Title of Invention

A PROCESS FOR PRODUCING ACTINOL FROM LEVODIONE

Abstract

The present invention relates to a process for producing actinol from levodione which comprises treating levodione with (i) an isolated and purified enzyme having levodione reductase activity or a cell- free extract of a microorganism belonging to the genus Corynebacterium and capable of producing a levodione reductase, said enzyme having the following physico- chemical properties: -a molecular weight of 142,000 to 155, 0003 + 10,000 for the whole enzyme; consisting of four known homologous subunits having a molecular weight of 36,000 +5,000, -a nicotinamide adenine dinucleotide (NAD/NADH) co-factor, -a substrate specificity for levodione, -an optimum temperature of 15&#730;C to 20&#730;C at a pH of7.0, -an optimum pH of 7.5 and wherein the enzyme is activated by a ion selected from the group consisting ofK<sup>+</sup>Cs<sup>+</sup>,Rb<sup>+</sup>,Na<sup>+</sup> and NH<sub>4</sub><sup>+</sup>, in the presence of a reduced form of nicotinamide adenine dinucleotide and recovering the actinol from the reaction mixture.

Full Text

The present invention relates to a process for producing actinol from levodione. In European Patent Application No.98115564.1, filed on August 19, 1998, and in the later European application No.99115723.1 filed on August 16, 1999 and claiming the priority of the earlier one, there is disclosed a process for the manufacture of actinol which comprises contacting levodione with a microorganism which is selected from the group consisting of microorganism of the genera Cellulomonas, Corynebecterium, Planococcus and Arthrobacter and which is capable of the selective asymmetric reduction of levodione to actinol, and recovering the resulting actinol from the reaction mixture, Coreynebacterium aquaticum AKU611 (PERM BP-6448) was found to be one of the best microorganism strains for this purpose. The microorganism strain Coreynebacterium aquaticum AKU 611 has the following taxonomical properties: 1) Growable temperature: 15-40°C 2) Optimum temperature for growth: 30°C 3) Obligatory aerobic and gram negativev microorganism 4) Spore formation None _ 5) Polymorphism and traditional rod-cocus cycles can be observed during cultivation. 6) Motility : _None__ Moreover, the strain Corynebacterium aquaticum AKU611 was identified as such based on assimilation of various carbon sources by the Biolog System (Biolog Inc., 3447 Investment Blvd., Suite 3, Hayward, California 94545, USA : Nature Vol. 339, 157-158, May 1 U 19S9) as follows: Cells of the strain were inoculated with 96-well microtiter-plates and incubated for 24 hours at 28oC- Each well contained one of 96 kinds of carbon sources in BUGM+B medium (Biolog Universal Growth Media + Blood; Biolog Inc.). After incubation, the strain showed the following assimilation of carbon sources; C source C source A1 - A2 - A3 - A4 - A5 - A6 - A7 - A8 + A9 + A10 - All - A12 + B1 - B2 + 33 - B4 - 85 + B6 - B7 + B8 - B9 + B10 + B11 + B12 - C1 - C2 - C3 - C4 + C5 + C6 + C7 - C8 + C9 - C10 - C11 - C12 - D1 - D2 D3 + D4 - D5 + D6 - D7 - D8 + D9 - D10 - D11 + D12 + El - E2 - E3 + E4 - E5 - E6 - E7 - E8 - E9 - E10 - E11 - E12 - Fl - F2 - F3 - F4 - F5 - F6 + F7 - F8 - F9 - F10 - F11 - F12 - G1 - G2 - G3 - G4 - G5 - G6 - G7 - G8 - G9 - G10 - G11 - G12 - H1 - H2 - H3 - H4 - H5 - H6 - H7 - H8 - H9 - HIO - HU - H12 - A1 : water A2 : a-cyclodextrin A3 : p-cyclodextrin A4 : dextrin A5 : glycogen A6 ; inulin A7 : mannan AS : Tween'=40 A9 : Tween® 80 A10 : N-acetyl-D-glucosamine A11 : N-acetyl-D-mannosamine A12 : amygdalin Bl : L-arabinose B2 : D-arabitol B3 : arbutin B4 : cellobiose B5 : D-fructose B6 : L-fucose B7 : D-galactose B8 : D-galacturonic acid 89 : gentiobiose B10 : D-gluconic acid Bll ; α-D-glucose B12 : m-inositol C1 :α-D-lactose C2 : lactulose C3 : maltose C4 : maltotriitrose C5 ; D-mannitol C6 : D-mannose C7 : D-melezitose C8 : D-melibiose C9 : α-methyl-D-galactoside C10 : α-methyl-D-galactoside C11 : 3-methyl-glucose C12 : α-methyl-D-glucoside D1 : β -methyl-D-glucoside D2 : α-methyl-D-mannoside D3 : palatinose D4 : D-psicose D5 D-raffmose D6 : L-rhamnose D7 : D-ribose D8 : salicin D9 : sedoheputulosan D10 : D-sorbitol D11 : stachyose D12 : sucrose El : D-tagatose E2 : D-trehalose E3 : turanose E4 : xylitol E5 : D-xylose E6 : acetic acid E7 : α-hydroxybutyric acid E8 : β-hydroxybutyric acid E9 : γ-hydroxybutyric acid E10 : β-hydroxy-phenylacetic acid E11: α-keto-glutaric acid E12 : α-keto-valeric acid Fl : lactamide F2 : D-lactic acid methyl ester F3 : L-lactic acid F4 : D-malic acid F5 : L-malic acid F6 : methyl pyruvate F7 : monomethyl succinate F8 : propionic acid F9 : pyruvic acid FIG : succinamic acid Fll : succinic acid F12 : N-acetyl-L-glutamic acid Gl : alaninamide G2 : D-alanine G3 : L-alanine G4 : L-alanyl-glycine G5 : L-asparagine G6 : L-glutamic acid G7 : glycyl-L-glutamic acid G8 : L-pyloglutamic acid G9 : L'Serine GIO : putrscine Gl 1 : 2,3-butanediol G12 : glycerol HI : adenosine H2 : 2'-deoxy-adenosine H3 : inosine H4 : thymidine H5 : uridine H6 : adenosine-5'-monophosphate H7 : thymidine-5'-monophosphate H8 ; uridine-5'-monophosphate H9 : fructose-6-phosphate H10 : glucose-1-phosphate HI 1 : glucose-6-phosphate H12 : DL-a-glycerol phosphate It is an object of the present invention to provide the novel LR which acts on levodione to produce actinol. LR has the following physico-chemical properties: a) Molecular weight: 142,000 - 155,000 i 10,000 (consisting of four homologous subunits having a molecular weight of 36,000 ± 5,000) b) Co-factor nicotinamide adenine dinucleotide (NAD/N.ADH) c) Substrate specificity: active on levodione d) Optimumtemperature: 15 - 20 T at pH 7.0 e) Optimum pH: 7.5 f) Activator: K', Cs', Rb', Na' and Nt It is another object of the present invention to provide a process for producing the novel LR as defined above by cultivation of a microorganism belonging to the genus Corynebacterium which is capable of producing the LR having the above physico-chemical properties, in an aqueous nutrient medium under aetobic conditions, disrupting the cells of the microorganism and isolating and purifying the LR from the cell-free e of the disrupted cells of the microorganism. A still further object of the present invention is to provide a process for producing actinol from levodione utilizing the_LR, which comprises contacting levodione with (i) an LR as defined above in the presence of the , reduced form of nicotinamide adenine dinucleotide (NADH), or (ii) a cell-free extract_of said microorganism, and in each of the cases (i) and (ii) isolating the resulting actinol from the reaction mixture. Accordingly the present invention provides a process for producing actinol from levodione which comprises treating levodione with (i) an isolated and purified enzyme having levodione reductase activity or a cell-free extract of a microorganism belonging to the genus Corynebacterium and capable of producing a levodione reductase, said enzyme having the following physico-chemical properties: - a molecular weight of 142,000 to 155,000 +10,000 for the whole enzyme; consisting of four known homologous subunits having a molecular weight of 36,000+5,000, - a nicotinamide adenine dinucleotide (NAD/NADH) co-factor, - a substrate specificity for levodione, - an optimum temperature of 15°C to 20°C at a pH of 7.0, - an optimum pH of 7.5 and wherein the enzyme is activated by a ion selected from the group consisting of K+,Cs+,Rb+,Na+ and NH4+, in the presence of a reduced form of nicotinamide adenine dinucleotide and recovering the actinol from the reaction mixture. The physico-chemical properties of the purified sample of the LR prepared according to the Examples presented below are as follows: 1 ) Enzyme activity: The novel LR of the present invention catilyzes the reduction of levodione to actinol in the presence of a co-factor according to the following formula: Levodione + NADH Actinol + NAD The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) does not work as an