WO2011147453A1

WO2011147453A1 - Enzymatic synthesis of nootkatone

Info

Publication number: WO2011147453A1
Application number: PCT/EP2010/057277
Authority: WO
Inventors: Holger Zorn; Marco Alexander Fraatz; Stephanie Johanna Luise Riemer; Meike Takenberg; Ulrich Krings; Ralf Günter Berger; Stefan Marx; Kateryna Zelena
Original assignee: N-Zyme Biotec Gmbh
Priority date: 2010-05-26
Filing date: 2010-05-26
Publication date: 2011-12-01

Abstract

The invention relates to oxygenases from Pleurotus sapidus for the biocatalytic oxidation of valencene to form nootkatone (terpenes).

Description

Enzvatic synthesis of nootkatone

The present invention relates to processes for the oxidation of terpene carbons and polypeptides usable therefor.

While terpene hydrocarbons are often removed as undesirable excess components from essential oils, synthetic oxyfunctionalized derivatives are widely used as flavorings and fragrances. This mismatch, in combination with the trace occurrences of terpenoids of interest in plant sources, has given the microbial terpene biotransformation a fifty-year history (Schrader and Berger 2001). (+) - Nootkatone has a scent reminiscent of citrus and grapefruit, a slightly bitter taste and an extremely low sensory threshold of about one pg L ^"1 water (Ohloff 1994) This combination of properties has made Nootkaton a highly sought after organic product worldwide The first biotransformation of valencene mentioned in the literature dates back to 1973 (Dhavlikar and Albroscheit 1973) .Valencene was transformed into (+) - nootkatone with two isolated Enterobacter species with a maximum yield of 12% (w / w) of valencene with cell cultures of grapefruit (Citrus paradisi) resulted in nootkatone levels of approximately 1 mg L ^"1 after six hours of incubation (Drawert et al., 1984). Del Rio et al. used various C / trus species for the biosynthesis of nootkatone and achieved the highest nootkatiosis yields with nine-month-old callus cultures of Citrus paradisi (1.6 μg ^"1 wet biomass.) In 1994, the biosynthesis of nootkatone by a Rhodococcus strain (KSM-5706 ) (Okuda et al., 1994), however, yields were also low.

Among the recent approaches to novel solutions are experiments with recombinant microorganisms and with plant cell cultures or plant preparations. Microsomal preparations from the root of chicory (Cichorium intybus L.) were characterized by a negligible formation of by-products (Bouwmeester et al., 2007; de Kraker et al., 2003), but for the foreseeable future there will be no viable route to sufficient quantities of this biocatalyst. The oxyfunctionalization of (+) -valences with recombinant P450 _ca m enzymes from Pseudomonas putida with a maximum yield of 9% (w / w) was published in 2005 (Sowden et al., 2005). The reaction with recombinant P450 _B M-3 enzymes was also described, but resulted in a number of other products besides (+) - nootkatone.

Submerser cultures of the ascomycete Chaetomium globosum were also used to display (+) - nootkatone from (+) - valencene (Kaspera et al., 2005). After transformation for ³ days, 8 mg L ^"1 (+) -nautaton was obtained, which in turn generated numerous volatile and non-volatile byproducts, and plant cell cultures of Gynostemma pentaphyllum, Caragana chamlagu, and Hibiscus cannabinus were able to produce nootkatone from (+) - valencene (Sakamaki et al., 2005), but maximal yields of (+) -nottkaton were only obtained after 20 days transformation with Gynostemma pentaphyllum (Cucurbitaceae) (600 mg L ^"1 ). Long incubation times were also found in submerged cultures of Mucor sp. and Chlorella pyrenoidosa (Hashimoto et al., 2003b, Hashimoto et al., 2003a; Furusawa et al., 2005).

