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Item Open Access Impact of remote mutations on metallo-beta-lactamase substrate specificity : implications for the evolution of antibiotic resistance(2005) Ölschläger, Peter; Mayo, Stephen L.; Pleiss, JürgenMetallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP 1 from IMP 6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants, with IMP-1 being the most active. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket and lead to enhanced activity. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion and increased sensitivity to mutations. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.Item Open Access How to find soluble proteins : a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli(2005) Koschorreck, Markus; Fischer, Markus; Barth, Sandra; Pleiss, JürgenBackground: In screening of libraries derived by expression cloning, expression of active proteinsin E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443-448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results: The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion: A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used to identify attractive target genes for expression using protein sequences published in databases. This analysis also directs the design of degenerate, family- specific primers to amplify new members from homologous families or superfamilies with a high probability of soluble alpha/beta hydrolases.Item Open Access "In gel patch electrophoresis" : a new method for environmental DNA purification(2005) Roh, Changhyun; Villatte, Francois; Kim, Byung-Gee; Schmid, Rolf D.Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and PCR amplifications.Item Open Access Development of a diagnostic microarray for the rapid detection of extended spectrum beta-lactamases for the use in clinical microbiology(2005) Grimm, Verena Ulrike; Schmid, Rolf D. (Prof. Dr.)Among the most important types of resistances to be detected are the extended spectrum beta-lactamases (ESBLs). ESBLs are found in many different species of the family Enterobacteriacae. Most ESBLs are mutants of TEM- or SHV-type beta-lactamases. The TEM- and SHV- subtypes are derived from parental sequences (TEM-1, SHV-1) and differ from them by a variable number of amino acid substitutions. These mutations lead to an extended spectrum of activity against newer lactams, especially against 3rd generation cephalosporins. Other derivatives of the classical TEM or SHV enzymes also show an inhibitor resistant TEM (IRT) phenotype conferring resistance to beta-lactamase inhibitors. ESBL producing organisms are difficult to detect in standard phenotypic screening tests, mainly because of their widely varying levels of activity against various cephalosporins. For an improved accuracy confirmatory susceptibility tests have to be performed resulting in a response time of three days until the ESBL phenotype can be identified unequivocally. Since infections with ESBL producing organisms are registered with increased prevalence and are associated with significantly longer hospital stays and higher costs, more accurate tests to detect ESBLs in clinical isolates are necessary. The microarray technology allows the genotypic identification of resistance traits in less than one day of analysis time. Furthermore, the identification of the beta-lactamase variant on a molecular level will define for most ESBL isolates a specific substrate pattern, which can be considered for the determination of the appropriate antibiotic treatment. Additionally, the genotyping of resistances can be used for the reliable surveillance of multiresistant bacteria in wards or hospitals. In the present study a diagnostic microarray was developed for the rapid identification of mutations of the majority of the currently known TEM or SHV beta-lactamase variants, which are related to the ESBL and/or IRT phenotype. The assay enabled the detection and identification of 99 % of the relevant polymorphisms for TEM beta-lactamases and 100 % of the mutations of SHV beta-lactamases. This allows the detection of 96 % of