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The very high radiation fluence expected at LHC (Large Hadron Collider) at CERN will induce serious changes in the electrical properties of the silicon detectors that will be used in the internal layers of the experiments (ATLAS, CMS, LHCb). To understand the influence of the fabrication technology in the radiation-induced degradation, silicon detectors were fabricated simultaneously with the three different possible technologies, P-in-N, N-in-N, and N-in-P, on standard and oxygenated float-zone silicon wafers. The diodes were irradiated with protons to fluences up to [10.sup.15] [cm.sup.-2]. The measurements of the electrical characteristics, current-voltage and capacitance-voltage, are presented for the detectors manufactured with the three technologies. In terms of alpha factor (leakage current) the three technologies behave similarly. In terms of beta factor (effective doping concentration) N-in-P devices show the best performances. Index Terms--Detector technology, N-in-N, N-in-P, P-in-N, radiation hardness, silicon radiation detectors.

Abstract: Aims: The purpose of the study was to demonstrate feasibility of the Conserved Domains Database (CDD) for identification of novel biocatalysts with desirable properties from a class of well‐characterized biocatalysts. Methods and Results: The thermostable ADH from Sulfolobus solfataricus with a broad substrate range was applied as a template for the search for novel thermostable ADHs via CDD. From the resulting hits, a putative ADH gene from the thermophilic organism Chloroflexus aurantiacus was cloned and expressed in Escherichia coli. The resulting enzyme was purified and characterized. With a temperature activity optimum of 70°C and a broad substrate spectrum especially for diketones, a versatile new biocatalyst was obtained. Conclusions: Database‐based mining in CDD is a suitable approach to obtain novel biocatalysts with desirable properties. Thereby, the available diversity of similar but not equal enzymes within this class can be increased. Significance and Impact of the Study: For industrial applications, there is a demand for larger diversity of similar well‐characterized enzymes in order to test them for a given process (biodiversity screening). For fundamental science, the comparison of enzymes with similar function but different sequence can provide insight into structure function relationships or the evolution of enzymes. This study gives a good example on how this demand can be efficiently met. [ABSTRACT FROM AUTHOR]

Alcohol dehydrogenases (ADH) are important tools for generating chiral α-hydroxyketones. Previously, only the ADH of Thauera aromatica was known to convert cyclic α-diketones with appropriate preference. Here, we extend the spectrum of suitable enzymes by three alcohol dehydrogenases from Citrifermentans bemidjiense (CibADH), Deferrisoma camini (DecADH), and Thauera phenylacetica (ThpADH). Of these, DecADH is characterized by very high thermostability; CibADH and ThpADH convert α-halogenated cyclohexanones with increased activity. Otherwise, however, the substrate spectrum of all four ADHs is highly conserved. Structural considerations led to the conclusion that conversion of diketones requires not only the expansion of the active site into a large binding pocket, but also the circumferential modification of almost all amino acid residues that form the first shell of the binding pocket. The constellation appears to be overall highly specific for the relative positioning of the carbonyl functions and the size of the C-ring., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Enzyme-mediated processes have proven to be a valuable and sustainable alternative to traditional chemical methods. In this regard, the use of multi-enzymatic systems enables the realization of complex synthetic schemes, while also introducing a number of additional advantages, including the conversion of reversible reactions into irreversible processes, the partial or complete elimination of product inhibition problems, and the minimization of undesirable by-products. In addition, the immobilization of biocatalysts on magnetic supports allows for easy reusability and streamlines the downstream process. Herein we have developed a cascade system for cladribine synthesis based on the sequential action of two magnetic biocatalysts. For that purpose, purine 2'-deoxyribosyltransferase from Leishmania mexicana ( Lm PDT) and Escherichia coli hypoxanthine phosphoribosyltransferase ( Ec HPRT) were immobilized onto Ni 2+ -prechelated magnetic microspheres (MagReSyn ® NTA). Among the resulting derivatives, M Lm PDT3 (activity: 11,935 IU/g support , 63% retained activity, operational conditions: 40 °C and pH 5-7) and M Ec HPRT3 (12,840 IU/g support , 45% retained activity, operational conditions: pH 5-8 and 40-60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the M Lm PDT3/M Ec HPRT3 system was biochemically characterized and successfully applied to the one-pot synthesis of cladribine under various conditions. This methodology not only displayed a 1.67-fold improvement in cladribine synthesis (compared to M Lm PDT3), but it also implied a practically complete transformation of the undesired by-product into a high-added-value product (90% conversion of Hyp into IMP). Finally, M Lm PDT3/M Ec HPRT3 was reused for 16 cycles, which displayed a 75% retained activity.

