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Shan-Chi Hsieh, Jung-Hao Wang, Yu-Chen Lai, Ching-Yeuh Su, Kung-Ta Lee
Medium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A from Marinobacter aquaeolei VT8 (CYP153A M...
February 15, 2018: Applied and Environmental Microbiology
Mukul G Jain, Daniela Lalli, Jan Stanek, Chandrakala Gowda, Satya Prakash, Tom S Schwarzer, Tobias Schubeis, Kathrin Castiglione, Loren B Andreas, P K Madhu, Guido Pintacuda, Vipin Agarwal
Very fast magic-angle spinning (MAS > 80 kHz) NMR combined with high-field magnets has enabled the acquisition of proton-detected spectra in fully protonated solid samples with sufficient resolution and sensitivity. One of the primary challenges in structure determination of protein is observing long-range1 H-1 H contacts. Here we use band-selective spin-lock pulses to obtain selective1 H-1 H contacts (e.g., HN -HN ) on the order of 5-6 Å in fully protonated proteins at 111 kHz MAS. This approach is a major advancement in structural characterization of proteins given that magnetization can be selectively transferred between protons that are 5-6 Å apart despite the presence of other protons at shorter distance...
June 1, 2017: Journal of Physical Chemistry Letters
Tom Sebastian Schwarzer, Maria Hermann, Swati Krishnan, Friedrich C Simmel, Kathrin Castiglione
The outer membrane of gram-negative bacteria constitutes an important hurdle for the transport of hydrophobic molecules into the cell. Mass flux is often facilitated by various outer membrane proteins. These proteins are of biotechnological importance because they could help to improve the performance of whole-cell biocatalysts or be incorporated into artificial cell-like systems. The characterization and understanding of their transport properties greatly benefits from the possibility to express and purify these proteins...
April 2017: Protein Expression and Purification
Marvin Kadisch, Mattijs K Julsing, Manfred Schrewe, Nico Jehmlich, Benjamin Scheer, Martin von Bergen, Andreas Schmid, Bruno Bühler
It is a common misconception in whole-cell biocatalysis to refer to an enzyme as the biocatalyst, thereby neglecting the structural and metabolic framework provided by the cell. Here, the low whole-cell biocatalyst stability, that is, the stability of specific biocatalyst activity, in a process for the terminal oxyfunctionalization of renewable fatty acid methyl esters was investigated. This reaction, which is difficult to achieve by chemical means, is catalyzed by Escherichia coli featuring the monooxygenase system AlkBGT and the uptake facilitator AlkL from Pseudomonas putida GPo1...
April 2017: Biotechnology and Bioengineering
Youri M van Nuland, Gerrit Eggink, Ruud A Weusthuis
UNLABELLED: The enzyme system AlkBGT from Pseudomonas putida GPo1 can efficiently ω-functionalize fatty acid methyl esters. Outer membrane protein AlkL boosts this ω-functionalization. In this report, it is shown that whole cells of Escherichia coli expressing the AlkBGT system can also ω-oxidize ethyl nonanoate (NAEE). Coexpression of AlkBGT and AlkL resulted in 1.7-fold-higher ω-oxidation activity on NAEE. With this strain, initial activity on NAEE was 70 U/g (dry weight) of cells (gcdw), 67% of the initial activity on methyl nonanoate...
July 1, 2016: Applied and Environmental Microbiology
Toby P Call, M Kalim Akhtar, Frank Baganz, Chris Grant
BACKGROUND: In recent years, there have been intensive efforts to develop synthetic microbial platforms for the production, biosensing and bio-remediation of fossil fuel constituents such as alkanes. Building predictable engineered systems for these applications will require the ability to tightly control and modulate the rate of import of alkanes into the host cell. The native components responsible for the import of alkanes within these systems have yet to be elucidated. To shed further insights on this, we used the AlkBGT alkane monooxygenase complex from Pseudomonas putida GPo1 as a reporter system for assessing alkane import in Escherichia coli...