electron donor in this reaction system. The standard enzyme assay was performed as follows: The basal reaction mixture of total volume 500µl and consisting of 100 µl of 1 M potassium phosphate buffer (pH 7.0), 20µ1 of 8mMM NADH in 0,2 mM KQH, 10 - 40 µl of the enzyme solution, and water up to a total of 500µl, was incubated for 1 minute at 37 °C. Then 2 ul of 0.5 M levodione solution were added to give a final concentration of 2 mM, and the whole was incubated for 1 minute at 37 °C- The enzyme activity was monitored with the decrease of the absorbance of NADH at 340 nm. One unit of the enzyme activitv was defined as the amount of the enzyme which catalyzes the oxidation of 1 µmole of NADH per minute, NAD, NADH and NADPH are available from Oriental Yeast, 3-6-10 Azusawa, Itabashi-ku, Tokyo, Japan. The protein concentration was determined by using a Bio-Rad protein assay kit (Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547, USA) 2) Molecular weight The molecular weight (MW) of the enzyme was measured with a gel filtration HPLC column Cosmosil 5Diol'300 (nacalai tesque: Nishi-iru, Karasuma, Nijodohri, Nakagyou-ku, Kyoto-fu, Japan). The apparent molecular weight of the (whole) enzyme was calculated to be 1.42,000- 155,000 ± 10,000 in comparison with the molecular weight marker proteins; LMW + HMW gel filtration calibration kit, Amersham Pharmacia Biotech (SE-75184 Uppsala, Sweden); ferritin (MW 440,000), aldolase (MW 158,000), bovine serum albumin (MW 67,000), ovalbumin (MW 43,000), and ribonuclease A (MW 13,700). SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) gave a single band with a molecular weight of 36,000 ± 5,000 in comparison with the molecular weight marker proteins: LMW Electrophoresis calibration kit, Amersham Pharmacia Biotech; bo\ine serum albumin (MW 67,000), ovalbumin (MW 43,000), carbonic anhydrase (MW 30,000), soybean trypsin inhibitor (MW 20,100), and a-lactalbumin (MW 14,400). This indicates that the enzyme is composed of four homologous subunits. The values of the molecular weight of the whole enzyme (142,000-155,000 + 10,000) and of each subunit (36,000 + 5,000) were determined as accurately as the respective methods, i.e. the gel filtration column method and the SDS-PAGE method, allowed. 3) Co-factor The co-factor requirement of the enzyme to convert levodione to actinol was investigated. As a result, it was established that NADH could serve as a co-factor for this reductive reaction, but that NADPH could not. 4) Substrate specificity The substrate specificity of the enzyme was determined using the same enzyme assay method as described under 1), except that various substrate solutions (2mM, final concentration in the reaction mixture) were used instead of levodione. It was shown that levodione was the only substrate for which the enzyme exhibited activity. 5) Optimum temperature The enzyme activities were measured at temperatures from 2 to 45°C . The optimum temperature of the enzyme activity was 15 -20°C 6) Optimum pH The correlation between the enzyme activity and the pH values of the reaction mixture was determined by using the same enzyme assay method as described under 1), except that various pHs and buffers were used and 40µl of 2.5M KCl solution were added to the reaction mixture. The optimum pH of the enzyme reaction was found to be 7.5. 7) Effect of metal ions The effect of metal ions on the enzyme activity was investigated by using the same enzyme assay method as described under 1), except that 100 µl of 1 M Tris-HCl buffer (pH 7.5) were used instead of 100 µl of I M potassium phosphate buffer (pH 7.0), and various metal solutions were added to the reaction mixture to give a final concentration of metal between 100 mM and 3 M. As a result, it was established that the enzyme activity was increased about 250-fold in the presence of 3 M RbCl and 1,8 M CsCl. Table 4-1 8) Temperature stability The enzyme solution was treated at various temperatures for 10 minutes, and the remaining enzyme activities were measured by using the same enzyme assay method as described under 1). It was established that the enzyme was stable up to 35°C and de¬activated with increasing temperature, becominng completely de-activated at 55°C . 