Enzymatic evaluation of different Aspergillus strains by biotransformation of cyclic ketones

The potential of different Aspergillus strains in carrying out the biotransformation of cyclic ketones was investigated. All the strains employed showed alcohol dehydrogenase and Baeyer–Villiger monooxygenase activities. trans-2-Methylcyclohexanol and trans-4-methylcyclohexanol were prepared in a single isomeric form by the use of Aspergillus terreus SSP 1498 and the corresponding ketones. Baeyer–Villiger oxidation of cyclic ketones by all the fungi Aspergillus led to chiral lactones in good enantioselectivity.     

Evaluation of acetophenone monooxygenase and alcohol dehydrogenase activities in different fungal strains by biotransformation of acetophenone derivatives

Experimental conditions using whole cells to select fungal strains for specific bioreduction of ketones and formation of Baeyer–Villiger oxidation products were studied. Epicoccum nigrum SSP 1498 was effective in the bioreduction leading to the chiral alcohols in up to 98% enantiomeric excess. High acetophenone monooxygenase activity was observed by the use of the fungus Emericella nidulans CCT 3119 as biocatalyst.     

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.     

Microbiological transformations 52.: Biocatalysed Baeyer–Villiger oxidation of 1-indanone derivatives

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.     

Biocatalytic properties of Baeyer–Villiger monooxygenases in aqueous–organic media

The biocatalytic properties of three Baeyer–Villiger monooxygenases (phenylacetone monooxygenase, 4-hydroxyacetophenone monooxygenase and ethionamide monooxygenase) in a variety of aqueous–organic media were studied using organic sulfides as substrates. The influence of the nature and the concentration of the solvents, as well as of the substrates, on the activity and enantioselectivity of the enzymes was investigated in detail. Solvents were found to decrease, to a different extent, enzyme activity. High increases of enantioselectivity and also reversal of enantiopreference were observed depending on the enzyme and on the nature of the solvent and the substrate employed.     

Comparison of cyclohexanone monooxygenase as an isolated enzyme and whole cell biocatalyst for the enantioselective oxidation of 1,3-dithiane

Both whole cells of recombinant Escherichia coli TOP10, overexpressing cyclohexanone monooxygenase (CHMO) and isolated cyclohexanone monooxygenase, were used to carry out the enantioselective oxidation of 1,3-dithiane (1) to (R)-1,3-dithiane-1-oxide (2). The two biocatalysts were evaluated under various experimental conditions (e.g., shaken flask or bioreactor; non-bound or resin-adsorbed substrate; different substrate concentrations) in terms of volumetric productivity and enantioselectivity. While productivity was similar in the two cases (up to 0.58 g L⁻¹ h⁻¹), the optical purity of the product was much higher with the isolated enzyme (up to 98% e.e.) than with the whole cell biocatalyst (up to 85% e.e.).     

Ferredoxin reductase enhances heterologously expressed cytochrome CYP105D1 in Escherichia coli and Streptomyces lividans

The ability of two different ferredoxin reductases from Streptomyces coelicolor, to enhance the amount of active recombinant Streptomyces griseus soyC (CYP105D1) was investigated in both Escherichia coli and Streptomyces lividans. In E. coli a two-plasmid system and a single operon construct were used for expression of the CYP105D1 and the ferredoxin reductase(s) under the control of T7 promoters. Expression levels of CYP105D1 were found to range between 85 and 280 nmol l⁻¹ cell culture after prolonged growth. In S. lividans the CYP105D1 and its ferredoxin were cloned downstream of the Pact1 promoter in the E. coli/Streptomyces shuttle vector pBW160. The recombinant E. coli and S. lividans cells converted 7-ethoxycoumarin into 7-hydroxycoumarin efficiently. Expression of a ferredoxin reductase as an operon with CYP105D1 and its ferredoxin enhances the o-dealkylation of 7-ethoxycoumarin. Ferredoxin NADPH reductase was found to enhance the level of the active form of CYP105D1 monooxygenase when no substrate was present.     

