KR20010109632A - Enantioselective lipase and burkholderia cocovenenans sy-01 strain producing the same - Google Patents

Abstract

The present invention relates to a new strain Burkholderia Cocovenenans SY-01 and a lipase having stereoselective substrate specificity produced therefrom, and more particularly, racemic as an intermediate of itraconazole used as a raw material of an antifungal agent. A lipase capable of stereoselectively decomposing an ester bond of cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3-dioxolane-4-methyl acetate as a mixture. .

Description

Stereoselective lipase and Vulcolderia cocobenenans strains producing the same {ENANTIOSELECTIVE LIPASE AND BURKHOLDERIA COCOVENENANS SY-01 STRAIN PRODUCING THE SAME}

The present invention relates to Vulcolderia cocovenenance producing lipase having stereoselective substrate specificity and lipase produced by the strain, more specifically cis- which is a racemic mixture as an intermediate of itraconazole used as a raw material of an antifungal agent. A novel lipase that steric-selectively hydrolyzes 2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3-dioxolane-4-methyl acetate.

In the selective organic synthesis of fine organic compounds, the chemical method has been used in many countries all over the world, and many studies have been conducted in Korea to narrow the technology gap at home and abroad. On the other hand, researches using biocatalysts are being actively conducted as a field of selective organic synthesis. Such studies are mainly conducted in advanced countries, and domestic research is insignificant. Many biochemical studies have been conducted on hydrolase in these biocatalysts, and studies on preparing precise compounds using the reaction specificity of hydrolase have been reported.

In particular, lipase among hydrolases has been studied biochemically and is widely used because it has a wide spectrum of reaction specificity. There are about 70 types of lipases known so far, and their reaction specificities vary slightly depending on each other. Therefore, pure isomers can be obtained if appropriate lipases can be selected and applied to the required substrate (Jackson, MA et al. , 1995., Enz. Microb. Technol. 17: 175-179.

In general, compared with racemic drugs, monoisomer drugs have the effect of increasing pharmacological activity in vivo and reducing side effects of racemic compounds in vivo. In particular, itraconazole, which is used as an antibacterial or anti-inflammatory agent, is chemically prepared by using cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl acetate as an intermediate. However, since the intermediate is manufactured in a racemic form, the subsequent steps are complicated and the final product is provided in a racemic form, which may cause problems such as concern about various side effects and low biological activity. There was an attempt to provide. However, when chemically synthesized, there are many difficulties in commercialization, such as an additional cost for separation of racemic forms. Therefore, by separating the intermediate into a single isomer and carrying out the subsequent reaction, it will be possible to simplify the manufacturing process, reduce manufacturing costs, increase biological activity and minimize side effects.

Itraconazole is a well known antifungal agent that is commercially available in the form of cis isomers, but is sold in the form of racemic mixtures. However, this racemic form of itraconazole is toxic to the liver and can cause side effects such as arrhythmia, nausea, vomiting, hypersensitivity, urticaria, abdominal pain, headache, and dizziness. Has been attempted, but there are no methods using a biocatalyst (US Pat. Nos. 5,952,502 and 5,474,997).

The present invention provides Burkholderia Cocovenenans SY-01, which produces a lipase having stereoselective specificity for a substrate, to solve the above problems with chemical organic synthesis.

It is also an object of the present invention to provide a lipase having stereoselective specificity for substrates produced by Burkholderia Cocovenenans SY-01.

Another object of the present invention is cis-2- (bromomethyl) -2, an intermediate of itraconazole, which is used as an anti-inflammatory and antifungal agent using lipase produced by Burkholderia Cocovenenans SY-01. To provide a single isomer separation from a racemic mixture of-(2,4-dichloro phenyl) -1,3-dioxolane-4-methyl acetate.

1A and 1B show cis-2- (bromomethyl) -2- (2,4-dichloro phenyl before and after hydrolysis by stereoselective lipases produced by Burkholderia Cocovenenans SY-01 Analyze) -1,3-dioxolane-4-methyl acetate and cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3-dioxoran-4-methyl alcohol One result.

FIG. 2 shows cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3- using stereoselective lipases produced by Burkholderia Cocovenenans SY-01 When dioxolane-4-methyl acetate was hydrolyzed, it showed the decrease rate of two isomers of the substrate and the increase ratio of the product to the two isomers over time.

