JP2009515541A - Citronellal production method - Google Patents

Abstract

The present invention relates to a method for producing optically active citronellal by enzymatic reduction of citral using a reductase from Zymomonas mobilis.
[Selection] Figure 1

Description

  Citral is an antibacterial terpene that gives a characteristic lemon scent to plants such as lemongrass and Australian lemon myrtle, and is also an industrial intermediate, for example, for the synthesis of vitamins A and E. Available. Several biotransformation reactions of citral have been reported: reduction or oxidation of aldehyde groups (see Non-Patent Document 1), acyloin formation (see Non-Patent Document 2) and lyase activity (see Non-Patent Documents 1 and 3). It is.

  The object of the present invention is to screen the enzymatic activity of reducing the α, β-unsaturated carbon bond of citral to obtain the chiral product citronellal (FIG. 1). Citral is a mixture of the trans isomer geranial and the cis isomer neral, but since the amino acids can catalyze the isomerization, the substrate specificity for either isomer is presumed to be unimportant (Refer nonpatent literature 4).

  The useful use of the product (R)-(+)-citronellal is to form isopulegol by ring-closing reaction via the Prins reaction and then to (lR, 2S, 5R)-(-)-menthol by hydration. (Refer nonpatent literature 5). Due to its limited stereoselectivity (citral has three double bonds) and enantioselectivity, nonbiological strategies are still difficult (see Non-Patent Document 6 and FIG. 1).

  Enzymes that reduce the carbon double bond of other α, β-unsaturated carbonyl compounds have been reported in the literature. These belong primarily to the “Old Yellow Enzyme” family of flavins and NADPH-dependent reductases, reviewed for possible biotechnological applications by Williams and Bruce (see Non-Patent Document 7). Yes.

  Another example is a carboxyl reductase obtained from Rhodococcus erythropolis, which utilizes an unidentified thermostable cofactor and does not depend on added NADH or NADPH (see Non-Patent Document 8). ).

In order to detect the biotransformation of citral to citronellal, the corresponding enzyme must be active, electrons for reduction must be available, and citral, a very hydrophobic substrate, is sufficient The enzyme must be reached at the correct concentration, and citronellal formation must proceed faster than any conversion reaction. Therefore, screening strategies have been developed to test various microorganisms for citral conversion.
Wolken and van der Werf, Appl. Microbiol. Biotechnol., 2001, 57, 731 Stumpf and Kieslich, Appl. Microbiol. Biotechnol., 1991, 34, 598 Wolken et al., Eur. J. Biochem., 2002, 269, 5903 Wolken et al., J. Agric. Food Chem., 2000, 48, 5401 Trasarti et al., J. Catal., 2004, 224, 484 Maki-Arvela et al., J. Mol. Cat. A: Chem., 2005, 240, 72 Williams and Bruce, Microbiology, 2002, 148, 1607 van der Werf and Boot, Microbiology, 2000, 146, 1129

  A first embodiment of the present invention comprises a reductase selected from the group consisting of Zymomonas, Citrobacter, Candida, Saccharomyces, Kluyveromyces, Candida, Issatchenkia and Rhizopus, wherein the reduction reaction is achieved in a liquid two-phase system. This is a method for producing optically active citronellal by enzymatically reducing citral.

  In a preferred embodiment of the method, Zymomonas mobilis, Citrobacter freundii, Candida rugosa, Saccharomyces bayanus, Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida utilis, Issatchenkia orientalis and Rhizopus javanicus are used.

  The above microorganisms are well known to those skilled in the art and can be isolated by well known methods or obtained from public depositories such as ATCC and DSMZ.

  Zymomonas mobilis has been deposited in particular as ATCC 10998, ATCC 29192, ATCC 31821, ATCC 35001, ATCC 39985. Citrobacter freundii has been deposited in particular as ATCC 8090D, ATCC 11811. Candida rugosa is specifically deposited as DSM 70761. Issatchenkia orientalis has been deposited in particular as DSM 3433, DSM 6128, DSM 11956, DSM 70075, 70079.

