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Bremen, Germany

Igressa A.,Medical Center Cologne Merheim | Defosse J.,Witten/Herdecke University | Disque C.,Molzym GmbH and Co. KG | Wappler F.,Witten/Herdecke University | Sakka S.G.,Witten/Herdecke University
International Journal of Infectious Diseases | Year: 2014

The early detection and treatment of sepsis in patients is essential for a positive outcome. Microbiological analysis of blood cultures, as the gold standard for diagnosis, is rather slow. However, more rapid methods like PCR have become available recently and are being evaluated clinically. We present data from the monitoring of a patient with sepsis who was on anti-infective treatment. The patient was positive for Streptococcus pneumoniae by broad-range PCR and sequence analysis in a blood sample and resected lung tissue specimen, the latter embedded in paraffin, while blood culture diagnostics remained negative. © 2014 The Authors.

Li Z.,RWTH Aachen | Li Z.,Jacobs University Bremen | Roccatano D.,Jacobs University Bremen | Lorenz M.,Molzym GmbH and Co. KG | And 2 more authors.
Journal of Biotechnology | Year: 2014

A subtilisin E variant (M4) showing high activity and resistance towards guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS) was previously identified after three rounds of directed evolution [Li et al., ChemBioChem 2012, 13(5), 691-699.]. In this report, 10 additional positions, identified during directed subtilisin E evolution, were saturated on the previously reported SeSaM1-5 variant (S62/A153/G166/I205). Screening confirmed that chaotolerant variants included amino acid substitutions either in the active site, or the substrate binding pocket. Two variants, M5 (S62I/A153V/G166S/T224A/T240S) and M6 (S62I/A153V/G166S/I205V/N218S/T224A) were finally generated to maximize activity and stability in the presence of GdmCl or SDS. The inactivation concentration (IC50) of M6 using Suc-AAPF-pNA as substrate was significantly increased compared to M4 in the presence of GdmCl (IC50 (M4): 2.7M; IC50 (M6): 4.6M) and SDS (IC50 (M4): 1.5%; IC50 (M6): 4.0%). The half-life in 5M GdmCl was also significantly improved for M6 compared to M4 (t 1/2 (M4): 2min; t 1/2 (M6): 15min). M5 retained resistance towards GdmCl or SDS as in M4. The activity of M5 towards a complex protein substrate (Azocasein) was increased by ~1.5 fold compared to M4 and M6. Circular dichroism (CD) analysis for subtilisin E wild type (WT) and three variants (M4, M5 and M6) indicated that secondary structures of all variants including wild type at 1-2M GdmCl (except M4) were not significantly perturbed, with unfolding occurring for WT and all three variants above 3M GdmCl. In SDS, the secondary structures of WT and all three variants remained intact at concentrations of 0.5 to 2.0% (w/v) SDS. Results suggest that subtilisin E inactivation occurred most likely due to inhibitory effect, since a general unfolding of the enzyme was not observed through circular dichroism. Such inhibition could be avoided by limiting the access of GdmCl and SDS to the active site and/or to residues involved in substrate binding. © 2013 Elsevier B.V.

Li Z.,RWTH Aachen | Li Z.,Jacobs University Bremen | Roccatano D.,Jacobs University Bremen | Lorenz M.,Molzym GmbH and Co. KG | Schwaneberg U.,RWTH Aachen
ChemBioChem | Year: 2012

Proteases have niche applications in diagnostic kits that use cell lysis and thereby require high resistance towards chaotropic salts and detergents, such as guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS). Subtilisin E, a well-studied serine protease, was selected to be re-engineered by directed evolution into a "chaophilic" protease that would be resistance to GdmCl and SDS, for application in diagnostic kits. In three iterative rounds of directed evolution, variant SeSaM1-5 (S62I/A153V/G166S/I205V) was generated, with improved activity (330%) and increased half life in 1M GdmCl (<2 min to 4.7 h) or in 0.5% SDS (<2 min to 2.7 h). Saturation mutagenesis at each site in the wild-type subtilisin E revealed that positions 62 and 166 were mainly responsible for increased activity and stability. A double mutant, M2 (S62I/G166M), generated by combination of the best single mutations showed significantly improved kinetic constants; in 2M GdmCl the K m value decreased (29-fold) from 7.31 to 0.25 mM, and the k cat values increased (fourfold) from 15 to 61 s -1. The catalytic efficiency, k cat/K m, improved dramatically (GdmCl: 247 mM -1s -1 (118-fold); SDS, 179 mM -1s -1 (13-fold)). In addition, the SeSaM1-5 variant showed higher stability in 2.0% SDS when compared to the wild-type (t 1/2 54.8 min (>27-fold)). Finally, molecular dynamics simulations of the wild-type subtilisin E showed that Gdm + ions could directly interact with active site residues, thereby probably limiting access of the substrate to the catalytic centre. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Koch T.,IWT - Foundation Institute of Materials Engineering | Rabenstein A.,Amtliche Material Prufungsanstalt Bremen | Muhl H.,Molzym GmbH and Co. KG
Tribologie und Schmierungstechnik | Year: 2012

The spreading and proliferation of microbes is a common incident in water miscible metal-working fluids (MWF). Bacteria and fungi degrade the ingredients of the MWF according to their bioavailability and biodegra-dabilify, leading to a progressive imbalance in the chemical equilibrium and composition of the MWF. In consequence to the depletion of certain components a decrease in the technical properties of the fluid is obtained, e.g. decrease in corrosion protection, loss in the lubricating and cutting performance, increased tool wear and an elevated health risk. To allow for a quick counteraction, reliable and rapid monitoring applications are needed. This paper presents new techniques for monitoring the microbial and chemical properties of water miscible MWF tested and developed at the IWT-Bremen in collaboration with partners from industry and science. Results of well known analysis techniques such as ATP-monitoring will be compared to cell counts like CFU, new molecular-biological applications and the MALDI-TOF. First findings of gassensor measurements to characterize the technical and chemical state of a MWF will be introduced as well.

Shehzad A.,RWTH Aachen | Panneerselvam S.,German Electron Synchrotron | Linow M.,Molzym GmbH and Co. KG | Bocola M.,RWTH Aachen | And 4 more authors.
Chemical Communications | Year: 2013

Solved crystal structures of P450 BM3 variants in complex with styrene provide on the molecular level a first explanation of how a positively charged surface residue inverts the enantiopreference of styrene epoxidation. The obtained insights into productive and non-productive styrene binding modes deepened our understanding of enantioselective epoxidation with P450 BM3. © The Royal Society of Chemistry 2013.

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