Baeyer-Villiger oxidations of representative heterocyclic ketones by whole cells of engineered Escherichia coli expressing cyclohexanone monooxygenase

Whole cells of an Escherichia coli strain overexpressing Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase (CHMO; E.C. 1.14.13.22) have been used for the Baeyer-Villiger oxidation of representative heterocyclic six-membered ketones to probe the potential impact of nitrogen, sulfur and oxygen on the chemoselectivity of these reactions. The fact that all of these heterocyclic systems were accepted as substrates by the enzyme and gave normal Baeyer-Villiger products broadens the synthetic utility of the engineered E. coli strain and emphasizes the chemoselectivity achievable with enzymatic oxidation catalysts.     

Nucleoside synthesis by immobilised bacterial whole cells

Biocatalysed synthesis of nucleosides was carried out using immobilised whole cells of Escherichia coli ATCC 47092, Enterobacter gergoviae CECT 857 and Citrobacter amalonaticus CECT 863. The synthesis of adenosine from uridine was used as reaction model to test the biocatalysts. Reactions were carried out using non-growing cells. Maximum activity was obtained with cells harvested at the beginning of the stationary phase. Immobilization by whole cell entrapment was employed using different matrix such as alginate, agar, agarose and polyacrylamide. The percentage of monomer, the shaking speed, the catalyst load and nature of the matrix were optimized. In the first reutilization cycle, similar yields (80–95%) were obtained with both free and immobilized cells in the reaction model, although in the last case, longer reaction times were necessary. The immobilized cells can be reused at least for more than 30 times without significant loss of the catalytic activity. The immobilized biocatalysts have been used in the synthesis of different nucleosides.     

Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining

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 (k_cat = 1.9 s^–1, K_M = 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.     

Expanding the biocatalytic toolbox of flavoprotein monooxygenases from Rhodococcus jostii RHA1

With the aim to enlarge the set of available flavoprotein monooxygenases, we have cloned 8 unexplored genes from Rhodococcus jostii RHA1 that were predicted to encode class B flavoprotein monooxygenases. Each monooxygenase can be expressed as soluble protein and has been tested for conversion of sulfides and ketones. Not only enantioselective sulfoxidations, but also enantioselective Baeyer–Villiger oxidations could be performed with this set of monooxygenases. Interestingly, in contrast to known class B flavoprotein monooxygenases, all studied biocatalysts showed no nicotinamide coenzyme preference. This feature coincides with the fact that the respective sequences appear to form a discrete group of sequence related proteins, distinct from the known class B flavoprotein monooxygenases subclasses: the so-called flavin-containing monooxygenases (FMOs), N-hydroxylating monooxygenases (NMOs) and Type I Baeyer–Villiger monooxygenases (BVMOs). Taken together, these data reveal the existence of a new subclass of class B flavoprotein monooxygenases, which we coined as Type II FMOs, that can perform Baeyer–Villiger oxidations and accept both NADPH and NADH as coenzyme. The uncovered biocatalytic properties of the studied Type II FMOs make this newly recognized subclass of monooxygenases of potential interest for biocatalytic applications.     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

Effects of geometry on transmission and sensing potential of tapered fiber sensors

Geometry of tapered fiber sensors critically affects the response of an evanescent field sensor to cell suspensions. Single-mode fibers (nominally at 1300 nm) were tapered to symmetric or asymmetric tapers with diameters in the range of 3–20 μm, and overall lengths of 1–7 mm. Their transmission characteristics in air, water and in the presence of Escherichia coli (JM101 strain) at concentrations of 100, 1000, 7000 and 7 million cells/mL were measured in the 400–800 nm range and gave rich spectral data that lead to the following conclusions. (1) No change in transmission was observed due to E. coli with tapers that showed no relative change in transmission in water compared to air. (2) Tapers that exhibited a significant difference in transmission in water compared to air gave weak response to the presence of the E. coli. Of these, tapers with low waist diameters (6 μm) showed sensitivity to E. coli at 7000 cells/mL and higher concentration. (3) Tapers that showed modest difference in water transmission compared to air, and those that had small waist diameters gave excellent response to E. coli at 100–7000 cells/mL. In addition, mathematical modeling showed that: (1) at low wavelength (470 nm) and small waist diameter (6 μm), transmission with water in the waist region is higher than in air. (2) Small changes in waist diameter (0.05 μm) can cause larger changes in transmission at 470 nm than at 550 nm at waist diameter of 6 μm. (3) For the same overall geometry, a 5.5 μm diameter taper showed larger refractive index sensitivity compared to a 6.25 μm taper at 470 nm.     

