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Now middle-aged and completely oblivious to the clinic's old error, our hero is an over-grown adolescent, living with a lovely young policewoman who just found out she's pregnant. He drives a delivery truck for his father's butcher shop and mostly ignores all of his parking tickets and any temptation to grow up. Her news is NOT welcome.
Out of the blue, 142 of his offspring file a class-action lawsuit to meet their biological father. They include: a manicurist, a soccer player, a bartender, an actor, a drug addict, a busker, etc., etc., etc. They are male and female, gay and straight. One is institutionalized because he has cerebral palsy. Of course the press has a field day trying to uncover the mysterious "Starbuck."
We have:
* Patrick Huard ("Funkytown") is Starbuck, our lusty hero, who quietly starts tracking down some of his children and is surprised by what he finds.
* Antoine Bertrand ("The Necessities of Life") is his best friend, who is (almost) a lawyer and volunteers to defend him in court to keep his identity a secret.Read more ›
"Starbuck" (2011 release from Quebec; 110 min.) brings the story of David (played by Patrick Huard), who in the late 80s made frequent donations to the local sperm bank (why? because he needed the money! Starbuck? his alias at the sperm bank!) and now 20+ years later learns from the sperm bank's lawyer that his sperm was used on many occasions, resulting in no less than 533 biological kids (!), and that 142 of them now have filed a class action law suit to find out the identity of their biological father. As it is, Davis is a good-for-nothing who slacks on the job (at his father's butcher shop) and not very nice to his girlfriend (whom we learn has just become pregnant). David gets a package with the profiles of the 142 kids in the class action, and he secretly starts out to observe them (without revealing his identity). They are in all walks of life: a pool lifeguard, a bartender, a foot manucurist, a guitarist playing in the subway for money and even a handicapped young man. Then one day one of the 142 has pieced things together and confronts David, threatening to "out" him. At this point we are about half-way into the movie. Will David's identity be revealed to these kids? Will he tell his girlfriend about his situation? To tell you more of the plot would surely ruin your viewing experience, you'll just have to see for yourself how it all plays out.Read more ›
Then he gets approached by a lawyer who says he is representing a group action of 142 young adults who are all the result of artificial insemination from a particular clinic, where he made a vast number of `deposits', it turns out he had the best `material' and has fathered 533. He is shocked at first but then curiosity gets the better of him and he decides to see what his `kids' are really like and if he should reveal his identity.
This is a funny, sweet, warm and sometimes moving piece of cinema; I was reaching for the hankies on a couple of occasions. He had made the `deposits' under the fake name of Starbuck, hence the title and is played by Patrick Huard who also made `Funky Town' which is another little gem. All of the cast are excellent and so much is crammed in here that you really are getting great value in a great feel good film. It is nigh on all in French, lots of English in the soundtrack which includes `Tuung' and the brilliant `The National'. The sub titles are all good too, the framing of the shots is adequate nothing award winning but still it has a wonderful feel to the whole thing and I really loved it, Ken Scott's next one is `Delivery Man' if it is half as good as this it will be worth the wait.
Most Recent Customer Reviews
Funny film, and great for showcasing Quebecois French. BUT, topics are mature (drugs, sex, etc.) It's clean as most of the hot topics are suggested rather than shown, but I would... Read morePublished 1 month ago by review98
My girlfriend made me watch this. It was actually a very fun movie to watch and read.Published 1 month ago by Matt
Such a great, feel-good movie. Worth reading subtitles for any non-french speakers.Published 4 months ago by CJ1234
A great feel good movie. As a private sperm donor myself, it was something that I reflect on, and this movie addressed a lot of my thoughts, especially as the kids grow up. Read morePublished 10 months ago by Ice cream samarai salami


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Featuring talented clinicians from major publishers, Stanton's summer choral clinics are a fantastic way to jump-start… read moreJuly 22nd
recommended by Barb M., Keyboard & Folk Music Specialist Fans of Rosanne Cash will be aware of "The River… read more
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Title: Irving Berlin's Christmas
Voicing/Format: Concert Band
Publisher: Irving Berlin Music Co
Arranger: Brymer/Brown
Catalog #: 04001237
Grade: 3
Description: W/Opt Choir & Strings Price: $70.00

subset: commoncrawl


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Rated 5 out of 5 by 4 reviewers.
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Canon: Murderer
Canon VB-C50iR
Surveillance Camera
Advertising Agency: Dentsu, Bangkok, Thailand
Creative Directors: Warunee Tanakijchumroon, Somkiat Kittipinijnanta
Art Director: Ditdanai Nopparat
Copywriter: Navanut Phlainoi
Photographer: Chub Nokkaew
Computer Artist: Chub Nokkaew
Producer: Yuppadee Thongprasert
Published:March, 2010
11 comments
shortafellaSep 06, 2010 - 7:43
actually this is pretty funny
GuestOct 08, 2010 - 5:30
i don't think it is funny, and it doesn't need to be funny either! this is a serious issue, finally. The ad is ok in terms of revealing out the benefit of the product directly. messege is clear. i like it although I wasn't charmed!


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Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca
Dudek, Hanna M.
Torres Pazmiño, Daniel E.
Rodríguez, Cristina
de Gonzalo, Gonzalo
Gotor, Vicente
Fraaije, Marco W.
Applied Microbiology and Biotechnology
Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.
Phenylacetone monooxygenase; Baeyer–Villiger oxidation; Coenzyme specificity; Enantioselectivity
Related Articles
Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis▿†
Dudek, Hanna M.
de Gonzalo, Gonzalo
Torres Pazmiño, Daniel E.
Stępniak, Piotr
Wyrwicz, Lucjan S.
Rychlewski, Leszek
Fraaije, Marco W.
Applied and Environmental Microbiology
Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.