the currently known TEM-variants and 100 % of the known SHV-variants. Consensus primers were developed and used for target amplification covering the majority of the known variant sequences. The sensitivity, reproducibility and identification capability of the developed arrays was determined with a set of reference samples. Furthermore, the TEM-array was validated by testing 72 clinical isolates collected in diverse institutions in Germany, Croatia and Russia. The SHV-array was validated by testing 30 clinical isolates collected in Croatia. The simultaneous detection of an extended spectrum-variant in presence of a narrow spectrum-variant was shown in a model system for TEM up to a ratio of 1:10, as well as in clinical isolates for SHV. Starting from the isolated DNA, the assay could be performed in less than 3.5 hours. The discrimination level, the sensitivity and the reproducibility were enhanced by automation of the hybridization procedure. The development of a marketable diagnostic ESBL microarray based on the presented prototypes and the extension of the developed system towards the detection of other relevant beta-lactamase families is in progress. In conclusion, the diagnostic test developed in this study offers a promising approach for the rapid identification and epidemiologic monitoring of TEM or SHV ESBL and IRT beta-lactamases.Item Open Access Ein GFP-basierter in-vivo-Assay für das Hochdurchsatz-Screening nach Hydrolaseaktivität(2005) Schuster, Sascha; Schmid, Rolf D. (Prof. Dr.)Im Rahmen dieser Arbeit wurde ein effektives intrazelluläres Testsystem zur Identifizierung von Hydrolaseaktivität entwickelt. Die Grundlage dieses in vivo Assays beruht auf dem Auftreten von pH-Änderungen bei Hydrolase-katalysierten Substratspaltungen. Ratiometrisches pHluorin, eine pH-sensitive Mutante von Green Fluorescent Protein (GFP) fungierte als Detektor zur Bestimmung dieser pH-Änderungen. pHluorin besitzt im Gegensatz zu GFP zwei pH-abhängige Peaks bei 395 bzw. 475 nm, deren Maxima sich innerhalb eines pH-Bereichs von 8,0 bis 5,5 reversibel zueinander verschieben. Das aus den beiden Peaks gebildete Fluoreszenzemissionsverhältnis R475/395 kann - gemessen bei einer maximalen Emission bei 508 nm - direkt als Maß für intrazelluläre pH-Änderungen herangezogen werden. Die praktische Anwendung dieses Prinzips wurde zunächst durch Koexpression von pHluorin und der Esterase aus Geobacillus stearothermophilus (BSE) als Modellhydrolase in Escherichia coli (E. coli) Zellen und anschließender Hydrolyse verschiedener Ester untersucht. So zeigten Zellen nach Expression von pHluorin und Esterase, in Abhängigkeit von der Enzymaktivität gegenüber den untersuchten Substraten eine mehr oder weniger starke Änderung des Fluoreszenzemissionsverhältnisses. Daraufhin konnte die Anwendung des Assays auch erfolgreich auf Hydantoinase- sowie Amidase-katalysierte Substrathydrolysen erweitert werden. Außerdem wurden verschiedende Experimente im Mikrotiterplattenformat sowie in der Durchflusszytometrie im Hinblick auf eine erfolgreiche Assayapplikation im Hochdurchsatz-Screening (HTS) von Enzymbibliotheken durchgeführt. Zur Etablierung im Hochdurchsatz-Screening wurde mittels error-prone PCR eine Mutantentenbibliothek der Esterase I aus Pseudomonas fluorescens (PFE I) aufgebaut und mit Hilfe des im Rahmen dieser Arbeit entwickelten Nachweissystems auf erhöhte hydrolytische Enzymaktivität gegenüber g-Butyrolacton (GBL) untersucht. Auf diese Weise wurde eine Mutante mit einer 5,6-fach gesteigerten Umsatzrate von GBL im Vergleich zum PFE I-Wildtyp gefunden. Somit konnte gezeigt werden, daß eine schnelle und zuverlässige Untersuchung von Biokatalysatoren bei Anwendung des pHluorin-Assays zur Durchmusterung von Enzymbibliotheken möglich ist.Item Open Access Biotransformation of ionones by engineered cytochrome P450 BM-3(2005) Urlacher, Vlada B.; Makhsumkhanov, Akhmadjan; Schmid, Rolf D.Wild type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has low activity for the hydroxylation of beta-ionone (>1 min-1). Substitution of phenylalanine by valine at position 87 increased the beta-ionone hydroxylation