KR100532627B1 - Alcohol dehydrogenase -producing microorganisms and the method of making optically active 2-phenyl-1-propanol by these microorganisms - Google Patents

Abstract

The present invention is an optically active 2-phenyl-1-propanol (2-phenyl-1-propanol) in racemic 2-phenylpropionaldehyde using a microorganism having the ability to produce alcohol dehydrogenase (alcohol dehydrogenase) It relates to a method of manufacturing. More specifically, the present invention relates to a method of reducing racemic 2-phenylpropionaldehyde to optically active 2-phenyl-1-propanol using one microorganism having both an alcohol dehydrogenase and a coenzyme.

In the method for producing optically active 2-phenyl-1-propanol, the method of using the co-enzyme of the same microorganism with the alcohol dehydrogenase produced by the microorganism is a new process technology that has not been reported before, and the racemic 2-phenylpropionaldehyde (R)-or (S) -2-phenyl-1-propanol can be prepared.

Description

Microorganisms having the ability to produce alcohol dehydrogenases and a method for preparing optically active 2-phenyl-1-propanol {alcohol dehydrogenase -producing microorganisms and the method of making optically active 2-phenyl-1-propanol by these microorganisms}

The present invention relates to a method for preparing optically active 2-phenyl-1-propanol by microorganisms. More specifically, screening microorganisms that reduce 2-phenylpropionaldehyde to 2-phenyl-1-propanol among microorganisms having an alcohol dehydrogenase-producing ability, and screening the microorganisms using the formula 1 in Scheme 1 A method for producing racemic 2-phenylpropionaldehyde represented by (S) -2-phenyl-1-propanol or (R) -2-phenyl-1-propanol.

Until now, the method for preparing optically active 2-phenyl-1-propanol has been performed by transesterification of racemic 2-phenyl-1-propanol using lipase enzyme or by racemic 2-phenyl-1-propanol. It has been reported to hydrolyze esters of panol to obtain optically active 2-phenyl-1-propanol and ester derivatives thereof.

Bianchi et al. (Journal of Organic Chemistry, 1988, 53: 5531-5534) catalyzed esterification using lipase P (amanosa) on benzene and acetic anhydride as acyl donor. (R) -2-phenyl-1-propanol (28% ee) and ester (S) -2-phenyl-1-propanol (8% ee) were obtained from Mic 2-phenyl-1-propanol. In addition, (R) -2-phenyl-1-prop from racemic 2-phenyl-1-propanol using vinyl acetate as acyl donor on hexane and porcine pancreatic lipase (PPL) enzyme as catalyst. Preparation of ester derivatives of panol (95% ee) and (S) -2-phenyl-1-propanol (48% ee) is described by Chen et al. (Journal of Organic Chemistry, 1991, 56: 1966-1968). It has been reported. Kawasaki et al. (Tetrahedron Letters, 1999, 40: 5223-5226) use PPL enzyme as a catalyst with 3- (2-naphthyl) -vinyl propionate as an acyl donor on cyclohexane The ether reaction was carried out to obtain 96.3% ee (S) -2-phenyl-1-propanol ester derivative and 28.8% ee (R) -2-phenyl-1 in racemic 2-phenyl-1-propanol. Propanol was prepared. Also, under the same process, (R) -alcohol (23.6% ee) and its (I) using acyl donor 3- (paratoryl) vinyl propionate and lipase PS (Amanosa) enzymes R) -ester derivative (95.8% ee) was prepared. Goto et al. (Journal of Molecular Catalysis B: Enzymatic, 2000, 9: 245-250) are also known as 2-phenyl-1- (3-phenylpropionyloxy) -propane (2-phenyl-1- (3-phenylpropionyloxy)- propane) was hydrolyzed with PPL (Sigma) enzyme to prepare (S) -2-phenyl-1-propanol having a conversion of 35% and 97% ee.

Meanwhile, a process for preparing optically active 2-phenyl-1-propanol from 2-phenylpropionaldehyde by an enzymatic reduction reaction has been reported. Yuan et al. (Journal of Organic Chemistry, 1997, 62: 2494-2499) 98% ee of (S) -2-phenyl-1 using Equine liver alcohol dehydrogenase and methyl viologen, an electron mediator, as an assistant. -Propanol was obtained. Kelemen-Horvath et al. (Chemicke Zvesti, 2002, 6: 52-56) and the like obtained 96% ee (S) -2-phenyl-1-propanol from 2-phenylpropionaldehyde using the same enzyme. Both reactions are characterized by the use of β-nicotinamide adenine dinucleotide (NAD) as a cofactor.

