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1.
N M Kamerbeek M J Moonen J G Van Der Ven W J Van Berkel M W Fraaije D B Janssen 《European journal of biochemistry》2001,268(9):2547-2557
A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of approximately 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximately 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger monooxygenases: cyclohexanone monooxygenase (27-33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding. 相似文献
2.
Baeyer–Villiger biooxidation of 4-methylcyclohexanone–5-methyloxepane-2-one catalysed by recombinant Escherichia coli overexpressing cyclopentanone monooxygenase encapsulated in polyelectrolyte complex capsules was used to investigate effect
of substrate conversion on the viability of cells. Confocal laser scanning microscopy (CLSM) was used to assess cell viability
using propidium iodide fluorescence marker for necrosis, and flavin autofluorescence to identify living bacteria. Viability
of encapsulated cells decreased with increasing substrate concentration from 99 ± 1 to 83 ± 4%, while substrate conversions
from decreased 100 to 6 ± 1%. Storage stabilization of encapsulated cells was observed by increased substrate conversion form
68 ± 2 to 96 ± 3%. Measurements by CLSM with standard deviations up to 5% may be regarded as powerful tool for recombinant
cell viability determination during Baeyer–Villiger biooxidations. 相似文献
3.
Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase 总被引:2,自引:2,他引:0 下载免费PDF全文
Nanne M. Kamerbeek Arjen J. J. Olsthoorn Marco W. Fraaije Dick B. Janssen 《Applied microbiology》2003,69(1):419-426
The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee >99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications. 相似文献
4.
Hanna M. Dudek Daniel E. Torres Pazmiño Cristina Rodríguez Gonzalo de Gonzalo Vicente Gotor Marco W. Fraaije 《Applied microbiology and biotechnology》2010,88(5):1135-1143
Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate
the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase
(PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with
NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed
that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes
to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme
specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic
efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant
and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results
indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex
catalytic mechanism and the vital role of the coenzyme in BVMOs. 相似文献
5.
Eun-Ju Kim Jong-Rok Jeon Young-Mo Kim Kumarasamy Murugesan Yoon-Seok Chang 《Applied microbiology and biotechnology》2010,87(4):1569-1577
The aerobic metabolism of monofluorophenols (mono-FPs) by the actinomycete, Pseudonocardia benzenivorans, was studied. This strain was able to grow on 4-fluorophenol (4-FP) and readily transform 2- and 3-fluorophenol to the corresponding
metabolites. The detailed mechanism of mono-FPs degradation by P. benzenivorans was elucidated from enzymatic assays and the identification of reaction intermediates by high-performance liquid chromatography
(HPLC) and gas chromatography–mass spectrometry. Two types of fluorocatechols (i.e., 3- and 4-fluorocatechol) were identified
as the key transformation products. During 4-FP degradation, only 4-fluorocatechol was detected, and a stoichiometric level
of fluoride was released. Both fluorocatechols were observed together in cultures containing 3-fluorophenol (3-FP), while
only 3-fluorocatechol was found to accumulate in 2-fluorophenol (2-FP)-containing cultures. Whole-cell extracts of P. benzenivorans expressed catechol 1,2-dioxygenase activity, indicating that the transformation of the three tested mono-FPs proceeded via
ortho-cleavage pathway. The results presented in this paper provide comprehensive information regarding the metabolism of mono-FPs
by a single bacterium. 相似文献
6.
The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally
transformed to 4-fluoro-4′-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts
by gas chromatography–mass spectrometry. Additionally, fluorinated water-soluble products were detected by 19F NMR and were identified as sulphate and β-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4′-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl)
were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4′-difluorobiphenyl only. 相似文献
7.
Coulombel L Nolan LC Nikodinovic J Doyle EM O'Connor KE 《Applied microbiology and biotechnology》2011,89(6):1867-1875
Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols
as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation
of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was
undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In
a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The
rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and
4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation
decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved
within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed
to 4-halocatechols at 2 mM within a 1–2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5–20-fold
decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol
products followed by column chromatography resulted in the production of purified products with a final yield of between 33%
and 38%. 相似文献
8.
Rehdorf J Behrens GA Nguyen GS Kourist R Bornscheuer UT 《Applied microbiology and biotechnology》2012,93(3):1119-1126
An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and
ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni–NTA-assisted affinity chromatography,
the kinetic parameters K
M and k
cat were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue.
Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally
the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity
was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product
of a Baeyer–Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting
a physiological role in P. putida JD1. 相似文献
9.
Kirschner A Altenbuchner J Bornscheuer UT 《Applied microbiology and biotechnology》2007,75(5):1095-1101
The genes encoding an alcohol dehydrogenase, Baeyer–Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were
cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expression levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (Cn) to the corresponding primary alcohols (Cn−2) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at
30°C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved
in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons. 相似文献
10.