Figure 3 shows the relationship between the growth of bacteria and the production of lipase enzyme in the culture process of Burkholderia Cocovenenans SY-01.

Figure 4 shows the result of coincidence of lipase enzyme activity peak and protein peak as a result of purification using Sephadex G-100 from the crude enzyme solution.

Figure 5 shows the result of SDS-PAGE electrophoresis and molecular weight measurement of the purified lipase enzyme solution.

Figure 6 shows the effect of pH on the activity and safety of the lipase of the present invention.

Figure 7 shows the effect of temperature on the activity and safety of the lipase of the present invention.

The present invention provides Burkholderia Cocovenenans SY-01 strain and lipase having stereoselective substrate specificity produced by the strain. More specifically, the lipase produced by the strain can be used to selectively separate only isomers from racemic mixtures of various substrates.

Specifically, the present invention relates to a single isomer in a racemic mixture of cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3-dioxolane-4-methyl acetate, an intermediate of itraconazole. It can be used to selectively separate only bays, and also to synthesize a single stereoisomer drug using this single isomer as an intermediate. Itraconazole is a well-known antifungal agent that is commercially available in the form of cis isomers and is marketed in racemic mixtures. There are problems that can cause side effects such as irritability, hives, abdominal pain, headache, dizziness. Therefore, the intermediate cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3-dioxolane-4-methyl acetate is separated from the racemic mixture into a single isomer to form a single isomer. It can serve as a single intermediate for producing itraconazole.

The chemical formula of cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3-dioxolane-4-methyl acetate is as follows.

In order to obtain lipase producing strains for separating racemic mixtures into homoisomers, the inventors have conducted racemic substrates, such as cis-2- (bromomethyl) -2- (2, In order to search for lipase-producing microorganisms showing high stereoselectivity to 4-dichlorophenyl) -1,3-dioxolane-4-methyl acetate, lipase enzymes having high activity and high stereoselectivity to the substrate in stream sludge around the satin The strain producing was isolated by the following procedure. That is, strains forming an opaque halo around colonies were purified in a separation medium containing Tween 80. Here, the colonies forming the halo are microorganisms that break down ester bonds contained in the Tween 80 and thus can be judged as microorganisms producing lipase or esterase. Each strain thus isolated was inoculated in a medium containing olive oil and rhodamine B, and when cultured, irradiated with UV light, strains showing orange fluorescence around the colonies were purely separated. In order to fluoresce orange in Rhodamine B-containing medium, glycerol, fatty acid and rhodamine B produced by decomposing the ester bond of olive oil contained in the medium must form a complex, so the colonies isolated here May be considered a lipase-producing microorganism.

After culturing each of the isolated strains in an olive-containing liquid medium, the supernatant was recovered by centrifugation, and the crude enzyme solution was separated, which was reacted for 3 hours at 40 ° C using an emulsion containing 50% olive oil as a substrate. After the titration of the fatty acid produced by the enzymatic reaction with 0.05M NaOH solution to measure the enzyme activity was finally isolated strain was confirmed that the enzyme reaction was carried out to secure a total of 200 lipase production strains. (Hou, C. T. et al., 1992., JAOCS. 69 (11): 1088-1097 .: Watanabe, N. et al., 1977., Agric. Biol. Chem. 41 (8): 1353-1358).

In order to screen lipase producing strains having excellent stereoselectivity among 200 species identified as lipase producing strains, 60 kinds of the lipase producing microorganisms isolated above were first cultured, and the crude enzyme solution and trisma isolated from each culture medium ( TRIZMA) 100 μl of a racemic substrate (10 g / l concentration) dissolved in normal hexane was added to the buffer solution to carry out the enzymatic reaction to recover only the normal hexane layer, and then to obtain cis-2- (bromomethyl) -2 using an HPLC analyzer. Stereoselective hydrolysis of-(2,4-dichloro phenyl) -1,3-dioxolane-4-methylacetate was confirmed.

