroxymethylfurfural in Comparative Example 1. DETAILED DESCRIPTION

[0033] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0034] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0035] Detection method of 5-hydroxymethylfurfural and 5-hydroxymethyl-2-furancarboxylic acid: The content was determined by high performance liquid chromatography (Agilent 1200). The chromatographic column was Bio-Rad Aminex HPX-87H (7.8 mm × 300 mm), the column temperature was 55°C, the detection was carried out by a differential refractive index detector, 5 mM H2SO4 was used as the mobile phase, and the flow rate was 0.6 mL / min.

[0036]

[0037]

[0038] The theoretical yield of 5-hydroxymethyl-2-furancarboxylic acid is the yield obtained when all 5-hydroxymethylfurfural in the reaction system is converted into 5-hydroxymethyl-2-furancarboxylic acid.

[0039] Example 1: Preparation of Agar-agar Acid Hydrolyzate

[0040] Wash the commercially available agar with tap water, chop it with scissors, and lay it flat to dry. Use a micro-plant grinder to crush the dried agar. Weigh 1 gram of the crushed agar with 10 mL of 2% oxalic acid solution, mix them evenly, place them in a pressure-resistant reaction tube, seal them, and keep them warm at 120°C for 40 minutes. After the reaction is completed, separate the solid and liquid to obtain the agar acid hydrolyzate.

[0041] Example 2: Biocatalyst preparation

[0042] The agar hydrolyzate obtained in Example 1 was measured, and calcium oxide solid was added at a ratio of 10 g calcium oxide per liter of agar hydrolyzate. After oscillating for 1.5 hours, the precipitate was removed by centrifugation, and then the pH of the supernatant was adjusted to 7.0 with sodium hydroxide solution, and the precipitate was removed by centrifugation again.

[0043] The agar-agar acid hydrolysate culture medium was prepared according to the following formula: 100 mL of agar-agar acid hydrolysate after pH adjustment, 5 g of corn steep liquor powder, 0.5 g of Na2HPO4, and 0.2 g of KH2PO4 in 1 L of distilled water. The solvent was water, and the pH value was 7.0.

[0044] According to the inoculation amount of 1% by volume, Pseudomonas hallii NL2019 (CCTCC NO: M 2021356) was inoculated from the glycerol tube storing the strain into GMM medium (the formula is that 1L distilled water contains: 5g galactose, 3.5g Na2HPO4, 1.5g KH2PO4, 0.5g NH4Cl, 0.25g NaCl, 0.24g MgSO4, 2.5mL trace element solution, the solvent is water, pH 7.0. The formula of the trace element solution is that 1L distilled water contains: 0.3g H3BO3, 0.05g ZnCl2, 0.03g MnCl2·4H2O, 0.2g CoCl2, 0.01g CuCl2·2H2O, 0.02g NiCl2·6H2O, 0.03g NaMoO4·2H2O 0.03g), cultured at 30°C, 200 rpm for 10h to activate the bacteria; the activated bacterial solution was transferred to the previously prepared agar acid hydrolyzate medium at a volume percentage of 2% to expand the culture, and cultured at 30°C, 200 rpm for 12h ( Figure 1 ), collect the bacterial cells by centrifugation, wash the bacterial cells twice with physiological saline, and separate the intact microbial cells by centrifugation as biocatalysts.

[0045] Example 3: Conversion of 5-hydroxymethylfurfural (HMF)

[0046] The biocatalyst collected in Example 2 was mixed with a phosphate buffer solution (100 mM, pH 7.0) containing HMF, so that the concentration of HMF in the mixture was 25 g / L, the dry weight concentration of the biocatalyst was 10 g / L, and light calcium carbonate powder was added to make its concentration 10 g / L, and the reaction was carried out at 30°C and 200 rpm to convert 5-hydroxymethylfurfural into 5-hydroxymethyl-2-furancarboxylic acid, and samples were taken at intervals of 1 hour to detect the concentration changes of 5-hydroxymethylfurfural and 5-hydroxymethyl-2-furancarboxylic acid. The data showed that after 5 hours of reaction, the conversion rate of 5-hydroxymethylfurfural was 98.6%, and the yield of the target product 5-hydroxymethyl-2-furancarboxylic acid was 92.6% ( Figure 2 ).