María C. Gutirrez Vronique Alphand Roland Furstoss 《Journal of Molecular Catalysis .B, Enzymatic》2003,21(4-6):231-238
The microbiological Baeyer–Villiger oxidation of various substituted 1-indanones is described. Three bacterial strains have been explored: an E. coli TOP10 [pQR 239] constructed to overexpress the cyclohexanone monoxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871, an E. coli TOP10 [hapE] strain recently constructed to overexpress 4-hydroxyacetophenone monoxygenase (HAPMO) of Pseudomonas fluorescens ACB and the wild type Pseudomonas sp. NCIMB 9872 strain known to metabolise cyclopentanone. This last strain oxidised some of the proposed substrates, leading to the corresponding lactones with good to excellent yields depending on the aromatic ring substituents. 相似文献
11.
Two strains, Alcaligenes sp. strain ACA and Pseudomonas fluorescens ACB, isolated from acetophenone and 4′-hydroxyacetophenone enrichments, respectively, cometabolize a range of chlorinated acetophenones (CAs). A biological Baeyer-Villiger reaction converts the CA to chlorophenyl acetate. This is evident only in the presence of an esterase inhibitor, since the CA is normally rapidly hydrolyzed to a chlorophenol which has the same substitution pattern as the original ketone. The oxygenase that attacks the ketone uses NADPH in the incorporation of one atom of 18O2 and is strongly inhibited by phenols that bear an ortho or meta chlorine or bromine, but much less by cresols or phenol itself. A feedback phenomenon may thus account for the inability of strain ACA to grow on CAs, which also fail to induce the cells for their own metabolism. 相似文献
12.
M. A. Ivanov G. S. Ludva A. V. Mukovnya S. N. Kochetkov V. L. Tunitskaya L. A. Alexandrova 《Russian Journal of Bioorganic Chemistry》2010,36(4):488-496
4′-Fluoro-2′,3′-O-isopropylidenecytidine was synthesized by the treatment of 5′-O-acetyl-4′-fluoro-2′,3′-O-isopropylideneuridine with triazole and 4-chlorophenyl dichlorophosphate followed by ammonolysis. The interaction of 4′-fluoro-2′,3′-O-isopropylidenecytidine with hydroxylamine resulted in 4′-fluoro-2′,3′-O-isopropylidene-5′-O-acetyl-N
4-hydroxycytidine. The removal of the 2′,3′-O-isopropylidene groups led to acetyl derivatives of 4′-fluorouridine, 4′-fluorocytidine, and 4′-fluoro-N
4-hydroxycytidine. 4′-Fluorouridine 5′-O-triphosphate was obtained in three steps starting from 4′-fluoro-2′,3′-O-isopropylideneuridine. 4′-Fluorouridine 5′-O-triphosphate was shown to be an effective inhibitor of HCV RNA-dependent RNA polymerase and a substrate for the NTPase reaction
catalyzed by the HCV NS3 protein, the hydrolysis rate being similar to that of ATP. It could also activate a helicase reaction
with an efficacy of only threefold lower than that for ATP. 相似文献
13.
Kirschner A Altenbuchner J Bornscheuer UT 《Applied microbiology and biotechnology》2007,73(5):1065-1072
A gene encoding a Baeyer–Villiger monooxygenase (BVMO) identified in Pseudomonas fluorescens DSM 50106 was cloned and functionally expressed in Escherichia coli JM109. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed an estimated
56 kDa-size protein band corresponding to the recombinant enzyme. Expression in BL21 (DE3) resulted mainly in the formation
of inclusion bodies. This could be overcome by coexpression of molecular chaperones, especially the DnaK/DnaJ/GrpE complex,
leading to increased production of soluble BVMO enzyme in recombinant E. coli. Examination of the substrate spectra using whole-cell biocatalysis revealed a high specificity of the BVMO for aliphatic
open-chain ketones. Thus, octyl acetate, heptyl propionate, and hexyl butyrate were quantitatively formed from the corresponding
ketone substrates. Several other esters were obtained in conversion >68%. Selected esters were also produced on preparative
scale. 相似文献
14.
Kamerbeek NM Olsthoorn AJ Fraaije MW Janssen DB 《Applied and environmental microbiology》2003,69(1):419-426
The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications. 相似文献
15.
Mohammad Ali Faramarzi Mojtaba Tabatabaei Yazdi Mohsen Amini Farzaneh Aziz Mohseni Gholamreza Zarrini Amir Amani Abbas Shafiee 《World journal of microbiology & biotechnology》2004,20(7):657-660
Two metabolites were obtained by microbial transformation of androstendione in the culture of Aspergillus terreus PTCC 5283, a fungus isolated from soil. Their structures were established as testosterone and testololactone on the basis of the spectral data including 1H NMR, 13C NMR, FTIR, MS and physical constants such as melting point and optical rotation. Aspergillus terreusproduced both metabolites after 3 days incubation at 27 °C. The bioconversion reactions observed were 17-carbonyl reduction and biological Baeyer–Villiger oxidation. 相似文献
16.
Whole-cell conversion of cyclohexanone to ɛ-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and ɛ-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone
to ɛ-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of ɛ-caprolactone
from cyclohexanone. A glucose-limited fed-batch Baeyer–Villiger oxidation where a cyclohexanone level was maintained less
than 6 g/l resulted in a maximum ɛ-caprolactone concentration of 11.0 g/l. The maximum ɛ-caprolactone concentration was improved
further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in ɛ-caprolactone concentration compared with the control experiment performed
under the same conditions. 相似文献
17.
