AU2001270612A1 - Microbial production of r-phenylacetylcarbinol by biotransformation of benzaldehyde by filamentous fungi - Google Patents
Microbial production of r-phenylacetylcarbinol by biotransformation of benzaldehyde by filamentous fungiInfo
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- AU2001270612A1 AU2001270612A1 AU2001270612A AU2001270612A AU2001270612A1 AU 2001270612 A1 AU2001270612 A1 AU 2001270612A1 AU 2001270612 A AU2001270612 A AU 2001270612A AU 2001270612 A AU2001270612 A AU 2001270612A AU 2001270612 A1 AU2001270612 A1 AU 2001270612A1
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- benzaldehyde
- filamentous fungi
- biotransformation
- rhizopus
- process according
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- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 title claims description 62
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 title claims description 31
- 241000233866 Fungi Species 0.000 title claims description 21
- 230000036983 biotransformation Effects 0.000 title claims description 18
- 238000004519 manufacturing process Methods 0.000 title claims description 10
- 230000000813 microbial effect Effects 0.000 title description 4
- 108010011939 Pyruvate Decarboxylase Proteins 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 17
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims description 12
- 241000588264 Rhizopus javanicus Species 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 10
- 241000235527 Rhizopus Species 0.000 claims description 8
- 241000235395 Mucor Species 0.000 claims description 6
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims description 5
- 241000223218 Fusarium Species 0.000 claims description 5
- 241001149952 Amylomyces rouxii Species 0.000 claims description 4
- 241001363490 Monilia Species 0.000 claims description 2
- 241000221960 Neurospora Species 0.000 claims description 2
- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 claims description 2
- 241000222640 Polyporus Species 0.000 claims description 2
- ZBFFNPODXBJBPW-UHFFFAOYSA-N 1-hydroxy-1-phenylpropan-2-one Chemical compound CC(=O)C(O)C1=CC=CC=C1 ZBFFNPODXBJBPW-UHFFFAOYSA-N 0.000 description 23
- 230000000694 effects Effects 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 241000221961 Neurospora crassa Species 0.000 description 6
- 240000005384 Rhizopus oryzae Species 0.000 description 6
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 6
- 239000000287 crude extract Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- ZBFFNPODXBJBPW-VIFPVBQESA-N (R)-phenylacetylcarbinol Chemical compound CC(=O)[C@H](O)C1=CC=CC=C1 ZBFFNPODXBJBPW-VIFPVBQESA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- 230000002538 fungal effect Effects 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 240000006439 Aspergillus oryzae Species 0.000 description 3
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 3
- 241000134719 Aspergillus tamarii Species 0.000 description 3
- 241000235646 Cyberlindnera jadinii Species 0.000 description 3
- 244000070804 Neurospora sitophila Species 0.000 description 3
- 241001465752 Purpureocillium lilacinum Species 0.000 description 3
- 238000006657 acyloin condensation reaction Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000008170 thiamine pyrophosphate Nutrition 0.000 description 3
- 239000011678 thiamine pyrophosphate Substances 0.000 description 3
- KWGRBVOPPLSCSI-WPRPVWTQSA-N (-)-ephedrine Chemical compound CN[C@@H](C)[C@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WPRPVWTQSA-N 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 241000223197 Fusarium lateritium Species 0.000 description 2
- 241001149959 Fusarium sp. Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 241000408048 Piptoporus portentosus Species 0.000 description 2
- 125000002015 acyclic group Chemical group 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 244000291564 Allium cepa Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000351920 Aspergillus nidulans Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000228230 Aspergillus parasiticus Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 241000306281 Mucor ambiguus Species 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000588902 Zymomonas mobilis Species 0.000 description 1
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 150000002009 diols Chemical class 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229960002179 ephedrine Drugs 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- KWGRBVOPPLSCSI-WCBMZHEXSA-N pseudoephedrine Chemical compound CN[C@@H](C)[C@@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WCBMZHEXSA-N 0.000 description 1
- 229960003908 pseudoephedrine Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229960002363 thiamine pyrophosphate Drugs 0.000 description 1
- YXVCLPJQTZXJLH-UHFFFAOYSA-N thiamine(1+) diphosphate chloride Chemical compound [Cl-].CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N YXVCLPJQTZXJLH-UHFFFAOYSA-N 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000007220 yepg medium Substances 0.000 description 1
Description
Microbial production of .R-phenylacetylcarbinol by biotrans- formation of benzaldehyde by filamentous fungi
5 The present invention relates to pocess for the production of .R-phenylacetylcarbinol ( R-PAC ) by biotransformation of benzaldehyde by filamentous fungi.
