Burkholderia and applications thereof

Abstract

A strain of Burkholderia is Burkholderia glathei ECU0712, with an accession number of CGMCC NO. 14464. With the strain or its extract as the biocatalyst, thioether is catalyzed to be oxidized asymmetrically to chiral sulfoxide, with significant advantages that the obtained product has a high optical purity, and benefits of a simple reaction system, short preparation time of the catalyst and a high yield of the product.

Claims

1. An immobilized Burkholderia glathei extract from the, wherein, the Burkholderia glathei is Burkholderia glathei ECU0712, preserved in the China General Microbiological Culture Collection Center, with an accession number of CGMCC NO. 14464; the immobilized Burkholderia glathei extract is prepared by immobilizing a cell free extract, or a lyophilized product of the cell free extract of Burkholderia glathei ECU0712; the cell free extract is obtained by crushing and separating the Burkholderia glathei; and the immobilizing is performed with a physical process or a chemical process to make the cell free extract or the lyophilized product of the cell free extract nonsoluble in water, while still in a state of catalytic activity.

2. The immobilized Burkholderia glathei extract of claim 1, wherein, the cell free extract or the lyophilized product of the cell free extract is immobilized with polyethyleneimine (PEI) by a chemical process with glutaraldehyde as a crosslinking agent.

3. A method of preparing a chiral sulfoxide by an asymmetrical oxidation of a thioether, comprising a step of using an immobilized extract of the Burkholderia glathei as a catalyst for catalyzing the asymmetrical oxidation of the thioether to the chiral sulfoxide, wherein the Burkholderia glathei has an accession number of CGMCC NO. 14464, the extract is a cell free extract, or a lyophilized product of the cell free extract; and the cell free extract k obtained by crushing and separating the Burkholderia glathei.

4. The method of claim 3, wherein, the extract of the Burkholderia glathei is immobilized in polyethyleneimine with glutaraldehyde as a crosslinking agent.

5. The method of claim 4, wherein, the thioether is selected from the following compounds or pharmaceutically acceptable salts of the following compounds: ##STR00006## a first compound, having the structure of the formula I, wherein R.sub.1 is CH.sub.3O—, R.sub.2 is CH.sub.3—, R.sub.3 is CH.sub.3O—, and R.sub.4 is CH.sub.3—; a second compound, having the structure of the formula I, wherein R.sub.1 is H, R.sub.2 is CH.sub.3—, R.sub.3 is CF.sub.3CH.sub.2O—, and R.sub.4 is H; a third compound, having the structure of the formula I, wherein R.sub.1 is F.sub.2CHO—, R.sub.2 is CH.sub.3O—, R.sub.3 is CH.sub.3O—, and R.sub.4 is H; a fourth compound, having the structure of the formula I, R.sub.1 is H, R.sub.2 is CH.sub.3—, R.sub.3 is CH.sub.3—O—CH.sub.2—CH.sub.2—CH.sub.2O—, and R.sub.4 is H; and a fifth compound, having the structure of the formula I, R.sub.1 is ##STR00007## R.sub.2 is CH.sub.3—, R.sub.3 is CH.sub.3O—, and R.sub.4 is H.

6. The method of claim 5, wherein: when the thioether is the first compound or a pharmaceutically acceptable salt of the first compound, the chiral sulfoxide is 5-methoxy-2-((S)-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole or a pharmaceutically acceptable salt of 5-methoxy-2-((S)-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole; when the thioether is the third compound or a pharmaceutically acceptable salt of the third compound, the chiral sulfoxide is 5-difluoromethoxy-2-[[(S)-(3,4-dimethoxy-2-pyridyl)-methyl]sulfinyl]-1H-benzimidazole or a pharmaceutically acceptable salt of 5-difluoromethoxy-2-[[(S)-(3,4-dimethoxy-2-pyridyl)-methyl]sulfinyl]-1H-benzimidazole; when the thioether is the fifth compound or a pharmaceutically acceptable salt of the fifth compound, the chiral sulfoxide is 5-(1H-pyrrol-1-yl)-2-[(R)-[(4-methoxy-3-methyl-2-pyridyl)-methyl]sulfinyl]-1H-benzimidazole or a pharmaceutically acceptable salt of 5-(1H-pyrrol-1-yl)-2-[(R)-[(4-methoxy-3-methyl-2-pyridyl)-methyl]sulfinyl]-1H-benzimidazole.