Four strains showing stereoselectivity were isolated through stereoselective hydrolysis of the racemic substrate of the lipase produced by the 60 isolates thus separated, and among them, microorganisms showing the highest stereoselectivity were selected and characterized. Was investigated. The isolated bacteria were Gram-negative bacteria, microorganisms with a coccus shape under the microscope, and the oxidase activity test showed a positive reaction, indicating that the strain was an aerobic microorganism. To identify using Biolog, Biolog's 95-well negative gram negative 96-well microplate (trade name: GN2 Microplate) was used. Metabolic fingerprinting is a method using the principle that the inoculated cells oxidize the carbon source contained in the 96-well microplate, and the tetrazolium dye contained in the well is purple while reducing. Inoculating and inoculating the 96 well micro plate incubated with the isolated bacterial suspension, the results of measuring substrate utilization and oxidation ability for 95 carbon sources of the isolated bacteria were identified by the Biolog company's identification program (trade name: Biolog MicroLog3). 4.01A) was identified as a microorganism of the present invention Burkholderia Cocovenenans (Burkholderia Cocovenenans). It was named Burkholderia Cocovenenans SY-01. Table 1 shows the 95 carbon source compositions of the 96-well microplate (GN2 Microplate), Table 2 shows the results of identification of metabolic fingerprinting method for the isolated strain of the present invention in the microplate.

From the results of Tables 1 and 2, isolated microorganisms of the present invention are Tween 40, Tween 80, methyl pyruvate, mono-methyl succinate, acetic acid, Cis-succinic acid, formic acid, α-ketobutyl acid, α-keto glutaric acid, propionic acid, succinic acid, bromosuccinic acid, alanineamide, L-alanine ), L-asparagine, L-glutamic acid, L-histidine, L-proline, L-serine It turns out that you can.

Burkholderia Cocovenenans SY-01 was deposited with the Korean microbiological conservation center under accession no. KFCC-11111 dated November 15, 1999.

Stereoselective lipases produced by Burkholderia Cocovenenans SY-01 were cultured in tryptone-glucose-yeast extract liquid medium containing olive oil for 3 days, followed by centrifugation and supernatant. By recovering, a crude enzyme solution can be obtained. Enzymatic hydrolysis using the crude enzyme solution using cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl acetate as a substrate as a substrate After carrying out the reaction, cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methylacetate and cis-2- produced by hydrolysis (Bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl alcohol was analyzed and produced by Burkholderia Cocovenenans SY-01 Enzymes have stereoselective substrate specificity. Using a fermentation broth or coenzyme solution produced by Burkholderia Cocovenenans SY-01, the racemic compound cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1, It can be used to separate from 3-dioxolane-4-methyl acetate into a single isomer and this single isomer can be used as an intermediate to synthesize a single stereoisomeric drug.

1A, which shows the results of decomposition of cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl acetate of the lipase of the present invention; In 1b, the two ester form isomer peaks before hydrolysis by lipase appear as one ester form isomer and two alcohol form isomer peaks after hydrolysis, so that the lipase produced by the present invention is cis-2- ( A stereoselective lipase that selectively acts on bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl acetate, and as shown in FIG. 2, the hydrolysis reaction proceeds. The ratio of the remaining amount of the two racemic substrates to the increased amount of the two product isomers is determined by the conversion of 75% of the substrates, while one isomer of the substrate is lost, while the other isomer remains 25%. Therefore, a single isomer is present so that only one isomer can be selectively separated from the racemic mixture of the two isomers.

The molecular weight of the lipase of the present invention was SDS-PAGE of the purely isolated enzyme with the standard protein, the molecular weight was about 39KD, the optimum pH range was 7.0-9.0 (FIG. 6A), and stable from pH 6 to 9 (FIG. 6b). In addition, the optimum activity temperature of the lipase ranges from 45 to 50 ° C. (FIG. 7A), and is stable from 40 ° C. to 60 ° C. (FIG. 7B). Lipase of the present invention is inhibited by mercury, copper, nickel, calcium ions (Table 6), while sodium dodecyl sulfate was strongly inhibited by the activity of the enzyme, but Tween 80, Triton X-100, and bile salts ( Gall Powder), sodium deoxycholate was added to increase the activity significantly (Table 7).

