Biomedical subjects

Bernard Witholt

Publications and source records attributed to Bernard Witholt.

At least 37 records · Page 2Linked to original sources

PubMed2003

Enantioselective trans dihydroxylation of nonactivated C-C double bonds of aliphatic heterocycles with Sphingomonas sp. HXN-200.

The bacterial strain Sphingomonas sp. HXN-200 was used to catalyze the trans dihydroxylation ofN-substituted 1,2,5,6-tetrahydropyridines 1 and 3-pyrrolines 4 giving the corresponding 3,4-dihydroxypiperidines 3 and 3,4-dihydroxypyrrolidines 6, respectively, with high enantioselectivity and high activity. The trans dihydroxylation was sequentially catalyzed by a monooxygenase and an epoxide hydrolase in the strain with epoxide as intermediate. While both epoxidation and hydrolysis steps contributed to the overall enantioselectivity in trans dihydroxylation of 1, the enantioselectivity in trans dihydroxylation of the symmetric substrate 4 was generated only in the hydrolysis of meso-epoxide 5. The absolute configuration for the bioproducts (+)-3 and (+)-6 was established as (3R,4R) by chemical correlations. Preparative trans dihydroxylation of 1a and 4b with frozen/thawed cells of Sphingomonas sp. HXN-200 afforded the corresponding (+)-(3R,4R)-3,4-dihydroxypiperidine 3a and (+)-(3R,4R)-3,4-dihydroxy pyrrolidine 6b in 96% ee both and in 60% and 80% yield, respectively. These results represent first examples of enantioselective trans dihydroxylation with nonterpene substrates and with bacterial catalyst, thus significantly extending this methodology in practical synthesis of valuable and useful trans diols. Enantioselective hydrolysis of racemic epoxide 2a with Sphingomonas sp. HXN-200 gave 34% of (-)-2a in >99% ee, which is a versatile chiral building block. Further hydrolysis of (-)-2a with the same strain afforded (-)-(3S,4S)-3a in 96% ee and 92% yield. Thus, both enantiomers of 3a can be prepared by biotransformation with Sphingomonas sp. HXN-200.

Catalysis↗

PubMed2003

Stereospecific biocatalytic epoxidation: the first example of direct regeneration of a FAD-dependent monooxygenase for catalysis.

Catalysis for chemical synthesis by cell-free monooxygenases necessitates an efficient and robust in situ regeneration system to supply the enzyme with reducing equivalents. We report on a novel approach to directly regenerate flavin-dependent monooxygenases. The organometallic complex [CpRh(bpy)(H(2)O)](2+) catalyzes the transhydrogenation reaction between formate and isoalloxazine-based cofactors such as FAD and FMN. Coupling this FADH(2) regeneration reaction to the FADH(2)-dependent styrene monooxygenase (StyA) resulted in a chemoenzymatic epoxidation reaction where the organometallic compound substitutes for the native reductase (StyB), the nicotinamide coenzyme (NAD), and an artificial NADH regeneration system such as formate dehydrogenase. Various styrene derivatives were converted into the essentially optically pure (S)-epoxides (ee > 98%). In addition, StyA was shown to be capable of performing sulfoxidation reactions. The productivity of the chemoenzymatic epoxidation reaction using 6.5 microM StyA reached up to 6.4 mM/h, corresponding to approximately 70% of a comparable fully enzymatic reaction using StyB, NADH, and formate dehydrogenase for regeneration. The coupling efficiency of the nonenzymatic regeneration reaction to enzymatic epoxidation was examined in detail, leading to an optimized reaction setup with minimized quenching of the electron supply for the epoxidation reaction. Thus, up to 60% of the reducing equivalents provided via [CpRh(bpy)(H(2)O)](2+) could be channeled into epoxide rather than hydrogen peroxide formation, allowing selective synthesis with high yields.

Biomimetic Materials↗

PubMed2003

Chemical biotechnology for the specific oxyfunctionalization of hydrocarbons on a technical scale.

Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means. Many enzymatic oxygenations have been described, but few of these have been scaled up to industrial scales, due to the complexity of oxygenase based biocatalysts and demanding process implementation. We have combined recombinant whole-cell catalysis in a two-liquid phase system with fed-batch cultivation in an optimized medium and developed an industrially feasible process for the kinetically controlled and complex multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde using the xylene monooxygenase of Pseudomonas putida mt-2 in Escherichia coli. Successful scale up to 30 L working volume using downscaled industrial equipment allowed a productivity of 31 g L(-1) d(-1) and a product concentration of 37 g L(-1). These performance characteristics meet present industry requirements. Product purification resulted in the recovery of 469 g of 3,4-dimethyl- benzaldehyde at a purity of 97% and an overall yield of 65%. This process illustrates the general feasibility of industrial biocatalytic oxyfunctionalization.

Benzaldehydes↗

PubMed2003

Highly enantioselective hydrolysis of alicyclic meso-epoxides with a bacterial epoxide hydrolase from Sphingomonas sp. HXN-200: simple syntheses of alicyclic vicinal trans-diols.