R-phenylacetyl carbinol is an intermediate in the production of 10 the pharmaceutical compound ephedrine and pseudoephedrine and is currently produced via a biotransformation of benzaldehyde by yeast cultures. The biotransformation is catalyzed by the enzyme pyruvate decarboxylase . This catalysis can be conducted using either whole microorganisms (for example Saccharomyces 15 cerevisiae, Candida utilis) or cell free extracts of microorganisms (for example Saccharomyces cerevisiae, Candida utilis, Zymomonas mobilis) .
Genes of pyruvate decarboxylases have been isolated from the 20 filamentous fungi Neurospora crassa (Alvarez et al . 1993), Aspergillus parasi ticus (Sanchis et al . 1994) and Aspergillus nidulans (Lockington et al . 1997).
In literature the following strains of filamentous fungi are 25 reported to conduct acyloin condensations: in a fermentation of benzaldehyde by Aspergillus niger a diol was detected after treatment with NaBH4 (Cardillo et al . 1991). Mucor circinelloides is reported for acyloin condensations with acyclic unsaturated aldehydes but not benzaldehyde as substrate (Stumpf and Kieslich 30 1991) .
It was the object of the present invention to provide a process for the microbial production of .R-phenylacetylcarbinol by bio- transformation of benzaldehyde that with respect to overall 35 yield, enantiomeric purity, stability and safety of microbial catalyst or costs of process should be advantagious over the prior art processes .
A first embodiment of the invention is a process for the 40 production of .R-phenylacetylcarbinol by biotransformation of benzaldehyde by filamentous fungi.
45
Filamentous fungi are classified according to Alexopoulos and Mims (Alexopoulos and Mims, 1979).
Preferred for the present invention are filamentous fungi of the subdivisions Ascomycotina, Zygomycotina and Basidiomycotina, es- pecially those selected from the group of Rhizopus, Neurospora, Polyporus, Fusarium, Monilia, Paecilomyces, Mucor. Especially preferred are those of the species Rhizopus javanicus, Neurospora crassa, Polyporus eucalyptorum, Fusarium lateritium, Monilia sitophila, Paecilomyces lilacinus, Mucor rouxii, which are further defined in the experimental section below.
These filamentous fungi are well known to the skilled person and can easily be isolated by known techniques (Onions et al . 1981), or can be obtained from public depositories.
A preselection for suitable filamentous fungi can be made on the capacity of the respective fungus to produce ethanol from sugar (Singh et al . , 1992; Skory et al,, 1997).
The biotransformation of benzaldehyde to R-PAC needs the presence of a source of acetaldehyde, which can be acetaldehyde itself or pyruvate. Preferred is the addition of pyruvate, especially in an amount of 1-2, preferred 1,5 mol pyruvate per mol of benzaldehyde .
The filamentous fungi can be used for the biotransformation as whole fungal mycelia or in the form of extracts which contain pyruvate decarboxylase . Extracts means soluble or solubilised forms of enzymes of the fungi. The extracts usually contains enzymes with a higher specific enzymatic activity than the whole fungal mycelia, because of a higher grade of purification.
The enzymes of the extract especially the pyruvate decarboxylase can optionally be stabilised by addition of e.g. natural co- factors of the enzymes, buffers, salts. The pyruvate decarboxylase of the extract can also be used in immobilised form.