Related Articles
Cloning, expression and characterization of a versatile Baeyer-Villiger monooxygenase from Dietzia sp. D5
Bisagni, Serena
Hatti-Kaul, Rajni
Mamo, Gashaw
AMB Express
A novel BVMO encoding gene was identified from a draft genome sequence of a newly isolated strain of Dietzia. Analysis of the protein sequence revealed that it belongs to a group of BVMOs whose most characterized member is cyclopentadecanone monooxygenase (CPDMO). The gene was PCR amplified, cloned and successfully expressed in E. coli. The expressed recombinant enzyme was purified using metal affinity chromatography. Characterization of the purified enzyme revealed that it has a broad substrate scope and oxidized different compounds including substituted and unsubstituted alicyclic, bicyclic-, aliphatic-ketones, ketones with an aromatic moiety, and sulfides. The highest activities were measured for 2- and 3-methylcyclohexanone, phenylacetone, bicyclo-[3.2.0]-hept-2-en-6-one and menthone. The enzyme was optimally active at pH 7.5 and 35°C, a temperature at which its half-life was about 20 hours. The stability studies have shown that this enzyme is more stable than all other reported BVMOs except the phenylacetone monooxygenase from the thermophilic organism Thermobifida fusca.
Baeyer-Villiger monooxygenase; Biocatalysis; Enzyme stability; Protein expression
Related Articles
Pseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic Ketones†
Iwaki, Hiroaki
Wang, Shaozhao
Grosse, Stephan
Bergeron, Hélène
Nagahashi, Ayako
Lertvorachon, Jittiwud
Yang, Jianzhong
Konishi, Yasuo
Hasegawa, Yoshie
Lau, Peter C. K.
Applied and Environmental Microbiology
Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ∼60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 μmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ∼1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (Km = 8 μM versus Km = 24 μM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C11 to C15 ketones, methyl-substituted C5 and C6 ketones, and bicyclic ketones, such as decalone and β-tetralone. CPDMO has the highest affinity (Km = 5.8 μM) and the highest catalytic efficiency (kcat/Km ratio of 7.2 × 105 M−1 s−1) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.
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Two Enzymes of a Complete Degradation Pathway for Linear Alkylbenzenesulfonate (LAS) Surfactants: 4-Sulfoacetophenone Baeyer-Villiger Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1
Weiss, Michael
Denger, Karin
Huhn, Thomas
Schleheck, David
Applied and Environmental Microbiology
Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C4-SPC). Second, these SPCs are mineralized. 3-C4-SPC is mineralized by Comamonas testosteroni KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C4-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria.
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Cloning, Expression, Characterization, and Biocatalytic Investigation of the 4-Hydroxyacetophenone Monooxygenase from Pseudomonas putida JD1▿ †
Rehdorf, Jessica
Zimmer, Christian L.
Bornscheuer, Uwe T.
Applied and Environmental Microbiology
While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E ≫100).
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Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase
Kamerbeek, Nanne M.
Olsthoorn, Arjen J. J.
Fraaije, Marco W.
Janssen, Dick B.
Applied and Environmental Microbiology
The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.
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Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening
Riebel, A.
Dudek, H. M.
de Gonzalo, G.
Stepniak, P.
Rychlewski, L.
Fraaije, M. W.
Applied Microbiology and Biotechnology
To expand the available set of Baeyer–Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-011-3823-0) contains supplementary material, which is available to authorized users.
Biocatalysis; Baeyer–Villiger monooxygenase; Sulfoxidation; Enantioselectivity
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Identification of a novel Baeyer‐Villiger monooxygenase from Acinetobacter radioresistens: close relationship to the Mycobacterium tuberculosis prodrug activator EtaA
Minerdi, Daniela
Zgrablic, Ivan
Sadeghi, Sheila J.
Gilardi, Gianfranco
Microbial biotechnology
This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long‐chain alkanes as the sole energy source expresses almA gene coding for a Baeyer‐Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer‐Villiger reactions as well as oxidation of the prodrug ethionamide.
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Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453
Leisch, Hannes
Shi, Rong
Grosse, Stephan
Morley, Krista
Bergeron, Hélène
Cygler, Miroslaw
Iwaki, Hiroaki
Hasegawa, Yoshie
Lau, Peter C. K.
Applied and Environmental Microbiology
A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105 M−1 s−1) as a substrate, although its affinity (Km = 32 μM) was lower than that of the CoA-activated substrate (Km = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.
Related Articles
Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007
Kadow, Maria
Saß, Stefan
Schmidt, Marlen
Bornscheuer, Uwe T
AMB Express
Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit.
Baeyer-Villiger monooxygenases; camphor; Pseudomonas putida NCIMB 10007; 2,5-diketocamphane 1,2-monooxygenase; bicyclic ketones
Related Articles
Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue
van Beek, Hugo L.
Wijma, Hein J.
Fromont, Lucie
Janssen, Dick B.
Fraaije, Marco W.
FEBS Open Bio
•Cyclohexanone monooxygenase was stabilized by an in silico designed disulfide bond.•Stabilizing disulfide bonds were successfully designed based on a model structure.•The half-life at 30 °C was increased 12-fold for the mutant enzyme.•The apparent melting point was increased by 6 °C for the mutant enzyme.•The most stabilizing disulfide bond spans only one residue.
Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer–Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C–A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.
BVMO, Baeyer–Villiger monooxygenase; CHMO, cyclohexanone monooxygenase; DTT, dithiothreitol; MD, molecular dynamics; PAMO, phenylacetone monooxygenase; Baeyer–Villiger monooxygenase; Computational design; Disulfide bonds; Thermostability; Flavoprotein; Enzyme engineering
Related Articles
Cloning, overexpression and biocatalytic exploration of a novel Baeyer-Villiger monooxygenase from Aspergillus fumigatus Af293
Mascotti, Maria Laura
Juri Ayub, Maximiliano
Dudek, Hanna
Sanz, Marcela Kurina
Fraaije, Marco W
AMB Express
The presence of several putative Baeyer-Villiger Monooxygenases (BVMOs) encoding genes in Aspergillus fumigatus Af293 was demonstrated for the first time. One of the identified BVMO-encoding genes was cloned and successfully overexpressed fused to the cofactor regenerating enzyme phosphite dehydrogenase (PTDH). The enzyme named BVMOAf1 was extensively characterized in terms of its substrate scope and essential kinetic features. It showed high chemo-, regio- and stereoselectivity not only in the oxidation of asymmetric sulfides, (S)-sulfoxides were obtained with 99% ee, but also in the kinetic resolution of bicyclo[3.2.0]hept-2-en-6-one. This kinetic resolution process led to the production of (1S,5R) normal lactone and (1R,5S) abnormal lactone with a regioisomeric ratio of 1:1 and 99% ee each. Besides, different reaction conditions, such as pH, temperature and the presence of organic solvents, have been tested, revealing that BVMOAf1 is a relatively robust biocatalyst.
