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Bacterial cell appendix formation supports cell-cell interaction, cell adhesion and cell movement. Additionally, in bioelectrochemical systems (BES), cell appendages have been shown to participate in extracellular electron transfer. In this work, the cell appendix formation of Clostridium acetobutylicum in biofilms of a BES are imaged and compared with conventional biofilms. Under all observed conditions, the cells possess filamentous appendages with a higher number and density in the BES. Differences in the amount of extracellular polymeric substance in the biofilms of the electrodes lead to the conclusion that the cathode can be used as electron donor and the anode as electron acceptor by C. acetobutylicum. When using conductive atomic force microscopy, a current response of about 15 nA is found for the cell appendages from the BES. This is the first report of conductivity for clostridial cell appendices and represents the basis for further studies on their role for biofilm formation and electron transfer.
NVS123 is a poorly water-soluble protease 56 inhibitor in clinical development. Data from in vitro hepatocyte studies suggested that NVS123 is mainly metabolized by CYP3A4. As a consequence of limited solubility, NVS123 therapeutic plasma exposures could not be achieved even with high doses and optimized formulations. One approach to overcome NVS123 developability issues was to increase plasma exposure by coadministrating it with an inhibitor of CYP3A4 such as ritonavir. A clinical boost effect was predicted by using physiologically based pharmacokinetic (PBPK) modeling. However, initial boost predictions lacked sufficient confidence because a key parameter, fraction of drug metabolized by CYP3A4 (ƒₘCYP3A4), could not be estimated with accuracy on account of disconnects between in vitro and in vivo preclinical data. To accurately estimate ƒₘCYP3A4 in human, an in vivo boost effect study was conducted using CYP3A4-humanized mouse model which showed a 33- to 56-fold exposure boost effect. Using a top-down approach, human ƒₘCYP3A4 for NVS123 was estimated to be very high and included in the human PBPK modeling to support subsequent clinical study design. The combined use of the in vivo boost study in CYP3A4-humanized mouse model mice along with PBPK modeling accurately predicted the clinical outcome and identified a significant NVS123 exposure boost (∼42-fold increase) with ritonavir.
A microfluidic chip integrating amperometric enzyme sensors for the detection of glucose, glutamate and glutamine in cell-culture fermentation processes has been developed. The enzymes glucose oxidase, glutamate oxidase and glutaminase were immobilized by means of cross-linking with glutaraldehyde on platinum thin-film electrodes integrated within a microfluidic channel. The biosensor chip was coupled to a flow-injection analysis system for electrochemical characterization of the sensors. The sensors have been characterized in terms of sensitivity, linear working range and detection limit. The sensitivity evaluated from the respective peak areas was 1.47, 3.68 and 0.28 μAs/mM for the glucose, glutamate and glutamine sensor, respectively. The calibration curves were linear up to a concentration of 20 mM glucose and glutamine and up to 10 mM for glutamate. The lower detection limit amounted to be 0.05 mM for the glucose and glutamate sensor, respectively, and 0.1 mM for the glutamine sensor. Experiments in cell-culture medium have demonstrated a good correlation between the glutamate, glutamine and glucose concentrations measured with the chip-based biosensors in a differential-mode and the commercially available instrumentation. The obtained results demonstrate the feasibility of the realized microfluidic biosensor chip for monitoring of bioprocesses.
Characterizing volcanic ash elements from the 2015 eruptions of bromo and raung volcanoes, Indonesia
(2020)
The volcanic eruptions of Mt. Bromo and Mt. Raung in East Java, Indonesia, in 2015 perturbed volcanic materials and affected surface-layer air quality at surrounding locations. During the episodes, the volcanic ash from the eruptions influenced visibility, traffic accidents, flight schedules, and human health. In this research, the volcanic ash particles were collected and characterized by relying on the detail of physical observation. We performed an assessment of the volcanic ash elements to characterize the volcanic ash using two different methods which are aqua regia extracts followed by MP-AES and XRF laboratory test of bulk samples. The analysis results showed that the volcanic ash was mixed of many materials, such as Al, Si, P, K, Ca, Ti, V, Cr, Mn, Fe, Ni, and others. Fe, Si, Ca, and Al were found as the major elements, while the others were the trace elements Ba, Cr, Cu, Mn, P, Mn, Ni, Zn, Sb, Sr, and V with the minor concentrations. XRF analyses showed that Fe dominated the elements of the volcanic ash. The XRF analysis showed that Fe was at 35.40% in Bromo and 43.00% in Raung of the detected elements in bulk material. The results of aqua regia extracts analyzed by MP-AES were 1.80% and 1.70% of Fe element for Bromo and Raung volcanoes, respectively.