9) pH stability The enzyme vvas treated in 1 M buffers of various pHs for 10 minutes at 30°C , and its remaining activity was measured by using the same enzyme assay method as described under 1). The enzyme was found to be most stable in the pH range between 8.0 and 8.5 10) Michaelis constant (Km) and Maximum velocity (Vmax) values The Km and Vmax values of the enzyme were measured by using levodione and actinol as the substrates. The basic enzyme assay method is the same as described under 1), but the substrate and the enzyme concentrations were varied. The Km and Vmax. values against levodione as the substrate were 8.5 mM and 101.26 unit/mg, respectively. On the other hand, the Kni and Vmax values against actinol as the substrate were 1.36 mM and 15.91 unit/mg, respectively. The Km and Vmax values were calculated on the basis of the known Michaelis-Menten equation. Km is the concentration of the substrate that gives 50% of the Vmax of the enzyme reaction. The values give a useful indication of the catalytic properties of the enzyme for the involved substrate. 11) Purification procedure The purification of the LR may in principle be effected by any combination.of known purification methods, such as fractionation with previpitants e.g. ammonium sulfate,polyethylene glycol and the like, ion exchange chromatography,_adsorption chromatography, gel-filtration chromatography, gel electrophoresis and sal ting out and dialysis. As mentioned above, the LR provided by the present invention can be prepared by cultivating an appropriate microorganisrn in an aqueous nutrient medium under aerobic, conditions, disrupting the cells of the microorganism and isolating and purifying the LR from the cell-free-extract of the disrupted cells of the microorganism. The microorganisms used for the present invention are microorganisms belonging to the genus Corymebacterium which are capable of producing LR as defined hereinbefore. Functional equivalents, subcultures, mutants and variants of said microorganism can also be used in the present invention. A preferred strain is Corynehacterium aquaticum. The specific strain most preferably used in the present invention is Corynebacterium aquaticum AKU6J1 (PERM BP-6448), a sample of which was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, on August 4, 1998, under the Budapest Treaty. Furthermore, European Patent Application No. 98115564.1, filed on August 19, 1998, and the later European application, No. 99115723.1, filed on August 16, 1999 and claiming the priority of the earlier one, disclose some characteristics of this strain. The microorganism may be cultured in a nutrient medium containing saccharides such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids such as oleic acid and stearic acid, or esters thereof, or oils such as rapeseed oil and soybean oil as carbon sources; ammonium sulfate, sodium nitrate, peptone, amino acids, com steep liquor, bran, yeast extract and the like as, nitrogen sources;.magnesium sulfate, sodium chloride, calcium carbon pptassium monohydrogen phosphate, potassium dihydrogen phosphate and the like as inorganic salt sources; and malt extract, meat extract_and the like as other nutrient sources. The cultivation can be carried out aerobically, normally for a period of 1 to 7 days at a medium pH of 3 to 9 and a cultivation temperature of 10 to 40"C. An embodiment for isolation and purification of the LR from the microorganism after the cultivation is as follows: (1) Cells are harvested from the liquid culture broth by centrifugation or filtration. (2) The harvested cells are washed with water, physiological saline or a buffer solution having an appropriate pH. (3) The washed cells are suspended in the buffer solution and disrupted by means of a homogenizer, sonicator, French press or