Eukaryotic BVMO; Aspergillus; Baeyer-Villiger oxidation; Kinetic resolution; Sulfide oxidation
Related Articles
Novel Acetone Metabolism in a Propane-Utilizing Bacterium, Gordonia sp. Strain TY-5▿
Kotani, Tetsuya
Yurimoto, Hiroya
Kato, Nobuo
Sakai, Yasuyoshi
Journal of Bacteriology
In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C3 to C10) and cyclic ketones (C4 to C8). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane → 2-propanol → acetone → methyl acetate → acetic acid + methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism.
Related Articles
The Substrate-Bound Crystal Structure of a Baeyer–Villiger Monooxygenase Exhibits a Criegee-like Conformation
Yachnin, Brahm J.
Sprules, Tara
McEvoy, Michelle B.
Lau, Peter C. K.
Berghuis, Albert M.
Journal of the American Chemical Society
The Baeyer–Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer–Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP+ and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP+ and FAD, to 2.4 Å resolution. This structure shows a drastic rotation of the NADP+ cofactor in comparison to previously reported NADP+-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP+ permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme's chemospecificity, to a location above the flavin–peroxide adduct where catalysis occurs.
Related Articles
Crystal Structure of Baeyer-Villiger Monooxygenase MtmOIV, the Key Enzyme of the Mithramycin Biosynthetic Pathway†
Beam, Miranda P.
Bosserman, Mary A.
Noinaj, Nicholas
Wehenkel, Marie
Rohr, Jürgen
Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kD homo-dimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIV's structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9Å. MtmOIV cleaves a C-C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments implicate several residues to participate in co-factor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis.
Related Articles
Isolation and initial characterization of a novel type of Baeyer–Villiger monooxygenase activity from a marine microorganism
Willetts, Andrew
Joint, Ian
Gilbert, Jack A.
Trimble, William
Mühling, Martin
Microbial biotechnology
A novel type of Baeyer–Villiger monooxygenase (BVMO) has been found in a marine strain of Stenotrophomonas maltophila strain PML168 that was isolated from a temperate intertidal zone. The enzyme is able to use NADH as the source of reducing power necessary to accept the atom of diatomic oxygen not incorporated into the oxyfunctionalized substrate. Growth studies have establish that the enzyme is inducible, appears to serve a catabolic role, and is specifically induced by one or more unidentified components of seawater as well as various anthropogenic xenobiotic compounds. A blast search of the primary sequence of the enzyme, recovered from the genomic sequence of the isolate, has placed this atypical BVMO in the context of the several hundred known members of the flavoprotein monooxygenase superfamily. A particular feature of this BVMO lies in its truncated C‐terminal domain, which results in a relatively small protein (357 amino acids; 38.4 kDa). In addition, metagenomic screening has been conducted on DNA recovered from an extensive range of marine environmental samples to gauge the relative abundance and distribution of similar enzymes within the global marine microbial community. Although low, abundance was detected in samples from many marine provinces, confirming the potential for biodiscovery in marine microorganisms.
Related Articles
Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH
Mädje, Katharina
Schmölzer, Katharina
Nidetzky, Bernd
Kratzer, Regina
Microbial Cell Factories
Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(P)H. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 22.214.171.124; FDH), presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S)-1-(2-chlorophenyl)ethanol from o-chloroacetophenone.
As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3) that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR) resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C) brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by just 19% (1140 U/gCDW). The E. coli whole-cell catalyst optimized for intracellular FDH activity showed improved performance in the synthesis of (S)-1-(2-chlorophenyl)ethanol, reflected in a substantial, up to 5-fold enhancement of productivity (0.37 g/gCDW) and yield (95% based on 100 mM ketone used) as compared to the reference catalyst. For xylitol production, the benefit of enhanced FDH expression was observed on productivity only after elimination of the mass transfer resistance caused by the cell membrane.
Expression engineering of C. boidinii FDH is an important strategy to optimize E. coli whole-cell reductase catalysts that employ intracellular formate oxidation for regeneration of NADH. Increased FDH-activity was reflected by higher reduction yields of D-xylose and o-chloroacetophenone conversions provided that mass transfer limitations were overcome.
Related Articles
Role of L-alanine for redox self-sufficient amination of alcohols
Klatte, Stephanie
Wendisch, Volker F
Microbial Cell Factories
In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578–5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed.
The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-taCv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily responsible for pyruvate catabolism during redox self-sufficient amination of alcohols using this whole cell biocatalyst.
The replacement of the transaminase TaVf by TaCv, which showed higher activity at 42°C, in the artificial operon ald-adh-ta improved amination of alcohols in whole cell biotransformation. The addition of L-alanine, which was consumed by E. coli via pyruvate catabolism, was required for 100% product formation possibly by providing maintenance energy. Metabolic engineering revealed that pyruvate catabolism occurred primarily via oxidative decarboxylation to acetate by PoxB under the chosen biotranformation conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-014-0189-x) contains supplementary material, which is available to authorized users.
Redox self-sufficient amination; Whole cell biotransformation; Escherichia coli; Transaminase; Chromobacterium violaceum; Energy maintenance; Acetate formation; Pyruvate oxidase; Phosphate acetyltransferase; Acetate kinase
Related Articles
Engineering NAD+ availability for Escherichia coli whole-cell biocatalysis: a case study for dihydroxyacetone production
Zhou, Yongjin J
Yang, Wei
Wang, Lei
Zhu, Zhiwei
Zhang, Sufang
Zhao, Zongbao K
Microbial Cell Factories
Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them.
In order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD+-dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD+ oxidase (NOX) and a NAD+ transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD+ uptake, respectively. We found that cellular cofactor level, NAD+/NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5α had the highest DHA specific titer of 0.81 g/gDCW with the highest NAD+/NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37°C for 8 h compared with those at 30°C for 8 h and 18 h. When cells were transformed with the ntt4 gene, feeding NAD+ during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13 g/gDCW. Supplementing NAD+ during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76 g/gDCW/h. Cellular NAD(H) level, NAD+/NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process.