Hydrolysis of N-benzyloxycarbonyl-3,4-epoxy-pyrrolidine and cyclohexene oxide with the epoxide hydrolase of Sphingomonas sp. HXN-200, respectively, gave the corresponding vicinal trans-diols in high ee and yield, representing the first example of enantioselective hydrolysis of a meso-epoxide with a bacterial epoxide hydrolase.

Alcohols↗

PubMed2003

Crystallization and preliminary X-ray analysis of native and selenomethionine 2-hydroxybiphenyl 3-monooxygenase.

2-hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 was produced in Escherichia coli both as native and SeMet-labelled protein. The two enzymes were purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 108.6, b = 196.8, c = 79.3 A, beta = 97.7 degrees for the native protein and a = 108.3, b = 196.8, c = 79.0 A, beta = 97.8 degrees for SeMet HbpA. Crystal-packing considerations led to the assumption of two HbpA subunits per asymmetric unit, which corresponds to a V(M) value of 3.3 A(3) Da(-1) and a solvent content of 62%. The crystals were radiation-sensitive and only had a lifespan of about 120 s when exposed to synchrotron radiation on an undulator beamline. To obtain complete data sets, data were collected from 23 native and 26 derivative crystals. The high-resolution limit was 2.0 A for native and 2.25 A for SeMet HbpA.

Crystallization↗

PubMed2003

Use of the two-liquid phase concept to exploit kinetically controlled multistep biocatalysis.

The two-liquid phase concept was used to develop a whole cell biocatalytic system for the efficient multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde. Recombinant Escherichia coli cells were employed to express the Pseudomonas putida genes encoding xylene monooxygenase, which catalyzes the multistep oxygenation of one methyl group of toluene and xylenes to corresponding alcohols, aldehydes, and acids. A fed-batch based two-liquid phase bioconversion was established with bis(2-ethylhexyl)- phthalate as organic carrier solvent and a phase ratio of 0.5; the product formation pattern, the impact of the nutrient feeding strategy, and the partitioning behavior of the reactants were studied. On the basis of the favorable conditions provided by the two-liquid phase system, engineering of the initial pseudocumene concentration allowed exploiting the complex kinetics of the multistep reaction for the exclusive production of 3,4-dimethyl- benzaldehyde. Further oxidation of the product to 3,4-dimethylbenzoic acid could be inhibited by suitable concentrations of pseudocumene or 3,4-dimethylbenzyl alcohol. The optimized biotransformation setup includes a completely defined medium with high iron content and a nutrient feeding strategy that avoids severe glucose limitation as well as high inhibitory glucose levels. Using such a system on a 2-liter scale, we were able to produce, within 14.5 h, 30 g of 3,4-dimethylbenzaldehyde as predominant reactant in the organic phase and reached a maximal productivity of 1.6 g per liter liquid volume per hour. The present study implicates that the two-liquid phase concept is an efficient tool to exploit the kinetics of multistep biotransformations in general.

Benzaldehydes↗

PubMed2003

Synthesis of 3-tert-butylcatechol by an engineered monooxygenase.

Recombinant Escherichia coli JM101 was used for the in vivo biocatalytic synthesis of 3-tert-butyl- catechol. The bacterial strain synthesized the laboratory-evolved variant HbpA(T2) of 2-hydroxybiphenyl 3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica HBP1. The mutant enzyme HbpA(T2) is able to hydroxylate 2-tert-butylphenol to the corresponding catechol, a reaction that is not catalyzed by the wild-type enzyme. The biotransformation was performed in a 3-L bioreactor for 24 h. To mitigate the toxicity of the 2-tert-butylphenol starting material, we applied a limited substrate feed. Continuous in situ product removal with the hydrophobic resin Amberlite XAD-4 was used to separate the product from culture broth. In addition, binding to the resin stabilized the product, which was important because 3-tert-butylcatechol is very labile in aqueous solution. The productivity of the process was 63 mg L(-1) h(-1) so that after 24 h, 3.0 g of 3-tert-butylcatechol were isolated. Down-stream processing consisted of two steps. First, bound 2-tert-butylphenol and 3-tert-butylcatechol were eluted from Amberlite XAD-4 with methanol. Second, the two compounds were separated over neutral aluminum oxide, which selectively binds the produced catechol but not the phenol substrate. The final purity of 3-tert-butylcatechol was greater than 98%.

Bioreactors↗

PubMed2003

Practical issues in the application of oxygenases.

Oxygenases carry out the regio-, stereo- and chemoselective introduction of oxygen in a tremendous range of organic molecules. This versatility has already been exploited in several commercial processes. There are, however, many hurdles to further practical large-scale applications. Here, we review various issues in biocatalysis using these enzymes, such as screening strategies, overoxidation, uncoupling, substrate uptake, substrate toxicity, and oxygen mass transfer. By addressing these issues in a systematic way, the productivity of promising laboratory scale biotransformations involving oxygenases may be improved to levels that allow industry to realise the full commercial potential of these enzymes.

Bioreactors↗

PubMed2003

Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa.

