Lysinibacillus sp. And application thereof in catalytic synthesis of 5-hydroxymethyl furoic acid

Abstract

The invention discloses lysinibacillus fusiformis, which is classified and named as lysinibacillus fusiformis B2P, is preserved in the China Center for Type Culture Collection on November 10, 2020, and has the preservation number of CCTCC NO: M 2020723. The invention also discloses an application of the Lysinibacillus sp. B2P in catalytic synthesis of 5-hydroxymethyl furoic acid, the strain has high tolerance to HMF, can be used as a biocatalyst to selectively convert HMF into a target product HMFCA, and the yield is high. By adopting the lysinibacillus sp. B2P disclosed by the invention, not only can higher substrate concentration and higher reaction efficiency than those of a previously reported biological catalysis method be realized, but also the selectivity is improved. In addition, mild conditions are convenient to control, and subsequent separation and purification of a target product are simplified.

Description

Technical Field

[0001] The invention relates to a lysine bacillus and application thereof in catalytic synthesis of 5-hydroxymethyl furoic acid, belonging to the field of biocatalysis and chemical engineering. Background Art

[0002] In the 21st century, as green energy gradually replaces petroleum resources and the chemical industry transforms to environmentally friendly alternatives, the trend of pursuing sustainable development is increasing. Biomass energy and biotechnology are particularly important in the context of resource and environmental challenges. 5-Hydroxymethylfurfural (HMF) has become an important raw material for the production of a variety of high-end liquid fuels and chemical products due to the active aldehyde, hydroxymethyl and furan ring functional groups contained in its molecules. It is regarded as one of the bio-based platform chemicals that replaces traditional petrochemical industry basic chemicals. The furan ring of HMF carries aldehyde and hydroxymethyl groups that are easily catalyzed, and a variety of furan aromatic compounds can be generated through catalytic oxidation reactions. Depending on the oxidation position and degree, it can be oxidized to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 2,5-furandicarboxaldehyde (DFF), 5-formyl-2-furancarboxylic acid (FFCA), 2,5-furandicarboxylic acid (FDCA), and when the aldehyde group on the ring is reduced to a hydroxyl group, the reduction product of HMF, 2,5-furan dimethanol (BHMF), is obtained. As key intermediates, these redox products are widely used in agriculture, energy, medicine, polymer materials and other fields. For example, DFF can be used as a precursor for the preparation of polymer materials, organic conductors, luminophores, bactericides and drugs, and is also used to synthesize new biomass-based resins, fluorescent materials and polyesters. HMFCA, due to its carboxyl and hydroxymethyl properties, can be used for self-polymerization or copolymerization with other compounds to form a variety of polyesters, and can also be used as an interleukin inhibitor.

[0003] At present, the main ways to convert HMF to HMFCA include chemical conversion and biological conversion. Although the chemical method of HMF oxidation using metal catalysts has made some progress, there are problems such as harsh reaction conditions, low product selectivity and poor substrate tolerance. In contrast, biocatalytic oxidation, as a mild conversion method, uses biocatalysts to convert HMF to HMFCA under milder conditions, with the advantages of high yield, high selectivity and low by-product production. However, HMF has certain toxicity to microbial cells, and most microorganisms have low tolerance to HMF and poor utilization efficiency. Therefore, screening out new microbial strains with high tolerance to HMF to achieve effective and highly selective oxidation of HMF to HMFCA has become an urgent problem that needs to be solved. Summary of the invention

[0004] The object of the present invention is to provide a lysinic Bacillus, which can be used as a catalyst to efficiently catalyze the selective oxidation of HMF to synthesize HMFCA, in order to solve the problems existing in the prior art.

[0005] The technical solution of the present invention is as follows:

[0006] A lysinibacillus strain, whose classification name is Lysinibacillus fusiformis B2P, has been deposited in the China Center for Type Culture Collection on November 10, 2020, with a deposit number of CCTCC No: M2020723, and the deposit address is Wuhan University, Wuhan, China. The strain was isolated and screened from a soil sample contaminated by nuclear radiation. After microbial classification and identification, it was determined that the strain belongs to a new species of Lysinibacillus. Studies have shown that the strain has a strong tolerance to aldehyde compounds.