2016: Journal of Biological Engineering
Nadine Ladkau, Miriam Assmann, Manfred Schrewe, Mattijs K Julsing, Andreas Schmid, Bruno Bühler
The expansion of microbial substrate and product scopes will be an important brick promoting future bioeconomy. In this study, an orthogonal pathway running in parallel to native metabolism and converting renewable dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to 12-aminododecanoic acid methyl ester (ADAME), a building block for the high-performance polymer Nylon 12, was engineered in Escherichia coli and optimized regarding substrate uptake, substrate requirements, host strain choice, flux, and product yield...
July 2016: Metabolic Engineering
Chris Grant, Dawid Deszcz, Yu-Chia Wei, Rubéns Julio Martínez-Torres, Phattaraporn Morris, Thomas Folliard, Rakesh Sreenivasan, John Ward, Paul Dalby, John M Woodley, Frank Baganz
Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C7-C16 n-alkanes. Without alkL expression, native E.coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C7-C16 n-alkanes and in the activation of a whole-cell alkane biosensor by C10 and C11 alkanes...
July 28, 2014: Scientific Reports
Manfred Schrewe, Mattijs K Julsing, Kerstin Lange, Eik Czarnotta, Andreas Schmid, Bruno Bühler
The oxyfunctionalization of unactivated C−H bonds can selectively and efficiently be catalyzed by oxygenase-containing whole-cell biocatalysts. Recombinant Escherichia coli W3110 containing the alkane monooxygenase AlkBGT and the outer membrane protein AlkL from Pseudomonas putida GPo1 have been shown to efficiently catalyze the terminal oxyfunctionalization of renewable fatty acid methyl esters yielding bifunctional products of interest for polymer synthesis. In this study, AlkBGTL-containing E. coli W3110 is shown to catalyze the multistep conversion of dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to the acid, exhibiting Michaelis-Menten-type kinetics for each reaction step...
September 2014: Biotechnology and Bioengineering
Daniel Scheps, Sumire Honda Malca, Sven M Richter, Karoline Marisch, Bettina M Nestl, Bernhard Hauer
A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated...
November 2013: Microbial Biotechnology
Sjef Cornelissen, Mattijs K Julsing, Jan Volmer, Ole Riechert, Andreas Schmid, Bruno Bühler
Living microbial cells are considered to be the catalyst of choice for selective terpene functionalization. However, such processes often suffer from side product formation and poor substrate mass transfer into cells. For the hydroxylation of (S)-limonene to (S)-perillyl alcohol by Pseudomonas putida KT2440 (pGEc47ΔB)(pCom8-PFR1500), containing the cytochrome P450 monooxygenase CYP153A6, the side products perillyl aldehyde and perillic acid constituted up to 26% of the total amount of oxidized terpenes. In this study, it is shown that the reaction rate is substrate-limited in the two-liquid phase system used and that host intrinsic dehydrogenases and not CYP153A6 are responsible for the formation of the undesired side products...
May 2013: Biotechnology and Bioengineering
Mattijs K Julsing, Manfred Schrewe, Sjef Cornelissen, Inna Hermann, Andreas Schmid, Bruno Bühler
The outer membrane of microbial cells forms an effective barrier for hydrophobic compounds, potentially causing an uptake limitation for hydrophobic substrates. Low bioconversion activities (1.9 U g(cdw)(-1)) have been observed for the ω-oxyfunctionalization of dodecanoic acid methyl ester by recombinant Escherichia coli containing the alkane monooxygenase AlkBGT of Pseudomonas putida GPo1. Using fatty acid methyl ester oxygenation as the model reaction, this study investigated strategies to improve bacterial uptake of hydrophobic substrates...
August 2012: Applied and Environmental Microbiology
J B van Beilen, G Eggink, H Enequist, R Bos, B Witholt
The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes...
November 1992: Molecular Microbiology
H Matras, W Jesch, G Kletter, H P Dinges
A new clot-suturing Technique (using high-concentration fibrinogen solutions) for water-tight closure of the dura is reported. Six dogs underwent laminectomy of the thoracic spine with medial longitudinal incision in the chordal dura. After the dural split had been sealed with natural tissue adhesive and closure of the wound in layers, the animals were sacrificed at intervals of 1 to 21 days postoperatively and the chordal segments involved were removed and histologically examined. Early fibrinolysis of the clot was prevented by adding a natural proteinase inhibitor and factor XIII concentrate to the clotting substances...
June 9, 1978: Wiener Klinische Wochenschrift
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