The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 μM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages., Competing Interests: The authors have declared that no competing interests exist.

The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α-diketone 1,2-cyclohexanedione to the corresponding α-hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α-substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.

AICA (5'-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Thermal alkaline pretreatment (TAP) of waste activated sludge (WAS) before anaerobic digestion (AD) was reviewed. Focus of the review was on impact of TAP process parameters on biomethane yield (BY) and kinetics of AD and downstream dewatering. With higher initial biodegradability of untreated WAS, effect of TAP on BY decreases. Depending on initial biodegradability, BY increase of 22-97% is expected. Treatment temperatures below 100 °C showed to be as effective as temperatures higher than 100 °C in terms of BY increase. Alkali dosage and resulting initial pH have a significant effect on BY increase and showed to have an optimum range of 40-60 mg NaOH per g total solids (TS) of sludge. It is advised that alkali is dosed based on solids content in WAS and monitored by pH. Treatment time of 1.5-5 h is sufficient for an effective low temperature TAP (T < 100 °C), with longer treatment times showing no positive impact on BY increase. Load of sludge liquor with organics and nutrients increases with more intensive TAP conditions. Despite kinetic enhancement of hydrolysis step in AD, more research is needed to clarify if TAP improves kinetics of entire AD process which determines required digester volume. Impact of TAP on dewaterability of digestate is ambiguous and needs more investigation using standardized methods, also with regards to potential effects on polymer demand. Findings of experimental studies were reflected against available data from commercialized TAP process of Pondus®, throughout review. Finally, important process design parameters of TAP such as input TS and point of alkali dosage are discussed and recommendations for future research are presented., (Copyright © 2021. Published by Elsevier Ltd.)

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cladribine triphosphate is the active compound of the anti-cancer and multiple sclerosis drug Mavenclad (cladribine). Biosynthesis of such non-natural deoxyribonucleotides is challenging but important in order to study the pharmaceutical modes of action. In this study, we developed a novel one-pot enzyme cascade for the biosynthesis of cladribine triphosphate, starting with the nucleobase 2Cl-adenine and the generic co-substrate phosphoribosyl pyrophosphate. The cascade is comprised of the three enzymes, namely, adenine phosphoribosyltransferase (APT), polyphosphate kinase (PPK), and ribonucleotide reductase (RNR). APT catalyzes the binding of the nucleobase to the ribose moiety, followed by two consecutive phosphorylation reactions by PPK. The formed nucleoside triphosphate is reduced to the final product 2Cl-deoxyadenonsine triphosphate (cladribine triphosphate) by the RNR. The cascade is feasible, showing comparative product concentrations and yields to existing enzyme cascades for nucleotide biosynthesis. While this study is limited to the biosynthesis of cladribine triphosphate, the design of the cascade offers the potential to extend its application to other important deoxyribonucleotides.

