A xylene monooxygenase-producing strain Arthrobacter woluwensis and its application

Abstract

The present invention belongs to microbial technology field and relates to a strain producing toluene o-xylene monooxygenase (Arthrobacter woluwensis) HW-1 and its application in preparation of 5-methylpyrazine-2-carboxylic acid by microbial fermentation. The present invention providing a new strain HW-1 which could produce toluene o-xylene monooxygenase, and the strain is identified as Arthrobacter woluwensis. The strain is firstly found to convert 2, 5-dimethylpyrazine by bio-fermentation to obtain the medicine intermediate 5-methylpyrazine-2-carboxylic acid. The concentration of accumulated product could reach 34.19 g/L and the yield rate is 81.4% by shake flask fermentation. Compared with 20.41 g/L reported in the literature, this method has a greater advantage and could be industrialized. The conditions to prepare 5-methylpyrazine-2-carboxylic acid provided in the present invention is mild, the reaction process is controllable and has good performance in environmental protection and energy saving. So this invention has great value in industry.

Claims

1. A xylene monooxygenase-producing strain Arthrobacter woluwensis HW-1 deposited at China General Microbiological Culture Collection Center with access number CGMCC 12833.

2-6. (canceled)

7. A method of preparing 5-methylpyrazine-2-carboxylic acid comprising subjecting 2-5-dimethylpyrazine as a starting material to fermentation reaction in the presence of the strain Arthrobacter woluwensis HW-1 of claim 1 as a catalyst.

8. The method of claim 7 wherein the fermentation reaction is carried out at 2040 C., pH 5.08.0.

9. The method of claim 8 wherein the pH value is maintained between 6.0 and 7.0 during the fermentation reaction.

10. The method of claim 7 wherein additional 2-5-dimethylpyrazine is fed during the fermentation reaction by fed-batch to increase concentration of 5-methylpyrazine-2-carboxylic acid.

11. The method of claim 10 wherein the additional 2-5-dimethylpyrazine is fed into fermentation when the staring material 2-5-dimethylpyrazine has a conversion rate of greater than 50%.

12. The method of claim 7 comprising preparing a seed liquid of the strain Arthrobacter woluwensis HW-1.

13. The method of claim 12 wherein the preparing of the seed liquid of the strain Arthrobacter woluwensis HW-1 comprises: inoculating the strain Arthrobacter woluwensis HW-1 into a seed medium comprising 10 g/L of peptone, 5 g/L of yeast extract, 10 g/L of NaCl, and water as a solvent, and culturing the strain Arthrobacter woluwensis HW-1 in a shake flask under conditions of 100180 r.Math.min.sup.1, 2040 C. for 1224 hours.

14. The method of claim 12 comprising inoculating the seed liquid of the strain Arthrobacter woluwensis HW-1 in a fermentation medium with 15% of vaccination quantity and culturing the strain Arthrobacter woluwensis HW-1 in a shake flask in the presence of xylene as an inducer for 1028 hours under conditions of 100180 r.Math.min1, pH 6.08.0, 2040 C., wherein the fermentation medium comprises: yeast extract 0.52.0 g/L; peptone 1.04.0 g/L; (NH.sub.4).sub.2SO.sub.4 1.03.0 g/L; NaHCO.sub.3 1.05.0 g/L; KH.sub.2PO.sub.4 0.54.0 g/L; NaCl 1.05.0 g/L; MgCl.sub.2 0.10.5 g/L; CaCl.sub.2 1.05.0 g/L; FeCl.sub.3 0.050.3 g/L; ZnSO.sub.4 0.020.1 g/L; MnCl.sub.2 0.050.1 g/L; CuCl.sub.2 0.050.02 g/L; NiCl.sub.2 0.010.03 g/L; EDTA.Na.sub.2.2H.sub.2O 1.05.0 g/L; FeSO.sub.4 1.03.0 g/L; xylene 0.1-2 mL/L, and water as a solvent.

15. The method of claim 14 comprising adding 2, 5-dimethylpyrazine into the fermentation medium at an initial concentration of 0.5 to 2 mL/L.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1: Agarose gel electrophoresis of the 16S rDNA sequence by PCR amplification of strain HW-1;

[0031] FIG. 2: HPLC chromatogram of the fermentation broth

[0032] FIG. 3: Accumulation concentration of 5-methylpyrazine-2-carboxylic acid during the fermentation process;

[0033] FIG. 4: Effect of initial pH on fermentation and biotransformation;

[0034] FIG. 5: Effect of the amount of substrate on the transformation (.diamond-solid. represents conversion rate, .box-tangle-solidup. represents accumulation concentration of 5-methylpyrazine-2-carboxylic acid;

[0035] FIG. 6: Effect of substrate addition time on transformation;

[0036] FIG. 7: Effect of fermentation and conversion temperature on transformation.