  The reductase can be used in isolated and purified form or as a “whole-cell-enzyme” which means that the enzyme is used in the method of the invention with a microorganism. Microorganisms can also be permeabilized to improve the translocation of substrates, products and cofactors into and out of the cell. The term “reductase” means not only an isolated enzyme, but also an extract, solution or suspension containing the enzyme, a carrier on which the enzyme is immobilized, and the entire cell containing the enzyme. .

  An improved embodiment of the present invention is the use of a cofactor that is oxidized by the simultaneous reduction of citral. Suitable cofactors are all substances that can be oxidized under the conditions of the process according to the invention.

  Preferred cofactor systems can be used in equimolar or higher amounts or regenerated by well known systems such as electrochemical means or enzymatic regeneration systems such as glucose-6-phosphate dehydrogenase. NADH or NADPH can be. If the cofactor is regenerated, an amount less than equimolar is preferred for the present invention.

  A liquid two-phase system means a system containing two liquid phases that do not mix with each other in large quantities. Preferably, the first phase includes an organic liquid, and the second phase is a liquid two-phase system including water or a water-based solvent. More preferably, the organic phase is a liquid two-phase system consisting of ethers such as ethyl ether, methyl-tert-butyl ether (MTBE), or aromatic hydrocarbons such as benzene and toluene. A buffered aqueous solution is suitable as the second liquid phase.

  In the present invention, we study biological strategies that lead to citronellal by steric and enantioselective reduction of the α, β-unsaturated carbon bond of the terpene citral (an isomer of geranial and neral). A conventional aqueous screen for 46 prokaryotic and eukaryotic microorganisms revealed that only the gram-negative bacterium Zymomonas mobilis can form citronellal. The disadvantage of the aqueous system was the low solubility of the substrate citral in aqueous media.

  In the two-phase bioconversion system using Zymomonas mobilis cells, citronellal was formed at a concentration 10 times higher than that of the system without addition of organic solvent. The best results have been achieved with the organic solvents toluene, methyl-tert-butyl ether (MTBE), isoamyl alcohol and diethyl ether. Organic media in biocatalytic reactions using whole cells offer several advantages (Leon et al., Enzyme Microb. Technol., 1998, 23, 483). The first advantage is improved substrate and / or product solubility. The organic phase can keep the substrate as a whole at a high concentration during the reaction, while maintaining low levels of toxic or inhibitory compounds in the aqueous phase. Cell permeabilization is another advantage of organic solvents. In addition to the “freeze-thaw” cycle, the use of organic solvents such as toluene for permeabilization has been used in many organisms, such as yeast Kluyveromyces (Decleire et al., Enzyme Microb. Technol., 1987, 9, 300). And Zymomonas mobilis (Chun and Rogers, Appl. Microbiol. Biotechnol., 1988, 29, 19) are reported to cause an increase in passive flow across the cell membrane. As shown in E. coli (Marvin et al., J. Bacteriol., 1989, 171, 5262), the presence of EDTA can significantly increase the permeability of the outer membrane.

  When 20 volume% (% v / v) toluene or MTBE was used as the organic solvent, 10 of 46 strains that were negative before were found to be positive. Compared to aqueous systems, this approach has been a very successful strategy in screening for effective microorganisms for biotransformation from citral to citronellal. The highest product concentrations were obtained by the prokaryotic cell lines Zymomonas mobilis and Citrobacter freundii, and the enantiomeric excess (e.e.) was greater than 99% and 75% for the (S) -enantiomer, respectively. In contrast, in the case of eukaryotic cell lines, Candida rugosa, Saccharomyces bayanus and Saccharomyces cerevisae have> 98%, 97% and 96% ee for the (R) -enantiomer, respectively, and the opposite enantioselection Showed sex.