The biotransformation process is usually made in water as solvent, preferred in a range of pH between 6.5 and 7.0. The temperature can be varied in a broad range from 0 to 60, preferred from 10 to 40 and especially preferred from 20 to 30°C.
The process can be performed either continuously or as a batch process .
The following examples provide further embodiments and details of the invention.
Example 1
Determination of pyruvate decarboxylase activity
Pyruvate decarboxylase activity (carboligation activity) was determined by phenylacetyl carbinol' formation from the substrates pyruvate and benzaldehyde in 20 min at 25°C. The samples contained 200 μl enzyme solution and 200 μl 2-fold concentrated substrate solution (80 mM benzaldehyde, 200 mM pyruvate, 3 M ethanol, 2 mM thiamine pyrophosphate , 20 mM MgS04 in 50 mM MES/KOH pH 7.0) . One unit (U) was defined as the amount of enzyme that produces 1 μ ol phenylacetyl carbinol per minute . Protein concentrations were estimated according to Bradford. Phenylacetyl carbinol concentrations were determined by HP C, based on peak areas with reference to phenylacetyl carbinol standards using an Alltima C8 column. For the determination of the phenylacetyl carbinol enantiomers a Chiracel OD column was used.
Example 2
Biotransformations with extracts from fungal mycelia
Crude extracts of the following strains of filamentous fungi were tested for their capability of transforming benzaldehyde. and pyruvate into phenylacetyl carbinol :
Rhizopus javanicus NRRL 13161
Rhizopus javanicus NRRL 2871
Rhizopus oryzae NRRL 6201
Rhizopus oryzae NRRL 1501
Aspergillus oryzae NRRL 694 Aspergillus tamarii NRRL 429
Neurospora crassa ATCC 9277
Neurospora crassa ATCC 9683
Polyporus eucalyptorum UNS 805400
Fusarium lateritium UNSW 807100 Fusarium sp. UNSW 871900
Monilia sitophila NRRL1275
Paecilomyces lilacinus NRRL 1746
Mucor rouxii ATCC 44260
NRRL means Northern Regional Research Laboratory (now the National Center For Agricultural Utilization Research) UNSW means University of New South Wales
Strains were grown in cotton stoppered Erlenmeyer-flasks at 30°C in liquid medium composed of 10 g/1 yeast extract, 20 g/1 peptone, 90 g/1 glucose with an initial pH of 6. Shaking at 230 rpm for 20-70 hours provided oxygen for fast biomass production. The flasks were then covered with parafilm and shaken at 60 rpm for 23-29 hours.
The mycelia were harvested in a Buchner funnel and washed twice with buffer. The frozen mycelium was ground to a powder in a mortar using glass beads as the grinding agent. Breakage buffer was added and the extracts were clarified by centrifugation and adjusted to a set volume. Thus, the crude extracts were about 4-fold concentrated in relation to the culture volume. They were stored in aliquots at -70 °C.
Biotransformations were carried out at a scale of 1.2 ml in 2 ml screwed glass vials with 80 % v/v crude extract and substrate concentrations of 100 mM benzaldehyde and 150 mM pyruvate in the presence of 20 mM MgS04, 1 mM TPP, 1 tablet Complete protease inhibitor (Boehringer) / 25 ml and 50 mM MES/KOH pH 7.0.
The vials were rotated vertically at 35 rpm and 22.5°C. After 20 min and after 20 h samples of 300 μl were taken and added to 30 μl 100 % [w/v] trichloric acid. After removal of protein by centrifugation, the supernatants were analysed for phenylacetyl carbinol by HPLC.
As shown in figure 1, highest specific carboligation activities were obtained from the Rhizopus, Fusarium and Mucor with 0.27 to 0.45 U/mg protein The Rhizopus strains also yielded the highest total amount of pyruvate decarboxylase (8.1-15.5 U) that could be recovered from a 20 ml culture.
