Biomedical subjects

Wouter A Duetz

Publications and source records attributed to Wouter A Duetz.

6 recordsLinked to original sources

PubMed2005

Biocatalytic production of perillyl alcohol from limonene by using a novel Mycobacterium sp. cytochrome P450 alkane hydroxylase expressed in Pseudomonas putida.

A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is (-)-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate l-limonene in the 7 position to produce enantiopure (-)-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams.

Amino Acid Sequence↗

PubMed2004

Rapid identification of new bacterial alcohol dehydrogenases for (R)- and (S)-enantioselective reduction of ss-ketoesters.

New bacterial alcohol dehydrogenases with high and complementary enantioselectivity for the reduction of ethyl 3-keto-4,4,4-trifluorobutyrate 1 and methyl 3-keto-3-(3'-pyridyl)-propionate 3 have been rapidly identified by use of a new methodology consisting of preselection of microorganisms based on degradation ability and high-throughput screening with a miniaturized system coupled with fast analysis of enantioselectivities.

Acinetobacter↗

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

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

Induction and repression of the sty operon in Pseudomonas putida CA-3 during growth on phenylacetic acid under organic and inorganic nutrient-limiting continuous culture conditions.

The effects of various nutrient-limiting conditions on expression of the sty operon in Pseudomonas putida CA-3 were investigated. It was observed that limiting concentrations of the carbon source phenylacetic acid, resulted in high levels of phenylacetyl coenzyme A (CoA) ligase activity, this was accompanied also by upper pathway styrene monooxygenase enzyme activity. The introduction of inorganic nutrient limitations, (nitrate, sulfate and phosphate), caused a dramatic reduction in detectable levels of phenylacetyl CoA ligase activity, particularly in the presence of the primary carbon source, succinate. Under these conditions it was no longer possible to detect styrene monooxygenase activity. Reverse transcription PCR analysis of total RNA, isolated under each of the continuous culture conditions examined, revealed that variations in the levels of enzyme activity coincided with altered patterns of corresponding paaK (phenylacetyl CoA ligase) and styA (styrene monooxygenase) gene expression. Transcription of the upper pathway regulatory sensor kinase gene styS was also observed to be growth condition-dependent. These observations suggest that induction/repression of the sty operon in P. putida CA-3, during growth on phenylacetic acid under continuous culture conditions, involves regulatory mechanisms coordinately affecting both the upper and lower pathways and acting at the level of gene transcription.

Carbon↗

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↗