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example

Example 1 Isolation of Lipase Producing Strains

Separation media of industrial complexes around Korea, 1 ml of river, wastewater, and soil samples were diluted in physiological saline (Tween 80 1.0%, Peptone 0.5% Beef extract 0.3%, CaCl ₂ · 2H ₂ O 0.001%, Agar 2.0%, pH 7.0) or medium containing olive oil (Olive oil 2.0%, NH ₄ Cl 0.1% K ₂ HPO ₄ 0.44%, NaH ₂ PO ₄ 2H ₂ O 0.39%, MgSO ₄ 7H ₂ O 0.05%, CaCl ₂ 2H ₂ O 0.0003% FeSO ₄ 7H ₂ O 0.0001%, MnCl ₂ 4H ₂ O 0.0001% CoCl ₂ 6H ₂ O 0.00001% Na ₂ MoO ₄ 2H ₂ O 0.00001% After repeated repeated incubation in citric acid monohydrate 0.0004%, pH 7.0), diluted and plated in a separation medium containing Tween 80, incubated at 30 ° C. for 4-5 days, and then opaque halo around colonies. Strains forming halo were first isolated. Each isolated strain was inoculated in a separation medium containing rhodamine B (olive oil 1.0%, peptone 0.5%, beef extract 0.3%, Rhodamine B 0.0001%, agar 2.0%, pH 7.0) at 30 ° C. After culturing for 3 days, when the UV irradiation, the strain showing the orange fluorescence around the colony was isolated secondary. Each strain finally isolated was tested in tubes containing 5 ml of lipase production medium (1.0% olive oil, 0.5% tryptone, 0.1% dextrose, 0.5% yeast extract, 0.1% K ₂ HPO ₄ , pH 7.0.) 20 ml dose), incubated at 30 ° C. and 150 rpm for 3 days, and then centrifuged to give 3 ml of culture supernatant, 50 ml of olive oil (Sigma Catalog No. 800-1), 3 ml, 200 mM Trisma buffer solution (pH 8.0). 1 ml and 2.5 ml of distilled water were mixed and reacted at 40 ° C. for 6 hours. After the reaction, 3 ml of 95% ethanol and 0.9% thymolphthalein indicator dissolved in ethanol were added and titrated with 0.05M NaOH solution to measure the lipase activity to finally separate the lipase producing strain. 200 lipase producing strains were obtained.

Of the 200 lipase-producing strains isolated, 60 were lipase-producing medium (1% olive oil, tryptone 0.5%, dextrose 0.1%, yeast extract 0.5%, K ₂ HPO ₄ 0.1%, pH 7.0). Inoculate each test tube containing 20 ml (20 ml volume), incubate at 30 ° C and 150 rpm for 3 days, and then centrifuge to dissolve 10 m / l of racemic substrate cis-2- (bromine) in 1 m of culture supernatant and normal hexane. Mother methyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl acetate, 10 mu l, 200 ml Trisma buffer solution (pH8.0) 1 ml, distilled water 4 ml was mixed and 40 ℃ After reacting at 250 rpm for 6 hours at 1 ml of normal hexane was added and mixed, only the normal hexane layer was recovered and analyzed by HPLC to confirm the stereoselectivity of the substrate. As such, four strains having excellent stereoselectivity were isolated from the substrate, and one strain showing the highest stereoselectivity was selected.

The results of the HPLC analysis are shown in FIGS. 1A and 1B, cis-2- (bromomethyl) -2- hydrolyzed by stereoselective lipase produced by Burkholderia Cocovenenans SY-01. As a result of analyzing (2,4-dichloro phenyl) -1,3-dioxolane-4-methyl acetate, two ester form isomers before the hydrolysis reaction, one ester form isomer and two The lipase produced by the present invention is shown in cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3-dioxolane-4-methyl acetate as indicated by two alcoholic isomer peaks. It has been demonstrated that it is a stereoselective lipase that acts selectively.

In addition, the stereoselectivity was measured by time in the enzymatic hydrolysis of the racemic substrate in the same manner as described above.

Figure 2 shows the ratio of the residual amount of the two racemic substrates and the increase of the two product isomers as the hydrolysis reaction proceeds when the hydrolysis reaction is carried out using the lipase produced by the present invention. When 75% was converted, one isomer of the substrate was lost, whereas the other isomer remained 25%, proving that a single isomer exists.