7. The method of claim 3, further comprising: adding the Burkholderia glathei or the extract of the Burkholderia glathei into a reaction system together with the thioether.

8. The method of claim 7, wherein: (a) the asymmetrical oxidation is carried out in a buffer solution at pH 7.5-9.0; (b) a reaction temperature is 15-35° C.; (c) a reaction time is 4-48 hours; (d) a concentration of the thioether in the reaction system is 1-100/L; and (e) the thioether is dissolved in a cosolvent, the cosolvent is a water soluble organic solvent, and the cosolvent makes up 2-15% of the reaction system by volume.

9. The method of claim 8, wherein, the pH of the buffer solution is 8.0-9.0.

10. The method of claim 3, wherein, the thioether is selected from the following compounds or pharmaceutically acceptable salts of the following compounds: ##STR00008## a first compound, having the structure of the formula I, wherein R.sub.1 is CH.sub.3O—, R.sub.2 is CH.sub.3—, R.sub.3 is CH.sub.3O—, and R.sub.4 is CH.sub.3—; a second compound, having the structure of the formula I, wherein R.sub.1 is H, R.sub.2 is CH.sub.3—, R.sub.2 is CF.sub.3CH.sub.2O—, and R.sub.4 is H; a third compound, having the structure of the formula I, wherein R.sub.1 is F.sub.2CHO—, R.sub.2 is CH.sub.3O—, R.sub.3 is CH.sub.3O—, and R.sub.4 is H; a fourth compound, having the structure of the formula I, R.sub.1 is H, R.sub.2 is CH.sub.3—, R.sub.3 is CH.sub.3—O—CH.sub.2—CH.sub.2—CH.sub.2O—, and R.sub.4 is H; and a fifth compound, having the structure of the formula I, R.sub.1 is R.sub.2 is CH.sub.3—, ##STR00009## R.sub.3 is CH.sub.3O—, and R.sub.4 is H.

11. The method of claim 4, wherein, the thioether is selected from the following compounds or pharmaceutically acceptable salts of the following compounds: ##STR00010## a first compound, having the structure of the formula I, wherein R.sub.1 is CH.sub.3O—, R.sub.2 is CH.sub.3—, R.sub.3 is CH.sub.3O—, and R.sub.4 is CH.sub.3—; a second compound, having the structure of the formula I, wherein R.sub.1 is H, R.sub.2 is CH.sub.3—, R.sub.3 is CF.sub.3CH.sub.2O—, and R.sub.4 is H; a third compound, having the structure of the formula I, wherein R.sub.1 is F.sub.2CHO—, R.sub.2 is CH.sub.3O—, R.sub.3 is CH.sub.3O—, and R.sub.4 is H; a fourth compound, having the structure of the formula I, R.sub.1 is H, R.sub.2 is CH.sub.3—, R.sub.3 is CH.sub.3—O—CH.sub.2—CH.sub.2—CH.sub.2O—, and R.sub.4 is H; and a fifth compound, having the structure of the formula I, R.sub.1 is ##STR00011## R.sub.2 is CH.sub.3—, R.sub.3 is CH.sub.3O—, and R.sub.4 is H.

12. The method of claim 3, further comprising: adding the extract of the Burkholderia glathei into a reaction system together with the thioether.

13. The method of claim 4, further comprising: adding the extract of the Burkholderia glathei into a reaction system together with the thioether.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: The phylogenetic tree of species of strains obtained from the screening of Embodiment 1 which were capable of asymmetrically oxidizing the compound OME; wherein Lysinibacillus sp. is the strain reported in the prior art which was capable of asymmetrically oxidizing the compound OME; in this figure, the underlined strain indicated that the product of asymmetrical oxidation was 5-methoxy-2-((S)- ((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole.

(2) FIG. 2: Process curve of immobilized Burkholderia glathei ECU0712 extract for catalyzing the oxidation of the compound OME.