  One of the problems faced in biotransformation using whole cells is the formation of by-products due to the large number of catalysts present in the cells. Interestingly, most organic solvents tested with Zymomonas mobilis reduced the alcohol byproducts geraniol, nerol and citronellol as a whole. Similarly, EDTA decreased alcohol formation in both prokaryotic and eukaryotic cell lines and in most cases increased citronellal concentrations. The opposite effect on these product and by-product formation is speculated to indicate that the reduction of the aldehyde group and the reduction of the carbon-carbon double bond in citral are catalyzed by different enzymes.

  The enzymatic reduction of double bonds in α, β-unsaturated carbonyl compounds has already been reported. In particular, an oxidoreductase belonging to the "Old Yellow Enzyme" family (Kitzing et al., J. Biol. Chem., 2005), which is a flavin-dependent enzyme commonly present in yeast, plants and bacteria. 280, 27904) tolerate many α, β-unsaturated aldehydes and ketones (Williams and Bruce, Microbiology, 2002, 148, 1607; Vaz et al., Biochemistry, 1995, 34, 4246 Stott et al., J. Biol. Chem., 1993, 268, 6097). However, no conversion of citral by OYE has been reported so far. A BLAST P (www.ncbi.nlm.nih.gov) search for the yeast OYE 1, 2, and 3 protein sequences was performed on the recently published translated open reading frame of the genome of Zymomonas mobilis ZM4 (Seo et al., Nature Biotechnol., 2005, 23, 63). The highest homology was observed for a putative NADH: flavin oxidoreductase (ZM01885) of unknown function. However, the amino acid homology to yeast OYE was less than 50%. Whether citronellal formation is related to enzymes belonging to the OYE family is not yet known, and overexpression of the genes involved will be required for the development of high-productivity biological production methods.

Microbial culture
Twenty yeast strains, nine filamentous strains and 17 bacterial strains were obtained from the University of New South Wales Cultured Cell Storage Organization (World Culture Collection number 248). Yeast strains, filamentous strains and bacteria Zymomonas mobilis and Zymobacter palmae were grown in YPG medium (3 g / l yeast extract, 5 g / l peptone and 10 g / l D-glucose, pH 6.9). Lactic acid bacteria were grown in MRS (de Man, Rogosa, Sharpe) broth (pH 6.2) from Oxoid. Oxoid nutrient broth (pH 7.4) was used for all other bacteria. For Planococcus citreus and Vibrio harveyi, 3% w / v sodium chloride (% w / v) was added to the nutrient broth. Growth temperatures were 30 ° C. or 37 ° C., depending on being closer to the reported optimal growth temperature. Culturing was carried out with stirring at 150 rpm in a rotary shaker, except for non-stirring cultures of Lactobacillus casei, Leuconostoc mesenteroides and Propionibacterium freundenreichii. All cultures were grown until they reached stationary phase. To create permeabilized cells, the organism was washed twice in buffer (50 mM MOPS / KOH, pH 7) and then half volume (yeast and filamentous fungi) or 1/4 volume (bacteria). Resuspended in the buffer. The suspension was freeze-thawed three times using liquid nitrogen and a 25 ° C. constant temperature water bath, and then dispensed and stored at −20 ° C.

Culture screening citral (5 mM) in aqueous system was added in the form of a 0.2 M isopropanol solution. 2.5% by volume (% v / v) isopropanol played two roles: increased solubility of citral, a hydrophobic substrate, and a potential substrate for intracellular alcohol dehydrogenase that regenerates NAD (P) H. It is to act on. Each culture was subjected to three experiments.

Activity assay (GC assay to determine citral reductase activity)
procedure:
500 μl sample or water (blank)
100 μl 1 M potassium phosphate, pH 7.0
100 μl of 1 M glucose solution
100 μl each of 10 M NADPH / NADH solution
E. coli cell suspension containing 100 μl glucose dehydrogenase
DMSO solution incubation with 25 μl of 10% cis- / trans-citral: Stir vigorously at 35 ° C. for at least 15 hours.