To circumvent the problems associated with the use of intact cells, the use of isolated enzymes has been proposed. A Swiss flavor producer describes the addition of lipoxygenase and unsaturated fatty acids to valencene (Muller et al., 1998). Laccases required synthetic cosubstrates (Hitchman et al., 2005) or a second reaction step (heating / heavy metal catalysis) (Huang et al., 2001). Good space-time yields were achieved with lyophilizates from Basidiomycten without the relevant patent application describing a concrete method example (Müller et al., 2005). There is therefore still a need for processes which make it possible to convert terpene hydrocarbons with high efficiency.

The object of the present invention was to provide such a method. The object is achieved by the provision of a polypeptide which is suitable for the oxidation of terpene hydrocarbons, as well as the information necessary to produce the enzyme recombinantly.

The subject is therefore on the one hand a polypeptide having an amino acid sequence according to SEQ ID NO. 1 or SEQ ID NO. 3 or a variant in which up to 10% of the amino acids are altered by insertions, deletions or substitution. Preferably, a sequence is used which corresponds exactly to SEQ ID. NO 1 or No. 3 has.

Insertion means the insertion of exactly one amino acid; Deletion the removal of exactly one amino acid. Substitution substitutes one amino acid for another amino acid. The invention also includes peptides and proteins which contain the sequence according to SEQ ID 1 or a variant in which up to 10% of the amino acids are modified by insertions, deletions or substitution, but are additionally extended N- or C-terminally as well as peptides and proteins which contain the sequence according to SEQ ID 3 or a variant in which up to 10% of the amino acids are modified by insertions, deletions or substitution, but are additionally extended N- or C-terminally.

Amino acids are in addition to the 20 naturally occurring amino acids and amino acid derivatives such. As hydroxyproline, derivatives which can be obtained by esterification of carboxylic acids or by amide formation or the like. In addition to the natural L-amino acids, D-amino acids can also be used. Preferably, a peptide is used in which less than 10% of the amino acids are modified by insertions, deletions or substitution, in particular not more than 8%, not more than 5%, not more than 3% and particularly preferably not more than 1%. In one embodiment, the peptides have C-terminal and / or N-terminal truncations.

A preferred embodiment of the protein is a protein represented by SEQ. ID. No. 2 or SEQ. ID. No. 4 or a nucleic acid which hybridizes with them under stringent conditions. The peptide according to the invention exhibits the properties of an enzyme, namely an oxygenase.

The enzyme is biochemically and molecular biologically characterized and shows only small sequence homologies to previously known oxygenases, be it from fungi or from other microorganisms. The invention furthermore relates to a nucleic acid which codes for the polypeptide according to the invention and a particularly preferred embodiment of which is the sequence according to Seq. ID No. 2 or Seq. ID No. 4 or hybridize with this under stringent conditions.

A further subject of the invention is a vector which contains the nucleic acid according to the invention and a transformed organism which contains the vector according to the invention.

The subject of the process is furthermore a process for the oxidation of terpene hydrocarbons comprising the step of

- Contacting terpene hydrocarbons with a polypeptide according to claim 1 or an enzyme preparation containing the polypeptide according to claim 1. So can be used a purified enzyme from natural sources or recombinant production or an enzyme preparation. Such may be obtained from the mycelium of a basidiomycete when physically, chemically or enzymatically digested. In one embodiment, the digestion is chemical. In another embodiment, the digestion is physical. In a further embodiment, the digestion is carried out enzymatically.

Preference is given to carrying out at least one step selected from chromatographic separation, precipitation, adsorption, crystallization, extraction and filtration, in particular on a membrane having an exclusion volume of 100 kDa or more.

Such digestion preferably takes place by dispersing, ball milling or high pressure homogenization.

Freeze-drying or lyophilization is not digestion; the sole lyophilization therefore does not lead to the desired result. Excluded is therefore the sole lyophilization without further homogenization steps.

Suitable basidiomycetes are in particular Basidiomycetes from the family Pleurotaceae, in particular Pleurotus sapidus, PI. Ostreatus and P. eryngii.

