Microbial Baeyer–Villiger oxidation of 4,4-disubstituted cyclohexan- and cyclohexenones by recombinant whole-cells expressing monooxygenases of bacterial origin

Screening of 4,4-disubstituted and 3,4,5-polysubstituted cyclohexan- and cyclohexenones with eight different overexpression systems of microbial monooxygenases in recombinant Escherichia coli provided valuable information about substrate acceptance and enantioselectivity of this enzyme family, which are responsible for the stereoselective Baeyer–Villiger biooxidation of ketones. For this purpose whole-cell mediated biotransformations were realized to overcome some limitations in the application of cofactor dependent biocatalysts. The different behavior of various enzymes reflects a recent hypothesis about two distinct clusters of biooxidation catalysts. In contrast to isolated enzyme biooxidations, recombinant cells did not yield unsaturated lactone products derived from cycloalkenones. They rather displayed reductase activity to reduce such precursors to saturated ketones, which were subsequently oxidized to the corresponding Baeyer–Villiger products in a sequential two-step biotransformation.     

Synthesis and cytotoxic activity of some novel polycyclic gamma-butyrolactones

Baeyer–Villiger oxidation of 5-aryl-7,11,11-trimethyltricyclo[5.4.0.0^3,6]-undec-1-en-4-ones 4a–h by H₂O₂ and formic acid in methanol yields mixtures of 3b,7,7-trimethyl-3-phenyl-3,3a,3b,4,5,6,7,8a-octahydro-1H-indeno-[1,2-c]furan-1-ones 8a–h and 3b,7,7-trimethyl-3-phenyl-3,3a,3b,4,5,6,7,8a-octahydro-1H-indeno-[1,2-c]furan-2-ones 9a–h in high yields. The obtained butyrolactones 8a–h display cytotoxic activity against a number of human cancer cells.     

19F NMR study on the biological Baeyer-Villiger oxidation of acetophenones

The biological Baeyer–Villiger oxidation of acetophenones was studied by ¹⁹F nuclear magnetic resonance (NMR). The ¹⁹F NMR method was used to characterise the time-dependent conversion of various fluorinated acetophenones in either whole cells of Pseudomonas fluorescens ACB or in incubations with purified 4′-hydroxyacetophenone monooxygenase (HAPMO). Whole cells of P. fluorescens ACB converted 4′-fluoroacetophenone to 4-fluorophenol and 4′-fluoro-2′-hydroxyacetophenone to 4-fluorocatechol without the accumulation of 4′-fluorophenyl acetates. In contrast to 4-fluorophenol, 4-fluorocatechol was further degraded as evidenced by the formation of stoichiometric amounts of fluoride anion. Purified HAPMO catalysed the strictly NADPH-dependent conversion of fluorinated acetophenones to fluorophenyl acetates. Incubations with HAPMO at pH 6 and 8 showed that the enzymatic Baeyer–Villiger oxidation occurred faster at pH 8 but that the phenyl acetates produced were better stabilised at pH 6. Quantum mechanical characteristics explained why 4′-fluoro-2′-hydroxyphenyl acetate was more sensitive to base-catalysed hydrolysis than 4′-fluorophenyl acetate. All together, ¹⁹F NMR proved to be a valid method to evaluate the biological conversion of ring-substituted acetophenones to the corresponding phenyl acetates, which can serve as valuable synthons for further production of industrially relevant chemicals. Journal of Industrial Microbiology & Biotechnology (2001) 26, 35–42. Received 20 April 2000/ Accepted in revised form 16 September 2000     

高等植物乙醇酸氧化酶与黄素单核苷酸的松弛结合

普查了 7个科 11种植物的乙醇酸氧化酶 (GO) ,发现其酶蛋白与黄素单核苷酸 (FMN)的结合均是松弛的 ;并据此特征找到了一种温和的制备完全脱FMNGO的新方法 ,酶液总活性回收可达 87.5 % ;外加FMN可使脱辅因子GO不同程度地恢复活性 ,恢复 5 0 %活性所需FMN的浓度为 8× 10 -7mol/L ,而当浓度大于 5× 10 -6mol/L时其复活作用达到 10 0 % ,表明两者间存在一个可逆的解离平衡。推测植物体内的FMN浓度可能是乙醇酸氧化酶活性的一个调节因子。     

高等植物乙醇酸氧化酶与黄素单核苷酸的松弛结合

The binding condition between flavin mononucleotide (FMN) and protein moiety of glycollate oxidase (GO) was investigated in 11 species of higher plants from 7 families. A loose binding of FMN with GO was universally observed in all the plants. According to this character, a new efficient but mild method was established for preparation of FMN-free GO, which could produce 87.5% recovery of activity . FMN-free GO could be reactivated by adding FMN. For half reactivation 8×10 mol/L FMN was needed, and more than 5×10 mol/L for 100%. The result indicates that there exists a reversible dissociation balance between FMN and protein moiety of GO. Therefore, the concentration of FMN may act as a factor to regulate GO activity in higher plants.     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.     

An unusual ring—A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

A series of steroids (progesterone, testosterone acetate, 17β-acetoxy-5α-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17β side-chain.This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17β-acetoxy-5α-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17β-alcohol of testosterone.All steroidal metabolites were isolated by column chromatography and were identified by ¹H, ¹³C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.     

Cloning,expression, and characterization of a Baeyer–Villiger monooxygenase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 50106 in <Emphasis Type="Italic">E. coli</Emphasis>

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.     

Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity

The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E approximately 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E approximately 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E approximately 75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E approximately 86).