[0007] The application of the above-mentioned Bacillus lysinic acid B2P in catalytic synthesis of 5-hydroxymethyl furoic acid comprises the following steps:

[0008] (1) Bacillus lysininus B2P was inoculated into TB medium for activation, and then inoculated into fresh TB medium at a 1% inoculum volume for cultivation, and bacterial cells were collected;

[0009] (2) dissolving 5-hydroxymethylfurfural in a buffer solution with a pH of 5.0 to 10.0, adding the bacterial cells obtained in step (1), and reacting at a temperature of 15 to 45° C. to obtain 5-hydroxymethylfuronic acid.

[0010] In step (2), the buffer is any one of phosphate buffer, Tris-HCl buffer or Gly-NaOH buffer.

[0011] In step (1), the preparation method of the TB culture medium is as follows: 24g yeast extract, 12g peptone and 4mL glycerol are mixed and dissolved in deionized water, and the volume is fixed to 900mL with deionized water to obtain solution A; 2.31g KH2PO4 and 12.54g K2HPO4 are dissolved in 90mL deionized water, and after complete dissolution, the volume is fixed to 100mL with deionized water to obtain solution B; solution A and solution B are steam sterilized at a high pressure of 15psi for 20min, and when solution A is cooled to below 60°C, solution B is added and mixed evenly to obtain solution B.

[0012] Furthermore, in step (1), the activation temperature is 30° C., the rotation speed is 200 r / min, and the activation time is 24 h.

[0013] Furthermore, in step (1), the culture temperature is 30°C, the rotation speed is 200r / min, and the time is 24h.

[0014] Furthermore, in step (2), the amount of bacterial cells added is 20 to 200 mg / mL.

[0015] Furthermore, in step (2), the concentration of 5-hydroxymethylfurfural in the buffer solution is 50 to 500 mM.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention discloses a lysinic Bacillus B2P, which has a high tolerance to HMF and can be used as a biocatalyst to selectively convert HMF into a target product HMFCA with a yield of up to 86%. The use of the lysinic Bacillus B2P of the present invention can not only achieve a higher substrate concentration and a higher reaction efficiency than previously reported biocatalytic methods, but also improve selectivity. In addition, its mild conditions are easy to control, simplifying the subsequent separation and purification of the target product. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the colony morphology of Bacillus lysinicus B2P;

[0018] Figure 2 It is the liquid chromatogram of the synthetic product in Example 2. DETAILED DESCRIPTION

[0019] The technical scheme of the present invention is further explained below through the accompanying drawings and specific embodiments. The listed embodiments are only for illustrative purposes and show that the spirit and scope of the present invention are not limited to the details and modifications thereof.

[0020] In the following examples, the preparation method of the TB medium used is as follows: the preparation method of the TB medium is as follows: 24g yeast extract, 12g peptone and 4mL glycerol are mixed and dissolved in deionized water, and the volume is fixed to 900mL with deionized water to obtain solution A; 2.31g KH2PO4 and 12.54g K2HPO4 are dissolved in 90mL deionized water, and after complete dissolution, the volume is fixed to 100mL with deionized water to obtain solution B; solution A and solution B are mixed and heated at 15psi (1.05kg / cm 2 ) Steam sterilize under high pressure for 20 minutes. When solution A cools to below 60°C, add solution B and mix well.

[0021] Example 1 Isolation, screening and identification of Lysinibacillus B2P

[0022] (1) Isolation and screening of strains:

[0023] Collect samples from soil contaminated by nuclear radiation, take 10g of soil sample and add 90mL of sterile water to shake thoroughly, mix evenly, and let it stand to settle to obtain soil leaching solution. Inoculate 3mL of soil leaching solution into 50mL of LB medium containing 30mM HMF. The purpose of adding HMF to the medium is to promote the growth of target microorganisms and inhibit the growth of other microorganisms. After shaking and culturing at 30℃ and 200rpm for 48h, take 0.5mL of the original culture solution and inoculate it into 50mL of LB medium containing 40mM HMF, and shake and culture at 30℃ and 200rpm for 48h; take the diluted culture solution, subculture it for many times, and increase the HMF concentration in the LB medium at the same time, observe the turbidity of each culture solution, and isolate the strain that can still grow normally in the LB medium containing 80mM HMF (named B2P). Prepare LB solid medium containing 40mM HMF, use a disposable culture dish to pour the plate, let it stand on the clean bench overnight, and use it after confirming sterility. Dilute the bacterial solution in a gradient and apply it to the plate. Place in a 28°C constant temperature incubator and culture until a single colony grows. Use the glycerol tube method to preserve the B2P strain.

