Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation

AbstractWe report here oxidation of propylbenzene and 3-chlorostyrene by wild-type cytochrome P450 BM-3 with high turnover (479 nmol 1-phenyl-1-propanol/min/nmol P450 and 300 nmol 3-chlorostyrene oxide/min/nmol P450). Furthermore, the residue size at position 87 of P450 BM-3 was found to play critical roles in determining stereoselectivity in oxidation of propylbenzene and 3-chlorostyrene. Replacement of Phe87 with Val, Ala and Gly resulted in decreases in optical purity of produced (R)-(+)-1-phenyl-1-propanol from 90.0 to 37.4, 26.0 and −15.6% e.e., respectively, and in increases in those of produced (R)-(+)-3-chlorostyrene oxide from −61.0 to −38.0, 67.0 and 94.6% e.e., respectively

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system

<p>Abstract</p> <p>Background</p> <p>(+)-Nootkatone (<b>4</b>) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (<b>1</b>) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (<b>4</b>) from (+)-valencene (<b>1</b>) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (<b>1</b>) at allylic C2-position to produce (+)-nootkatone (<b>4</b>) via <it>cis</it>- (<b>2</b>) or <it>trans</it>-nootkatol (<b>3</b>). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from <it>Pseudomonas putida </it>in <it>Escherichia coli</it>.</p> <p>Results</p> <p>Addressing the whole-cell system, the cytochrome CYP109B1 from <it>Bacillus subtilis </it>was found to catalyze the oxidation of (+)-valencene (<b>1</b>) yielding nootkatol (<b>2 </b>and <b>3</b>) and (+)-nootkatone (<b>4</b>). However, when the <it>in vivo </it>biooxidation of (+)-valencene (<b>1</b>) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents – isooctane, <it>n</it>-octane, dodecane or hexadecane – were set up, resulting in accumulation of nootkatol (<b>2 </b>and <b>3</b>) and (+)-nootkatone (<b>4</b>) of up to 97% of the total product. The best productivity of 120 mg l^-1of desired products was achieved within 8 h in the system comprising 10% dodecane.</p> <p>Conclusion</p> <p>This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (<b>4</b>), as it is safe and can easily be controlled and scaled up.</p

Neurotoxin detection in food using disposable AChE-biosensors

The extensive use of pesticides to protect agricultural crops necessitates reliable tools for the detection of residues in food and water, thus ensuring environmental protection and consumer safety. Neuroinhibitors such as organophosphates and carbamates are widely used in agriculture because of their high insecticidal activity and their rapid mineralisation in the environment. Since they do not only inhibit insect acetylcholinesterase (AChE) but also interfere with neural transmission in other organism, including humans, they represent a potential hazard for human health and environmental food chains, and thus require continuous assessment. Recent reports on organophosphate contamination in baby food have initiated the reassessment of permitted residual concentrations of these compounds in view of the US Food Quality Protection Act. Here we present a rapid detection method for neurotoxins in food, which can increase the number of tested foodstuff and thus improve the consumer safety. We developed a highly sensitive and rapid food analysis biosensor based on disposable screen-printed thickfilm electrodes. Matrix problems were solved by using isooctane as extraction solvent. The performance of this amperometric test was checked by three different food matrixes, namely orange juice, apple and peach baby food. The detection limit was comparable to chemical standard methods and met the requirements for monitoring the maximum residue levels in baby food set by the EU

Ein mehrkanaliges Biosensormesssystem zur Überwachung der Nitrifikation in Abwasserreinigungsanlagen

Bei der biologischen Abwasserreinigung treten häufig Störungen der Nitrifikationsstufe (biochemische Oxidation von Ammonium über Nitrit zu Nitrat) auf, die durch Hemmstoffe sowie durch Stossbelastungen hoher Stickstofffrachten (N-BSB) verursacht werden. Dadurch gelangen erhöhte Mengen an sauerstoffzehrenden reduzierten Stickstoffverbindungen in die Oberflächengewässer. Dies ist besonders kritisch bei Vorflutern mit einem bereits niedrigem Sauerstoffgehalt, da durch diese reduzierten Stickstoffverbindungen die Konzentration an gelöstem Sauerstoff in Folge mikrobieller Oxidation stark absinken kann und somit die Biocönose im Gewässer nachhaltig gestört wird. Da es sich bei Nitrit und Ammoniak zudem um starke Fischgifte handelt, ist die Elimination dieser Stickstoffverbindungen aus dem Abwasser nicht zuletzt auch gesetzlich vorgeschrieben. Zur Erfassung von Störungen der Nitrifikationsstufe wurde deswegen in den vergangenen Jahren am ISWA ein Nitrifikanten-Einzelbiosensor entwickelt. Dabei wird über den Sauerstoffverbrauch des Immobilisates die bakterielle Stoffwechselaktivität überwacht, wobei dies ein Maß für das Vorhandensein von Hemmstoffen bzw. Nitrifikationssubstraten in einer Probe ist. Der Vorteil dieses Systems ist vor allem darin zu sehen, dass die damit durchgeführten Messungen sowohl schnell als auch reproduzierbar durchführbar sind. So ist mit diesem Geräteprototyp über die Durchführung von ca. 10 – 15 Einzelmessungen verschiedener Probenverdünnungen die Quantifizierung der Hemmwirkung von einer Probe pro Messtag möglich. Schlussfolgerung und Ausblick Die Untersuchungen zur Bestimmung der nitrifikationshemmenden Wirkung von Standardhemmstoffen, Abwasserproben und des N-BSB ergaben, dass die neu entwickelten Nitrifikanten-Biosensoren schnell und mit hoher Signalstabilität auf sich ändernde Hemmstoff- und Substratkonzentrationen reagieren und somit für die Klärwerksüberwachung prinzipiell einsetzbar sind

The Laccase Engineering Database: a classification and analysis system for laccases and related multicopper oxidases

Laccases and their homologues form the protein superfamily of multicopper oxidases (MCO). They catalyze the oxidation of many, particularly phenolic substances, and, besides playing an important role in many cellular activities, are of interest in biotechnological applications. The Laccase Engineering Database (LccED, http://www.lcced.uni-stuttgart.de) was designed to serve as a tool for a systematic sequence-based classification and analysis of the diverse multicopper oxidase protein family. More than 2200 proteins were classified into 11 superfamilies and 56 homologous families. For each family, the LccED provides multiple sequence alignments, phylogenetic trees and family-specific HMM profiles. The integration of structures for 14 different proteins allows a comprehensive comparison of sequences and structures to derive biochemical properties. Among the families, the distribution of the proteins regarding different kingdoms was investigated. The database was applied to perform a comprehensive analysis by MCO- and laccase-specific patterns