The best initial productivities of 3.8 - 6.5 g/1 phenylacetyl carbinol in 20 minutes were obtained with crude extracts from
Rhizopus and Mucor (see figure 3) . Rhizopus and Fusarium resulted in the highest final phenylacetyl carbinol concentrations of 78 - 84 mM (11.7 - 12.6 g/1, see figure 4). This was 78 - 84 % of the theoretical yield based on the initial benzaldehyde concentration. These results were obtained without any optimisation of the experimental conditions .
The enantiomeric excess of i?-phenylacetyl carbinol from the final biotransformation samples are shown in the following table.
strain enantiomeric excess of fl-PAC [%]
Rhizopus javanicus NRRL 13161 90.4 Rhizopus javanicus NRRL 2871 93.0
Rhizopus oryzae NRRL 6201 92.9
Rhizopus oryzae NRRL 1501 91.4
Aspergillus oryzae NRRL 694 92.6
Aspergillus tamarii NRRL 429 92.2
Neurospora crassa ATCC 9277 73.4 Neurospora crassa ATCC 9683 not determined
Polypous eucalyptorum UNSW 805400 98
Fasarium lateritium UNSW 807100 91
Fusarium sp . UNSW 871900 92
Monilia sitophila NRRL1275 82 Paecilomyces lilacinus NRRL 1746 93
Mucor rouxii ATCC 44260 91
Example 3
Biotransfor ations with whole fungal mycelia
The following strains of filamentous fungi were tested for their capability of transforming benzaldehyde into phenylacetyl carbinol using whole mycelia:
Rhizopus javanicus NRRL 13161
Rhizopus javanicus NRRL 2871
Rhizopus oryzae NRRL 6201
Rhizopus oryzae NRRL 1501
Aspergillus oryzae NRRL 694 Aspergillus tamarii NRRL 429
The strains were grown in YEPG medium (90 g/1 glucose, 10 g/1 yeast extract, 20 g/1 peptone, initial pH 6) in cotton stoppered Erlenmeyer flasks at 30°C. The Rhizopus strains were shaken at 230 rpm for 12 hours, the Aspergillus strains for 48 hours. In order to induce pyruvate decarboxylase, the cultures were transferred into sterile screwed glass vials and were left standing at 30°C for 3.5 h. Gas was produced at a high rate, indicating a high activity of pyruvate decarboxylase.
The culture broth was discarded and an equal amount of YEPG including 100 mM benzaldehyde was added. The cultures were shaken in the screwed glass vials at 30°C and 230 rpm.
Only 0.2 - 0.7 mM phenylacetyl carbinol was produced from 100 mM benzaldehyde in 12 hours and the phenylacetyl carbinol concentrations were not increased after further 12 hours. Despite of the low amounts, it is shown, that phenylacetyl carbinol can be produced from benzaldehyde without prior disruption of the mycelia.
Example 4
Biotransformation of benzaldehyde by Rhizopus javanicus PDC
The PDC of Rhizopus javanicus was partially purified by acetone precipitation .
Reaction composition:
0.6 - 2 M (preferable 2 M) MOPS / KOH, pH 7
20 mM MgS04
1 mM TPP
150-600 mM pyruvate (ratio pyruvate / benzaldehyde = 1.5) 100-394 mM benzaldehyde
7.2 U/ml PDC carboligase activity
(1 Unit carboligase activity is defined as the amount of enzyme that produces 1 μmol PAC from 40 mM benzaldehyde and 100 mM pyruvate in 1 min at pH 7 and 25°C)
The reaction was started by adding PDC enzyme. After mixing at 6°C for 18 hours the reaction was stopped by diluting samples 20-fold with 10 % [w/v] trichloroacetic acid. Protein was removed by centrifugation and PAC concentrations were analysed by HPLC.
Results
The results are shown in Figure 5. PAC concentrations of up to 43 g/1 were obtained with Rhizopus javanicus PDC. The yields of PAC on initial benzaldehyde were 86 % for 295 mM initial benzaldehyde and 73 % for 394 mM initial benzaldehyde. The enantio- meric excess (ee-value) was 98.7.