Characterization and evaluation of lignocellulosic biomass 130 hydrolysates for ABE fermentation
(2016)
Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC–MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec- and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed.
An acetoin biosensor based on a capacitive electrolyte–insulator–semiconductor (EIS) structure modified with the enzyme acetoin reductase, also known as butane-2,3-diol dehydrogenase (Bacillus clausii DSM 8716ᵀ), is applied for acetoin detection in beer, red wine, and fermentation broth samples for the first time. The EIS sensor consists of an Al/p-Si/SiO₂/Ta₂O₅ layer structure with immobilized acetoin reductase on top of the Ta₂O₅ transducer layer by means of crosslinking via glutaraldehyde. The unmodified and enzyme-modified sensors are electrochemically characterized by means of leakage current, capacitance–voltage, and constant capacitance methods, respectively.
C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli
(2010)
Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477.
This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in KM = 0.23 ± 0.01 mM and kcat = 167.5 ± 3.6 s⁻¹. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.
For several thousand years, biotechnology and its associated technical processes have had a great impact on the development of mankind. Based on empirical methods, in particular for the production of foodstuffs and daily commodities, these disciplines have become one of the most innovative future issues. Due to the increasing detailed understanding of cellular processes, production strains can now be optimized. In combination with modern bioprocesses, a variety of bulk and fine chemicals as well as pharmaceuticals can be produced efficiently. In this article, some of the current trends in biotechnology are discussed.
The terms bioeconomy and biorefineries are used for a variety of processes and developments. This short introduction is intended to provide a delimitation and clarification of the terminology as well as a classification of current biorefinery concepts. The basic process diagrams of the most important biorefinery types are shown.
Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T. The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0–12.0 and temperature 20–80 °C, optimally at pH 9.0–9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H2O2 for 1 h while stimulated at 1% (v/v) H2O2. Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future.
Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822ᵀ (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H₂O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂O₂, suggest it has potential for biotechnological applications.
Bio-feedstocks
(2011)
Lignin is a promising renewable biopolymer being investigated worldwide as an environmentally benign substitute of fossil-based aromatic compounds, e.g. for the use as an excipient with antioxidant and antimicrobial properties in drug delivery or even as active compound. For its successful implementation into process streams, a quick, easy, and reliable method is needed for its molecular weight determination. Here we present a method using 1H spectra of benchtop as well as conventional NMR systems in combination with multivariate data analysis, to determine lignin’s molecular weight (Mw and Mn) and polydispersity index (PDI). A set of 36 organosolv lignin samples (from Miscanthus x giganteus, Paulownia tomentosa and Silphium perfoliatum) was used for the calibration and cross validation, and 17 samples were used as external validation set. Validation errors between 5.6% and 12.9% were achieved for all parameters on all NMR devices (43, 60, 500 and 600 MHz). Surprisingly, no significant difference in the performance of the benchtop and high-field devices was found. This facilitates the application of this method for determining lignin’s molecular weight in an industrial environment because of the low maintenance expenditure, small footprint, ruggedness, and low cost of permanent magnet benchtop NMR systems.
Bacillus pumilus reveals a remarkably high resistance to hydrogen peroxide provoked oxidative stress
(2014)
Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.
The Monte Carlo code FLUKA is used to simulate the production of a number of positron emitting radionuclides, ¹⁸F, ¹³N, ⁹⁴Tc, ⁴⁴Sc, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁵²Mn, ⁶¹Cu and ⁵⁵Co, on a small medical cyclotron with a proton beam energy of 13 MeV. Experimental data collected at the TR13 cyclotron at TRIUMF agree within a factor of 0.6 ± 0.4 with the directly simulated data, except for the production of ⁵⁵Co, where the simulation underestimates the experiment by a factor of 3.4 ± 0.4. The experimental data also agree within a factor of 0.8 ± 0.6 with the convolution of simulated proton fluence and cross sections from literature. Overall, this confirms the applicability of FLUKA to simulate radionuclide production at 13 MeV proton beam energy.
Air- and water-stable phenyl complexes with nitridotechnetium(V) cores can be prepared by straightforward procedures. [TcNPh2(PPh3)2] is formed by the reaction of [TcNCl2(PPh3)2] with PhLi. The analogous N-heterocyclic carbene (NHC) compound [TcNPh2(HLPh)2], where HLPh is 1,3,4-triphenyl-1,2,4-triazol-5-ylidene, is available from (NBu4)[TcNCl4] and HLPh or its methoxo-protected form. The latter compound allows the comparison of different Tc–C bonds within one compound. Surprisingly, the Tc chemistry with such NHCs does not resemble that of corresponding Re complexes, where CH activation and orthometalation dominate.