An Expanded Set of Fluorogenic Sulfatase Activity Probes

Fluorogenic probes that are activated by an enzymatic transformation are ideally suited for profiling enzyme activities in biological systems. Here, we describe two fluorogenic enzyme probes, 3‐O‐methylfluorescein‐sulfate and resorufin‐sulfate, that can be used to detect sulfatases in mycobacterial lysates. Both probes were validated with a set of commercial sulfatases and used to reveal species‐specific sulfatase banding patterns in a gel‐resolved assay of mycobacterial lysates. The fluorogenic probes described here are suitable for various assays and provide a starting point for creating new sulfatase probes with improved selectivity for mycobacterial sulfatases.     

Chemically Synthesized 58‐mer LysM Domain Binds Lipochitin Oligosaccharide

Recognition of carbohydrates by proteins is a ubiquitous biochemical process. In legume–rhizobium symbiosis, lipochitin oligosaccharides, also referred to as nodulation (nod) factors, function as primary rhizobial signal molecules to trigger root nodule development. Perception of these signal molecules is receptor mediated, and nod factor receptor 5 (NFR5) from the model legume Lotus japonicus is predicted to contain three LysM domain binding sites. Here we studied the interactions between nod factor and each of the three NFR5 LysM domains, which were chemically synthesized. LysM domain variants (up to 58 amino acids) designed to optimize solubility were chemically assembled by solid‐phase peptide synthesis (SPPS) with microwave heating. Their interaction with nod factors and chitin oligosaccharides was studied by isothermal titration calorimetry and circular dichroism (CD) spectroscopy. LysM2 showed a change in folding upon nod factor binding, thus providing direct evidence that the LysM domain of NFR5 recognizes lipochitin oligosaccharides. These results clearly show that the L. japonicus LysM2 domain binds to the nod factor from Mesorhizobium loti, thereby causing a conformational change in the LysM2 domain. The preferential affinity for nod factors over chitin oligosaccharides was demonstrated by a newly developed glycan microarray. Besides the biological implications, our approach shows that carbohydrate binding to a small protein domain can be detected by CD spectroscopy.     

O6‐Alkylguanine‐DNA Alkyltransferase‐Mediated Repair of O4‐Alkylated 2′‐Deoxyuridines

O⁶‐Alkylguanine‐DNA alkyltransferases (AGTs) are responsible for the removal of O⁶‐alkyl 2′‐deoxyguanosine (dG) and O⁴‐alkyl thymidine (dT) adducts from the genome. Unlike the E. coli OGT (O⁶‐alkylguanine‐DNA‐alkyltransferase) protein, which can repair a range of O⁴‐alkyl dT lesions, human AGT (hAGT) only removes methyl groups poorly. To uncover the influence of the C5 methyl group of dT on AGT repair, oligonucleotides containing O⁴‐alkyl 2′‐deoxyuridines (dU) were prepared. The ability of E. coli AGTs (Ada‐C and OGT), human AGT, and an OGT/hAGT chimera to remove O⁴‐methyl and larger adducts (4‐hydroxybutyl and 7‐hydroxyheptyl) from dU were examined and compared to those relating to the corresponding dT species. The absence of the C5 methyl group resulted in an increase in repair observed for the O⁴‐methyl adducts by hAGT and the chimera. The chimera was proficient at repairing larger adducts at the O⁴ atom of dU. There was no observed correlation between the binding affinities of the AGT homologues to adduct‐containing oligonucleotides and the amounts of repair measured.     

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.     

Probing Ambiguous Base‐Pairs by Genetic Transformation with XNA Templates

The templating potential of anhydrohexitol oligonucleotides bearing ambiguous bases was studied in vivo, by using a selection screen for mosaic heteroduplex plasmids in Escherichia coli. 1,5‐Anhydro‐2,3‐dideoxy‐2‐(5‐nitroindazol‐1‐yl)‐D ‐arabino‐hexitol showed the greatest ambiguity among the three nucleosides tested. At most two successive ambiguous bases could be tolerated on hexitol templates read in bacterial cells. Hexitol nucleosides bearing simplified heterocycles thus stand as promising monomers for generating random DNA sequences in vivo from defined synthetic oligonucleotides.     

Vergleich des Stofftransports von hängenden und akustisch levitierten Wassertropfen in CO2

High‐pressure acoustic levitation devices allows a contact‐free investigation of liquids. Here, acoustically levitated water droplets and pendant water droplets were examined in regards to the mass transfer coefficient in a static CO₂‐atmosphere under elevated pressures. Based on the droplet geometry and the decrease of the droplet volume over time, it was possible to calculate the mass transfer coefficient of water into CO₂ at any given time. Additionally, open questions regarding possible differences between pendant and acoustically levitated water droplets were discussed.     

Modularisierung von Gaswäschern für die CO2‐Entfernung aus Biogas

Modularization has been identified as one of the research fields of the ?50 % idea”?. A development methodology for modules must consider both the economies of scale for investment costs and costs of operation and maintenance. In this paper, the impact of an absorber module, which is offered as discretized diameter scaling, on the total process is investigated at the example of the CO₂ separation from biogas. The simulation shows the effect of this approach to the stripper diameter and the energy demand of the process. The calculations form the basis for applying cost models.