[0047] Example 4: Biocatalyst preparation and conversion of 5-hydroxymethylfurfural

[0048] The biocatalyst was prepared according to the method of Example 2, and only the formula of the agar agar acid hydrolyzate medium was adjusted (inorganic salts were increased and organic nitrogen sources were reduced). The agar agar acid hydrolyzate medium was prepared according to the following formula: 100 mL of agar agar acid hydrolyzate after pH adjustment, 2 g of corn syrup dry powder, 2.0 g of Na2HPO4, 1.0 g of KH2PO4, 0.05 g of NaCl, 0.1 g of MgSO4, and 1.0 mL of trace element solution in 1 L of distilled water, and the solvent was water, pH 7.0. The formula of the trace element solution was: 0.3 g of H3BO3, 0.05 g of ZnCl2, 0.03 g of MnCl2·4H2O, 0.2 g of CoCl2, 0.01 g of CuCl2·2H2O, 0.02 g of NiCl2·6H2O, and 0.03 g of NaMoO4·2H2O in 1 L of distilled water. The cell growth at this time was similar to that in Example 2. Then, HMF was converted according to the method of Example 3. The results showed that the catalytic ability of the biocatalyst cultured under such conditions was consistent with that of the biocatalyst in Example 2.

[0049] Comparative Example 1:

[0050] The biocatalyst was prepared according to the method of Example 2, wherein the culture medium used for cell expansion culture was not a Gelidium agar hydrolysate culture medium, but a LB culture medium commonly used in the literature (components: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, solvent is water, pH 7.0). At this time, the cell growth was slightly better than that in Example 2. Then, HMF was converted according to the method of Example 3. The results showed that the catalytic ability of the biocatalyst cultured in LB culture medium was significantly lower than that of the cell catalyst cultured in the culture medium in Example 2. After 5 hours of reaction, the conversion rate of 5-hydroxymethylfurfural was 69.0%, and the yield of the target product 5-hydroxymethyl-2-furancarboxylic acid was 64.9% ( Figure 3).

[0051] Comparative Example 2:

[0052] The biocatalyst was prepared according to the method of Example 2, wherein the culture medium used for cell expansion culture did not use agar-agar acid hydrolysate culture medium, and galactose was used as the carbon source instead of agar-agar acid hydrolysate. The agar-agar acid hydrolysate culture medium was prepared according to the following formula, 1L of distilled water contained: galactose 5g, corn syrup dry powder 5g, Na2HPO4 0.5g, KH2PO4 0.2g, the solvent was water, pH 7.0. At this time, the cell growth was similar to that in Example 2. Then, HMF was converted according to the method of Example 3. The results showed that the catalytic ability of the biocatalyst cultured in this culture medium was significantly lower than that of the cell catalyst cultured in the culture medium in Example 2, which was consistent with the catalytic ability of the biocatalyst in Comparative Example 1.

[0053] Comparative Example 3:

[0054] The biocatalyst was prepared according to the method of Example 2, wherein the medium used for cell expansion culture was not a Gelidium agar hydrolyzate medium, but a commercial carbon source and organic nitrogen source (the formula was: 1L distilled water containing: 5g / L glucose, 5g / L yeast powder, the solvent was water, pH 7.0). At this time, the cell growth was similar to that in Example 2. Then, HMF was converted according to the method of Example 3. The results showed that the catalytic ability of the biocatalyst cultured in this medium was significantly lower than that of the cell catalyst cultured in the medium in Example 2, and was consistent with the catalytic ability of the biocatalyst in Comparative Example 1.

[0055] The reagent prices in the above examples and comparative examples are estimated according to the following standards:

[0056] Agar: 50 yuan / kg; oxalic acid: 46 yuan / kg; corn steep liquor powder: 45 yuan / kg; Na2HPO4: 160 yuan / kg; KH2PO4: 180 yuan / kg; trypsin: 900 yuan / kg; yeast powder: 500 yuan / kg; sodium chloride: 28 yuan / kg; galactose: 1500 yuan / kg; glucose: 52 yuan / kg. The specific culture cost is shown in Table 1 below. It can be seen that the cost of Agar acid hydrolyzate culture medium is much lower than that of other culture media (the cost is rounded to one decimal place).

[0057] Table 1 Cost accounting table

[0058]

[0059] In summary, the present invention uses agar-agar acid hydrolysate as a carbon source, and a small amount of cheap organic nitrogen source and inorganic salt as a culture medium to culture Pseudomonas hallii. The catalytic ability of the obtained cells to oxidize 5-hydroxymethylfurfural is much higher than that of cells cultured using expensive carbon sources and expensive organic nitrogen source culture medium. While ensuring the conversion rate of 5-hydroxymethylfurfural and the yield of 5-hydroxymethyl-2-furancarboxylic acid, the overall catalytic cost is greatly reduced.