  Extraction: For each sample, add 250 μl of chloroform and mix well for at least 5 minutes. This is then centrifuged for 3 minutes. The lower organic phase is removed, dried with molecular sieve 4A, transferred to an HPLC glass vial fitted with a micro inset and measured by GC. The enantiomeric excess (e.e.) due to the purified enzyme is a value exceeding 98% (determined by the peak area ratio).

a. Whole cells in culture
5 ml of fresh medium was added to 5 ml of stationary phase culture medium so that there was no temperature change, and the mixture was stirred at 150 rpm for 1 hour, and then a citral solution in isopropanol was added. After 3 and 24 hours, 1 ml of broth was extracted with 0.2 ml of a chloroform solution containing 0.3% by volume of 1-octanol (internal standard for GC analysis) prepared freshly. After collecting the organic phase, it was diluted with isopropanol and subjected to GC analysis.

b. Permeabilized cells + NADH
2 ml volumes containing 0.5 ml of thawed permeabilized cells, each injected with a total volume of 1 ml containing 50 mM MOPS buffer (pH 7), 10 mM NADH, 5 mM citral and 2.5 vol% isopropanol as final concentrations. The screw cap glass vial was shaken and stirred at 30 ° C. and 150 rpm with a rotary shaker. After 3 hours, all the samples were extracted with a chloroform / octanol solution according to the above method.

c. Permeabilized cells + NADPH regenerating system
The procedure is to replace NADH with a final concentration of NADPH regeneration system containing 1.5 mM NADP +, 10 mM glucose-6-phosphate, 3.3 mM magnesium chloride and 0.4 U / ml glucose-6-phosphate dehydrogenase, respectively. The same as b above.

Culture Screening in Biphasic System Prior to bioconversion, 0.5 ml of permeabilized cells were vortexed with 0.15 ml of toluene in a 2 ml screw cap glass vial and allowed to stand at room temperature for 10 minutes. Subsequently, other components were added, and 1 ml of an aqueous phase having the same composition as the water / NADPH system was obtained except for citral and isopropanol. The reaction was started by adding 0.05 ml of a toluene solution containing 0.4 M citral, and the vials were turned vertically in a rotary incubator at 30 ° C. After 3 hours, all the samples were extracted with 0.4 ml of a chloroform solution containing 0.15% by volume of 1-octanol (an internal standard for GC analysis) prepared newly. After centrifugation, 0.4 ml of the lower organic phase (toluene / chloroform mixture) was collected for use in GC analysis. The cell lines that formed citronellal were compared for the two-phase system of toluene and methyl-tert-butyl ether (MTBE) by the method described in detail below.

Bioconversion The bioconversion shown in FIGS. 2-4 follows a culture screening procedure in a two-phase system, but in a 4 ml screw cap glass vial, 0.8 ml of aqueous phase and 0.2 ml of organic solvent are added in the aqueous phase. It was carried out with stirring with a magnetic stir bar so that the organic solvent maintained the emulsion. Details of the composition and conditions are given in the corresponding figure descriptions. Prior to the start of the reaction, 0.5 ml of washed cells prepared to the prescribed organism concentration was stirred with 0.15 ml of the corresponding organic solvent for 30 minutes on an ice bath. The reaction was started by adding the same organic solvent containing 0.05 ml of 0.4 M citral.

Analytical method The concentration of terpenes was determined by gas chromatography (GC) connected with a capillary column (Chrompack CP-SIL 5B, Varian, 50 mx 0.25 mm, phase thickness 0.12 μm) and carrier gas (nitrogen: 0.98 ml / min) and It was determined according to the conditions of a flame ionization detector (250 ° C., hydrogen: 20 ml / min, air: 300 ml / min). The sample injection volume was 1 μl and the split ratio was 29: 1. The temperature of the injector section was 250 ° C., the column temperature was constant at 100 ° C. for 9 minutes, and then the temperature was raised to 240 ° C. at 50 ° C./min. 1-Octanol was used as an internal standard substance, and the terpene concentration was calculated based on a sample according to the same extraction procedure as the sample. For separation of citronellal and isopulegol, the initial column temperature was 75 ° C. for 12 minutes. Nerol and citronellol did not separate under any of the above conditions. These separations were achieved using a Supelco Beta Dex 225 column (30 mx 0.25 mm, phase thickness 0.25 μm) as in chiral analysis. The split ratio was 1:35, the column flow rate was 2.77 ml / min, the column temperature was constant at 95 ° C for 35 minutes, and then the temperature was increased to 160 ° C at 5 ° C / min. Citronellal was identified by coelution with the standard citronellal in any GC column.