The process according to the invention is particularly preferably usable in order to obtain nootkatone from valencene. However, any further terpene hydrocarbons can also be used.

The invention is explained in more detail by the following examples.

FIG. 1 shows the product yields after transformation of (+) -valences with digested biomass; Transformation of 2 μΙ_ (+) -valences with bio-wet mass (BFM, digested) in 1.5 ml of 50 mM TRIS-HCl, pH 7.5 for 16 hours. FIG. 2 shows the product yields after fed-batch transformation of (+) - valencene with digested biopump in 100 ml of 50 mM TRIS-HCl, pH 7.5 for 50 hours.

FIG. 3 shows a FPLC chromatogram detail of the third stage of the 3-stage purification of separated supernatant on a Superdex 200 column; Running buffer: 200 mM TRIS-HCl, pH 7.5, flow rate: 0.5 mL min ^-1 , sample volume 200 pL, fraction size 1.0 mL.

FIG. 4 shows the product yields after transformation with FPLC fractions of the third purification stage; Transformation of 1 pL (+) -valencene in a volume of 1.5 mL for 20 hours; Buffer: 20 mM TRIS-HCl, pH 7.5.

Figure 5 shows a gel (12%) of SDS-PAGE after 3-step protein purification by FPLC followed by silver staining; MW = molecular weight, hrs. = Standard (1 pL).

FIG. 6A shows a gel gel (12%) of the SDS-PAGE after 3-stage protein purification by means of FPLC with specific staining for heme / metal enzymes (3,3 ', 5,5'-tetramethylbenzidine); MW = molecular weight of the standard, Std. = Standard (4 pL), MPO = horseradish peroxidase (15 pL, 345 mU), Over. = separated supernatant (15 pL), GF = combined active fractions from the 3rd purification stage (gel filtration, 15 pL), XXX = corresponding sample heated to 95 ° C. for 5 min before SDS-PAGE.

Figure 6B shows a gel (12%) of SDS-PAGE followed by staining for heme / metal enzymes; SLOX = soybean lipoxygenase (20 pL, 85 mU).

Figure 7 shows an alignment of the peptide sequences 66-1 and 66-3 from P. sapidus with the sequence of A. fumigatus lipoxygenase (XP_746844.1) using ClustalW (Thompson et al., 1994). FIG. 8 shows the cDNA and amino acid sequence of the polypeptide according to the invention according to SEQ ID. No. 1 or 2 from P. sapidus.

FIG. 9 shows the cDNA sequence of the polypeptide according to the invention according to SEQ ID. No. 4 from P. sapidus. FIG. 10 shows the amino acid sequence of the polypeptide according to the invention according to SEQ ID. No. 3 from P. sapidus.

Examples Example 1: Microorganism and cultivation

Pleurotus sapidus (DSMZ 8266) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig. a) nutrient solutions

The individual components (Table 1) were dissolved in distilled water and adjusted to pH 6.0 with 0.5 M sodium hydroxide solution.

Table 1: Composition of nutrient solutions; Glc = D - (+) - glucose monohydrate, Asn = L-asparagine monohydrate, SE-Lsg = trace element solution (Table 2) = concentration, * = modified according to Sprecher 1959

Table 2: Composition of the trace element solution

Substance concentration [g L ^" ']

FeCl ₃ · 6H ₂ 0 0.080

ZnS0 ₄ · 7H ₂ 0 0.090

MnS0 ₄ - H ₂ 0 0.030

CuS0 ₄ · 5 H ₂ 0 0.005

EDTA 0.400 b) Tribe Cultivation

Stocks of Pleurotus sapidus were grown on agar plates with SNL-H agar medium. To this was added one each agar plate inoculated with a 1 cm ² large, overgrown with mycelium agar piece, sealed with Parafilm ^® and in an incubator at 24 ° C cultivated (Taubert et al. 2000). After the plates were half-covered with mycelium, the culture was stored at 4 ° C. These stock cultures were inoculated at least every 6 months by the same procedure.

