Molzym GmbH and Co. KG

Bremen, Germany

Molzym GmbH and Co. KG

Bremen, Germany
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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.

Bwanga F.,Makerere University | Bwanga F.,MBN Clinical Laboratories | Disque C.,Molzym GmbH and Co. KG | Lorenz M.G.,Molzym GmbH and Co. KG | And 5 more authors.
BMC Infectious Diseases | Year: 2015

Blood stream tuberculosis (TB), caused by (MTB) is common among HIV-positive patients, turning rapidly fatal unless detected and treated promptly. Blood culture is currently the standard test for the detection of MTB in whole blood but results take weeks; patients deteriorate markedly and often die before a diagnosis of blood stream TB is made. Rapid molecular tests on whole blood, with potential for same day diagnosis of blood stream TB usually show low sensitivity due to the problem of insufficient MTB DNA template when extraction is performed directly on low blood volumes. This study assessed the influence of blood volume on the sensitivity of a HyBeacon PCR assay-the FluoroType ® MTB (Hain Lifescience, Nehren, Germany) on direct detection of MTB in whole blood. Methods: Prospective recruitment of HIV-positive patients with clinical suspicion of blood stream TB but not on anti-TB or HIV drug treatment was done. Venous blood samples were collected and DNA extracted using the MolYsis (Molzym, Bremen, Germany) methods; for study A, from duplicate 1 ml ( 42 patients) and for study B ( 31 patients) from 9 ml EDTA blood samples. The FluoroType ® MTB PCR assay targeting an IS 6110 sequence was performed and results compared with blood culture. Results: The diagnostic sensitivity and specificity of the FluoroType ® MTB PCR in study A was 33% and 97%, respectively. Corresponding values in study B were 71% and 96%, respectively. In both studies, one case each of blood culture-negative blood stream TB was detected with the FluoroType ® MTB PCR assay. The median time to positivity of blood culture was 20.1 (range 12 - 32 ) for study A and 19.9 days (range 15 - 30 ) for study B. Conclusion: Larger blood volumes ( 9 ml) improved and gave acceptable sensitivity of direct PCR diagnosis of blood stream TB. © Bwanga et al.

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.

Orszag P.,Hannover Medical School | Disque C.,Molzym GmbH and Co. KG | Keim S.,Molzym GmbH and Co. KG | Lorenz M.G.,Molzym GmbH and Co. KG | And 5 more authors.
Journal of Clinical Microbiology | Year: 2014

The rRNA gene PCR and sequencing test, SepsiTest, was compared with blood culture (BC) regarding the diagnosis of pathogens in 160 blood samples drawn from 28 patients during extracorporeal membrane oxygenation. With 45% of positive samples, SepsiTest was 13 to 75 h faster than BC. SepsiTest indicated bacteremias in 25% of patients who were BC negative.Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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.

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.

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.

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