(3) FIG. 3: Liquid chromatograph spectrum of the product obtained from Embodiment 12.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiment 1. Screening of Strains

(4) (1) 252 soil samples from different environments were harvested, including Shanghai Fengxian Chemical District, Xinhua Hospital, the Vicinity of Orchard and River, Greenbelts, Schoolyard, Residential Greening, Shanghai Botanical Garden, etc. The screening process employed four cycles of enrichment culture, the preliminary poor medium formulation was yeast powder 2 g/L, (NH.sub.4).sub.2SO.sub.4 1.0 g/L, K2HPO.sub.4.3H.sub.2O 6.0 g/L, KH.sub.2PO.sub.4 3.0 g/L, NaCl 0.5 g/L, MgSO.sub.4.7H.sub.2O 0.5 g/L, CaCl.sub.2) 0.05 g/L, pH 7.0, the concentration of yeast powder in each cycle of passage enrichment culture was reduced by half, while the concentration of the substrate (compound OME) (the first-run concentration was 0.1 mM) doubled in each cycle of cultivation, upon screening, to obtain the target strains capable of using the substrate as the carbon source and transforming the substrate. After culturing for 1-2 days, soil sample tubes in which the culture solution was cloudy and the bacteria grew well were chosen, from which was sucked up 500 μL bacteria solution, into which was added 4 mL fresh poor medium, and then the next cycle of enrichment was started. Upon the completion of four cycles of enrichment culture, they were analyzed by thin-layer chromatography, samples with the substrate significantly reduced were isolated using plate streaking, and single colonies were selected.

(5) (2) Single colonies were inoculated into 10 ml rich medium (glucose 1.5 g/L, peptone 0.5 g/L, yeast powder 0.5 g/L, Na.sub.2HPO.sub.4.2H.sub.2O 0.05 g/L, NaH.sub.2PO.sub.4 0.05 g/L, NaCl 1.0 g/L, MgSO.sub.4 0.05 g/L), cultivated at 30° C. for 24 h and then the bacteria was harvested by centrifugation, into which was added 1 mL potassium phosphate buffer (100 mM, pH 9.0), and the substrate (compound OME, DMSO cosolvent, the final concentration 1.0 mM) was added to react. The reaction was stopped after 24 h, and extracted with 700 μL ethyl acetate, the extract was dried and then the conversion ratio was determined by the high-performance liquid chromatography, in which the strains with the conversion ratio greater than 1% were selected for second-screening.

(6) (3) Single colonies were inoculated into 100 ml of the above rich medium, cultivated at 30° C. for 24 h and then the bacteria was harvested by centrifugation, into which was added 5 mL potassium phosphate buffer (KPB, pH 9.0), and the substrate (compound OME, DMSO cosolvent, the final concentration 1.0 mM) was added to react. The reaction was stopped after 24 h, and a sample of 1 ml was taken and extracted with 700 μL ethyl acetate, the extract was dried and then the optical purity of the product was analyzed with a chiral column.

(7) (4) Upon repeated comparisons, a batch of bacteria capable of oxidizing compound OME were screened and obtained, including 7 strains which may oxidize compound OME to produce 5-methoxy-2-((S)-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole, 10 strains which may oxidize thioether to produce 5-methoxy-2-((R)-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole. The phylogenetic tree of these microorganism species was drawn according to 16S rDNA, as shown in FIG. 1, from which it was found that there were great differences between the Burkholderia glathei obtained by screening and the Lysinibacillus sp. reported in the literatures. The optical purity results of the products of the catalytic oxidation of compound OME by these microorganisms were shown in Table 1.

(8) TABLE-US-00001 TABLE 1 Screening results of compound OME oxidation strains Strain ee (%) Gordonia neofelifaecis 99.9 (R) Bradyrhizobium oligotrophicum 99.9 (R) Aeromicrobium marium 99.9 (R) Gordonia polyisoprenivorans 90.6 (R) Gordonia amarae 90.3 (R) Rhodococcus sp. ECU0066 80.2 (R) Gordonia sihwensis 79.5 (R) Saccharothrix espanaensis 70.5 (R) Acinetobacter sp. 67.4 (R) Acinetobacter baumannii 66.6 (R) Burkholderia glathei 99.1 (S) Pseudomonas putita 87.2 (S) Gordonia terrae 86.8 (S) Staphylococcus epidermidis 85.1 (S) Pseudonocardia dioxanivorans 83.4 (S) Starkeya novella 81.8 (S) Fluviicola taffensis 76.1 (S)

Embodiment 2. Identification of Burkholderia glathei ECU0712

(9) (1) Genomic DNA of strains was extracted by general methods, and PCR amplification was carried out using 16S rDNA amplification universal primers, with the genomic DNA of strains as the template. After detection by agarose gel electrophoresis, target fragments of about 1400 bp were amplified. PCR products were purified and recycled with gel purification kits (Agarose Gel DNA Extraction Kit from Beijing Tiangen Biochemical Co.). Finally, the recycled DNA fragments were sequenced, with the sequencing results shown in SEQ ID No. 1.