Thermal alkaline pretreatment (TAP) of waste activate sludge (WAS) was carried out in pilot-scale over a year to investigate its seasonal effects on anaerobic digestion and its impact on dewaterability, sludge liquor quality and formation of soluble refractory COD (sCOD ref ). Temperature of TAP was set at 65-70 °C and pH was increased by initial dosing of sodium hydroxide [NaOH] (50% w/w, 1-2.5 mL/L sludge) as alkali agent following 2-2.5 h reaction time. Pilot digesters were fed with primary sludge (PS) and hydrolyzed WAS (HWAS) and compared to a reference digester fed with PS and untreated WAS. Biogas yield increase due to TAP of WAS showed a sinusoidal trend throughout the year with maximum in summer (+42%), minimum in winter (+3%) and average of +20%, indicating a strong seasonal effect on TAP efficiency. Ammonium [NH 4 + -N], orthophosphate [PO 4 3- -P] and sulphate [SO 4 2- ] in sludge liquor increased by 34.6%, 17.0% and 21.6% with TAP, respectively. Centrifugation tests showed no significant difference in dewaterability of both digestates with respect to total solids of sludge cake. Normalized capillary suction time of digestate increased due to TAP, indicating a lower capability for water release. Furthermore, detected sCOD ref after batch aerobic biodegradation tests showed an increase of 30.3% with TAP. Hence, implementation of TAP of WAS in full-scale will potentially lead to an increase of 0.8-1.1 mg/L of sCOD ref in effluent of six wastewater treatment plants (WWTP) in Berlin. In conclusion, TAP of WAS leads to increase in biogas production with a slighter negative impact on effluent COD quality than high-temperature thermal hydrolysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bce-like systems mediate resistance against antimicrobial peptides in Firmicutes bacteria. Lactobacillus casei BL23 encodes an "orphan" ABC transporter that, based on homology to BceAB-like systems, was proposed to contribute to antimicrobial peptide resistance. A mutant lacking the permease subunit was tested for sensitivity against a collection of peptides derived from bacteria, fungi, insects, and humans. Our results show that the transporter specifically conferred resistance against insect-derived cysteine-stabilized αβ defensins, and it was therefore renamed DerAB for de fensin r esistance ABC transporter. Surprisingly, cells lacking DerAB showed a marked increase in resistance against the lantibiotic nisin. This could be explained by significantly increased expression of the antimicrobial peptide resistance determinants regulated by the Bce-like systems PsdRSAB (formerly module 09) and ApsRSAB (formerly module 12). Bacterial two-hybrid studies in Escherichia coli showed that DerB could interact with proteins of the sensory complex in the Psd resistance system. We therefore propose that interaction of DerAB with this complex in the cell creates signaling interference and reduces the cell's potential to mount an effective nisin resistance response. In the absence of DerB, this negative interference is relieved, leading to the observed hyperactivation of the Psd module and thus increased resistance to nisin. Our results unravel the function of a previously uncharacterized Bce-like orphan resistance transporter with pleiotropic biological effects on the cell. IMPORTANCE Antimicrobial peptides (AMPs) play an important role in suppressing the growth of microorganisms. They can be produced by bacteria themselves-to inhibit competitors-but are also widely distributed in higher eukaryotes, including insects and mammals, where they form an important component of innate immunity. In low-GC-content Gram-positive bacteria, BceAB-like transporters play a crucial role in AMP resistance but have so far been primarily associated with interbacterial competition. Here, we show that the orphan transporter DerAB from the lactic acid bacterium Lactobacillus casei is crucial for high-level resistance against insect-derived AMPs. It therefore represents an important mechanism for interkingdom defense. Furthermore, our results support a signaling interference from DerAB on the PsdRSAB module that might prevent the activation of a full nisin response. The Bce modules from L. casei BL23 illustrate a biological paradox in which the intrinsic nisin detoxification potential only arises in the absence of a defensin-specific ABC transporter., (Copyright © 2020 American Society for Microbiology.)

Thermal hydrolysis (TH) increases the anaerobic biodegradability of waste activated sludge (WAS), but also refractory organic and nutrient return load to a wastewater treatment plant (WWTP). This could lead to an increase in effluent chemical oxygen demand (COD) of the WWTP. The aim of this study was to investigate the trade-off between increase in biogas production through TH and anaerobic digestion and increase in refractory COD in dewatered sludge liquors at different temperatures of TH in lab-scale. WAS was thermally hydrolyzed in temperature range of 130-170 °C for 30 min to determine its biomethane potential (BMP). After BMP test, sludge was dewatered and sludge liquor was aerated in Zahn-Wellens test to determine its non-biodegradable soluble COD known as refractory soluble COD (sCOD ref ). With increasing temperature in the range of 130-170 °C, BMP of WAS increased by 17-27%, while sCOD ref increased by 3.9-8.4%. Dewaterability was also enhanced through relative increase in cake solids by 12-30%. A conversion factor was defined through mass balance to relate sCOD ref to volatile solids of raw WAS. Based on the conversion factor, expected increase in effluent CODs of six WWTPs in Berlin were predicted to be in the range of 2-15 mg/L after implementation of TH at different temperatures. It was concluded that with a slight decrease in temperature, formation of sCOD ref could be significantly reduced, while still benefiting from a substantial increase in biogas production and dewaterability improvement., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bacterial biohybrid microswimmers aim at exploiting the inherent motion capabilities of bacteria (carriers) to transport objects (cargoes) at the microscale. One of the most desired properties of microswimmers is their ability to communicate with their immediate environment by processing the information and producing a useful response. Indeed, bacteria are naturally equipped with such communication skills. Hereby, two-component systems (TCSs) represent the key signal transducing machinery and enable bacteria to sense and respond to a variety of stimuli. We engineered a natural microswimmer based on the Gram-positive model bacterium Bacillus subtilis for the development of biohybrids with sensing abilities. B. subtilis naturally adhered to silica particles, giving rise to different motile biohybrids systems with variable ratios of carrier(s)-to-cargo(es). Genetically engineered TCS pathways allowed us to couple the binding to the inert particles with signaling the presence of antibiotics in their surroundings. Activation of the antibiotic-induced TCSs resulted in fluorescent bacterial carriers as a response readout. We demonstrate that the genetically engineered TCS-mediated signaling capabilities of B. subtilis allow for the custom design of bacterial hybrid microswimmers able to sense and signal the presence of target molecules in the environment. The generally recognized as safe (GRAS) status of B. subtilis makes it a promising candidate for human-related applications of these novel biohybrids.

