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Biomedical subjects

Jan B van Beilen

Publications and source records attributed to Jan B van Beilen.

At least 19 recordsLinked to original sources
PubMed2007

Alkane hydroxylases involved in microbial alkane degradation.

This review focuses on the role and distribution in the environment of alkane hydroxylases and their (potential) applications in bioremediation and biocatalysis. Alkane hydroxylases play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Environmental studies demonstrate the abundance of alkane degraders and have lead to the identification of many new species, including some that are (near)-obligate alkanotrophs. The availability of a growing collection of alkane hydroxylase gene sequences now allows estimations of the relative abundance of the different enzyme systems and the distribution of the host organisms.

Alkanes↗
PubMed2006

Selection of biocatalysts for chemical synthesis.

To determine whether microbial chemosensors can be used to find new or better biocatalysts, we constructed Escherichia coli hosts that recognize the product of a biocatalytic conversion through the transcriptional activator NahR and respond by expression of a lacZ or tetA reporter gene. Equipped with a benzaldehyde dehydrogenase (XylC from Pseudomonas putida), the lacZ-based host responded to the oxidation of benzaldehyde and 2-hydroxybenzaldehyde to the corresponding benzoic acids by forming blue colonies, whereas XylC- cells did not. Similarly, the tetA-based host was able to grow under selective conditions only when equipped with XylC, enabling selection of biocatalytically active cells in inactive populations at frequencies as low as 10(-6).

Aldehyde Oxidoreductases↗
PubMed2006

Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases.

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.

Alkanes↗
PubMed2006

CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation.

The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C6 to C11) to 1-alkanols with a maximal turnover number of 70 min(-1) and has a regiospecificity of > or =95% for the terminal carbon atom position. Spectroscopic binding studies showed that C6-to-C11 aliphatic alkanes bind in the active site with Kd values varying from approximately 20 nM to 3.7 microM. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.

Alkanes↗
PubMed2005

Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida.

We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.

Acyltransferases↗
PubMed2005

Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation.

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.

Acyltransferases↗
PubMed2005

Expanding the alkane oxygenase toolbox: new enzymes and applications.

As highly reduced hydrocarbons are abundant in the environment, enzymes that catalyze the terminal or subterminal oxygenation of alkanes are relatively easy to find. A number of these enzymes have been biochemically characterized in detail, because the potential of alkane hydroxylases to catalyze high added-value reactions is widely recognized. Nevertheless, the industrial application of these enzymes is restricted owing to the complex biochemistry, challenging process requirements, and the limited number of cloned and expressed enzymes. Rational and evolutionary engineering approaches have started to yield more robust and versatile enzyme systems, broadening the alkane oxygenase portfolio. In addition, metagenomic approaches provide access to many novel alkane oxygenase sequences.

Alkanes↗
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↗
PubMed2005

Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases.

Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.

Alkanes↗
PubMed2004

Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2.

Alcanivorax is an alkane-degrading marine bacterium which propagates and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented. To identify the genes responsible for alkane degradation in this organism, two putative genes for alkane hydroxylases were cloned from Alcanivorax borkumensis SK2. They were named alkB1 and alkB2. These genes were subsequently disrupted in A. borkumensis SK2, and the growth phenotypes of the disruptants were examined. The results indicate that the alkB1 gene is responsible for the degradation of short-chain n-alkanes. A double mutant defective in both alkB1 and alkB2 was still able to grow on medium-chain n-alkanes, indicating that genes other than alkB1 and alkB2 are also involved in n-alkane hydroxylation by A. borkumensis SK2.

Alkanes↗
PubMed2004

Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis.

The marine gamma-Proteobacterium Alcanivorax borkumensis is highly specialized in the assimilation of aliphatic hydrocarbons, and makes up a large part of the biomass in oil-polluted marine environments. In addition to the previously identified alkane hydroxylase AlkB1, a second alkane hydroxylase (AlkB2) showing 65% identity to the Pseudomonas aeruginosa AlkB2 alkane hydroxylase was identified. Unlike alkB1, alkB2 is not flanked by genes involved in alkane metabolism. Heterologous expression of the A. borkumensis AP1 alkB1 and alkB2 genes showed that they encode functional alkane hydroxylases with substrate ranges similar to those of their P. putida and P. aeruginosa homologues. The transcription initiation sites and levels of the alkB1, alkB2 and alkS mRNA transcripts were determined. Expression of both alkB1 and alkB2 was induced by alkanes, but transcripts corresponding to alkB1 were much more abundant than those of alkB2. An inverted repeat similar to the binding site for the P. putida GPo1 transcriptional activator AlkS was present upstream of the promoters for alkB1 and alkB2, although that of alkB2 was less well conserved, and only the transcriptional fusion of promoter PalkB1 to the reporter gene lacZ efficiently responded to n-octane. Contrary to what has been found for the P. putida GPo1 alkane degradation pathway, expression of the A. borkumensis AP1 alkS gene was not induced by alkanes, and an AlkS binding site was not present upstream of the promoter for alkS. This indicates that, in spite of the clear similarities, the A. borkumensis alk-genes are regulated by a strategy different from that of the P. putida GPo1 alk genes.

Alkanes↗
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↗
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

Enzyme technology: an overview.

Enzymes are being used in numerous new applications in the food, feed, agriculture, paper, leather, and textiles industries, resulting in significant cost reductions. At the same time, rapid technological developments are now stimulating the chemistry and pharma industries to embrace enzyme technology, a trend strengthened by concerns regarding health, energy, raw materials, and the environment.

Biotechnology↗
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↗