Culture screening in aqueous systems
For 20 yeast strains, 9 filamentous strains and 17 bacterial strains, use 3 types of aqueous systems, i.e. whole cells in culture, or add NADH to washed and permeabilized cells Citral conversion test was performed when the cells were used or NADPH was added to the cells. Citronellal was formed only when NADPH was added to permeabilized Zymomonas mobilis. Both NADPH regeneration system or direct addition of NADPH supported the formation of citronellal. Similar to the addition of NADH or NADPH to permeabilized cells, geraniol was the major product in many strains tested in whole cell cultures. Nerol and / or citronellol were also formed. Thus, the enzyme activity for the reduction of aldehyde groups competed with the activity required for the reduction of double bonds.

Effect of solvent on citral conversion by Zymomonas mobilis
Using Zymomonas mobilis, the feasibility of an organic / water two-phase system for the conversion of citral to citronellal was tested. Figure 2a shows the formation of citronellal in the absence and presence of various water-soluble (asterisk) and water-insoluble solvents, in the order of functional groups, two linear alkanes, nine aliphatic alcohols, three ketones, Two types of esters, two types of ethers, and three types of aromatic solvents are arranged in this order. Four solvents, i.e. isoamyl alcohol, MTBE, diethyl ether and toluene, supported about 10 times higher product amounts compared to the control without organic solvent. A further advantage was that in most solvents the amount of by-products geraniol, nerol and / or citronellol was reduced (FIG. 2b). In the control test, extraction of geraniol, nerol and citronellol preparations, it was confirmed that these extraction amounts relative to the internal standard were not affected by the presence of eg isopropanol, toluene or MTBE in the sample. Therefore, the reduction in the amount of by-products seems to have been due to a decrease in the enzymatic reduction of aldehyde groups. Toluene and MTBE were selected for further testing.

Culture screening in a biphasic system In the water / toluene screening system, citronellal was formed from citral in several strains, but these were not previously identified in the water screening system. For comparison, these cultures were adjusted for similar organism concentrations and citronellal production in toluene and MTBE two-phase systems is illustrated in FIG. In the toluene system, two bacterial strains achieved citronellal concentrations that were at least five times higher than nine eukaryotic cell lines. In MTBE system, Citrobacter freundii formed a small amount of citronellal compared to the presence of toluene. A positive result was obtained in Issatchenkia orientalis, which did not form citronellal in the toluene system. The addition of 5 mM EDTA resulted in higher concentrations of citronellal in most strains, but had the opposite effect in Citrobacter freundii and the filamentous fungus Rhizopus javanicus (FIG. 3b). Citronellal formation by Zymomonas mobilis was not affected by EDTA.

  Regarding the formation of by-products in the MTBE / EDTA system, the yeast strain and the bacterium Citrobacter freundii produced mainly geraniol and some nerol and a small amount of citronellol. In particular, the Saccharomyces strain formed geraniol reaching 13 mM. Excluding EDTA, alcohol by-product formation was increased by approximately 10% in the Saccharomyces strain and 9-fold in Zymomonas mobilis. Isopulegol was not detected in any experiment.

Table 1 shows the enantiomeric excess (ee) values of the product Citronellal. Bacterial enzymes had a preference to form (S) -enantiomers in Zymomonas mobilis> 99% ee and Citrobacter freundii in 75% ee. In contrast, the yeast strain preferentially produced (R) -citronellal, for example in Candida rugosa, with an enantiomeric excess of> 98% ee.