c) precultures

1 cm ^{2 of} the stock culture was transferred with the aid of a sterile spatula into an Erlenmeyer flask (500 ml) with 200 ml of SNL-H medium, homogenized (Ultra-Turrax homogenizer, TP 18/10, IKA, Staufen, about 20 s at low rotational speed) and 4 days at 24 ° C and 150 U min ^"1 incubated.

d) growth of biomass

In the stirred tank reactor, 2.3 L of NLMA medium were inoculated with 230 ml of homogenized preculture. After 4 days, the stirred tank reactor contents were harvested. For this purpose it was filtered through a cotton cloth. The resulting fungal mycelium was washed twice with 400 mL distilled water (m / v) each time and used for further experiments. The work was carried out under a sterile workbench and the equipment and solutions used were previously autoclaved at 121 ° C for 20 min.

Example 2: Cell disruption

a) Homogenization of organic moisture

3 g of wet biomass were added 7 mL of 50 mM TRIS-HCl, pH 7.5 and then treated at 15,600 rpm ^"1 for 5 min on ice with an Ultra-Turrax homogenizer (TP 18/10, IKA). The mixture was then the disrupted fungal mycelium was diluted to 16.7% (v / v) with the same buffer and used to transform (+) - valencene. b) Homogenization of biomaterial upscaling

50 g of wet biomass were added with 50 mL 50 mM TRIS-HCl, pH 7.5 and then treated at 20,000 rpm ^"1 for 15 min on ice with an Ultra-Turrax homogenizer (TP 18/10, IKA). The mixture was then the disrupted fungal mycelium is diluted to 25% (v / v) with the same buffer and used to transform (+) - valencene.

c) Homogenization of biosolids with high pressure

Fungal mycelial suspensions in DI water were digested by high-pressure homogenization (LAB 60/60 TBS, Gaulin APV, Switzerland). Cooled mycelium was digested with 1 to 3 passages (150/30 bar and 300/60 bar) and used directly for the biotransformation reaction. Alternatively, valencene was homogenized directly during cell disruption.

d) Preparation of lyophilisates

Up to 100 g of biomass was weighed into a glass petri dish, covered with aluminum foil and frozen at -20 ° C. The lyophilization was carried out for 3 to 7 days (VaCo 2, Zirbus Technology, Bad Grund). The shelf temperature was -20 ° C, the temperature of the cooling coil -45 ° C. The resulting lyophilizate was weighed, minced with a glass rod, transferred to sterile Falcon ™ tubes and stored at -70 ° C until use. Example 3: Biotransformation of valencene

a) Transformation with digested biomass

The transformation was carried out in screw-top vials (4 mL) in a horizontal position at 300 rpm ^"1 and 24 ° C. The biomass of Pleurotus sapidus (1.5 mL) was treated with an Ultra-Turrax homogenizer and used to transform 2 pL ( +) - valencene (Döhler, 70%) was used and extracted after the end of the transformation The determined content of (+) - nootkatone was 221 mg L ^"1 (FIG. b) Fed-batch transformation of valencene

Transformation was into glass bottles (500 mL) on a magnetic stirrer (Variomag poly 15, Thermo Fisher Scientific, Waltham, MA, USA) at 900 U min ^"1 and room temperature through out the transformation was performed by adding 333 pL (+) -. Valencene (Döhler,> 70%) was added to 100 mL cell suspension (homogenized), and after 6, 12, 18, 24, 30, 36 and 42 h, 156 pL (+) - valencene were added after 6, 23, 46 and 50 In each case 2 × 1.5 ml of sample were removed and extracted After 50 hours of transformation, the content of (+) - nootkatone was 603 mg L ^-1 (FIG. In total, 893 mg L ^"1 oc-, 3-nootkatol and (+) - nootkatone were formed c) Transformation with purified enzyme solution

