Stefaan De Wildeman

Stereoselective Microbial Dehalorespiration with Vicinal Dichlorinated Alkanes

ABSTRACT The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C2 groundwater pollutant on earth. However, a reductive in situ detoxification technology for this compound does not exist. Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reductive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and metabolically convert them into completely dechlorinated end products in defined growth media have been reported. Here we report on the isolation of Desulfitobacterium dichloroeliminans strain DCA1, a nutritionally defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible vicinal dichloropropanes and -butanes into completely dechlorinated end products. Menaquinone was identified as an essential cofactor for growth of strain DCA1 in pure culture. Strain DCA1 converts chiral chlorosubstrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energy-conserving anti mechanistic dichloroelimination. Unlike any known dehalorespiring isolate, strain DCA1 does not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates. This unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation purposes and fine-chemical synthesis.

Growth-Substrate Dependent Dechlorination of 1,2-Dichloroethane by a Homoacetogenic Bacterium

A rod shaped, gram positive, non sporulating Acetobacterium strain was isolated that dechlorinated 1,2-dichloroethane (1,2-DCA) to ethene at a dechlorination rate of up to 2 nmol Cl- min-1 mg-1 of protein in the exponential growth phase with formate (40 mM) as the substrate. Although with other growth substrates such as pyruvate, lactate, H2/CO2, and ethanol higher biomass productions were obtained,the dechlorination rate with these substrates was more than 10-fold lower compared with formate growing cells. Neither cell extracts nor autoclaved cells of the isolatedAcetobacterium strain mediated the dechlorination of 1,2-DCA at significant rates. The addition of 1,2-DCA to the media did not result in increased cell production. No significant differences in corrinoid concentrations could be measured in cells growing on several growth-substrates. However, these measurements indicated that differences in corrinoid structure might cause the different dechlorination activity. The Acetobacterium sp. strain gradually lost its dechlorination ability during about 10 transfers in pure culture, probably due to undefined nutritional requirements. 16S rDNA analysis of the isolate revealed a 99.7% similarity with Acetobacterium wieringae. However, the type strains of A. wieringae and A. woodii did not dechlorinate 1,2-DCA.

Biochemical properties and crystal structure of a β-phenylalanine aminotransferase from Variovorax paradoxus

ABSTRACT By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg−1 for (S)-β-phenylalanine at 30°C and 33 U mg−1 at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.

Reductive biodegradation of 1,2-dichloroethane by methanogenic granular sludge in lab-scale UASB reactors

Dechlorination of 1,2-dichloroethane (1,2-DCA) dosed to a model wastewater in lab-scale upflow anaerobic sludge blanket (UASB) reactors was examined. Anaerobic granular sludge was used as a biocatalyst. Ethanol served as the main methanogenic substrate. For 3 months, two types of UASB reactors were studied, the first type consisting of a sludge blanket and the second type containing an additional layer of activated carbon. When subjected to 1,2-DCA at an average volumetric loading rate of 87.6 mg l−1 day−1, the latter type obtained an average removal efficiency of 82%. Increasing the volumetric loading rate of ethanol from 5 to 15 g COD l−1 day−1 resulted in higher 1,2-DCA conversion rates. No chlorinated intermediates or residues were found. 1,2-DCA was converted mainly to ethene (65–80%) and ethane (<1%). Both autoclaved sludge and cell extracts were not able to degrade 1,2-DCA, which indicates the need for metabolic activity. The reactor effluents were less toxic relative to the influent when analyzed by Nitrox tests, indicating that such UASB treatments can protect a subsequent aerobic nitrifying system. The 1,2-DCA removal rates achieved, and the safe nature of the endproducts, warrant the combination of granular sludge and UASB technology for practical decontamination of waters containing such types of organochlorines.

Increasing the solubility range of polyesters by tuning their microstructure with comonomers

The solubility range of ω-pentadecalactone (ω-PDL) based polymers is increased by copolymerization with a smaller branched lactone, δ-undecalactone (δ-UDL). The copolyester microstructure was assessed by 13C NMR/MALDI-ToF MS and indicates a block-like or random-like structure depending on the feed ratio. DSC analysis reveals a considerable decrease in the crystallinity of the copolyesters which can be attributed to the lack of stereoselectivity of the alkyl substituent of δ-UDL hampering chain packing. Consequently, the ω-PDL-based copolyesters present a broader solubility range towards polar aprotic solvents as demonstrated by the Hansen solubility parameter analysis. Finally, the effect of the ring size and position of the substituents of the comonomer lactone on the solubility range of ω-PDL-based copolyesters was investigated by copolymerization with a β,δ-substituted-e-caprolactone. This broadening of the solubility range of ω-PDL-based copolyesters should enable the use of this biobased macrolactone in applications such as additives in coatings.