Biotransformation characteristics of citral Figure 4 illustrates the biotransformation characteristics of citral by Zymomonas mobilis in water / toluene and water / MTBE two-phase systems. For the former, the increase in citronellal concentration continued somewhat linearly over 3 hours. The same final citronellal concentration was achieved when the concentration of the NADPH regeneration system was doubled. This indicates that the reaction was not limited by the NADPH regeneration system. The concentration of both geranial and neral citral isomers decreased with time. The alcohols nerol, geraniol and citronellol were not detected. In the water / MTBE two-phase system, citral isomers geranial and neral were consumed linearly over time, but citronellal formation was observed to slow down over time. Of the alcohol by-products, 0.34 mM geraniol and less than 0.1 mM citronellol were formed. The molar concentration balance after 3 hours showed a 16% decrease in the initial substrate in the toluene system and a 10% decrease in the MTBE system. In contrast, the control without organisms showed no disappearance of citral or citronellal over 3 hours. However, controls containing boiling-treated organisms lost as much citral and citronellal as observed with the biotransformation.

Isolation of reductase from Zymomonas mobilis and investigation of its properties
Zymomonas mobilis culture:
The cells are in the medium with the following composition:
Bacto peptone 10 g / l
Yeast extract 10 g / l
Glucose monohydrate 20 g / l
Potassium dihydrogen phosphate 1 g / l
Ammonium sulfate 1 g / l
Magnesium sulfate heptahydrate 0.5 g / l
pH 7.0; Sterilization: 20 minutes, 121 ° C
Grow in.

  For this purpose, in each case 800 ml of medium is placed in a 1 L Erlenmeyer flask (without baffle) and cultured at 30 ° C. and 100 rpm for at least 48 hours (usually about 65-72 hours). Slight turbidity is removed by centrifugation. The yield of the organism is small (about 2.0-2.5 g / l organism wet weight).

Protein purification:
Homogenization
106 g of wet organism is resuspended in 500 ml of disruption buffer (20 mM Tris / HCl, 2 mM EDTA, 10 tablets / l complete protease inhibitor, pH 8.0), pH adjusted with sodium hydroxide Adjust to pH 6.6 to 7.5. The 100 ml fraction of the suspension is mixed with 100 ml glass beads (diameter 0.1-0.2 mm) and crushed in a ball mill at 5000 rpm for 12 minutes. The glass beads are removed and washed by suction through a G1 glass suction filter. The filtrate (710 ml) is centrifuged (Sorvall) for 30 minutes at 12000 rpm and 4 ° C. The supernatant (610 ml, 18.6 mg protein / ml, pH 7.4, conductivity 2.6 mS / cm) is dispensed into three 200 ml fractions and stored at -20 ° C.

Ion exchange chromatography (Q-Sepharose FF)
The homogeneous solution is injected into a Q-Sepharose FF chromatography column (diameter 5 cm, column volume 400 ml) (15 ml / min), 20 mM Tris / HCl, pH 8.0, 2 mM EDTA, 2 tablets / l complete protease Prewashed with inhibitor (buffer A). For this purpose, the thawed crude homogenate was diluted to 500 ml with buffer A (electric conductivity 1.6 mS / cm, pH 8.0). After sample introduction, the column was washed with 1000 ml of buffer A (15 ml / min). This was followed by a linear concentration gradient to 100% buffer B (buffer A containing 0.5 M sodium chloride, pH 8.0) (1500 ml). An additional 750 ml of 100% buffer B was used for washing. In each case, 10 fractions of 12 ml were combined and assayed.

Hydrophobic chromatography (TSK-Phenyl)
Predetermined fractions from the Q-Sepharose column were used for sample introduction (142 ml, pH 7.0, adjusted to 40% saturation with 32.2 g ammonium sulfate; protein amount 500 mg).