Microbiological transformations 44. Optimisation of a new Baeyer–Villigerase activity: application to the stereospecific oxidation of 3-phenylcyclobutanone

A screening was achieved out of 80 microbial strains in order to detect new “Baeyer–Villigerase” activities, using bicyclo[3.2.0]hept-2-en-6-one 1 as a test substrate. Such a new and interesting activity was discovered in the fungus Cunninghamella echinulata. Using this strain, oxidation of prochiral 3-phenyl-cyclobutanone 5 was examined. After an optimisation of the experimental conditions, the corresponding γ-butyrolactone 6 was obtained in 71% yield and 98% ee.     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNA^Phe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNA^Phe/(U)₉/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319–322).     

A MEMS based amperometric detector for E. coli bacteria using self-assembled monolayers.

We developed a system for amperometric detection of Escherichia coli (E. coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification. Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable. Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E. coli. Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au. The biotinylated thiol approach yielded the best results. High specificity for E. coli was achieved using ssDNA–rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica.     

Bacterial cell surface display of lipase and its randomly mutated library facilitates high-throughput screening of mutants showing higher specific activities

The thermostable lipase (TliA) from Pseuodmonas fluorescens was functionally displayed on the surface of Escherichia coli using the ice-nucleation protein (INP) as an anchor. The INP–TliA fusion proteins were correctly synthesized and localized on the surface, confirmed by flow cytometer and halo forming activity on tributyrin emulsion agar plate. Lipase-displaying cells were used as an alternative immobilized biocatalyst to hydrolyze olive oil in aqueous–organic solvent two phases reaction. Furthermore, the randomly generated library of TliA was also displayed on E. coli. In order to be able to screen mutants showing increased specific activities, we optimized culture conditions, induction condition and host cell types. From more than 10⁵ members of library, top four mutants were selected. Selected clones of T48, T54, T61, and T68 showed 29-, 24-, 2-, and 19-fold increases, respectively, in whole-cell activities compared to wild-type enzyme. The DNA sequencing showed that one or three amino acids were exchanged and positions critical for increased activities were random. These results demonstrate that surface display provide a useful technology for directed evolution of industrially important lipases.     

Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases

Three C terminal His₆-tagged recombinant microbial CMP–sialic acid synthetases [EC 2.7.7.43] cloned from Neisseria meningitidis group B, Streptococcus agalactiae serotype V, and Escherichia coli K1, respectively, were evaluated for their ability in the synthesis of CMP–sialic acid derivatives in a one-pot two-enzyme system. In this system, N-acetylmannosamine or mannose analogs were condensed with pyruvate, catalyzed by a recombinant sialic acid aldolase [EC 4.1.3.3] cloned from E. coli K12 to provide sialic acid analogs as substrates for the CMP–sialic acid synthetases. The substrate flexibility and the reaction efficiency of the three recombinant CMP–sialic acid synthetases were compared, first by qualitative screening using thin layer chromatography, and then by quantitative analysis using high performance liquid chromatography. The N. meningitidis synthetase was shown to have the highest expression level, the most flexible substrate specificity, and the highest catalytic efficiency among the three synthetases. Finally, eight sugar nucleotides, including cytidine 5′-monophosphate N-acetylneuraminic acid (CMP–Neu5Ac) and its derivatives with substitutions at carbon-5, carbon-8, or carbon-9 of Neu5Ac, were synthesized in a preparative (100–200 mg) scale from their 5- or 6-carbon sugar precursors using the N. meningitidis synthetase and the aldolase.     

Construction and Characterisation of a Genetically Engineered Escherichia coli Strain for the Epoxide Hydrolase-catalysed Kinetic Resolution of Epoxides

The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis, despite the formation of Eph1 inclusion bodies. Optimization of cultivation conditions and co-expression of molecular chaperones resulted in a further increase in activity and a reduction of the inclusion bodies formation, respectively. Compared to Rhodotorula glutinis cells and cell extracts, a total increase in Eph1 activity of over 200 times was found for both Escherichia coli cells and crude enzyme preparations of these cells. The improved conditions for recombinant Eph1 production were used to demonstrate the Eph1-catalysed kinetic resolution of a new Eph1 substrate, 1-oxaspiro[2.5]octane-2-carbonitrile.