activity up to 100-fold (115 min-1). For further activity improvement methods of site-directed and random mutagenesis were applied. The R47L Y51F F87V mutant, designed by site-directed mutagenesis and the A74E F87V P386S mutant, obtained after two rounds of error-prone PCR, exhibit an increase in activity up to 300-fold compared to the wild type enzyme. All mutants converted -ionone regioselectively to 4-hydroxy-beta-ionone.Item Open Access Biotransformation von 11-Desoxycortisol mit Schizosaccharomyces pombe und Aspergillus nidulans(2005) Appel, Daniel; Schmid, Rolf D. (Prof. Dr.)In diesem Projekt, welches in Kooperation mit dem Institut für Biochemie der Universität des Saarlandes und der Schering AG Bergkamen durchgeführt wurde, sollte mit Hilfe von rekombinanten Spalthefestämmen, welche die humane Monooxygenase P450 CYP11B1 exprimieren, eine nachhaltigere Bio-Produktion von Hydrocortison entwickelt werden, um das bestehende Verfahren der Hydrocortisonherstellung, mittels des filamentösen Pilz Curvularia lunata zu ersetzen. Um die Optimierung der Hydrocortisonproduktion im Fermenter zu verfolgen, wurde zunächst ein alternatives Nachweissystem für Hydrocortison entwickelt, welches eine schnellere semi-quantitative Bestimmung der Hydrocortisonmenge im Fermenter erlaubt. Der im ersten Teil dieser Arbeit entwickelte Assay basiert auf der Bildung eines Fluorophors bei Inkubation von Hydrocortison mit einer konzentrierten Schwefelsäure/Essigsäure-Mischung. Der so entwickelte Assay erlaubt eine direkte semi-quantitative Bestimmung des Hydrocortisons und dient dazu, die Veränderungen der Hydrocortison-Bildungsraten während der Fermentation zu Überwachen. Für die Bestimmung der exakten Hydrocortison-Mengen wurde eine bereits beschriebene HPLC-Methode verwendet. Die Optimierung der Fermentation wurde hauptsächlich mit den beiden rekombinanten S. pombe Stämmen CAD1 und CAD13 durchgeführt, welche die humane CYP11B1 Monooxygenase mitochondrial exprimieren. Beide Stämme wurden am Institut für Biochemie der Universität des Saarlandes entwickelt. Die ersten rekombinanten Stämme zeigten Umsatzraten von nur 3,1 μM*d-1. Diese Rate konnte für den CAD1-Stamm durch Fermentationsoptimierung auf 70,6 μM*d-1 erhöht werden. Durch Einbringen einer zusätzlichen Kopie des CYP11B1 auf einem autoso-mal replizierenden TOPO-Plasmid (CAD13-Stamm) ließ sich die Expressionsrate des P450 CYP11B1 und somit auch die Umsatzrate von Hydrocortison auf 168 µM*d-1 steigern. Durch die Optimierung der Fermentationsbedingungen bezüglich Umsatzrate und Biomasseproduktion wurde ein Fed-Batch-Prozess entwickelt, welcher eine gesteigerte Raum-Zeit-Ausbeute ermöglichte. Der im ersten Teil entwickelte Hydrocortison-Assay wurde zum Screening eingesetzt, um Stämme zu finden, welche in der Lage sind, eine Hydroxylierung von 11-Desoxycortisol an Position 11 des Steroidgrundgerüsts durchzuführen. Trotz der eigentlich unspezifischen Reaktion des Hydrocortison-Assays bilden nur Steroide mit einer Hydroxyl-Gruppe an Position 11 einen fluoreszierenden Farbstoff aus. Beim Screening einiger am Institut für Technische Biochemie vorhandenen Stämme konnte bei dem filamentösen Pilz Aspergillus nidulans eine hohe 11-Hydroxylierungs-Aktivität bei Verwendung von 11-Desoxycortisol als Substrat festgestellt werden. Die HPLC/MS- und 1H-NMR-Analyse zeigte die Bildung von epi-Hydrocortison (11alpha-Hydroxylierung), eines pharmakologisch unwirksamen Epimeren von Hydrocortison (11beta-Hydroxylierung). Durch die hohe Regioselektivität der gefundenen Reaktion wurde in weiteren Experimenten Progesteron als Substrat verwendet, welches in Säugetieren durch Hydroxylierung in 11alpha-Hydroxyprogesteron überführt wird. Nach Analyse mit 1H-NMR konnte gezeigt werden, dass Progesteron durch Aspergillus nidulans regioselektiv in 11alpha-Hydroxyprogesteron überführt wird. Es konnte in dieser Arbeit ein Steigerung der Hydrocortison-Bildungsrate durch Optimierung von diversen Fermentationsparametern von 3,1 µM*d-1 auf 168 µM*d-1 erreicht werden. Eine weitere Optimierung der verwendeten Stämme ist unbedingt erforderlich, da eine Nebenproduktbildung, wie