  The Phenyl-Sepharose column (Pharmacia) had a diameter of 2.6 cm, a column volume of 170 ml, and an operating flow rate of 10 ml / min. The column was equilibrated with buffer A (40% saturated ammonium sulfate, 2 mM EDTA, 2 tablets / l complete protease inhibitor, 20 mM sodium dihydrogen phosphate, pH 7.0). After sample introduction, the column was washed with buffer A until the absorption reached baseline. This was followed by a linear concentration gradient to 100% buffer B (buffer A without ammonium sulfate, 3 times the column volume). After completion of the operation, the column was eluted with 2.5 times the column volume of buffer C (buffer B containing 20% isopropanol). In each case, 10 fractions were combined and subjected to activity assay. Fractions 61-70 (92 ml) were collected.

Molecular sieve chromatography (Superdex)
A predetermined fraction (90 ml) from the Phenyl-Sepharose column was precipitated using 46.4 g of ammonium sulfate (80% saturation) and collected by centrifugation. Resuspend the pellet in separation buffer (20 mM sodium phosphate, pH 6.8, 1 tablet / l Complete, 1 mM EDTA) (9 ml) and inject onto a Superdex column (4 ml / min, 2 min fraction) did. The active fractions 27 and 28 were collected.

  Using all these procedures, all three homogeneous solutions were processed. A predetermined fraction of 67 ml containing 18.8 mg of total protein was obtained.

Affinity chromatography (Blue Sepharose FF)
A Blue Sepharose FF column (diameter 2.6 cm, column volume 50 ml, Pharmacia) was equilibrated with a buffer (20 mM sodium dihydrogen phosphate, pH 6.8, 1 tablet / l Complete, 1 mM EDTA). Predetermined fractions from this molecular sieve chromatography were combined and injected at a flow rate of 3 ml. Thereafter, the column was washed with 150 ml of the same buffer, and then with the same buffer (130 ml) containing 0.5 M sodium chloride. Elution was performed using a second wash buffer supplemented with 1 mM NADH and NADPH in each case. Prior to elution with the buffer, the column was filled with the buffer and incubated for 16 hours. Thereafter, the active fraction was collected. In the selected conditions, the protein does not bind to the Blue Sepharose column. However, other proteins were removed by this method, resulting in successful purification.

Affinity chromatography (4-hydroxybenzoic acid affinity column)
Affinity column preparation: Wash 50 ml of aminohexanoic acid Sepharose with 5 L of water. Resuspend the filter cake in 40 ml water. 4.2 g of 4-acetoxybenzoic acid (hydroxybenzoic acid introduced with protecting groups) is dissolved in 50 ml of DMF and adjusted to pH 4.7 (10 M sodium hydroxide). Dissolve 3.9 g EDC in 10 ml water and add to the binding mixture within 15 minutes. The pH is maintained at 4.7 with hydrochloric acid. Thereafter, an additional 50 ml of DMF is added and the mixture is stirred at room temperature for 16 hours. The column material is collected by suction through a G2 glass suction filter and washed with 1 L of 50% water / DMF solution. Then wash with 1 L of water. The column material is then resuspended in 200 ml of cold sodium bicarbonate solution and 2 ml of acetic anhydride is added. Deprotection is carried out on ice for 90 minutes, which causes a violent evolution of carbon dioxide. The reaction solution is removed by suction and the column material is washed with water. The column material is then stirred in 1 M imidazole solution at pH 7.9 for 1 hour and then removed again by suction, washed with water and stored in this state. Protein does not adsorb to this column.

Mono-Q chromatography
Mono-Q chromatography column (1 ml, Pharmacia) is equilibrated with buffer (20 mM sodium phosphate, pH 7.5, 1 tablet / l Complete, 1 mM EDTA). The non-adsorbed fraction (pH 7.5, 2.7 mS / cm) from a 130 ml Blue Sepharose FF column is injected into the column (1 ml / min). The column is then washed with the same buffer. Again, the protein is already eluted during sample introduction, but contaminants bind to the column.