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Therefore, the present inventors, unlike the conventional method using NAD as the alcohol dehydrogenase and coenzyme in the liver of the liver, using the microorganism capable of the reduction reaction with the alcohol dehydrogenase and the coenzyme together optical activity in 2-phenylpropionaldehyde 2 It was intended to synthesize -phenyl-1-propanol.

According to the aforementioned research by Yuan et al., In the preparation of optically active 2-phenyl-1-propanol, the stereoselective reduction reaction does not occur when the reaction is performed only with alcohol dehydrogenase and coenzyme obtained from horse liver. In this case, methyl viologen should be added as an auxiliary material to prepare optically active 2-phenyl-1-propanol. Kelemen-Horvath also adds coenzyme β-nicotinamide adenine dinucleotide (NAD) to help the enzyme's action. As in the case of alcohol dehydrogenase, enzymes involved in redox reactions are used to transport hydrogen ions. The NAD ^{+ /} NADH system is an essential NADH-dependent enzyme. Therefore, when the optically active 2-phenyl-1-propanol is prepared by a reduction reaction using an alcohol dehydrogenase, a coenzyme must be supplied separately or an auxiliary material must be supplied together with the coenzyme. However, the method according to the present invention is a technology that can overcome the existing disadvantages by using a microorganism having both the enzyme suitable for the reaction and the coenzyme necessary for the reduction reaction, the addition of additional expensive coenzyme is unnecessary and the process is simple and economical Has the advantage. In addition, there have been no reports on the preparation of optically active 2-phenyl-1-propanol using microorganisms or enzymes derived from microorganisms. Accordingly, an object of the present invention is to provide a racemic 2-phenylpropion using a microorganism as a biocatalyst. To provide a process for preparing (R)-or (S) -2-phenyl-1-propanol of high optical purity and yield from aldehyde.

The method of the present invention for achieving the above object consists of screening and culturing a microorganism having an optically active 2-phenyl-1-propanole generating ability in phenyl propionaldehyde, and reacting using it as a catalyst.

Hereinafter, the present invention will be described in more detail. As described above, the present invention relates to a process for preparing optically active 2-phenyl-1-propanol from racemic 2-phenylpropionaldehyde using microorganisms.

As the microorganism bacteria used in the present invention, Enterobacter cloacae, Erwinia herbicolar, Klebsiella pneumoiae , Micrococcus luteus, Pseudomonas putida, Rhodococcus as erythropolis and yeasts of Candida spp, or the like Candida parapsilosis and Candida rugosa, Geotrichum candidum, Kluyveromyces lactis and Kluyveromyces Microorganisms of the genus Kluyveromyces , including marxianus , Mucor racemosus , Mortierella ramanniana var., Penicillium pinophilum , Pichia pastoris , Rhodotorula mucilaginosa , Saccharomyces cerevisiae , Trigonopsis variabili s, etc. It doesn't happen.

In the present invention, the compound produced by the microorganism was quantified using gas chromatography (Donam Instruments, Model DS 6200) equipped with a capillary column BP-5 (SGE, 0.53 mm X 30 m). Analytical conditions were heated at 60 ° C. for 5 minutes, increased 7 ° C. per minute to 200 ° C., and maintained at 200 ° C. for 5 minutes. As a carrier gas, helium gas was flowed at a rate of 2 ml / min and detected using a flame ionization detector (FID) at 230 ° C. At this time, racemic 2-phenyl propionaldehyde was detected at 13.8 minutes and 2-phenyl-1-propanol at 16.2 minutes, respectively. Optically active 2-phenyl-1-propanol was analyzed using gas chromatography (Cheonnam Instruments, Model DS 6200) equipped with chiral column G-TA (Alltech, 0.32 mm × 30 m). The temperature was maintained at 80 ° C., head pressure at 22 psi, and detected at 200 ° C. using a flame ionization detector (FID). At this time, (R) -2-phenyl-1-propanol was detected at 30.8 minutes, and (S) -2-phenyl-1-propanol was detected at 29.8 minutes.