Therefore, the isolated strain of the present invention produces a lipase having stereoselective substrate specificity, cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1, Single isomers can be separated by enzymatic reaction using 3-dioxolane-4-methyl acetate as a substrate, and various single isomeric pharmaceuticals can be synthesized using this single isomer.

Example 2: Identification of Strains

In order to investigate the characteristics of the strain isolated in Example 1, Gram staining resulted in gram-negative, and observed under the microscope it was found that the microorganisms having a circular shape. In addition, oxidase activity test showed a positive reaction was confirmed to be aerobic microorganisms.

In addition, 200 µl of each of the isolated bacterial suspensions incubated in the method of Example 1 was previously inoculated on a gram-negative 96-well microplate (trade name: GN2 Microplate) coated with Biolog's 95 carbon sources, and incubated in a 30 ° C. incubator for 24 hours. Subsequently, the results of measuring substrate utilization for 95 carbon sources were inputted to Biolog's identification program (trade name: Biolog MicroLog 34.01A). As a result, isolates of the present invention were identified as Burcolderia cocobenenans.

Example 3: Production of Lipase

To produce lipase, 3 l of medium in 0.5% olive oil, 0.5% tryptone, 0.1% glucose, 0.5% yeast extract, 0.1% K ₂ HPO ₄ was adjusted to pH 7.5 and cooled by autoclaving at 121 ° C. for 15 minutes. Subsequently, 100 ml of the culture medium incubated with the Bulkholderia Cocovenenans SY-01 strain (KFCC-11111) of the present invention, which had been incubated in the same medium composition for 24 hours in advance, was inoculated with a culture temperature of 30 ° C, After culturing for 10 hours in a 5 L fermenter (Fermenter, Korea fermenter) at 600 rpm, lipase activity of 364 units / liter was obtained. Figure 3 shows the growth of the cells and lipase production, pH change with incubation time.

Example 4: Purification of Lipase

After centrifugation (12000 rpm, 10 minutes) of the culture solution of Burkholderia Cocovenenans SY-01 strain (KFCC-11111), 500 ml of the culture supernatant was charged with a 100 Dalton Pore size membrane. It was concentrated about 10 times by volume using the equipped ultrafiltration system (Amicon 8200 model). The concentrate was centrifuged again (12000 rpm, 10 minutes) to completely remove the remaining cells. This concentrate was placed in a Sephadex G-100 column (150 mm x 30 mm) equilibrated with Tris-HCl buffer (pH 8.0) and eluted with a buffer of 20 ml / hr. 5 ml of each fraction was collected to measure the lipase activity and the absorbance at the wavelength of 280 nm. The results showed that the enzyme activity peak and the protein peak coincided with the fractions 5-9 (FIG. 4). This purification process is summarized in Table 3. The specific activity of the lipase of the present invention was 2.44 unit / mg, the yield was 34.86%, and the purity was increased by 39.2 times. SDS-PAGE electrophoresis of this purified enzyme solution showed that the enzyme was purified because it appeared as a single band on electrophoresis (FIG. 5).

Example 5 Properties of Lipase of the Present Invention

The purified enzyme in Example 4 was used to investigate the properties of the lipase according to the following experimental example.

A. Determination of molecular weight and N-terminal amino acid sequence of enzyme

SDS-PAGE of the pure enzyme isolated with the standard protein showed a molecular weight of about 39 KD (FIG. 5). Extracting the protein on the gel and analyzing the amino acid sequence of the N-terminal was as shown in Table 4. The isolated enzyme is a novel enzyme significantly different from the lipase produced by the same Gram-negative bacteria Pseudomonas genus.

B. PH effect on lipase

In Example 5 the effect of pH and temperature on purely isolated lipase was investigated. As a result of measuring enzyme activity with varying pH, the optimum pH range was between 7.0 and 9.0 (FIG. 6A).

The pH was changed as shown in Table 5 using Britain-Robinson buffer.

The activity was measured by mixing 3 ml of an emulsion substrate (Sigma Catalog No. 800-1) containing 1 ml of Britain-Robinson buffer and 50% olive oil, 2.5 ml of distilled water, and 1 ml of enzyme solution, and reacting at 50 ° C. for 6 hours. After completion of the reaction, 3 ml of ethanol (95%) and 6 drops of thymolphthalein indicator were mixed and titrated with 0.05 M NaOH solution. A blank test was performed in the same manner except for the enzyme solution.