High NAD+/NADH ratio driving by NOX was very important for DHA production. Once cofactor was efficiently cycled, high cellular NAD(H) level was also beneficial for whole-cell redox biocatalysis. Our results indicated that NAD+ transporter could be applied to manipulate redox cofactor level for biocatalysis. Moreover, we suggested that genetically designed redox transformation should be carefully profiled for further optimizing whole-cell biocatalysis.
Cofactor engineering; NAD(H) level; NAD+ transporter; Escherichia coli; Dihydroxyacetone; Whole-cell biocatalysis
Related Articles
Metabolism of alkenes and ketones by Candida maltosa and related yeasts
Beier, Andy
Hahn, Veronika
Bornscheuer, Uwe T
Schauer, Frieder
AMB Express
Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known. In this study we show the degradation of the aliphatic ketone dodecane-2-one by Candida maltosa and the related yeasts Candida tropicalis, Candida catenulata and Candida albicans as well as Trichosporon asahii and Yarrowia lipolytica. One pathway is initiated by the formation of decyl acetate, resulting from a Baeyer-Villiger-oxidation of this ketone. Beyond this, an initial reduction to dodecane-2-ol by a keto reductase was clearly shown. In addition, two different ways to metabolize dodec-1-ene were proposed. One involved the formation of dodecane-2-one and the other one a conversion leading to carboxylic and dicarboxylic acids. Furthermore the induction of ketone degrading enzymes by dodecane-2-one and dodec-1-ene was shown. Interestingly, with dodecane no subterminal degradation products were detected and it did not induce any enzymes to convert dodecane-2-one.
Hydrocarbon; alkene; ketone; Candida; yeast; biotransformation
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Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli-Expressed Cyclopentanone 1,2-Monooxygenase†
Iwaki, Hiroaki
Hasegawa, Yoshie
Wang, Shaozhao
Kayser, Margaret M.
Lau, Peter C. K.
Applied and Environmental Microbiology
Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus Comamonas. Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as (orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orf3-orf4 [partial 3′ end]). The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ-Kmr cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of σ54-dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-β-thio-d-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.
Related Articles
Molecular Insight into Substrate Recognition and Catalysis of Baeyer–Villiger Monooxygenase MtmOIV, the Key Frame Modifying Enzyme in the Biosynthesis of Anticancer Agent Mithramycin
Bosserman, Mary A.
Downey, Theresa
Noinaj, Nicholas
Buchanan, Susan K.
Rohr, Jürgen
ACS chemical biology
Baeyer-Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C-C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed us to probe the substrate binding pocket of MtmOIV, and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues.
Related Articles
Crystallization and X-ray diffraction properties of Baeyer–Villiger monooxygenase MtmOIV from the mithramycin biosynthetic pathway in Streptomyces argillaceus
Wang, Chenchen
Gibson, Miranda
Rohr, Jurgen
Oliveira, Marcos A.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications
2005;61(Pt 11):1023-1026.
Crystals of the type I Baeyer–Villiger monooxygenase (BVMO) MtmOIV from the biosynthetic pathway of mithramycin were obtained; the crystals diffracted to 2.69 Å resolution and belong to the monoclinic space group C2 (a = 143.5, b = 114.2, c = 137.8 Å β = 102.5°). Light scattering indicates that MtmOIV is a dimer of 127 kDa in solution, while in the crystalline state the data are consistent with two dimers in the asymmetric unit.
The Baeyer–Villiger monooxygenase MtmOIV from Streptomyces argillaceus is a 56 kDa FAD-dependent and NADPH-dependent enzyme that is responsible for the key frame-modifying step in the biosynthesis of the natural product mithramycin. Crystals of MtmOIV were flash-cooled and diffracted to 2.69 Å resolution using synchrotron radiation on beamline SER-CAT 22-ID at the Advanced Photon Source. Crystals of MtmOIV are monoclinic and light-scattering data reveal that the enzyme forms dimers in solution. The rotation function suggests the presence of two dimers in the asymmetric unit. l-Selenomethionine-incorporated MtmOIV has been obtained. Structural solution combining molecular-replacement phases and anomalous phases from selenium is in progress.
mithramycin; MtmOIV; Baeyer–Villiger monooxygenase; FAD-dependent; NADPH-dependent
Related Articles
Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca
Schallmey, Anett
den Besten, Gijs
Teune, Ite G. P.
Kembaren, Roga F.
Janssen, Dick B.
Applied Microbiology and Biotechnology
Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-010-2965-9) contains supplementary material, which is available to authorized users.
Cytochrome P450 monooxygenase; Thermobifida fusca; Enzyme catalysis; Thermostable enzyme; Hydroxylation
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Revlon Colorsilk Beautiful Color Light Sun Blonde 95 Kit includes:
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Great color!
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Comments about Revlon Colorsilk Beautiful Color, Light Sun Blonde 95, 1 application:
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Essential Guide
Virtualization to cloud: Planning, executing a private cloud migration
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A private cloud vs. the standard data center: What's the difference?
Cloud computing expert Chris Moyer explains the difference between a private cloud and the standard data center.
Virtualization to cloud: Planning, executing a private cloud migration
What's the difference between a private cloud and the standard data center?
Clouds never refer to hardware or physical resources. Regardless of whether it's called a public, private or hybrid cloud, the term doesn't define anything about the physical hardware it's running on. Clouds can reside on any hardware, in any data center. Often, they're thought of as an abstraction layer above the physical hardware and data centers they reside in.
BYOC: Rethink How You're Supporting Mobile Apps
Gain expert advice on how to govern unrestricted technology and explore the risks to secured information posed by careless mobile use
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Here's a quick graph to help illustrate:
In any data center resides servers (as well as other network infrastructure, but for now we'll just focus on servers). Virtualization software runs on those servers, and above that resides the cloud application programming interface (API). The difference with a cloud is that you're providing access to all of the resources virtually, via an API. This API allows you to automatically "provision" hardware to scale your resources as needed. A cloud is based on a virtualization layer, similar to VMware (in fact, VMware is moving up in the cloud space, attempting to produce its own version of a private cloud).