(10) (2) 16S rDNA sequence was aligned in NCBI database. The 16S rDNA similarity between the strain obtained and Burkholderia glathei (also named Caballeronia glathei) was 99%, so the strain we obtained was named as Burkholderia glathei ECU0712.

(11) (3) Burkholderia glathei ECU0712 described in the present invention has the following morphological characteristics: Gram-negative bacterium, no sporulation, rod, width of 0.6-0.9 μm, length of 1.1-1.4 μm. The colony is round, white, humid, translucent, with regular margin.

Embodiment 3. Shake-Flask Cultivation of Burkholderia glathei ECU0712

(12) Inclined bacterial strains preserved at 4° C. were taken and inoculated into the LB fermentation medium in a tube (peptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, pH 7.2), cultivated with shaking at 200 rpm for 10 h at 30° C., forming the seed solution. 2 ml seed solution was inoculated into 100 ml fermentation medium (5 g/L peptone, 5.0 g/L yeast extract, 5.0 g/L sodium chloride, pH 6.5) with a proportion of 2%, cultivated for 24 hat 30° C., centrifugated, and washed to give the resting cells.

Embodiment 4. Fermentor Cultivation of Burkholderia glathei ECU0712

(13) Inclined bacterial strains preserved at 4° C. were taken and inoculated into the LB fermentation medium in a tube (peptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, pH 7.2), cultivated with shaking at 200 rpm for 10 h at 30° C., forming the seed solution. 300 ml seed solution was inoculated into a 5 L fermentor charged with 3 L fermentation medium (10.0 g/L peptone, 5.0 g/L yeast extract, 8.0 g/L sodium chloride, pH7.0), cultivated for 18 h at 30° C., and then centrifugated, and washed to give the resting cells which were ready for being used in the preparation reactions.

Embodiment 5. Preparation of Burkholderia glathei ECU0712 Lyophilized Cells

(14) Cells obtained from Embodiment 4 were first placed into a −80° C. refrigerator to prefreeze overnight, freeze dried for 24 h at the conditions of 0.1-0.2 mbar vacuum degree and −65° C. freezing temperature, to give the lyophilized cells, which can be stored at 4° C. ready for use.

Embodiment 6. Preparation of Burkholderia glathei ECU0712 Cell Free Extract and the Lyophilized Products of the Cell Free Extract

(15) Cells obtained from Embodiment 4 were resuspended with 1 L potassium phosphate buffer (10 mM, pH 7.0), the suspension was filtered through a 100-mesh sieve and then crushed consecutively at a pressure of 1000 bar for two times. The centrifugal speed of the crushed solution was 15000 rpm, and the centrifuge time was 30 min. The crushed supernatant was collected to give the cell free extract.

(16) The crushed supernatant was placed into a −80° C. refrigerator to prefreeze overnight, freeze dried for 48 h at the conditions of 0.1-0.2 mbar vacuum degree and −65° C. freezing temperature, to give the lyophilized products of the cell free extract, which can be stored at 4° C. ready for use.

Embodiment 7. Qualitative and Quantitative Analysis on the Substrates, Products, by-Products

(17) (1) Silica gel thin layer chromatography was employed as the qualitative analysis method for detecting whether there were products generated or not. Capillaries fed with extracted samples were dotted on a GF silica gel plate, placed in a developing bottle, in which the developer composition was ethyl acetate volume:ether volume=10:1, taken out when the leading edge of the solvent was apart from the top of the plate at 1 cm. The volatilization of the solvent was accelerated using a blower. Upon the volatilization of the solvent, it was observed by an ultraviolet analyzer and compared with a standard sample. Rf of the substrate (compound OME)=0.1, Rf of the product 5-methoxy-2-(-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole=0.42.