DESCRIPTION OF EMBODIMENTS

Example 1: Microbial Screening

[0037] Seed culture medium: 5.0 g/L of yeast extract, 10.0 g/L of peptone, 10.0 g/L of NaCl, and the solvent is water and the pH was 7.0.

[0038] Enrichment and screening of medium components: yeast extract 1.5 g/L; peptone 2 g/L; (NH.sub.4).sub.2SO.sub.4 2 g/L; NaHCO.sub.3 1.5 g/L; KH.sub.2PO.sub.4 2 g/L; NaCl 2 g/L; MgCl.sub.2 0.3 g/L; CaCl.sub.2 1.2 g/L; FeCl.sub.3 0.1 g/L; ZnSO.sub.4 0.05 g/L; MnCl.sub.2 0.06 g/L; CuCl.sub.2 0.06 g/L; NiCl.sub.2 0.02 g/L; EDTA.Na.sub.2.2H.sub.2O 2 g/L; FeSO.sub.4 1.03.0 g/L; xylene was added in an amount of 0.1 mL/L, the solvent is water.

[0039] Soil sampling: Soil samples were collected under 515 cm deep near the sewage outlet of chemical plant, and acquisition time and place were recorded in details. A total of 100 more soil samples were collected from Weihai, Yantai and Qingdao for screening of objective strains, the differences in geographical distribution of the soil samples could bring more successful opportunities for strains filtration.

[0040] 1 g of soil samples were added into flask and cultured for 40 minutes with addition of 20 mL of saline and glass beads. 0.5 mL of soil suspension was added into the enrichment medium and incubated for 36 h in shaker at 30 C. and 150 r.Math.min.sup.1. This operation was repeated three times. 0.1 mL of the culture liquid was coated on the solid plate for isolation and growing into single colony at 28 C. The components of solid plate culture medium were as follows: yeast extracts 5.0 g/L, peptone 10.0 g/L, NaCl 5.0 g/L, agar 20 g/L, pH 7.0, and the solvent was water. Single colony was then taken into seed culture medium, and cultured in 150 r.Math.min.sup.1 shaker at 28 C. for 16 hours to obtain the seed liquid of pure strain. The seed liquid was inoculated into the fermentation medium with 2% vaccination quantity, and incubated in 150 r.Math.min.sup.1 shaker for 16 h at 28 C. Adding 0.20 g of 2, 5-dimethylpyrazine into fermented liquor after the screened strain fermented for 12 hours. Fermentation liquor was detected by using high-performance liquid chromatography (Column: C.sub.18 column, acetonitrile:water:trifluoroacetic acid=12:88:0.5, flow rate 1 mL/min, detection wavelength 270 nm, column temperature 25 C.) after fermented for another 36 hours. FIG. 2 is the HPLC chromatogram. According to the detection result, HW-1 strain with the highest enzyme activity (Deposit No. CGMCC12833) was obtained.

Example 2: Fermentation Process

[0041] The single colony was inoculated into the seed medium and cultured in 150 r.Math.min.sup.1 shaker for 16 h at 28 C. to obtain the seed liquid of the pure strain. The period of 3 to 16 hours among culture process was the logarithmic period, so this period was the best time to inoculate.

[0042] The seed liquid was inoculated into the fermentation medium with 2% vaccination quantity, and incubated in 150 r.Math.min.sup.1 shaker for 12 h at 28 C. Then 0.20 g of 2, 5-dimethylpyrazine was added into fermentation liquor. The pH of the fermentation liquor was decreasing during the fermentation process and the pH was reduced to 5.1 at 46 hours, and the rate of fermentation was decreased too. During the fermentation process, the concentration of the product was continuously accumulated, and the specific trend was shown in FIG. 3.

Example 3: Effect of Initial pH on Fermentation and Biotransformation

[0043] Fermentation mediums were prepared with different initial pH which were 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, respectively. The seed liquid was inoculated into the fermentation medium with 2% vaccination quantity, and incubated in 150 r.Math.min.sup.1 shaker for 12 h at 28 C. Then 0.20 g of 2, 5-dimethylpyrazine was added into fermentation liquor. Fermentation liquor was detected after cultured for another 36 hours. The results were shown in FIG. 4. Too high or low pH was bad for the normal growth and metabolism of strains, and also affected the fermentation conversion efficiency. A pH around 7.0 was the best for enhancing the conversion activity and efficiency.