You can also have a private cloud that isn't in a data center that you own. The term "private" simply means that you're the only one that has access to it. The private cloud could also be hosted in a shared data center, as long as you're the only one that has access to that physical set of resources.
About the author
Chris Moyer is author of the book Building Applications in the Cloud and creator of two Web frameworks, Marajo and botoweb. He currently lives in New York, where he helps developers migrate applications to the cloud.
Follow us on Twitter at @CloudAppsTT.
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Chris Moyer asks:
Which does your company use: a standard data center or a private cloud?
0 ResponsesJoin the Discussion
Related Q&A from Chris Moyer
What are the key DevOps roles in an enterprise?
The DevOps model is taking off as cloud adoption grows. But what exactly are the key responsibilities of a DevOps team in the enterprise?continue reading
What is a cloud-based system?
Get an overview of what a cloud-based system is and what would work best in your enterprise.continue reading
How do incremental backups on Amazon EBS work?
Looking for the best way to back up Amazon EBS? Expert Chris Moyer clarifies how incremental backups work.continue reading
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text:
Trump says states should set wage
Says Donald Trump is "against minimum wage."
— PolitiFact Virginia
on Tuesday, July 19th, 2016
Mind the generational gap
Says British voters "under 50, especially millennials, overwhelmingly voted to stay," in the European Union. "It was older voters who voted to leave."
— PolitiFact Global News Service
on Sunday, June 26th, 2016
An overshoot, but broadly right
"For every extra year that a girl stays in secondary school, her chance of getting infected with HIV/AIDS decreases by half."
— PolitiFact Global News Service
on Friday, June 24th, 2016
Data more than 20 years old
"It's estimated that 40 percent of all the guns that are sold in the United States occur with no background records check."
— PolitiFact Virginia
on Tuesday, April 28th, 2015
But Obama's action is more sweeping
"Every president since Eisenhower has taken executive action to address immigration issues."
— PolitiFact Virginia
on Sunday, November 30th, 2014
There's nine; can you name them?
Pinnacle Overlook in Southwest Virginia is "closer to eight other state capitals than to Richmond."
— PolitiFact Virginia
on Monday, September 1st, 2014
Wild shot
Sens. Mark Warner and Tim Kaine voted "to let the U.N. take OUR guns."
— PolitiFact Virginia
on Sunday, June 15th, 2014
Not a record by all counts
Bill Clinton presided over "the biggest expansion of the American economy since World War II."
— PolitiFact Virginia
on Monday, October 29th, 2012
Includes cuts after he left office
"As governor, I cut $5 billion in spending."
— PolitiFact Virginia
on Wednesday, October 24th, 2012
Superficial claim
"The tax burden on Virginia families was lower under Tim Kaine than under George Allen."
— PolitiFact Virginia
on Monday, October 15th, 2012
Even so, the program would have still grown
George Allen "voted to end Pell Grants for 84,000 students."
— PolitiFact Virginia
on Monday, October 1st, 2012
"Tim Kaine announced he wants to raise taxes on everyone."
— PolitiFact Virginia
on Monday, September 24th, 2012
Lacking context
"Tim Kaine doubled down in his support of devastating defense cuts, saying the 'the deal was the right thing to do.'"
— PolitiFact Virginia
on Thursday, September 20th, 2012
Mixing apples and oranges
"When Tim Kaine was governor, spending soared, blowing holes in the budget every year."
— PolitiFact Virginia
on Friday, August 31st, 2012
Cherry-picked figures
The state government workforce grew when Tim Kaine was governor.
— PolitiFact Virginia
on Monday, August 27th, 2012
In Kaine's last budget
Tim Kaine, as governor, proposed tax increases on people earning "as little as $17,000."
— PolitiFact Virginia
on Friday, August 3rd, 2012
Three specific votes
Says former Sen. George Allen voted four times to raise his own pay.
— PolitiFact Virginia
on Sunday, July 29th, 2012
The recession required cuts
"I ended up my time as governor with a general fund budget that was smaller than the one I started with."
— PolitiFact Virginia
on Friday, July 27th, 2012
Many ways to look at pay gap
Virginia women earn "only 79 cents to each dollar earned by men..."
— PolitiFact Virginia
on Friday, June 15th, 2012
Updated with new ruling to reflect GDP
George Allen and his colleagues in the Senate "turned the biggest surplus in the history of the United States into the biggest deficit in the history of the United States."
— PolitiFact Virginia
on Tuesday, May 15th, 2012
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text:
- News tagged "graphics card"
Tuesday, 8 Mar 2011
HIS Unveils the HD 6990 Fan 4GB GDDR5
HIS unleashes the HIS 6990 Fan 4GB GDDR5. Loaded with stunning array of advanced technologies, such as AMD HD3D, Eyefinity, EyeSpeed and Eye-Definition, it delivers the superior gaming performance and flawless image quality.
Sapphire HD 6990 Delivers Top Performance and Features
The new Sapphire HD 6990 is a dual GPU card, featuring the latest architecture from AMD providing massively parallel computing power for graphics and other accelerated applications.
ASUS Launches the HD 6990 Graphics Card
ASUS introduces the new HD 6990 graphics card. It uses dual HD 6970 cores on one printed circuit board, doubling the power while maintaining streamlined energy draw requirements.
MSI Announces Radeon R6990-4PD4GD5
MSI announces the AMD Radeon R6990-4PD4GD5 graphics card. It is equipped with two AMD Radeon HD6900-series graphics processors and the world's first desktop card with 4GB GDDR5 memory.
Friday, 4 Mar 2011
The HIS 6950 Fan 1GB GDDR5 Has Landed on Earth, Offering You a More Affordable Alternative for a Premium Card
HIS launches its new graphics card, the HIS 6950 which has a core clock speed of 800 MHz and a 5000 MHz memory clock speed and its available at low costs.
Wednesday, 2 Mar 2011
The New HIS 6870 IceQ X TURBO X
The new top of the line HIS 6870 IceQ X Turbo X is 15 degrees cooler, 15 dB quieter and boasts a core clock speed of 975 MHz and has been released by HIS.