treatment with lysozyme and the like to give a solution of disrupted cells. (4) The LR is isolated and purified from the cell-free extract of disrupted cells. The LR provided by the present invention is useful as a catalyst for the production of actinol from levodione. The reaction of the LR-catalysed reduction of levodione to actinol is conveniently conducted at pH values of from about 6.0 to about 9.0 in the presence ofNADH in a solvent. As a solvent, any buffer which maintains the pH in the range of about 6.0 to about 9.0, such as Tris-HCl buffer, phosphate buffer, Bis-tris buffer, HEPES buffer and the like, is suitable. A preferred temperature range for carrying out the reaction is from about 2 to about 30°C. The reaction usually gives the bst results when the PH and the temperature are in the ranges about 7.0 to 8.0 and 10 to 25 °C, respectively. The concentration of levodione in the solvent depends on the other reaction conditions, but in general is from 1 mM to 2 M, preferably from 10 mM to 100 mM. The amount of the LR and NADH suitably present in the reaction mixture depends on the other reaction conditions, but in general is in each case independently about 10-1 to 10-6 of the amount of the substrate. When a regeneration system of NADH from NAD is coupled with the above reaction system, the reaction proceeds more efficiently. In the reaction, the LR may also be used in an immobilized sta with an appropriate carrier. Any means of immobilizing enzymes generally known in the art may be used. For instance, the enzyme may be bound directly to a membrane, granules or the like of a resin having one or more functional groups, or it may be bound to the resin through bridging compounds having one or more functional groups, e.g. glutaraldehyde. Such enzyme immobilizing means are described for example on pages 369-394 of the 2nd Edition of Microbial Enzymes and Biotechnology (Elsevier Applied Science 1990; Ed. W.M. Fogarty and C.T. Kelly). The following Examples further illustrate the present invention. Example 1 Preparation of LR AH the operations were performed at 4°C, and the buffer was 10 mM potassium phosphate buffer (pH 7,0) containing 0.1 mM dithiothreitol unless otherwise stated. Cultivation of Corynbacterium aquaticum AKU 611 (PERM BP-6448) One colony of Corynebacteriiim aquaticum AKU 611 (PERM BP-6448) on an agar plate was inoculated into 5ml of the medium (pH 7.0) consisting of D-glucose (1%), KH.PO4 (0.3%), MgSO4.7H2O (0.02%), Peptone (1.5 %), NaCl (0.2%) and yeast extract (0.1%) in a tube, and incubated for 20 hours at 30°C. This culture was inoculated into 500 ml of the same medium as above in a 2 1 Sakaguchi flask, and incubated for 20 hours at 30°C. A 250 ml portion of the seed culture was inoculated into 20 1 of the same medium in a jar fermenter MSJ-U3W (Marubishi Bioengineering, 2-20-15 Higashikanda, Chiyoda-ku, Tokyo, Japan). Cultivation was effected at 30°C for 20 hours with aeration at the rate of 20 l/min. and agitation at 300 rpm. The culture thus obtained was centrifuged at 8,000 rpm for 20 minutes at 4°C. In total, 133.8 g of wet cells were obtained. (2) Preparation of the cell-free extract The wet cells (30 g) were suspended in 90 ml of the buffer, and sonicated for 1 hour at 190 W using a Kubota Insonator 201 sonicator (Kubota, 3-29-9 Kongo, Bunkyo-ku, Tokyo, Japan). After sonication, the sample was centrifuged at 16,000 rpm for 20 minutes. As a result, 80 ml of the cell-free extract containing 2,444 mg of protein were obtained. (3) Ammonium sulfate precipitation To the cell-free extract (80 ml) obtained in the previous step was added ammonium sulfate until a 60% saturation concentration had been achieved. Then the resulting precipitate was collected by centrifugation, solubilized in 15 ml of the buffer, and dialyzed four times against 5 1 of the buffer. The total enzyme activity in this solution was 38.8 units. (4) Diethylaminoethyl (hereinafter referred to as DEAE)-Sephacel column chromatography The dialyzed sample prepared as