Polycycloacetals via polytransacetalization of diglycerol bisacetonide

Diglycerol bisacetonide was synthesized and isolated from acetone and diglycerol, which is renewable and available in large scale. DGA was directly used in polytransacetalization with 1,4-cyclohexanedione or 4,4′-bicyclohexanone as diketone monomers. Polycycloacetals were obtained with molecular weights (Mn) of up to 28 kg mol−1, broad dispersities (Đ = 1.5–4.0) and as semi-crystalline polymers with high melting points (Tm = 210–241 °C) and glass transition temperatures (Tg) of 48 °C or 65 °C. Introducing di(trimethylolpropane) (di-TMP) in the polymerization of DGA and 1,4-cyclohexanedione resulted in copolymers as confirmed by 1H-NMR and 13C-NMR spectroscopy. Increasing the di-TMP content from 10 to 50 mol% reduces the crystallinity of the polycycloacetals and increases the Tg, eventually yielding amorphous polymers (Tg = 60–71 °C). For the amorphous polycycloacetals, Youngs moduli could be determined by tensile strength testing (E = 1.1–1.4 GPa). The polycycloacetals with renewable carbon contents in the range of 33–100% cover a wide range in material properties and are stable against hydrolysis at pH > 1–3, depending on the polycycloacetal composition.

Structure–Property Relations in New Cyclic Galactaric Acid Derived Monomers and Polymers Therefrom: Possibilities and Challenges

In order to fully exploit the potential of carbohydrate-based monomers, different (and some new) functionalities are introduced on galactaric acid via acetalization, and subsequently, partially-biobased polyamides are prepared therefrom via polycondensation in the melt. Compared to nonsubstituted linear monomer, faster advancement of the reaction is observed for the different biacetal derivatives of galactaric acid. This kinetic observation is of great significance since it allows conducting a polymerization reaction at lower temperatures than normally expected for polyamides, which allows overcoming typical challenges (e.g., thermal degradation) encountered upon polymerization of carbohydrate-derived monomers in the melt. The polymers derived from the modified galactaric acid monomers vary in terms of glass transition temperature, thermal stability, hydrophilicity, and functionality.

Exploring the substrate scope of Baeyer-Villiger monooxygenases with branched lactones as entry towards polyesters

Baeyer–Villiger monooxygenases (BVMOs) are biocatalysts that are able to convert cyclic ketones into lactones by the insertion of oxygen. The aim of this study was to explore the substrate scope of several BVMOs with (biobased) cyclic ketones as precursors for the synthesis of branched polyesters. The product structure and the degree of conversion of several biotransformations were determined after conversions by using self‐sufficient BVMOs. Full regioselectivity towards the normal lactones of jasmatone and menthone was observed, whereas the oxidation of other substrates such as α,β‐thujone and 3,3,5‐trimethylcyclohexanone resulted in mixtures of regioisomers. This exploration of the substrate scope of both established and newly discovered BVMOs towards biobased ketones contributes to the development of branched polyesters from renewable resources.

Toward Upscaled Biocatalytic Preparation of Lactone Building Blocks for Polymer Applications

Although Baeyer–Villiger monooxygenases (BVMOs) have gained attention in recent years, there are few cases of their upscaled application for lactone synthesis. A thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) was applied to the oxidation of 3,3,5-trimethylcyclohexanone using a glucose dehydrogenase (GDH) for cofactor regeneration. The reaction progress was improved by optimizing the biocatalyst loading, with investigation into oxygen limitations. The product concentration and productivity were increased by keeping the substrate concentration below the inhibitory level via continuous substrate feeding (CSF). This substrate feeding strategy was evaluated against two biphasic reactions using either toluene or n-butyl acetate as immiscible organic solvents. A product concentration of 38 g L–1 and a space-time yield of 1.35 g L–1 h–1 were achieved during the gram-scale synthesis of the two regioisomeric lactones by applying the CSF strategy. These improvements contribute to the large-scale application of BVMOs in the synthesis of branched building blocks for polymer applications.

Application of a thermostable Baeyer-Villiger monooxygenase for the synthesis of branched polyester precursors: Enzymatic synthesis of branched lactones

Abstract BACKGROUND It is widely accepted that the poor thermostability of Baeyer–Villiger monooxygenases limits their use as biocatalysts for applied biocatalysis in industrial applications. The goal of this study was to investigate the biocatalytic oxidation of 3,3,5‐trimethylcyclohexanone using a thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) for the synthesis of branched ϵ‐caprolactone derivatives as building blocks for tuned polymeric backbones. In this multi‐enzymatic reaction, the thermostable cyclohexanone monooxygenase was fused to a phosphite dehydrogenase (PTDH) in order to ensure co‐factor regeneration. RESULTS Using reaction engineering, the reaction rate and product formation of the regio‐isomeric branched lactones were improved and the use of co‐solvents and the initial substrate load were investigated. Substrate inhibition and poor product solubility were overcome using continuous substrate feeding regimes, as well as a biphasic reaction system with toluene as water‐immiscible organic solvent. A maximum volumetric productivity, or space–time‐yield, of 1.20 g L‐1 h‐1 was achieved with continuous feeding of substrate using methanol as co‐solvent, while a maximum product concentration of 11.6 g L‐1 was achieved with toluene acting as a second phase and substrate reservoir. CONCLUSION These improvements in key process metrics therefore demonstrate progress towards the up‐scaled Baeyer–Villiger monooxygenase‐biocatalyzed synthesis of the target building blocks for polymer application.