MSI Releases the R6950 Twin Frozr III Power Edition Graphics Card
The new R6950 Twin Frozr III Power Edition from MSI has propeller blade technology and triple over-voltage support which makes it 11 degrees cooler and 13.9dB quieter than its predecessors while also gaining a 21 percent performance boost.
Tuesday, 1 Mar 2011
Sapphire Displays New High End Graphics Cards and Technologies
Sapphire introduces a new line of graphics cards along with a new range of mini-PCs. The graphics cards include the Eyefinity technology making them usable with multiple screens, while the mini-PCs are only half a liter in volume along with many more features.
Monday, 28 Feb 2011
Sparkle Announces GTX580 Thermal Guru Graphics Card
Sparkle announces the new Sparkle GTX580 Thermal Guru Graphics Card. Featuring a unique design for cooling which utilizes aluminum fins and copper pipes for a balance between heat efficiency and low wight. It also boasts a special ducting mechanism to optimize the airflow into the fan for cooling and longer usage life.
Friday, 18 Feb 2011
Sparkle Introduces Calibre X570 Graphics Card
Sparkle introduces the Calibre X570 graphics card, which delivers intense cooling performance and broadest set of features for gamers.


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By Catholic Online (NEWS CONSORTIUM)
4/2/2014 (2 years ago)
Catholic Online (www.catholic.org)
Some futuristic thinkers say that in the next 20 years or so, when people go to the market, they will pick up the items they need and leave - without the benefit of paying a cashier. Sensors will identify a consumer by a mark, their way of walking or facial recognition. Will cash money - paper and coins, become extinct, for a system that calls forth the "Mark of the Beast" as spoken in the Book of Revelations?
Theoretically, a unique identifier is attached to your digital wallet, could transmits payment for the groceries directly to the store.
By Catholic Online (NEWS CONSORTIUM)
Catholic Online (www.catholic.org)
4/2/2014 (2 years ago)
Published in Business & Economics
Keywords: Cash-less transactions, money, Bitcoins
LOS ANGELES, CA (Catholic Online) - Theoretically, a unique identifier is attached to your digital wallet, could transmits payment for the groceries directly to the store. Your virtual wallet has a dozen digital currencies in it, all with different values based on a variety of factors, including loyalty programs.
At some grocery stores, you might pay with their version of frequent-flier miles, while at others, you might pay with the equivalent of a virtual credit card.
It is better to light one tiny candle than to curse the darkness --
While this sounds fanciful, technologists and thinkers say that this is in fact the most conservative and likely of all future scenarios. "What was it like to see that old-fashioned building called a bank?" may be a question posed by our grand-children and great grandchildren.
The emergence of Bitcoin and the ecosystem growing around it is just one of the any symptoms of this New World Order.
And even if you believe Bitcoin is no more than a fad - and that may well be, given its volatility, security issues and potential regulatory challenges - it has raised the prospect of new virtual currencies and, at the least, cheaper and more efficient transaction mechanisms.
"Money is a very interesting philosophical idea in that we have all of humanity agreeing on this system," Ray Kurzweil, a futurist, inventor and author says. "So even though we may radically disagree on some things - like let's say the U.S. government and Al Qaeda - they both respect money. So it's remarkable how we have this universal respect for this very esoteric virtual construct."
Other technocrats such as Marc Andreessen, who led the team that invented the first commercial web browser, say they believe that new virtual currencies will come to dominate the way we pay for things in the future.
"Bitcoin offers a sweeping vista of opportunity to reimagine how the financial system can and should work in the Internet era, and a catalyst to reshape that system," he wrote recently.
Kurzweil, however, says that change may not be rapid as that. "We've built up respect for currencies associated with nations," he said. "People respect dollars, mostly, I think, because of the track record, relative stability."
By Marshall Connolly (CALIFORNIA NETWORK)
Something is wrong with our economy, and the future is more troubling that it has been in almost a century. A study reveals that British millennials are earning less than their parents. The implications are staggering. LOS ANGELES, CA (California Network) - A ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
A Pew survey reveals that newsprint is dying and more people are turning to the internet and video for their news. Newspapers have lost 7 percent of their daily circulation over the past year alone. LOS ANGELES, CA (California Network) - The 13th annual Pew Research ... continue reading
By Kenya Sinclair (CALIFORNIA NETWORK)
What does it mean when Americans have a failing sense of confidence in the very institutions that directly affect their daily lives? LOS ANGELES, CA (Catholic Online) - According to the latest Gallup survey, Americans lack confidence in the schools responsible for the ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
Americans owe at least $3.4 trillion in credit debt, and that number continues to rise as payment on the debt falls below borrowing. On average, each household owes more than $15,000 in debt. LOS ANGELES, CA (California Network) - Americans are falling deeper into debt ... continue reading
By Kenya Sinclair (CALIFORNIA NETWORK)
The poor are getting poorer and the rich don't give a damn - what have we done? LOS ANGELES, CA (Catholic Online) - According to Exodus 22:24, God tells us to be fair. "If you lend money to any of my people, to anyone poor among you, you will not play the userer with ... continue reading
By Kenya Sinclair (CALIFORNIA NETWORK)
China has been purchasing major American companies, resulting in nearly 100,000 U.S. citizens working to support the Chinese economy. LOS ANGELES, CA (Catholic Online) - State-owned companies and those closely-related to the Chinese government are responsible for the ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
There's growing concern that Buzzfeed doesn't have a very bright future, at least as far as news is concerned. The firm has missed its projected revenues, and it may look to the news for the place to cut. LOS ANGELES, CA (California Network) - It's hard to get news ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
Your favorite brands may be in trouble for tax evasion. Over a hundred police officers and another hundred support personnel have raided Google's headquarters in France as part of an investigation into the company's taxes. Google is one of many brands that may owe ... continue reading
By David Drudge (CALIFORNIA NETWORK)
Freedom of the press may be the most important fundamental right in a free society. This is why censorship, particularly on social networks such as Facebook, is particularly onerous. LOS ANGELES, CA (California Network) - Freedom of the press is enshrined in the First ... continue reading
By Kenya Sinclair (CALIFORNIA NETWORK)
Mark Zuckerberg is set to hold a meeting on Wednesday with Glenn Beck and Dana Perino to talk Facebook's censorship scandal. LOS ANGELES, CA (Catholic Online) - Recently a Gizmodo report accused Facebook of suppressing conservative news in the "trending" section.The ... continue reading


text:
With the Doc McStuffins Happy Smile Dentist playset, kids can help Doc help her pal Bronty get rid of his "Stinky-Salami-Breath-itis" with this mini dentist themed playset. This playset comes with miniature versions of Doc dressed in her dentist uniform, Bronty, dentist table, exam chair, Doc's Big Book of Boo Boos, and all the dentistry accessories Doc might need to help give Bronty a winning smile and fresh breath.
