KR100431516B1 - Method for preparing high concentration of phenylacetaldehyde from styrene oxide using microorganism without hydrogenation of styrene oxide - Google Patents

Abstract

PURPOSE: A method for preparing phenylacetaldehyde from styrene oxide using a microorganism is provided, thereby preparing high concentration of phenylacetaldehyde without hydrogenation of styrene oxide. CONSTITUTION: The method for preparing high concentration of phenylacetaldehyde from styrene oxide using a microorganism comprises the steps of: adding 1 to 10 mg of Pseudomonas putida(KCTC 0203BP) into 1 ml of styrene oxide and organic solvent; and culturing the microorganism at 30 to 70 deg. C, wherein the log p of the organic solvent is 3.5 or more; the organic solvent is hexane, heptane, octane or hexadecane; and Pseudomonas putida KCTC 0203BP produces styrene oxide isomerase and phenylacetaldehyde reductase, thereby converting styrene oxide into phenylacetaldehyde in the presence of the organic solvent.

Description

Method for producing phenylacetaldehyde using strains

The present invention relates to a method for preparing phenylacetaldehyde using a strain, and more particularly, to a phenyl oxide from styrene oxide in an organic solvent using a Pseudomonas putida strain having styrene oxide isomerase and phenylacetaldehyde reductase production ability. It relates to a process for preparing acetaldehyde.

In 1979 Shirai and Hisatsuka (Agric. Biol. Chem. 43: 1399-1406) confirmed the production of phenylethanol in styrene and styrene oxide by Pseudomonas strains and the reaction mechanism was styrene by styrene epoxygenase enzyme. Styrene oxide, and then phenylethanol is produced by the styrene oxide reductase enzyme. Baggi (System. Appl. Microbiol. 4: 141-147, 1983) proposed a metabolic pathway by Pseudomonas fluorescens, which is said to produce phenylacetic acid as an intermediate in styrene. Hartmans (Appl. Environ. Microbiol. 55: 2850-2855, 1989), isolated from Xanthobacter strain, a styrene degradation strain, were converted from styrene oxide to phenylacetaldehyde by styrene oxide isomerase enzyme, and phenyl It is proposed to convert to acetaldehyde dehydronase enzyme to phenylacetic acid. Warhurst (Appl. Environ. Microbiol. 60: 1137-1145, 1994) suggested that rhodeococide rhodochrous NCIMB 13259 proceeds from styrene to styrene cis-glycol. O'connor (Appl. Environ. Microbiol. 61: 544-548, 1995) recently used Pseudomonas putida CA-3 to convert styrene oxide to phenylacetaldehyde and phenylacetaldehyde to phenylacetic acid. Confirmed to proceed. However, enzymes produced by the Pseudomonas putida strain tended to have low temperature stability.

The present inventors found that a new Pseudomonas putida strain metabolizes styrene oxide to phenylacetic acid among styrene degrading strains isolated from Ulsan Oil Refinery Plant and PO / SM plant, Pseudomonas used by O'connor et al. (1995). Unlike putida CA-3 strain, it was confirmed that the phenylacetaldehyde metabolizes through phenylethanol to phenylacetic acid. In order to apply for a patent on a method and strain for producing phenylacetaldehyde and 2-phenylethanol in aqueous solution, Pseudomonas putida KCTC 0203BP was deposited with the Korea Advanced Institute of Science and Technology on October 19, 1995, and patented on October 26 Filed in application 95-37419.

On the other hand, the solubility of the substrate and the styrene oxide and phenylacetaldehyde or 2-phenylethanol, which is a substrate, is low in aqueous solution, which is disadvantageous in proceeding with the reaction. In addition, since 2-phenylethanol is produced from phenylacetaldehyde, there are many disadvantages in obtaining phenylacetaldehyde from styrene oxide.

In order to overcome this problem, in the present invention, the reaction was carried out in an organic solvent to block the path from phenylacetaldehyde to 2-phenylethanol, and only phenylacetaldehyde was obtained from styrene oxide.