The highest reported PAC concentrations from biotransformations are 28.6 g/1 using partially purified PDC from the yeast Candida utilis (Shin and Rogers, 1996; Rogers, Shin and Wang, 1997) and 30.2 g/1 in a fermentative process with the yeast Torulopsis (JP 2000-93189A) .
References
Alexopoulos, C.J., Mims, C.W.: Introductory Mycology, third edition 1979, John Wiley and Sons, USA
Alvarez, M.E., Rosa, A.L., Temporini, E.D., Wolstenholme, A., Panzetta, G., Patrito, L. Maccioni, H.J.F.: The 59-kDa polypep- tide constituent of 8-10-nm cytoplasmic filaments in Neurospora crassa is a pyruvate decarboxylase. Gene 130, 253-258 (1993)
Bradford, M.M. : A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anlal . Biochem. 72, 248-254 (1976)
Cardillo, R. , Servi, S., Tinti, C: Biotransformation of un- saturated aldehydes by microorganisms with pyruvate decarboxylase activity. Appl . Microbiol . Biotechnol . 36, 300-303 (1991)
Dalboge, H., Lange, L.: Using molecular techniques to identify new microbial biocatalysts . Tibtech 16,265-272 (1998)
Lockington, R.A., Borlace, G.N., Kelly, J.M. : Pyruvate Decarboxylase and anaerobic survival in Aspergillus nidulans . Gene 191, 61-67 (1997)
Onions, A.H.S., Allsopp, D. , Eggins , H.O.W.: Smith's Introduction to Industrial Mycology. Seventh edition 1981, Edward Arnold, GB
Sanchis, V., Vinas , I., Roberts, I.N., Jeenes, D.J., Watson, A.J., Archer, D.B.: A pyruvate decarboxylase gene from Aspergillus parasi ticus . FEMS Microbiol. Lett. 117, 207-210 (1994)
Shin, H.S. Rogers, P.L.: Production of L-Phenylacetylcarbinol (L-PAC) from benzaldehyde using partially purifiied pyruvate decarboxylase (PDC). Biotech. Bioeng. 49, 52-62 (1996)
Rogers, P.L., Shin H.S., Wang, B. : Biotransformation for L-ephedrin-production. Advances in Biochemical Engineering Biotechnology 56, 33-59 (1997)
Singh, A., Kumar, P.K.R., Schuegerl, K. : Bioconversion of cellulosic materials to ethanol by filamentous fungi. Adv. Biochem. Eng. / Biotech. 45, 30-55 (1992)
Skory, CD., Freer, S.N., Bothast, R. J. : Screening for ethanol- producing filamentous fungi. Biotech. Lett. 19, 203-206 (1997)
Stumpf, B., Kieslich, K. : Acyloin condensation of acyclic unsaturated aldehydes by Mucor species. Appl. Microbiol. Biotechnol. 34, 598-603 (1991)
JP 2000-93189A
Figure 1 shows specific carboligation activities in crude extracts. The error bars indicate minimum and maximum results from the three cultures per strain.
Figure 2 shows total carboligation activities per flask containing 20 ml culture. The error bars indicate minimum and maximum results from the three cultures per strain.
Figure 3 shows initial productivity for phenylacetyl carbinol (PAC) . The error bars indicate minimum and maximum results from the three cultures per strain.
Figure 4 shows initial phenylacetyl carbinol (PAC) concentrations and theoretical yields based on initial benzaldehyde concentra- tions. The error bars indicate minimum and maximum results from the three cultures per strain.
Figure 5 shows the effect of substrate concentration on PAC production with PDC of Rhizopus javanicus.
Claims (9)
1. Process for the production of .R-phenylacetylcarbinol by biotransformation of benzaldehyde by filamentous fungi
2. Process according to claim 1 where the filamentous fungi are selected from the group of Rhizopus, Neurospora, Polyporus, Fusarium, Monilia, Paecilomyces, Mucor.