Doc loves helping her friends feel better and learning about all the different fields of medicine. This playset taps into that, expanding classic roleplay with the character.
The Doc McStuffins Happy Smile Dentist playset is for Doc fans ages 3 and up.
Additional Doc McStuffins mini doll playsets and characters are sold separately.
Some of the pieces in this playset can pose a choking risk to kids under age 3.
Project Mc2 Camryn Coyle
from MGA Entertainment
Project Mc2 Adrienne Attoms
from MGA Entertainment
Barbie Crimp & Color Deluxe Styling Head
from Just Play
Littlest Pet Shop Pet Shop
from Hasbro
Project Mc2 Bryden Bandweth Smiley Face Shirt
from MGA Entertainment
Project Mc2 Bryden Bandweth Sharpest Pencil Shirt
from MGA Entertainment
DC Super Hero Girls Power Action Wonder Woman Doll
from Mattel
Monster High Dance the Fright Away Playset
from Mattel
Monster High Voltageous Hair Frankie Stein
from Mattel
Disney Princess Mini Toddler Aurora
from Jakks Pacific


text:
- Miss Florida 2017 stripped of crown days after win - US Weekly
- Lindsay Lohan steps out amid relationship drama - TMZ
- Was Taylor Swift snubbed by the VMAs? - Gossip Cop
- Rachel Platten sings "Fight Song" with lots of stars - Just Jared Jr
- Farrah Abraham sizzles in a revealing bikini - Radar
- New pics from the Clooneys' Italian summer - Lainey Gossip
- Fox News snubs major DNC moment - The Hollywood Reporter
11 April 2014 at 3:51 PM
Brad Pitt Looks Super Handsome While Checking Out an Art Gallery - See the Pics Here!
About this photo set: Brad Pitt is as handsome as ever as he leaves the Fahey/Klein Gallery on Friday morning (April 11) in Los Angeles. The 50-year-old actor reportedly had a parking… Read More Here
Photo #: 3089170
powered by ZergNet


text:
Comment Correct use of the term (Score 2, Interesting) 181
It's nice to see an example of correct use of "hacker" by the mainstream media, even if it's just by chance
Or he could punching an Arm in the Dick - much more effective if you ask me!
Intel however, has to date not yet managed to do this.
System checkpoint complete.


text:
Top Definition
1. when someone tries to interpret something as deep, but really its not at all.
Jim: How could she inturdpret that leaf falling down as a sign from God? What a fucking idiot.
door cuban luis 7 april 2008
Trending Now
De e-mails zijn afkomstig van email@example.com. We sturen nooit spam.×


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text:
"No exceptional circumstances whatsoever, whether a state of war or a threat of war, internal political instability, or any other public emergency, may be invoked as a justification of torture." -- U.N. Convention Against Torture
HTS costs up to $300 a month to operate. We need your help!
Lol, where has you been? IRC seems so desolate without my okie pal XD
poopingfish come on help me in basic 8|9 ok ;)
All the people in HTS are happy hack
so um.. yea.. i herd u liek mudkipz
Happy hacking :^)


text:
June Unemployment Statistics
On July 7th, the Central Statistics Office (CSO) published the Live Register for June 2016. According to these figures there were 315,883 people on the Live Register, of whom 45.5% or 143,832 were on the Register for more than a year. These figures were up on the month of May, but down annually by 11.4% and 13.6% respectively.
On the year the Live Register figures fell across the 8 regions the country is divided into, with the biggest decrease visible in Dublin: 9,534, and the smallest decrease seen in the Midlands: 2,826. These changes had no impact on their relative share of the Live Register: with Dublin still accounting for 24% and Midlands accounting for 8% of the register.
In an annex to the Live Register figures, the CSO also publishes data on participation on activation programmes, which lag a month behind the Live Register figures. So, in May 2016, there were 70,040 people participating on employment, education and training programmes: 13.4% fewer people than the same month last year.
A 25% drop in the numbers of people participating on education and training programmes accounts for 65% of the overall change. Over the same period, participation on employment programmes fell by 7.3%. The numbers of people on JobBridge fell by 33.6% to 3,836 participants. Community Employment, the largest activation programme, had 22,844 participants in May, an annual decrease of 7%. While, the Back to Work Enterprise Allowance, the second largest employment programme increased marginally to 11,550 participants.
On July 5th, the Central Statistics Office published the Monthly Unemployment (MU) figures for June 2016. The MU figures are based on the most recent Quarterly National Household Survey and changes in the Live Register. According to these figures, there were 169,100 people unemployed. The MU Rate was 7.8%, which is unchanged from May and 1.6% lower than June 2015. Looking at these figures from a gender perspective, there were 107,800 men and 61,300 women unemployed. The male unemployment rate was 9.1%, 0.1% lower than May; while the female unemployment rate remained unchanged at 6.2%.
Young people accounted for 16.9% of the Monthly Unemployment figure, but their unemployment rate was significantly higher than the national rate, at 15.3%, up 0.2% on May. People aged 25-74 accounted for 83.1% of the MU figure and their unemployment rate was 7.1%, down 0.1% on May. Please note that the unemployment rate captures people who are unemployed as a percentage of people who are in the Labour Force. The Labour Force includes people who are unemployed or employed, but it does not include people who are, for example, studying, working within the home, or retired.
Virtual Jobs Expo for the IT Sector
Virtual Expos online recruitment event for the IT Sector takes place on 28 July 2016. Virtual Expos are targeted to particular industries. This event is for jobseekers and employers working in the IT and computer sector
for more information on this event please click here
- INOU Pre-Budget Submission 2017 21 Jul 2016 - INOU Pre-Budget Submission to the Department of Social Protection Today the Irish National Organisation of the Unemployed is publi...