0.5 mL of purified protein fraction (fractions 08, 09 and 12 to 22) were mixed with 1.0 mL of 20 mM TRIS-HCl, pH 7.5. Subsequently, the transformation was started by adding 1 pL (+) - valencene (Fluka,> 90%). The transformation was carried out in screw-top vials (4 mL) in a horizontal position at 150 rpm ^"1 and 24 ° C. After the end of the transformation, extraction was carried out Significant contents of the transformation products oc, β-nootkatol and (+) - nootkatone were after reaction with fractions 15 and 16 detected (Figure 4).

d) Capillary gas chromatography (GC) - microextraction after transformation

The microextraction took place directly in the screw-cap vials used for the transformation. The batch was mixed with internal standard (67 mg L ^"1 or 200 mg L ^{" 1} thymol) and 2 mL pentane, vortexed for 10 s, shaken horizontally for 10 min at 150 rpm ^"1 After centrifugation (10 min, 3,313 g , 4 ° C), the organic phase was dried over sodium sulfate and analyzed by gas chromatography.

e) capillary gas chromatography (HRGC)

The quantification of oc, ß-Nootkatol and (+) - Nootkaton by FID was carried out with a cold feed system and polar separation column. Table 3: GC-FID (KAS) with polar separation column

Example 4: Enzyme purification

Fast Protein Liquid Chromatography (FPLC)

To remove impurities, the sample was centrifuged (10 min, 13,000 rpm ^"1 , 16,060 xg, 4 C) and the supernatant injected into the FPLC (Table 4). If necessary, the sample was additionally membrane filtered (0.45 μm pore size , 25 mm, PET, Carl Roth GmbH) a) Three-stage purification by FPLC

Four 5 mL separated supernatants from Pleurotus sapidus were injected into the FPLC and separated using the weak anion exchanger DEAE FF (see Table 5). Fractions 09 to 24 of the four FPLC runs were pooled (purification level 1, DEAE pool), buffered with 20 mM Na citrate, pH 3.0 (Centricon Plus-70, 10 kDa cutoff) and concentrated to 2.5 mL , Precipitated proteins were removed by centrifugation. 2.0 mL of the rebuffered DEAE pool was re-injected into the FPLC and separated using the strong cation exchanger SP Sepharose FF, Table 6). Fractions 07-11 were pooled (Purification Stage 2, SP-FF pool), buffered with 200 mM TRIS-HCl, pH 7.5 (Amicon Ultra-15, 10 kDa cut-off) and concentrated to 300 pL. 200 pL of the buffered SP-FF pool was injected into the FPLC and purified by gel filtration on a Superdex 200 Column (Table 7) separately (purification step 3). After calibration of the separation column used, the peak maximum (30 min, FIG. 3) corresponded to a molecular weight of 54 kDa.

For SDS analysis, fractions 12 to 19 were rebuffered with E-Pure water and concentrated to 80 μΙ_ (Figure 5). These concentrates were used for denaturing SDS-PAGE with subsequent silver staining, for non-denaturing SDS-PAGE with subsequent specific staining for heme / metal enzymes (FIG. 6) and for sequencing selected protein bands after denaturing SDS-PAGE and subsequent staining using Coomassie.

Table 4: FPLC with UV detector

FPLC systems biology Duo Flow Chromatography system (Bio-Rad) with Biology faction ^¬ collector Model 2128 (Bio-Rad)

Data Acquisition Biology Duo Flow Workstation (Bio-Rad),

Version 3.00

Detection wavelength 280 nm

Ion Exchange Chromatography (IEX)

Table 5: Enzyme purification by DEAE, step gradient

Column HiPrep 16/10 DEAE FF

(Amersham Pharmacia Biotech)