(18) (2) Determination of the conversion ratio by a reversed-phase high-performance liquid chromatography: The conversion ratio of the substrate (compound OME) being converted to the product 5-methoxy-2-(-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole was detected using a water phase chromatography HPLC, and the substrate and product were quantitatively analyzed. A C.sub.18 reversed-phase column (Elite Hypersil BDS C.sub.18, 5 μm, 4.6 mm×250 mm) was employed, the mobile phase was acetonitrile:water=53:47 (volume), with a flow rate of 1 ml.Math.min.sup.−1, the column temperature at 30° C., the feed amount at 10 μl, the detecting wavelength at 250 nm, the retention time of the product and the substrate was 3.9 min (the mixture of R configuration and S configuration) and 5.7 min, respectively.

(19) (3) Determination of the optical purity of the product and the proportion of the by-product using normal phase high-performance liquid chromatography: AS-H column was employed to analyze the optical purity of the product, the mobile phase was n-hexane:isopropanol=30:70 (volume), with a flow rate of 0.5 ml/min, the column temperature at 30° C. the feed amount at 10 μl, the detecting wavelength at 250 nm, the retention time of the substrate, sulfoxide in R-configuration, the product in S-configuration, and the by-product sulphone was about 8.3 min, 12.2 min, 15.3 min and 9.3 min, respectively.

Embodiment 8. pH Optimization of Oxidation of Compound OME

(20) To a 1 ml reaction system was added the substrate (compound OME) 1 g/L (solubilized with DMSO, 10% v/v; indicating that the substrate concentration in the reaction system was 1 g/L, the substrate was added into the reaction system after being solubilized with DMSO amounting 10% of the reaction system), the resting cells described in Embodiment 3, 1 g/L, glucose 1 g/L. The pH of the reaction was controlled with a potassium phosphate buffer (50 mM, pH 7.5, 8.0, 8.5 or 9.0). The reaction was carried out at 20° C. for 8 h, then samples were taken to analyze the conversion ratio of the reaction, as shown in Table 2.

(21) TABLE-US-00002 TABLE 2 pH Optimization of Oxidation of Compound OME No. pH of the Reaction Conversion Ratio (%) 1 7.5 78 2 8.0  88 3 8.5 100 4 9.0  96

Embodiment 9. Temperature Optimization of Oxidation of Compound OME

(22) To a 1 ml reaction system was added the substrate (compound OME) 2 g/L (solubilized with acetone, 5% v/v), the resting cells described in Embodiment 3, 1 g/L, glucose 1 g/L. The pH of the reaction was controlled with a potassium phosphate buffer (50 mM, pH 8.5). The reaction was carried out at different temperatures for 12 h, then samples were taken to analyze the conversion ratio of the reaction and the proportion of the by-products, as shown in Table 3.

(23) TABLE-US-00003 TABLE 3 Temperature Optimization of Oxidation of Compound OME Reaction Conversion Proportion of No. Temperature Ratio (%) By-products (%) 1 15 45 0 2 20 70 0 3 25 >99 0.1 4 30 >99 1.1 5 35 >99 8.5

Embodiment 10. Cosolvent Optimization of Oxidation of Compound OME

(24) To a 10 ml reaction system was added the substrate (compound OME) 5 g/L (solubilized with different cosolvents, 5% v/v), the lyophilized yeast powder 2 g/L, glucose 5 g/L, NADP.sup.+ 0.2 mM. The pH of the reaction was controlled with a potassium phosphate buffer (50 mM, pH 8.5). The reaction was carried out at 25° C. for 12 h, then samples were taken to analyze the conversion ratio of the reaction, as shown in Table 4. Wherein: “+” represents a conversion ratio of 1-10%, “++” represents a conversion ratio of 10.1-30%, “+++” represents a conversion ratio of 30.1-60%, “++++” represents a conversion ratio of 60.1-100%.