In order to measure the safety of the lipase pH, it was stabilized from pH 6 to 9 when the residual enzyme activity was measured at 50 ° C. after 24 hours of maintaining at 30 ° C. at various pHs. (FIG. 6B).

C. Temperature Effect on Lipase

The temperature change was reacted at 30, 40, 45, 50, 60, and 70 ° C in a temperature-controlled thermostat, and the activity was measured using an emulsion substrate (sigma) containing 1 ml of 200 mM TRIZA buffer (pH 8.0) and 50% olive oil. Catalog No. 800-1) 3 ml, 2.5 ml of distilled water, and 1 ml of enzyme solution were mixed and reacted at each temperature for 6 hours. After completion of the reaction, 3 ml of ethanol (95%) and 6 drops of thymolphthalein indicator were mixed and titrated with 0.05M NaOH solution. A blank test was carried out in the same manner except for the enzyme solution. The optimum activity temperature of the enzyme was found to be between 45 and 50 ° C. (FIG. 7A), and the enzyme activity rapidly decreased below 40 ° C. and above 50 ° C. In order to measure the thermal stability of the lipase, the enzyme activity was measured at 50 ° C. after 1 hour at each temperature. As a result, the enzyme was found to be stable up to 60 ° C. (7b). .

D. Effect of metal ions and various reagents on lipase

To see the effect on the metal ions, 1 ml of 200 mM TRIZA buffer (pH 8.0) and 3 ml of emulsion substrate (Sigma Catalog No. 800-1) containing 50% olive oil, 2.5 ml of distilled water, and 1 ml of enzyme solution were mixed. The metal ion concentration of 5mM was quantified and mixed and reacted for 6 hours in a 50 ℃ thermostat. After completion of the reaction, 3 ml of ethanol (95%) and 6 drops of thymolphthalein indicator were mixed and titrated with 0.05M NaOH solution. A blank test was performed in the same manner except for the enzyme solution. The effect on various reagents was also performed in the same way.

As a result of examining the effects of various metal ions on the lipase of the present invention, mercury, copper, nickel and calcium ions were inhibited (Table 6). In addition, as a result of investigating the effects of various reagents, the activity of this enzyme was strongly inhibited by sodium dodecyl sulfate, but when Tween 80, Triton X-100, Gall Powder, and sodium deoxycholate were added The activity was greatly increased (Table 7).

E. Degree of Resistance to Solvents of Lipase

Lipid hydrolysis titer of 10%, 20%, and 50% ethanol, DMSO, Dioxane, Hexane, and Decane in 1 ml of 956 units / l lipase activity As a result of the measurement, it was found that the enzyme of the present invention can be used as a biocatalyst because the stability of the solvent was very high as shown in Table 8.

The Burkholderia Cocovenenans SY-01 strain is steric in cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl acetate It selectively acts to produce a lipase that hydrolyzes the ester form, and cis-2- (bromomethyl) -2- (2,4-dichloro phenyl using lipase having stereoselective substrate specificity produced from the strain. Single isomers can be separated by enzymatic reactions based on) -1,3-dioxolane-4-methyl acetate, and these single isomers can be used to synthesize a variety of single isomeric pharmaceuticals.

Claims (5)

Lipase produced by Vulcolderia cocobenenance, has a molecular weight of about 39KD, has a stereoselective substrate specificity of 45 ~ 50 ℃ temperature range of enzyme activity, 7.0-9.0 optimum pH. The lipase according to claim 1, wherein the lipase is produced by the Bulcolderia cocobenenance SY-01 strain having accession number KFCC-11111. 3. The lipase according to claim 1 or 2, wherein the lipase is isolated from cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -13-dioxolane-4-methyl acetate into a single isomer. Characterized lipase. Burkholderia Cocovenenans SY-01 strain with accession no. KFCC-11111 to produce stereoselective lipase. Cultivation of the Vulcolderia cocobenenance SY-01 strain of claim 1 and the culture using the culture cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -13-dioxolane-4 A process for separating homoisomers from methyl acetate.