Column volume (CV) 20 ml_

Starting buffer (A) 20 mM TRIS-HCl, pH 7.5

Elution buffer (B) 20 mM TRIS-HCl + 0.2 M NaCl, pH 7.5

Rinse buffer (C) 20 mM TRIS-HCl + 2.0 M NaCl, pH 7.5

Sample loop 5.0 ml_

Flow rate 5.0 ml min. ¹

Elution program 2 CV 100% buffer A

Sample injection: 2 x sample volume (buffer A)

7 CV100% buffer A

6 CV35% buffer B

5 CV100% buffer B

4 CV100% buffer C

5 CV100% buffer A

Fraction volume 2.0 ml_

Fraction volume 2.0 mL

Table 7: Enzyme purification by means of Superdex 200

Column Superdex 200 10/300 GL

(Amersham Pharmacia Biotech)

Bed volume approx. 24 mL

Separation range 10 - 600 kDa

Sample loop 200 nL

Flow rate 0.5 mL min ¹

Running buffer 200 mM TRIS-HCl, pH 7.5

Elution program Sample injection: 2 x sample volume

(Running buffer)

30 mL 100% running buffer

Fraction size 1.0 mL

Calibration of the gel filtration column was performed using standard proteins of two gel filtration calibration kits (High Molecular Weight and Low Molecular Weight, Amersham Pharmacia Biotech) according to the manufacturer's instructions. b) SDS-PAGE under denaturing conditions

The proteins were separated in 12% separating gels (modified according to Laemmmli, 1970) and visualized by Coomassie staining or silver staining (Blum et al., 1987). Both methods are known to expert persons. c) SDS-PAGE under non-denaturing conditions

SDS-PAGE under non-denaturing conditions was carried out analogously to item 4b, but the composition of the application buffer (Table 8) was modified.

Table 8: Composition of the Auftra uffers

d) staining for heme and metal enzymes (heme staining)

Heme staining (modified according to Thomas et al., 1976 and Henne et al., 2001) was performed only on SDS-PAGE under non-denaturing conditions. The following solutions were used for the heme staining (Table 9):

Table 9: Composition of the solutions for heme dyeing

Solution I and III were each prepared shortly before use.

SDS gels were incubated in Solution III in the dark for 45 min -1 h, then H ₂ O ₂ (30%) was added to the final concentration of 30 mM and incubated for 1 min. The gels were washed three times for 20 seconds each in e-pure water. Separated lyophilizate from Pleurotus sapidus was compared with the enzyme sample purified over three chromatographic steps (see Example 3, purification over three chromatographic stages (IEX, IEX, GF)). The heme-containing enzyme horseradish peroxidase and a lipoxygenase from soybeans (FIG. 6B) served as positive controls. On the basis of the specific staining, two proteins were identified as metal enzymes, which could also be detected after the chromatographic purification over three stages. Heating the samples before each application resulted in loss of activity. Example 5: Peptide sequences

Analogous to Example 4a, a three-stage chromatographic purification of the separated supernatant of Pleurotus sapidus was performed. After protein purification, incubation of (+) - valencene by gas chromatography revealed that the purified fractions had activity. The active fractions 15 and 16 of the final gel filtration were separated on a SDS-gel under denaturing conditions and stained with Coomassie ^® R (compare analog SDS-PAGE with silver stain in Figure 5). The estimated 75 kDa molecular weight band was excised from the gel and sent to de novo sequencing by electrospray tandem mass spectrometry (ESI-MS-MS) after trypsin digestion (Table 10).