described above was applied to a DEAE-Sephacel column (2.8 cm in diameter and 18 cm in height; Amersham Pharmacia Biotech) which was equilibrated with the buffer. After washing the column with the same buffer, the enzyme was eluted with 600 ml of a linear gradient of NaCl (0 - 0.8 M). The active fractions were collected and concentrated by ultrafiltration (ultrafilter YM-10 with Amicon concentration apparatus (Amicon Inc., Beverly, MA 01915, USA) to 10 ml (5) Alkyl Superose column chromatography To the sample from the previous step was added (NH4)2SO4 to a final concentration of 2 M, and the mixture was filtered. An alkyl supersose 10/10 column (1 cm in diameter and 10 cm in height; Amersham Pharmacia Biotech) was equilibrated with the buffer containing 2 M (NH4)2SO4, and applied by the above sample. The enzyme was eluted by a 150 ml of linear gradient of the buffer [2 - 0 M (NH4)SO4]. The active fractions were collected, and dialyzed four times against 5 1 of the buffer. (6) MONO Q HR5/5 column chromatography The dialyzed sample from the previous step was applied to a MONO Q 5/5 column (5 mm in diameter and 5 cm in height; Amersham Pharmacia Biotech) which was equilibrated by the buffer. The enzyme was eluted with 21 ml of a linear gradient of NaCl (0 - 0,8 M). The specific activity of the enzyme was not increased due to the de-activation of the enzyme during the dialyzation step before this chromatography. But the enzyme gave a homogenous band on SDS-PAGE analysis. A summary of the purification steps of the enzyme is shown in Table 7. (7) Identification of the reaction product The reaction mixture (3.33 ml) containing 50 mg of NADH, 833 µl of 1 M potassium phosphate buffer (pH 7.0), 2 ml of the enzyme sample from the purification step of DEAE Sephace! column chromatography, and 550µl of distilled water was incubated at 39oC. To this reaction mixture, 10µl of 0.5 M levodione solution were added five times at 6 minute intervals. The reaction mixture was incubated for a further 5 minutes, and extracted with 1 ml of ethyl acetate. The extract was analyzed by gas chromatography [column: HR-20M (Shinwa, 50 Keisho-Machi, Fushimi-ku, Kyoto-shi, Kyoto, Japan) 0.25 mm 0 X 30m, column temperature: 160°C (constant), injector temperature: 250oC, carrier gas: He (about 1ml/min.)]. As a result, the product was identified as being actinol in comparison with a standard sample of actinol When NADH was replaced with NADPH, only a trace amount of actinol was detected. This application has been divided out of Indian Patent Application NO.65/MAS/2000 which relates to "a process for producing an enzyme having levodione reductase activity". WE CLAIM; 1. A process for producing actinol from levodione which comprises ireaimg levodione with (i) an isolated and purified enzyme having levodione reductase activity or a cell-free extract of a microorganism belonging to the genus Corynebacterium and capable of producing a levodione reductase, said enzyme having the following physico-chemical properties: - a molecular weight of 142,000 to 155,000 +10,000 for the whole enzyme; consisting of four known homologous subunits having a molecular weight of 36,000+5,000, - a nicotinamide adenine dinucleotide (NAD/NADH) co-factor, - a substrate specificity for levodione, - an optimum temperature of about 15°C to 20°C at a pH of 7.0, - an optimum pH of 7.5 and wherein the enzyme is activated by a ion selected from the group consisting of Kt,+ Cs+Rb+Na+ and NH4'; in the presence of a reduced form of nicotinamide adenine dinucleotide and recovering the actinol from the reaction mixture. 2. The process according to claim 1 wherein the enzyme is obtained from Corynebacterium. 3. The process according to claim 2 wherein the Corynebacterium is selected from the group consisting of Corynebacterium aquaticum AKU 611 (FERM BP- 6448), and mutants thereof. 4. A process for producing actinol from levodione substantially as herein described, and exemplified.

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