die eines dehydrogenierten 11-Desoxycortisol-Derivats durch S. pombe, bei der Herstellung pharmakologischer Wirkstoffe nicht tolerierbar ist. Durch das entwickelte alternative Nachweisverfahren von Hydrocortison konnte ein filamentöser Pilz gefunden werden, welcher eine hohe 11-Hydroxylierungs-Aktivität gegenüber 11-Desoxycortisol und Progesteron zeigt, und die gebildeten Produkte durch NMR identifiziert werden. Die vollständige Sequenzierung des Genoms von Aspergillus nidulans erlaubt, die für die gefundene Aktivität verantwortlichen P450-Isoenzyme zu identifizieren und in andere Hefestämme wie Pichia pastoris zu klonieren.Item Open Access Catalytic hydroxylation in biphasic systems using CYP102A1 mutants(2005) Maurer, Steffen Christian; Kühnel, Katja; Kaysser, Leonard A.; Eiben, Sabine; Schmid, Rolf D.; Urlacher, Vlada B.Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP+-dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD+-dependent FDH was used. Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.Item Open Access Screening, cloning and biochemical characterisation of novel esterases from bacillus sp. associated with the marine sponge aplysina aerophoba(2005) Karpushova, Anna Alexandrovna; Brümmer, Franz; Lange, Stefan; Schmid, Rolf D.Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp. associated with the marine sponge Aplysina aerophoba. EstB1 shows low identity (26-44 %)with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity (73-74 %) with the carboxylesterases from B. cereus and B. anthracis. Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+). Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC. A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme. Substrate specificity of the two enzymes towards pnitrophenyl and methyl esters and the respective kinetic parameters Km and Vmax were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30-50°C and 20-35°C, respectively. The pH optima were found to be in the range of 6.5-7.5 and 6.5-8.0, respectively. Both enzymes showed the highest stability in up to 50 % (v/v) DMSO followed by methanol, ethanol and 2-propanol. The influence of high NaCl and KCl concentrations was tested. The inhibition effect of 10-50 mM Zn2+ and 50 mM Mg2+ and Ca2+ ions was observed for both esterases. 1-5 mM PMSF deactivated the enzymes, whereas β-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity.Item Open Access Biocatalysts for the epoxidation and hydroxylation of fatty acids and fatty alcohols(2005) Maurer, Steffen Christian; Schmid, Rolf D.Whereas most products of the chemical industry are based on petrochemical feedstocks, considerable efforts have been made during the past few decades to use renewable resources as industrial raw materials. Besides polysaccharides and sugars, plant oils and animal fats play an important role in such programmes because of their ready availability (present production is >100 million t/a, which could be increased on demand) of which the lion’s share is used for nutrition (~85 million t/a), whereas ~ 15 – 20 million t/a are used for the synthesis of polymers, surfactants, emollients, lubricants, bio-diesel, emulsifiers, etc.). From a chemical point of view, most natural triglycerides offer just two reactive sites, the ester group and the double bonds of unsaturated fatty acids. In fact, the chemistry of fats and oils is largely focused on the ester group which can be hydrolyzed or catalytically reduced, leading to glycerol and fatty acids or fatty alcohols, respectively. Reactions involving the alkyl chain or double bonds of triglycerides, fatty acids, fatty alcohols or their derivatives represent far less than 10% of today’s oleochemistry, with the production of sulfonated fatty alcohols and their derivatives being a major process of this kind. Oxidation reactions at the alkyl or alkenyl chains would be highly desirable as they would lead to oleochemicals with new properties, but the methods available today lack selectivity and require harsh conditions. Notable exceptions are the epoxidation of unsaturated plant oils and the synthesis and use of a few hydroxy fatty acids.