Accordingly, it is an object of the present invention to provide a method for producing a high concentration of phenylacetaldehyde using a strain.

The method of the present invention for achieving the above object consists of adding Pseudomonas putida KCTC 0203BP to styrene oxide in the presence of an organic solvent, and then maintaining the temperature range of 30 ~ 70 ℃.

Looking at the present invention in more detail as follows.

Microorganism used in the present invention means Pseudomonas putida KCTC 0203BP itself and means the enzyme involved in the reaction. The strain converts styrene oxide to phenylacetaldehyde in the presence of an organic solvent. At this time, the amount of the cells added is preferably 1 to 10 mg with respect to 1 ml of the mixed amount of the organic solvent and styrene oxide. If the amount is less than 1 mg, the reaction proceeds slowly, and if the amount exceeds 10 mg, the reaction rate does not increase. . The reaction mechanism by the microorganisms identified and proposed by the inventors is shown in Scheme I below.

Scheme I

Styrene oxide isomerase (enzyme 1)

Styrene oxide → phenylacetaldehyde

Phenylacetaldehyde reductase (enzyme 2)

→ phenyl ethanol → phenyl acetate

+ 2H ⁺

In other words, when going from phenylacetaldehyde to phenylethanol, two hydrogen ions are required, but the hydrogen ions are present in the aqueous solution, but the hydrogen ions are not present in the organic solvent. Therefore, the use of an organic solvent has the advantage that a high concentration reaction is possible according to the increased solubility of the substrate and the product, and also 2-phenylethanol is not produced, thereby obtaining a high concentration of phenylacetaldehyde.

According to the present invention, the styrene oxide is used by dissolving in an organic solvent, in which the concentration of styrene oxide in the organic solvent is not particularly limited, but in consideration of economical efficiency is preferably 1% or more, self-toxicity of styrene oxide Less than 10% is preferred due to handling problems and economics.

On the other hand, Log p (where p is the partition coefficient dissolved in octanol and water) is an important factor in the organic solvent reaction. (Manjon et al., 1991 Biotechnol, Lett., 13: 339-334). In the present invention, any organic solvent having a log p of 3.5 or more can be used, and preferably hexane (Log p = 3.5), heptane, octane, or hexadecane.

Compounds produced according to the invention were quantified using gas chromatography (Hewlett-Packard, Model 5890). As analytical conditions, the HP-1 capital column (inner diameter 0.2 mm, length 25 m) was heated at 100 ° C. for 1 minute, increased to 10 ° C. per minute to 250 ° C., and stopped at 250 ° C. for 1 minute. Helium gas was flowed into the carrier at a rate of 2 ml per minute. Styrene oxide was detected at 5.7 minutes, phenylacetaldehyde at 5.3 minutes, phenylethanol at 6.3 minutes, and phenylacetic acid at 8.1 minutes, and the respective substances were identified by GC-MSD (gas chromatography; HP5890 II, MSD; HP5972). The compound was analyzed by the above experimental conditions, and the analysis method was applied to the following examples.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

Example 1

Inoculate 300 ml of medium solution into 1 l flask, inoculate bacteria (Pseudomonas putida KCTC 0203BP), incubate for about 2 days with styrene as the only energy source and carbon source, and isolate the cultured cells by centrifugation. Lyophilize. 5 mg of lyophilized Pseudomonas putida cells were placed in a 15 ml vial which blocked the flow of air with a silicone rubber stopper, 5 ml of a hexane solution containing 5% styrene oxide was injected into the vial, and then stirred at 30 ° C. using a stirring tank. Reacted. After a certain amount of contents were recovered from the vial at a predetermined time shown in Table 1, the reaction was stopped by quenching on ice, and the cells and products were separated by a centrifuge, and the supernatant was quantitatively analyzed by gas chromatography according to the above method. The results are shown in Table 1 below.

When reacting 5% styrene oxide / hexane Response time (hours) Phenylacetaldehyde (mM) 0.5 8.93 One 11.95 2 14.54 3 22.75 6 26.36 24 31.53

Examples 2-6

50 mg of the dried cells in the conditions of Example 1 was added to change the styrene oxide from 1% to 10% to proceed with the reaction. The results after 3 hours are shown in Table 2 below.