(25) TABLE-US-00004 TABLE 4 Cosolvent Optimization of Oxidation of Compound OME No. Cosolvent Conversion Ratio of the Reaction 1 blank + 2 dimethylsulfoxide ++++ 3 methanol ++++ 4 ethanol +++ 5 acetonitrile ++ 6 acetone ++++ 7 tert-butanol ++ 8 isopropanol +++ 9 dimethylformamide ++++ 10 tetrahydrofuran ++ 11 N-methylpyrrolidone +++

Embodiment 11. Immobilization and Catalytic Reactions of the Burkholderia glathei ECU0712 Extract

(26) The lyophilized product of the cell free extract prepared in Embodiment 6 has been immobilized by means of cross-linked enzyme aggregates. Polyethyleneimine (PEI) was chosen as the preferable enzyme sediment reagent, the best mass ratio between PEI and the lyophilized product of the cell free extract was 2:1; glutaraldehyde was chosen as the crosslinking agent for the sedimentation of aggregates, the most suitable concentration was 0.2% (w/v). The prepared aggregates suspension was suction filtered, and the resulting filter cake was washed repeatedly with KPB (100 mM, pH 7.0), with the residual glutaraldehyde being washed off, to obtain the immobilized Burkholderia glathei extract useful for the oxidation of compound OME. To a 100 ml reaction system were added the substrate (compound OME) 10 g/L (solubilized with DSMO, 5% v/v), the immobilized Burkholderia glathei extract 5 g/L, glucose 5 g/L, NADP.sup.+ 0.2 mM. The pH of the reaction was controlled with a potassium phosphate buffer (50 mM, pH 8.5). The reaction was carried out at 25° C. for different times, then samples were taken to detect the progress of the reaction, as shown in FIG. 2, the conversion ratio of reaction when reacted for 4 hours may be greater than 90%, the conversion ratio of reaction when reacted for 5 hours may be greater than 95%, and the conversion ratio of reaction when reacted for 8 hours may be greater than 99%.

Embodiment 12. Preparation of 5-methoxy-2-((S)-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole

(27) To a 1 L reaction system were added the substrate (compound OME) 20 g/L (solubilized with DSMO, 10% v/v), the lyophilized cells prepared in Embodiment 5 15 g/L, glucose 10 g/L, NADP.sup.+ 0.2 mM. The pH of the reaction was controlled with a potassium phosphate buffer (50 mM, pH 8.5), and the reaction was carried out at 25° C. for 16 h. After completion, the reaction was extracted with dichloromethane and then centrifugated, the organic phase was taken and dichloromethane was evaporated off to give the product 5-methoxy-2-((S)-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole 19.36 g, with a yield of 92.32%, the ee value of the product being greater than 99%, the proportion of the by-product sulphone being lower than 0.1%, with the liquid phase chromatogram as shown in FIG. 3.

Embodiment 13. Oxidation of Compound PAN

(28) To a 1 L reaction system were added the substrate (compound PAN) 5 g/L (solubilized with DSMO, 10% v/v), the lyophilized cells prepared in Embodiment 5 15 g/L, glucose 10 g/L, NADP.sup.+ 0.2 mM. The pH of the reaction was controlled with a potassium phosphate buffer (50 mM, pH 8.5), and the reaction was carried out at 25° C. for 10 h. After completion, the reaction was extracted with dichloromethane and then centrifugated, the organic phase was taken and dichloromethane was evaporated off to give the product 4.53 g, with a yield of 86.78%, the ee value of the product being greater than 99%. The product was 5-difluoromethoxy-2-[[(S)-(3,4-dimethoxy-2-pyridyl)-methyl]sulfinyl]-1H-benzimidazole.

Embodiment 14. Oxidation of Compound ILA

(29) To a 1 L reaction system were added the substrate (compound ILA) 5 g/L (solubilized with DSMO, 10% v/v), the lyophilized cells prepared in Embodiment 5 15 g/L, glucose 10 g/L, NADP.sup.+ 0.2 mM. The pH of the reaction was controlled with a potassium phosphate buffer (50 mM, pH 8.5), and the reaction was carried out at 25° C. for 10 h. After completion, the reaction was extracted with dichloromethane and then centrifugated, the organic phase was taken and dichloromethane was evaporated off to give the product 2.33 g, with a yield of 44.64%, the ee value of the product being greater than 99%. The product was 5-(1H-pyrrol-1-yl)-2-[(R)-[(4-methoxy-3-methyl-2-pyridyl)-methyl]sulfinyl]-1H-benzimidazole.