Piedad del Socorro Murdoch Zijian Guo John A. Parkinson P. J. Sadler 《Journal of biological inorganic chemistry》1999,4(1):32-38
Reaction of [Pt(dien)Cl]+ (1) with the 14-mer oligonucleotide 5′-d(ATACATGGTACATA) (I) gave rise to two major species which corresponded to the 5′-G and 3′-G platinated monofunctional adducts, and a minor amount
of the bis-platinated adduct formed during the later stages of the reaction. The reaction of (1) with the related octamer 5′-d(ATACATGG) (II) was also investigated. Kinetic data obtained by HPLC showed that the 5′-G and 3′-G bases of the 14-mer oligonucleotide were
platinated at similar rates: the second-order rate constant is 53×10–2 M–1 s–1 at 298 K in 0.1 M NaClO4. However, the platination rate of 5′-G of the octamer (II) (k=69×10–2 M–1 s–1) was enhanced by a factor of three compared to the rate of platination at 3′-G (k=22×10–2 M–1 s–1). All the adducts were separated by HPLC and characterized by NMR spectroscopy, enzymatic digestion and MALDI-TOF mass spectrometry.
1H and 15N NMR shifts suggest that there are distinct conformational differences between 14-mer duplexes platinated at 5′-G (I5′
ds) and 3′–G (I3′
ds). Molecular mechanics modelling indicates that rotation around the Pt-N7 bond is more restricted in the case of the 5′-G adduct than in that of the 3′-G adduct. The binding of {Pt(dien)}2+ to 5′-GN7 and 3′-GN7 in the monofunctional adducts of (I) was shown to be reversible upon the addition of high concentrations of chloride ions.
Received: 3 July 1998 / Accepted: 10 November 1998 相似文献
18.
Valerio Pieri Sonja Sturm Christoph Seger Chlodwig Franz Hermann Stuppner 《Metabolomics : Official journal of the Metabolomic Society》2012,8(2):335-346
The characterization of T. vulgaris plant material for quality control purposes was performed by NMR-based methods. Direct extraction of 141 T. vulgaris samples with DMSO-d
6 enabled the obtainment of crude extracts with a representative composition in terms of both volatile and non-volatile constituents.
The acquisition of 600 MHz 1H NMR spectra resulted in a dataset which was analyzed by a combination of metabolic profiling and target analysis approaches.
Preliminary analysis of the 1H NMR spectra was performed by principal component analysis, which revealed sample discrimination on a chemotype basis (thymol,
carvacrol and linalool chemotypes). Further minor discriminative constituents were identified as p-cymene, γ-terpinene, rosmarinic acid, and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl. Metabolite identification
was accomplished by 1D and 2D NMR techniques and supported by spiking experiments. Fast dereplication of constituents not
available as reference compounds was performed by HPLC–SPE–NMR experiments. A targeted approach based on qHNMR was validated
for quantification of the identified secondary metabolites. Validation was performed in terms of precision (intra-day RSD ≤ 4.51%,
inter-day RSD ≤ 4.18%), repeatability (RSD ≤ 2.30%), accuracy (recovery rates within 93.4 and 103.4%), linearity (correlation
coefficients ≥ 0.9990), robustness, and stability. The amount of the dominant monoterpene in thymol, carvacrol, and linalool
chemotypes was respectively found to be within 0.4–2.6, 0.7–2.3, and 1.1–3.6% (w/w). Variable amounts of the precursors p-cymene and γ-terpinene were found in thymol and carvacrol chemotypes. The highest amount of rosmarinic acid and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl
in the analyzed samples was respectively 4.6 and 0.4% (w/w). Since quantification is performed on a weight basis, the essential
oil content can be estimated based on the sum of the quantified monoterpenes. The NMR-based analysis of T. vulgaris represents a more comprehensive approach in comparison to traditional chromatographic methods such as GC and LC, respectively
employed for the analysis of volatile and non-volatile constituents. Further advantages lie in the simple sample preparation,
rapidity and reproducibility of the NMR analysis. 相似文献
19.
Fujii M Akita H Ida Y Nakagawa T Nakamura K 《Applied microbiology and biotechnology》2007,77(1):45-51
Amberlite XAD-7, a hydrophobic polymer, was used to change microbial reaction of ketones from reduction to Baeyer–Villiger
(BV) oxidation. Thus, D. magnusii NBRC 4600 and G. reessii NBRC 1112 could catalyze the BV reaction of ketones in the presence of the polymer while reduction of the substrates proceeded,
and BV oxidation was scarcely found in the absence of the polymer. 相似文献
20.
Fraaije MW Wu J Heuts DP van Hellemond EW Spelberg JH Janssen DB 《Applied microbiology and biotechnology》2005,66(4):393-400
Baeyer–Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (kcat = 1.9 s–1, KM = 59 M). The enzyme exhibits moderate enantioselectivity with -methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs. 相似文献