3. Process according to claim 2 where the filamentous fungi are selected from the group of Rhizopus, Fusarium, Mucor.
4. Process according to claim 3 where the filamentous fungi are Rhizopus javanicus or Mucor rouxii.
5. Process according to claim 1-4 where the biotransformation of benzaldehyde is made in the presence of pyruvate.
6. Process according to claim 5 where 1-2 mol pyruvat are added per mol of benzaldehyde.
7. Process according to claim 1-6 where the biotransformation is made by extracts of filamentous fungi.
8. Process according to claim 7 where the extracts contain pyruvate decarboxylase.
9. Process according to claim 8 where the pyruvate decarboxylase is stabilised.
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| DE10032058.9 | 2000-07-05 | ||
| DE10032058A DE10032058A1 (en) | 2000-07-05 | 2000-07-05 | Microbial production of R-phenylacetylcarbinol by biological conversion of benzaldehyde by filamentous fungi |
| PCT/EP2001/007641 WO2002002791A1 (en) | 2000-07-05 | 2001-07-04 | Microbial production of r-phenylacetylcarbinol by biotransformation of benzaldehyde by filamentous fungi |
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| JP (1) | JP2004502430A (en) |
| CN (1) | CN1440460A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP4679923B2 (en) * | 2005-02-15 | 2011-05-11 | 三菱化学フーズ株式会社 | New phospholipase C |
| EP1995313B1 (en) | 2006-03-10 | 2013-05-08 | Mitsubishi-Kagaku Foods Corporation | Novel phospholipase c |
| CN105154463B (en) * | 2015-09-30 | 2018-10-02 | 西北大学 | A kind of structure of bacterial strain and application thereof being overexpressed Thermotoga maritima acetolactate synthase catalytic subunit |
| CN107630050A (en) * | 2017-04-01 | 2018-01-26 | 武汉茵茂特生物技术有限公司 | The biological preparation method of pseudoephedrine |
| CN108165591B (en) * | 2017-12-18 | 2020-07-03 | 上海凌凯医药科技有限公司 | Enzymatic preparation method of L-xylose |
| CN111139185B (en) * | 2018-11-06 | 2023-03-10 | 广州中医药大学(广州中医药研究院) | Aspergillus fungi and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3875007A (en) * | 1972-11-03 | 1975-04-01 | Amano Pharma Co Ltd | Lipid metabolism improving and anti-atheromatic agent |
| AU4501989A (en) * | 1988-10-21 | 1990-05-14 | Synergen, Inc. | A process for producing l-phenyl acetyl carbinol (pac), an immobilized cell mass for use in the process and a method for preparing the cell mass |
| JP3593550B2 (en) * | 1991-07-01 | 2004-11-24 | ノルトマルク アルツナイミッテル ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディートゲゼルシャフト | Use of lipase for pharmaceutical manufacturing |
| DE19736104A1 (en) * | 1997-08-20 | 1999-02-25 | Basf Ag | Preparation of enantiomerically pure phenylacetycarbinol derivatives |
-
2000
- 2000-07-05 DE DE10032058A patent/DE10032058A1/en not_active Withdrawn
-
2001
- 2001-07-04 EP EP01949464A patent/EP1297171A1/en not_active Withdrawn
- 2001-07-04 AU AU7061201A patent/AU7061201A/en active Pending
- 2001-07-04 US US10/332,035 patent/US20030100085A1/en not_active Abandoned
- 2001-07-04 CN CN01812291.4A patent/CN1440460A/en active Pending
- 2001-07-04 WO PCT/EP2001/007641 patent/WO2002002791A1/en not_active Ceased
- 2001-07-04 CA CA002414742A patent/CA2414742A1/en not_active Abandoned
- 2001-07-04 JP JP2002508031A patent/JP2004502430A/en not_active Withdrawn
- 2001-07-04 AU AU2001270612A patent/AU2001270612B2/en not_active Ceased
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