Most clinical isolates identified as Pseudomonas aeruginosa grow on long-chain n-alkanes, while environmental P. aeruginosa isolates often grow on medium- as well as long-chain n-alkanes. Heterologous expression showed that the two alkane hydroxylase homologs of P. aeruginosa PAO1 (AlkB1 and AlkB2) oxidize C(12)-C(16)n-alkanes, while two rubredoxin (RubA1 and RubA2) and a rubredoxin reductase (RubB) homologs can replace their P. putida GPo1 counterparts in n-octane oxidation. The two long-chain alkane hydroxylase genes are present in all environmental and clinical isolates of P. aeruginosa strains tested in this study.

Alkanes↗

PubMed2003

Cloning of Baeyer-Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers.

To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.

Amino Acid Sequence↗

PubMed2003

Production of 6-phenylacetylene picolinic acid from diphenylacetylene by a toluene-degrading Acinetobacter strain.

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.

Acetylene↗

PubMed2002

Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase.

Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp. strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99%. This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion. The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5. At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited. The reactor had an estimated power input in the order of 5 W. L(-1), which is close to values typically obtained with commercially operating units. The product could be easily purified by fractional distillation to a purity in excess of 97%. The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products.

Cell Line↗

PubMed2002

Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase.

Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC ) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpA(ind)) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole. The wild-type protein does not catalyze these reactions. HbpA(ind) contains amino acid substitutions D222V and V368A. The activity for indole hydroxylation was increased 18-fold in this variant. Concomitantly, the K(d) value for indole decreased from 1.5 mm to 78 microm. Investigation of the major reaction products of HbpA(ind) with indole revealed hydroxylation at the carbons of the pyrrole ring of the substrate. Subsequent enzyme-independent condensation and oxidation of the reaction products led to the formation of indigo and indirubin. The activity of the HbpA(ind) mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme. In HbpA(ind), there was significantly increased uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation, which could be attributed to the substitution D222V. The position of Asp(222) in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp(222) is involved in substrate activation in HbpA.

Catalysis↗

PubMed2002

Practical syntheses of N-substituted 3-hydroxyazetidines and 4-hydroxypiperidines by hydroxylation with Sphingomonas sp. HXN-200.

[reaction: see text] Hydroxylation of N-substituted azetidines 11 and 12 and piperidines 15-19 with Sphingomonas sp. HXN-200 gave 91-98% of the corresponding 3-hydroxyazetidines 13 and 14 and 4-hydroxypiperidines 20-24, respectively, with high activity and excellent regioselectivity. High yields and high product concentrations (2 g/L) were achieved with frozen/thawed cells as biocatalyst. For the first time, rehydrated lyophilized cells were successfully used for the biohydroxylation.

Aldehydes↗

PubMed2002

Production of chiral R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters via hydrolytic degradation of polyhydroxyalkanoate synthesized by pseudomonads.

A novel and efficient method for the production of enantiomericaly pure R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters was developed. The described method is based on hydrolysis of poly(hydroxyalkanoate) copolymers synthesized by Pseudomonas putida. The polymer was isolated via solvent recovery and hydrolyzed by acid methanolysis. The obtained 3-hydroxyalkanoic acid methylester mixture was distilled into several fractions with an overall yield of 96.6% (w/w). Gas chromatography-mass spectrometry analysis of the fractions showed that 3-hydroxyhexanoic-, 3-hydroxyoctanoic-, 3 hydroxydecanoic-, and 3-hydroxydodecanoic acid methylesters were enriched to purities exceeding 96 mol%, with distillation yields of 99.9, 99.8, 88.4, and 56.8% (w/w), respectively. Subsequent saponification of the purified methylester fractions yielded the corresponding 3-hydroxyalkanoic acids, which were recovered up to 92.8% (w/w). Chiral gas chromatography analysis confirmed that both 3-hydroxyoctanoic acid and 3-hydroxyoctanoic acid methylester are present in the R-form at a very high enantiomeric excess (>99.9%).

Fermentation↗

PubMed2002

Oxidative biotransformations using oxygenases.

Considerable progress has been made in manipulating oxidative biotransformations using oxygenases. Substrate acceptance, catalytic activity, regioselectivity and stereoselectivity have been improved significantly by substrate engineering, enzyme engineering or biocatalyst screening. Preparative biotransformations have been carried out to synthesize useful pharmaceutical intermediates or chiral synthons on the gram to several-hundred-gram scale, by use of whole cells of wild type or recombinant strains. The synthetic application of oxygenases in vitro has been shown to be possible by enzymatic or electrochemical regeneration of NADH or NADPH.

Biotransformation↗

PubMed2002

Alkane hydroxylase homologues in Gram-positive strains.

We isolated Gram-positive alkane-degraders from soil and a tricking-bed reactor, and show using polymerase chain reaction (PCR) with degenerate alkane hydroxylase primers and Southern blots that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene. Two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologues in the remaining strains.

Alkanes↗

PubMed2002

Characterization and application of xylene monooxygenase for multistep biocatalysis.

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.

Benzene Derivatives↗