Ribonucleotide reductases (RNR) are essential enzymes for all known life forms. Their current taxonomic distribution suggests extensive horizontal gene transfer e.g., by processes involving viruses. To improve our understanding of the underlying processes, we characterized a monomeric class II RNR (NrdJm) enzyme from a Thermus virus, a subclass not present in any sequenced Thermus spp. genome. Phylogenetic analysis revealed a distant origin of the nrdJm gene with the most closely related sequences found in mesophiles or moderate thermophiles from the Firmicutes phylum. GC-content, codon usage and the ratio of coding to non-coding substitutions (dN/dS) suggest extensive adaptation of the gene in the virus in terms of nucleotide composition and amino acid sequence. The NrdJm enzyme is a monomeric B 12 -dependent RNR with nucleoside triphosphate specificity. It exhibits a temperature optimum at 60-70 °C, which is in the range of the growth optimum of Thermus spp. Experiments in combination with the Thermus thermophilus thioredoxin system show that the enzyme is able to retrieve electrons from the host NADPH pool via host thioredoxin and thioredoxin reductases. This is different from other characterized viral RNRs such as T4 phage RNR, where a viral thioredoxin is present. We hence show that the monomeric class II RNR, present in Thermus viruses, was likely transferred from an organism phylogenetically distant from the one they were isolated from, and adapted to the new host in genetic signature and amino acids sequence., Competing Interests: The authors declare there are no competing interests.

Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 Å crystal structure. We also present evidence for an Mn III Mn IV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex., Competing Interests: IR, DL, MH, MC, VJ, CL, MS, NM, IB, GB, AH, DL, BS No competing interests declared, (© 2017, Rozman Grinberg et al.)

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Molecular docking of substrates is more challenging compared to inhibitors as the reaction mechanism has to be considered. This becomes more pronounced for zinc-dependent enzymes since the coordination state of the catalytic zinc ion is of greater importance. In order to develop a predictive substrate docking protocol, we have performed molecular docking studies of diketone substrates using the catalytic state of carbonyl reductase 2 from Candida parapsilosis (CPCR2). Different docking protocols using two docking methods (AutoDock Vina and AutoDock4.2) with two different sets of atomic charges (AM1-BCC and HF-RESP) for catalytic zinc environment and substrates as well as two sets of vdW parameters for zinc ion were examined. We have selected the catalytic binding pose of each substrate by applying mechanism based distance criteria. To compare the performance of the docking protocols, the correlation plots for the binding energies of these catalytic poses were obtained against experimental Vmax values of the 11 diketone substrates for CPCR2. The best correlation of 0.73 was achieved with AutoDock4.2 while treating catalytic zinc ion in optimized non-bonded (NBopt) state with +1.01 charge on the zinc ion, compared to 0.36 in non-bonded (+2.00 charge on the zinc ion) state. These results indicate the importance of catalytic constraints and charge parameterization of catalytic zinc environment for the prediction of substrate activity in zinc-dependent enzymes by molecular docking. The developed predictive docking protocol described here is in principle generally applicable for the efficient in silico substrate spectra characterization of zinc-dependent ADH.