[0024] Qualitative screening of the ability to convert HMF:

[0025] The preserved strain was inoculated into 50 mL of LB medium without HMF, activated at 30°C and 200 rpm for 24 h, then transferred to 100 mL of LB medium containing 80 mM HMF at a 1% amount, and cultured in a shake flask at 30°C and 200 rpm for 24 h, and the medium gradually became turbid; the bacterial liquid was collected, centrifuged at 8000 rpm for 4 min, the supernatant was discarded, phosphate buffer (100 mM, pH 7.4) without HMF was added to wash the cells, and the washed cells were added to 5 mL of phosphate buffer (100 mM, pH 7.4) containing 100 mM HMF, shaken evenly, reacted at 30°C and 200 rpm for 10 h, sampled every 1 h, and centrifuged at 8000 rpm for 4 min. It can be observed that the supernatant gradually changes from slightly brown to light yellow.

[0026] Quantitative screening of strains for their ability to convert HMF:

[0027] The preserved strain was inoculated into 50 mL of LB medium without HMF, activated at 30°C and 200 rpm for 24 h, then transferred to 100 mL of LB medium without HMF at 1%, cultured in a shake flask at 30°C and 200 rpm for 24 h, the bacterial liquid was collected, centrifuged at 8000 rpm for 4 min, the supernatant was discarded, phosphate buffer (100 mM, pH 7.4) without HMF was added to wash the cells, 5 mL of phosphate buffer (100 mM, pH 7.4) containing 100 mM HMF was added to the washed cells, shaken evenly, reacted at 30°C and 200 rpm for 10 h, centrifuged at 12500 rpm for 10 min, the supernatant was filtered to remove impurities, a liquid sample was prepared, and the content of the product and substrate was detected by high performance liquid chromatography.

[0028] (2) Identification of strains

[0029] Total DNA was extracted from the B2P bacterial solution, and the variable region of the 16s rRNA gene was amplified by PCR using specific primers. The amplified products were sequenced, and the sequencing results were compared with the known 16s rRNA gene sequence database. The type and relationship of the microorganisms were determined by bioinformatics methods. After microbial classification and identification, it was determined that the strain belonged to a new species of Lysinibacillus (Lysinibacillus), named Lysinibacillus sp.B2P. The strain was deposited in the China Center for Type Culture Collection on November 10, 2020, with the deposit number CCTCC No: M 2020723, and the deposit address is Wuhan University, Wuhan, China.

[0030] Figure 1 The colony morphology of Bacillus lysinicus B2P is shown in Figure 1. Figure 1 It can be seen that the strain appears light yellow on the solid plate culture medium, the colonies are round, the edges are wavy, and the surface is smooth.

[0031] Example 2

[0032] The method for synthesizing 5-hydroxymethyl furoic acid by catalysis of Bacillus lysinicus B2P comprises the following steps:

[0033] (1) Bacillus lysinin B2P was inoculated into TB medium, activated at 30°C and 200 r / min for 24 h, then inoculated into fresh TB medium at a 1% inoculum amount, cultured at 30°C and 200 r / min for 24 h, and bacterial cells were collected;

[0034] (2) 0.1 mmol HMF (100 mM) was dissolved in 5 mL phosphate buffer (100 mM, pH 7.4), and the bacterial cells collected in step (1) were added at a concentration of 100 mg / mL (based on cell wet weight). The reaction was carried out at 20° C. and 800 r / min. The reaction was monitored by liquid chromatography. After 12 h, the synthetic product was obtained.

[0035] The synthetic product obtained in Example 2 was subjected to liquid chromatography detection. The method and conditions of liquid chromatography detection were as follows: the instrument was Thermo Fisher ultimate 3000, the detector was a UV detector; the detection wavelength was 230 nm; the chromatographic column was Sepax GP-C18 column (4.6 mm × 250 mm, 5 μm); the mobile phase was A: 20 mM KH2PO4; B: 100% acetonitrile; gradient elution (0 min: 10% B; 7 min: 24%; 10 min: 10% B); the flow rate was 1.0 mL min -1 ; Column temperature was 25°C; Injection volume was 5 μL.