- are you looking to develop a career in training and/or community development? 8 Jul 2016 - are you looking to develop a career in training and/or community development? if so, the INOU are offering an exciting programme d...
- Building Futures ART expo at Finglas Festival 2016 a great success 8 Jul 2016 - INOU working in collaboration with the Finglas Festival For a second year running the Finglas Festival kindly facilitated our B...


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THIS ARTICLE IS A STUB. You can learn more about this topic in the related articles below.
Juvenile delinquent, any young person whose conduct is characterized by antisocial behaviour that is beyond parental control and subject to legal action. See delinquency.
Learn More in these related articles:
criminal behaviour, especially that carried out by a juvenile. Depending on the nation of origin, a juvenile becomes an adult anywhere between the ages of 15 to 18, although the age is sometimes lowered for murder and other serious crimes. Delinquency implies conduct that does not conform to the...


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Kenneth Heiman
Kenneth L. Heiman, 64, Scranton, passed away on Saturday, March 2, 2013, at Stormont Vail Health Center.
Kenneth was born on Jan. 7, 1949, in Iola, to Mary (Kipp) and Frank Heiman. He was preceded in death by his parents; brother, Bill Heiman; and sister, Marie Sinclair.
He graduated from Humboldt High in 1967. He worked from 1967 to 1971 as a journeyman carpenter. He also worked for Williams Brothers Pipeline/Magellan Midstream Pipeline from 1971 to 2004, when he retired.
He was united in marriage to Bethenia Lee Dickey on Sept. 25, 1971. He was a member of St. Patrick's Parish in Scranton. He is survived by his loving wife, Bethenia Heiman, Scranton; two sons, Francis Heiman and wife Krystal, Scranton, and Jonathan Heiman and wife Kelly, Carbondale. He is also survived by four grandchildren, Samuel, Eli, Riley, and Quentin; two sisters, Sylvia York, Humboldt, and Betty Heppler, Wichita; three brothers, Robert Heiman, Humboldt, Daniel Heiman, Moran, and Charles Heiman, Piqua, as well as many nieces and nephews.
A memorial mass will be celebrated at 1 p.m. Thursday at St. Patrick's Catholic Church in Scranton.
A private family inurnment will take place at a later date.
Memorial contributions may be made to St. Patrick's Catholic Church-Scranton, 309 S. 6th St., Osage City, KS 66523.
To leave a message for the family online, please visit www.PenwellGabelTopeka.com.
Elpha Springeman
Elpha Izetta Springeman, 91, passed away, Wednesday, July 20, 2016 at the Life Care Center in Burlington. She was born March 10, 1925, on the family farm near Lebo, the daughter of Owen and Maude Robinson Sullivan. Elpha attended Lebo High School and graduated with the Class... [More]
Jack Serene
Melvin "Jack" Shirley Serene, 95, Garnett, died Wednesday, July 20, 2016 at Anderson County Hospital. Funeral service will be at 10:30 a.m. Tuesday at the First United Methodist Church, Garnett, with burial following in the Garnett Cemetery. Jack enjoyed riding around... [More]
Melvin Cole
Melvin Wayne Cole, 78, Iola, died Thursday, July 21, 2016, at Windsor Place in Iola. The family will host a memorial service at 10 a.m. Saturday at the Gas City School in Gas. Inurnment will be at LaHarpe Cemetery at a later date. Memorials are suggested to the... [More]
More Obituaries
Stigma of breastfeeding harms efforts
While dining in an area restaurant, a young mother's infant became fussy with hunger. So she did what comes naturally, draping herself and baby with a large cloth she began to breastfeed her child. Though discreet, the act offended another patron who notified management,... [More]
Project benefits Prairie Spirit Trail
N athan Wiltsey spent more than a year overseeing a project to put signs along the Prairie Spirit Trail, helping users identify animals, plants and birds they may spot along the converted rail corridor between Iola and Ottawa. He completed the project Thursday, part of his... [More]
County launches Next Gen 911 service
Next Generation 911, which moves Allen County's 911 system from the old telephone lines to an Internet-based communications system, will provide a more efficient system for residents to make emergency calls. Crews from the Kansas 911 Administrative Council were at the... [More]
More News
AP Video
Baseball rivalry fuels friendship
Two of the area's top baseball products didn't play high school ball together, but they teamed up this season to do great things at the college level. Levi Ashmore, a shortstop from Iola, and Nathan Whitcomb, a pitcher from Humboldt, began their college careers at... [More]
Iola's Splane, Bycroft lead Legion team past Garnett and into state playoffs
HUMBOLDT — Going into Wednesday's zone championship action, Daylon Splane and Derek Bycroft knew they faced tall tasks. The Indians needed to win two games against Garnett in order to win the American Legion Zone 4 championship and advance to the state playoffs... [More]


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Once a vehicle has been certified for roadworthiness by any member of the European Union, that vehicle can legally be sold in any fellow member country. Such is the case with the Th!nk City, which has just officially passed vehicle inspection in the Netherlands.
1 / 1
11Th!nk gets homologation in the Netherlands, ready to be sold in European Union
- Xavier Navarro
- May 11, 2009
- 11
AddZenn is now legal to drive on Canada's streets
- Lascelles Linton
- Nov 3, 2007
- Add
Less than a week ago, we told you about a CBC report with Zenn complaining Transport Canada was not allowing its low speed vehicle to sold in Canada. The report included a regulatory enforcement engineer that explained they were not being allowed to be sold yet because they were going to change the law. Turns outs the idea of ex post facto is still around, so they got a National Safety Mark.
19Never be late again: Street-legal Dallara Indy Car
- Alex Nunez
- Jul 11, 2007
- 19
Click image to view more shots of the street-legal Indy car
1 / 1
- Volvo shoots for self-drivers by 2021
- Jeep spends $1 billion on factories
- Find Parts & Accessories for your vehicle!
- Obama rolls out new EV plan
- Infiniti dealers ranked best, Tesla worst
- Compare Volvo XC90 and Lincoln MKX