Zinc-dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc-dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27-fold increase in specific activity for 4'-acetamidoacetophenone, and the double mutant CPCR2 B16-(C57A, L119M), with a 45-fold improvement in the kcat ⋅KM (-1) value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recently, a new type of wastewater treatment system became the focus of scientific research: the mesh filter activated sludge system. It is a modification of the membrane bioreactor (MBR), in which a membrane filtration process serves for sludge separation. The main difference is that a mesh filter is used instead of the membrane. The effluent is not of the same excellent quality as with membrane bioreactors due to the much lager pore sizes of the mesh. Nevertheless, it still resembles the quality of currently used standard treatment system, the activated sludge process. The new process shows high future potential as an alternative where a small footprint of these plants is required (3 times lower footprint than conventional activated sludge systems because of neglecting the secondary clarifier and reducing the biological stage). However, so far only limited information on this innovative process is available. In this study, the effect of different pore sizes and different mesh module configurations on the effluent quality was investigated varying the parameters cross-flow velocity (CFV) and flux rate. Furthermore the long-term filtration performance was studied in a pilot reactor system and results were compared to the full-scale conventional activated sludge process established at the same site. The results demonstrate that the configuration of the filter module has little impact on effluent quality and is only of importance with regard to engineering aspects. Most important for a successful operation are the hydrodynamic conditions within the filter module. The statement "the higher the pore size the higher the effluent turbidity" was verified. Excellent effluent quality with suspended solids between 5 and 15 mg L(-1) and high biological elimination rates (chemical oxygen demand (COD) 90-95%, biological oxygen demand (BOD5) 94-98%, total nitrogen (TN) 70-80%, and ammonium nitrogen (NH(4)-N) 95-99%) were achieved and also compared to those of conventional activated sludge systems. Regarding the air requirement for module aeration, which is the main cost factor in MBR technology, an astonishing optimization could be achieved. During the long-term filtration experiments only 4 N m(3)/m(3) was necessary to keep a stable filtration process for more than 3 weeks (MBR 20-50 N m(3)/m(3)).

Spirochaeta thermophila is a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on the S. thermophila genome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from the S. thermophila DSM 6192 genome, all putative β-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, β-glucanase, β-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected in Spirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes of Cytophaga fermentans and Mahella australiensis, both of which are phylogenetically very distant from S. thermophila and noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.

*At the end of the cell cycle, the plant cell wall is deposited within a membrane compartment referred to as the cell plate. Little is known about the biogenesis of this transient membrane compartment. *We have positionally cloned and characterized a novel Arabidopsis gene, CLUB, identified by mutation. *CLUB/AtTRS130 encodes a putative TRAPPII tethering factor. club mutants are seedling-lethal and have a canonical cytokinesis-defective phenotype, characterized by the appearance of bi- or multinucleate cells with cell wall stubs, gaps and floating walls. Confocal microscopy showed that in club mutants, KNOLLE-positive vesicles formed and accumulated at the cell equator throughout cytokinesis, but failed to assemble into a cell plate. Similarly, electron micrographs showed large vesicles loosely connected as patchy, incomplete cell plates in club root tips. Neither the formation of KNOLLE-positive vesicles nor the delivery of these vesicles to the cell equator appeared to be perturbed in club mutants. Thus, the primary defect in club mutants appears to be an impairment in cell plate assembly. *As a putative tethering factor required for cell plate biogenesis, CLUB/AtTRS130 helps to define the identity of this membrane compartment and comprises an important handle on the regulation of cell plate assembly.

Simultaneous nitrification-denitrification (SND) allows biological nitrogen removal in a single reactor without separation of the two processes in time or space but requires adapted control strategies (anoxic/aerobic conditions). In this study, the formation of gaseous nitric oxide (NO(G)) and nitrogen dioxide (NO(2G)) was monitored for SND in relation to the oxidation-reduction potential (ORP) and nitrogen removal in a lab batch reactor and a pilot membrane bio-reactor (MBR). In addition hospital wastewater (COD/N(tot)>6:1) was treated on site for 1 year. The highest total nitrogen removal rates of max 90% were reached at 220-240mV ORP (given as E(h)) with corresponding maximal NO(G) emissions rates of 0.9microgg(-1)VSSh(-1). The maximal emission rates of NO(2G) (0.2microgg(-1)VSSh(-1)) were reached at the same ORP level and the NO(2G) emissions correlated to the nitrite accumulation in the activated sludge up to 5mgl(-1)NO(2L)-N. It was shown that this correlation was due to biological production and not due to pH-dependent chemical conversion. Therefore, NO(2G) can be used as additional control loop for ORP-controlled SND systems to avoid the inhibition of denitrification and high nitrite concentrations in the plant effluent.