[0036] Figure 2 : is the liquid chromatogram of the synthetic product obtained in Example 2. In the figure, the retention times of HMF, BHMF and HMFCA are 2.948, 5.133 and 6.08 min, respectively. The measured HMF conversion rate is 100% and the HMFCA yield is 86%.

[0037] Example 3

[0038] The reaction temperature in step (2) was changed to 15° C., and the rest was the same as in Example 2. After 10 h of reaction, the HMF conversion rate was measured to be 77%, and the HMFCA yield was 58%.

[0039] Example 4

[0040] The pH of the phosphate buffer in step (2) was changed from pH 7.4 to pH 7.0, and the reaction temperature was changed to 30° C., and the rest was the same as in Example 2. After 10 h of reaction, the HMF conversion rate was measured to be 100%, and the HMFCA yield was 67%.

[0041] Example 5

[0042] The pH of the phosphate buffer in step (2) was changed from pH 7.4 to pH 8.0, and the reaction temperature was changed to 30° C., and the rest was the same as in Example 2. After 10 h of reaction, the HMF conversion rate was measured to be 100%, and the HMFCA yield was 71%.

[0043] Example 6

[0044] The 5 mL phosphate buffer (100 mM, pH 7.4) in step (2) was replaced with 5 mL Gly-NaOH buffer (50 mM, pH 9.0), the reaction temperature was changed to 30° C., and the rest was the same as in Example 2. After 10 h of reaction, the HMF conversion rate was measured to be 100%, and the HMFCA yield was 50%.

[0045] Example 7

[0046] The 5 mL phosphate buffer (100 mM, pH 7.4) in step (2) was replaced with 5 mL Gly-NaOH buffer (50 mM, pH 10.0), the reaction temperature was changed to 30° C., and the rest was the same as in Example 2. After 10 h of reaction, the HMF conversion rate was measured to be 100%, and the HMFCA yield was 55%.

[0047] Example 8

[0048] The amount of bacterial cells added in step (2) was changed from 100 mg / mL to 80 mg / mL, and the rest was the same as in Example 2. After 10 hours of reaction, the HMF conversion rate was measured to be 100%, and the HMFCA yield was 63%.

[0049] Example 9

[0050] In step (2), 100 mM HMF was changed to 120 mM HMF, the amount of bacterial cells added was changed from 100 mg / mL to 200 mg / mL, the reaction temperature was changed to 30° C., and the rest was the same as in Example 1. After 24 hours, the HMF conversion rate was measured to be 100%, and the HMFCA yield was 79%.

[0051] Example 10

[0052] The 100 mM HMF in step (2) was changed to 150 mM HMF, the amount of bacterial cells added was 100 mg / mL, the reaction temperature was changed to 30° C., and the rest was the same as in Example 2. After 24 hours, the HMF conversion rate was measured to be 99%, and the HMFCA yield was 60%.

Claims

1. A lysinibacillus, classified and named Lysinibacillus fusiformis B2P. The strain was deposited in the China Center for Type Culture Collection on November 10, 2020, with the deposit number CCTCC No: M2020723, and the deposit address is Wuhan University, Wuhan, China.

2. Use of the lysinic Bacillus B2P according to claim 1 in catalyzing the synthesis of 5-hydroxymethyl furoic acid.

3. The use according to claim 2, It is characterized in that The application method includes the following steps: (1) Bacillus lysininus B2P was inoculated into TB medium for activation, and then inoculated into fresh TB medium at a 1% inoculum volume for cultivation, and bacterial cells were collected; (2) Dissolve 5-hydroxymethylfurfural in a solution of pH Add the bacterial cells obtained in step (1) into a buffer solution having a concentration of 5.0 to 10.0, and react at a temperature of 15 to 45° C. to obtain 5-hydroxymethyl furoic acid; In step (2), the buffer is any one of phosphate buffer, Tris-HCl buffer or Gly-NaOH buffer.

4. The use according to claim 3, It is characterized in that In step (1), the activation temperature is 30° C., the rotation speed is 200 r / min, and the activation time is 24 h.

5. The use according to claim 3, It is characterized in that In step (1), the culture temperature is 30°C, the rotation speed is 200r / min, and the time is 24h.

6. The use according to claim 3, It is characterized in that In step (2), the amount of bacterial cells added is 20 to 200 mg / mL.

7. The use according to claim 3, It is characterized in that In step (2), the concentration of 5-hydroxymethylfurfural in the buffer solution is 50-500 mM.

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