Example 4: Effect of Substrate Amount on Fermentation and Biotransformation

[0044] The seed liquid was inoculated into 100 mL fermentation medium with 2% vaccination quantity, and incubated in 150 r.Math.min.sup.1 shaker for 12 h at 28 C. Then 2, 5-dimethylpyrazine was added into fermentation liquor. The additions were 0.05 g, 0.10 g, 0.15 g, 0.20 g and 0.25 g, respectively. Centrifugal fermentation liquor was detected by HPLC to analyze the concentration of 5-methylpyrazine-2-carboxylic acid after cultured for another 36 hours. The results were shown in FIG. 5. The conversion rate was decreased with the addition of substrate 2, 5-dimethylpyrazine increasing, while the concentration of 5-methylpyrazine-2-carboxylic acid in fermentation was increased. The amount of 2, 5-dimethylpyrazine exceeded 0.20 g, substrate inhibition was evident, and it led to the decrease of conversion efficiency and product concentration.

Example 5: Effects of Addition Time of Substrate on the Transformation

[0045] The seed liquid was inoculated into 100 mL fermentation medium with 2% vaccination quantity, and incubated in 150 r.Math.min.sup.1 shaker for 12 h at 28 C. Then 2, 5-dimethylpyrazine was added into fermentation liquor. The addition time were after 0 hour, 4 hours, 8 hours, 12 hours, 16 hours and 20 hours, respectively. Centrifugal fermentation liquor was detected by HPLC to analyze the concentration of 5-methylpyrazine-2-carboxylic acid after cultured for another 36 hours. The results were shown in FIG. 6. The suitable substrate addition time was 12 to 16 hours.

Example 6: Effect of Fermentation and Conversion Temperature on Transformation

[0046] The seed liquid was inoculated into 100 mL fermentation medium and incubated in 150 r.Math.min.sup.1 shaker for 12 h. Then 0.20 g of 2, 5-dimethylpyrazine was added into fermentation liquor. The fermentation temperatures were 15 C., 20 C., 25 C., 30 C., 35 C., 40 C. and 45 C., respectively. Centrifugal fermentation liquor was detected by HPLC to analyze the concentration of 5-methylpyrazine-2-carboxylic acid after cultured for another 36 hours. The results were shown in FIG. 7. The suitable substrate fermentation temperature was between 25 C. to 35 C.

Example 7: Additional Substrate Conversion

[0047] The seed liquid was inoculated into 100 mL fermentation medium and incubated in 150 r.Math.min.sup.1 shaker for 12 h at 28 C. Then 0.20 g of 2, 5-dimethylpyrazine was added into the fermentation liquor. According to the product and substrate concentration, 0.20 g of substrate 2, 5-dimethylpyrazine was added into the fermentation after a period of transformation. The transformation process took 372 hours and needed 21 substrate additions. The concentration of 5-methylpyrazine-2-carboxylic acid reached 34.19 g/L, and the yield was 81.4%.

INDUSTRY PRACTICAL APPLICABILITY

[0048] A new strain which could produce xylene monooxygenase was screened, and identified as Arthrobacter woluwensis. This strain could be used to convert 2,5-dimethylpyrazine by bio-fermentation to obtain the pharmaceutical intermediate, 5-methylpyrazine-2-carboxylic acid. The concentration of the accumulated product could reach 34.19 g/L, and the yield rate was 81.4%. Compared with 20.41 g/L reported in the literature, this method has a greater advantage and could be industrialized. This strain could convert 2,5-dimethylpyrazine to 5-methylpyrazine-2-carboxylic continuously and stably, and the reaction condition is mild, and the reaction process is controllable. Compared with the traditional chemical method, this method has the advantages of high specificity, low by-product, high yield, low energy consumption, environmental protection and energy saving. So this invention has great value in industry.

TABLE-US-00001 Genesequencetable CAGTCGAACGATGAAGCCTAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAACACG 060 TGAGTAACCTGCCCTTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATACG 120 ACCATTGCCCGCATGGGTTGGTGGTGGAAAGCTTTTGTGGTTTTGGATGGACTCGCGGCC 180 TATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAG 240 GGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG 300 GAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTT 360 CGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGC 420 GCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAAT 480 TATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAAGGCCAGGGCTCA 540 ACCCTGGTTCTGCAGTGGGTACGGGCAGACTTGAGTGATGTAGGGGAGACTGGAATTCCT 600 GGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTG 660 GGCATTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGT 720 AGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGT 780 AGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAA 840 TTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAAC 900 CTTACCAAGGCTTGACATGGACTGGATCGCATCAGAGATGGTGTTTCCCTTCGGGGCTGG 960 TTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC 1020 AACGAGCGCAACCCTCGTTCCATGTTGCCAGCGCGTAATGGCGGGGACTCATGGGAGACT 1080 GCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTT 1140 GGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGTTGCGATACTGTGAGGTGGAGCTA 1200 ATCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTTGGA 1260 GTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACA 1320 CCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGGTGGC 1365