[0060] The present invention provides a method for cultivating Pseudomonas holmesii to improve its 5-hydroxymethylfurfural oxidation ability. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for improving the 5-hydroxymethylfurfural oxidation ability of Pseudomonas hallii, characterized in that: The steps include: (1) crushing the agar-agar and mixing it with an organic acid solution, reacting it at high temperature, and separating the solid and liquid after the reaction to obtain the agar-agar acid hydrolyzate; (2) adjusting the pH of the agar-agar acid hydrolyzate obtained in step (1) to neutral, using the agar-agar acid hydrolyzate after pH adjustment as a carbon source, compounding with an organic nitrogen source and an inorganic salt, to prepare agar-agar acid hydrolyzate culture medium; (3) inoculating Pseudomonas hallii into GMM culture medium and culturing to obtain an activated solution; transferring the activated solution to the agar acid hydrolyzate culture medium prepared in step (2) for further expansion and culturing, collecting the cells and washing them with physiological saline, and obtaining a biocatalyst by solid-liquid separation; (4) catalyzing 5-hydroxymethylfurfural with the biocatalyst prepared in step (3); after the catalytic reaction is completed, collecting the supernatant by solid-liquid separation to obtain 5-hydroxymethyl-2-furancarboxylic acid; Wherein, in step (2), the agar-agar acid hydrolyzate culture medium has a formula of: 1L of distilled water contains: 50-200 mL of agar-agar acid hydrolyzate after pH adjustment, 2-5 g of an organic nitrogen source, 0.5-3 g of Na2HPO4, 0.2-1.5 g of KH2PO4, 0-0.5 g of NH4Cl, 0-0.25 g of NaCl, 0-0.2 g of MgSO4, and 0-2 mL of a trace element solution, the solvent is water, and the pH is 6.0-7.5; Wherein, in step (2), the pH of the agar-agar acid hydrolyzate obtained in step (1) is adjusted to neutral, and the neutralizing agents used are calcium oxide and sodium hydroxide, and the two neutralizing agents are added in sequence; Wherein, in step (2), the organic nitrogen source is any one or a combination of corn steep liquor powder, peanut cake powder, and soybean cake powder; Wherein, in step (3), the Pseudomonas hallii is Pseudomonas hallii NL2019, and its deposit number is CCTCCNO: M 2021356; Wherein, in step (4), 5-hydroxymethylfurfural is catalyzed by the biocatalyst prepared in step (3), wherein the biocatalyst has a dry weight concentration of 5-10 g / L; and the 5-hydroxymethylfurfural has a concentration of 2-40 g / L; Wherein, in step (4), the catalytic reaction has the following reaction conditions: reaction temperature is 25°C to 45°C, reaction pH is 5.5 to 8.0, and reaction time is 0.5 to 10 hours.

2. The culture method according to claim 1, characterized in that In step (1), the organic acid is any one of oxalic acid and citric acid, wherein the organic acid solution is a 1wt% to 10wt% organic acid aqueous solution, and the solid-liquid ratio of agar-agar to the organic acid solution is 1:5 to 1:20 g / mL.

3. The culture method according to claim 1, characterized in that In step (1), the high temperature reaction is carried out under the following reaction conditions: the reaction temperature is 110°C to 140°C, and the reaction time is 15 min to 90 min.

4. The culture method according to claim 1, characterized in that The formula of the trace element solution is that 1L of distilled water contains: H3BO3 0.1~0.5 g, ZnCl2 0.01~0.1 g, MnCl2·4H2O 0.01~0.05 g, CoCl2 0.1~0.5 g, CuCl2·2H2O 0.01~0.5 g, NiCl2·6H2O 0.01~0.1 g, and NaMoO4·2H2O 0.01~0.05 g.

5. The culture method according to claim 1, characterized in that In step (3), when the activation solution is transferred to the agar-agar acid hydrolyzate culture medium prepared in step (2) for further culture expansion, the transfer amount is 1% to 10% by volume.

6. The culture method according to claim 1, characterized in that In step (3), the activation culture and expansion culture are carried out under the following culture conditions: temperature 25-35°C, culture time 8-14 hours.

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