Influence of Styrene Oxide Concentration Example Styrene Oxide (%) Phenylacetaldehyde (mM) 2 One 2.47 3 3 6.79 4 5 10.22 5 7 17.82 6 10 21.03

Examples 7-10

The reaction was carried out by changing the amount of cells in a 5% styrene oxide / hexane solution of the conditions of Example 1. The results after 24 hours are shown in Table 3 below.

Changes in Phenyl Acetaldehyde Formation with Different Cell Sizes Example Cell mass (mg) Phenylacetaldehyde (mM) 7 5 31.53 8 10 51.89 9 20 73.32 10 50 92.14

Examples 11-13

5 mg of lyophilized cells were added to an octane solution containing 5% styrene oxide in the conditions of Example 1, and the reaction temperature was adjusted from 30 ° C. to 70 ° C., and the product was analyzed according to Example 1. The results are shown in Table 4 below.

Product concentration change with reaction temperature Example Reaction temperature (℃) Phenylacetaldehyde (mM) 11 30 9.48 12 50 9.86 13 70 13.41

Example 14

50 mg of dried cells under the conditions of Example 1 was placed in a 15 ml vial, and 5 ml of a hexane solution containing 0.1% styrene oxide was injected into the vial, followed by reaction. The results of the hexane layer after 24 hours are shown in Table 5 below.

Comparative Example 1

50 mg of dried cells under the conditions of Example 1 were placed in a 15 ml vial, and the volume ratio of hexane solution and water containing 0.1% styrene oxide was 1: 1, and the reaction was carried out in a vial. The results of the hexane layer and the water layer after 24 hours are shown in Table 5 below.

Comparative Example 2

50 mg of dried cells under the conditions of Example 1 were placed in a 15 ml vial, and 5 ml of an aqueous solution containing 0.1% styrene oxide was injected into the vial, followed by reaction. The results of the water layer after 24 hours are shown in Table 5 below.

Composition of Phenyl Acetaldehyde and Phenyl Ethanol According to Liquid Phase Examples and Comparative Examples Phase Phenyl Acetaldehyde (mM) Phenylethanol (mM) Remarks Example 14 Hexane layer 4.33 0 Comparative Example 1 Water layer 0 2.56 Hexane layer 0.37 0.60 Comparative Example 2 Water layer 0 2.24

Examples 15-18

In the conditions of Example 1, instead of hexane, heptane, octane, and hexadecane were used for the reaction. After 4 hours reaction the product was analyzed according to Example 1 above. The results are shown in Table 6 below.

Product concentration change with solvent Example menstruum Phenylacetaldehyde (mM) Log p 15 Hexane 21.21 3.5 16 Heptane 41.62 4.0 17 octane 47.17 4.5 18 Hexadecane 37.75 8.8

As described above, phenylacetaldehyde was synthesized from styrene oxide on an organic solvent using Pseudomonas putida KCTC 0203BP. It can be seen from the above examples and comparative examples that phenylacetaldehyde can be prepared at a high concentration because the hydrogenation does not occur when the styrene oxide is reacted on an organic solvent using a strain metabolized to phenylethanol via phenylacetaldehyde. Could.

Claims (5)

Pseudomonas putida KCTC 0203BP is added to the styrene oxide in the presence of an organic solvent, and then maintained in a temperature range of 30 ~ 70 ℃ phenylacetaldehyde production method using a strain. The method for producing phenylacetaldehyde using a strain according to claim 1, wherein the added amount of the strain is 1 to 10 mg based on 1 ml of the mixed amount of the organic solvent and styrene oxide. The method of claim 1, wherein the concentration of the styrene oxide is 1-10%. The method for producing phenylacetaldehyde using a strain according to claim 1, wherein the log p of the organic solvent is 3.5 or more. The method of claim 1 or 4, wherein the organic solvent is hexane, heptane, octane or hexadecane.