Hydrophobic chromatography (Resource-Phenylsepharose, FPLC)
Equilibrate a 1 ml Resource column (Amersham) with buffer (20 mM sodium phosphate, pH 7.0, 2 tablets / l Complete, 2 mM EDTA, 40% saturated ammonium sulfate). The non-adsorbed fraction (pH 7.5, 2.7 mS / cm) from a 140 ml Mono-Q-Sepharose column is adjusted to 40% saturation with 31.6 g ammonium sulfate and injected onto the column (160 ml, 1 ml / min) . The column is then washed with the same buffer. The column is then developed with a linear gradient to the same buffer without ammonium sulfate. The active fraction is collected and separated by SDS gel electrophoresis (FIG. 1). Blot the 40 kDa band and perform N-terminal sequence analysis.

The purification is summarized in Table 2.

Sequence analysis of the N-terminal N-terminal sequence shown below,
PSLFDPIRFGAFTAKNRIWMAPLTRGR
was gotten.

  By computer search of the identified N-terminal peptide sequence against the published Zymomonas mobilis genome (Seo et al., Nat. Biotechnol., 2005, 23, 63, EMBL AE008692), the protein was isolated from 358 amino acid residues. It was identified as NADH flavin oxidoreductase (EMBL AE008692, AAV90509.1, primary accession number Q5NLA1), which contains a group and has a molecular weight of 39489 Dalton.

Biotransformation from citral (geranial and neral) to citronellal. Biotransformation of citral by Zymomonas mobilis cells in the presence of NADPH regeneration system and various solvents: a) Citronellal product b) Sum of by-products geraniol, nerol and citronellol. Initial conditions: 20% organic solvent, 20 mM citral, 5 mM dithiothreitol, 3.3 mM magnesium chloride, 1.5 mM NADP +, 10 mM glucose-6-phosphate, 0.4 U / ml glucose-6-phosphate dehydrogenated Enzyme, approximately 14 g / l DCM Z. mobilis, 50 mM MOPS / KOH, pH 7.0, 30 ° C., 3 hours. ^* Solvents marked with an asterisk dissolved in water and all other solvents formed emulsions. MTBE = methyl-tert-butyl ether, nd = not detected. AU = arbitrary unit: A value obtained by dividing the GC area of the compound by the GC area of the internal standard. Average values for 3 replicates are shown, error bars indicate maximum and minimum values for the test. For toluene, the peak ratio corresponded to a concentration of 1.4 mM citronellal. Comparison of citronellal formation by bacteria, yeast and filamentous fungi in a) water / toluene biphasic system and b) water / methyl-tert-butyl ether (MTBE) biphasic system According to the composition shown in FIG. 2 except that the amount is about 12 g DCM / l). Average values of 2 replicates are shown, error bars indicate maximum and minimum values of the test. nd = not detected. Citronellal bioconversion by Zymomonas mobilis using NADPH regeneration system in a water / toluene two-phase system and b) water / methyl-tert-butyl ether (MTBE) two-phase system (at 30 ° C, organism concentration about 14 g) Other than using DCM / l, the composition is as shown in Fig. 2). Average values for 3 replicates are shown, error bars indicate maximum and minimum values for the test.

Claims (7)

Reduction is achieved by a liquid two-phase system, selected from the group consisting of Zymomonas mobilis, Citrobacter freundii, Candida rugosa, Saccharomyces bayanus, Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida utilis, Issatchenkia orientalis and Rhizopus javanicus A method for producing optically active citronellal by enzymatic reduction of citral using 2. The method of claim 1, wherein (S) -citronellal is produced by an enzyme selected from Zymomonas mobilis and Citrobacter freundii. The method according to claim 1, wherein (R) -citronellal is produced by an enzyme selected from Candida rugosa, Saccharomyces bayanus, Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida utilis, Issatchenkia orientalis and Rhizopus javanicus. 6. The method of claim 1, wherein the enzyme is selected from the group of enzymes shown in FIG. The method of claim 1, wherein the liquid two-phase system is formed by MTBE and water. 6. The method of claim 5, wherein a buffered aqueous solution is used as one phase of the liquid two-phase system. The method of claim 1, wherein the reductase is conjugated to a NADPH regeneration system.

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