Table 10: Detected peptide sequences (one-letter code) after ESI-MS-MS analysis of the 3-step purified enzymes; MW = molecular weight after SDS-PAGE, bold = secure identification

Homology comparisons were performed by database research with NCBI blast using blastp (Schäffer et al., 2001) and found hits on various lipoxygenases from ascomycetes. Starting from a comparison of peptide sequences 66-1 and 66-3 with the sequence of an Aspergillus fumigatus lipoxygenase (XP_746844.1; Figure 7), degenerate primers were derived and used in PCR screening (Example 6, molecular characterization, cDNA synthesis and PCR screening). Example 6: Molecular Characterization

a) cDNA synthesis and PCR screening

For the cloning of the oxygenase-encoding cDNA sequences, cDNA from P. sapidus was synthesized and screened by polymerase chain reaction. The mycelium of P. sapidus was harvested on the 4th day of culture. To isolate the total RNA, the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used according to the manufacturer's instructions. The quality of the isolated RNA was checked by denaturing formaldehyde-agarose gel electrophoresis and staining with ethidium bromide. For the synthesis of the cDNA, the SMART ™ PCR cDNA Synthesis Kit (Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France) was used according to the manufacturer's instructions. The first strand was synthesized by Superscript ™ II RNase H ^{"(Invitrogen,} Karlsruhe, Germany). PCR primers were produced by Eurofins MWG Operon (Ebersberg, Germany). 20 ng cDNA as template in 50 pL approaches were for the PCR lx CoralLoad buffer (Qiagen), 0.2mM dNTPs, 0.4μΜ primer and 1.25U HotStarTaq DNA polymerase (Qiagen) The amplification was performed with a PCR Mastercycler personal (Eppendorf, Hamburg, Germany) Primers were used for the overall sequence of the cDNA: forward (5 '> AAA CCT GAT GAG GAG CTG TTT <3'); reverse (5 '> ACA GGA TAC GGT GAT GAA TG <3').

b) Cloning and sequencing of PCR products

The PCR products were analyzed using the TA cloning kit (Invitrogen) cloned into the vector pCR ^® 2.1. Sequencing was performed with M13 reverse and forward primers and was performed by MWG Operon. The expected fragment size in the PCR screening when using the degenerate primer against each other was about 429 bp (equivalent to 143 amino acids). The corresponding band was isolated from the agarose gel, intermediately inserted into the vector pCR2.1-TOPO and then sequenced. The resulting sequence was again subjected to homology comparisons and identified as a fragment of an oxygenase. Primer walking was used to determine the coding sequence of the cDNA with a length of 1191 bp or 396 amino acids (FIG. 8). The translated protein sequence showed a ~ 50% homology to an oxygenase from the basidiomycete Laccaria bicolor (determined with blastp, Schäffer et al. 2001).

A highly homologous but much longer coding cDNA sequence with 1944 bases (plus stop codon) corresponding to 648 amino acids was also determined in this way (FIG. 9). This protein has a calculated molecular mass of about 75 kDa, which agrees well with the gel electrophoretic findings described in example 5.

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Claims

claims

Polypeptide containing an amino acid sequence according to SEQ ID no. 1 or SEQ ID NO. 3 or a variant in which up to 10% of the amino acids are altered by insertions, deletions or substitution.

Nucleic acid coding for a polypeptide according to claim 1, in particular having a sequence according to SEQ ID no. 2 or SEQ ID NO. 4th

Nucleic acid which hybridizes with the nucleic acid according to claim 2 under stringent conditions.

Vector containing a nucleic acid according to claim 2 or 3.

Transformed organism containing a vector according to claim 4.

Process for the oxidation of terpene hydrocarbons comprising the step of

- Contacting terpene hydrocarbons with a polypeptide according to claim 1 or an enzyme preparation containing the polypeptide according to claim 1.

The method of claim 6, wherein the enzyme preparation is obtained from the mycelium of a basidiomycete that is digested physically, chemically or enzymatically.

A process according to claim 5, which is disrupted by dispersing, ball milling or high pressure homogenization.

9. The method according to claim 7 or 8, wherein the Basidiomycetes come from the family of Pleurotaceae, in particular Pleurotus sapidus.

10. A process according to any one of claims 7 to 9, wherein the terpene hydrocarbon is valencene and the aroma active terpene is nootkatone.