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

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In a 1 L flask, the bacteria specified in Table 1 were incubated in 200 ml of glucose-added LB medium for 36 hours, followed by centrifugation of the cultured cells. In a 15 ml vial, 4.45 ml of 50 mM phosphate buffer (pH 7.0), 500 μl of 1M glucose and 50 μl of 2-phenylpropionaldehyde were injected, and then the separated cells were injected. . After reaction for a certain time, the reaction solution was extracted with ethyl acetate and analyzed according to the above analysis method. Reaction conversion and optical purity of 2-phenyl-propanol are shown in Table 1.

Example Strain name Response time (hours) % Conversion Optical purity (% ee) Arrangement One Enterobacter cloacae KCTC 2361 18 15.1 1.2 R 2 Erwinia herbicolar KCTC 2104 16 4.3 0 - 3 Klebsiella pneumoniae IFO 3319 2 23.2 100 S 4 Micrococcus luteus KCTC 1071 5 32.4 32.9 R 5 Pseudomonas putida ATCC 21244 43 10.2 44.4 R 6 Pseudomonas putida KCTC 0203BP 71 30.2 9.1 R 7 Rhodococcus erythropolis KCCM 40452 4 44.5 31 S 8 Rhodococcus erythropolis KCTC 1061 43 50.5 10.8 S 9 Rhodococcus erythropolis KCTC 1062 22 48.6 27.7 S

Example 10-26

The yeast specified in Table 2 was cultured using GYP medium under the same conditions as in Example 1, and the reaction was performed in the same manner as in Example 1, and the results are shown in Table 2. Example Strain name Response time (hours) % Conversion Optical purity (% ee) Arrangement 10 Candida parapsilosis IFO 0708 53 49.6 9.1 S 11 Candia parapsilosis KCTC 7653 53 27.7 100 S 12 Candida rugosa KCTC 7292 43 5.2 30.6 S 13 Geotrichum candidum IFO 4597 18 14.5 100 S 14 Geotrichum candidum IFO 5767 39 19.5 47.5 S 15 Kluyveromyces lactis KCTC 7133 2 63.5 57.1 S 16 Kluyveromyces marxianus KCTC 7524 6 13.7 10.7 S 17 Mucor racemosus KCTC 6119 21 13.7 55.6 R 18 Mortierella ramanniana var. KCTC 6137 3 51.8 18.1 S 19 Penicillium pinophilum KCTC 2124 68 3.8 6.9 S 20 Pichia pastoris KCTC 7190 23 17.5 53.7 R 21 Rhodotorula mucilaginosa KCTC 7117 22 54.8 27.8 S 22 Saccharomyces cerevisiae KCTC 1205 7 11.3 13.3 R 23 Saccharomyces cerevisiae KCTC 1218 18 48 24.1 S 24 Saccharomyces cerevisiae KCTC 1552 22 44.2 37.6 S 25 Saccharomyces cerevisiae KCTC 7917 4 19.1 23.2 S 26 Trigonopsis variabilis KCTC 7263 18 9.1 100 S

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As can be seen in the above examples, in the preparation of (R) -2-phenyl-1-propanol and (S) -2-phenyl-1-propanol, microorganisms having an alcohol dehydrogenase and coenzyme-producing ability (R)-and (S) -2-phenyl-1-propanol can be prepared by carrying out the reaction using the characteristic that the optical activity is different for each microorganism. In addition, the present invention is environmentally friendly in that it uses microorganisms rather than asymmetric synthesis.

Claims (3)

Enterobacter cloacae, Erwinia herbicolar, Klebsiella pneumoniae , Micrococcus luteus, Pseudomonas putida, Rhodococcus erythropolis, Candida spp in, Geotrichum candidum, the Kluyveromyces spp, Mucor racemosus, Mortierella ramanniana var. , Penicillium pinophilum, Pichia pastoris, Rhodotorula mucilaginosa, Saccharomyces cerevisiae Microorganism having the ability to produce alcohol dehydrogenase which synthesizes 2-phenyl-1-propanol from 2-phenylpropionaldehyde selected from Trigonopsis variabilis Among the microorganisms having an alcohol dehydrogenase-producing ability, the microorganism according to claim 1 for synthesizing 2-phenyl-1-propanol from 2-phenylpropionaldehyde is cultured, and using this as a catalyst, 2-phenyl-1 from 2-phenylpropionaldehyde to 2-phenyl-1 Method for preparing optically active 2-phenyl-1-propanol using microorganisms characterized in that -propanol is prepared delete