A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.     

Control of inverter-based micro-grids

The predicted growth of small-scale non-50/60 Hz power sources and the desire to be able to support loads independently of the public electricity grid requires the development inverter-based micro-grids. Power electronic interfaces have very different characteristics to conventional electrical machines and, therefore, different operation, control and protection schemes are required. Attention also needs to be given to the dominance of single-phase harmonically distorting loads in some networks and control schemes put in place that maintain voltage quality. A control scheme that exploits the controllability of inverters to operate a micro-grid and provide good power quality is examined and compared with both traditional power systems and with control of dc/dc power converters. The limitations of communication and control bandwidth are discussed. Experimental results are used to illustrate the performance that can be achieved with various combinations of linear and non-linear, three-phase and single-phase loads.     

Design of compensation network with variable inductance for wide variation of coupling coefficient in inductive power transfer

In this paper, a design of the compensation network with a variable inductor is proposed, considering a wide variation of the coupling coefficient in the inductive power transfer (IPT). The characteristics of the proposed compensation network are analyzed with respect to the variable antenna coupling conditions. The realization of the zero voltage switching (ZVS) and the reactive power demand are discussed through the theoretical deduction and numerical simulation. Influence of the variable inductor on the compensation network characteristics is examined in detail, and the control algorithm for achieving the coupling independent constant output voltage is proposed. Detailed steps to design a prototype fulfilling all performance requirements are given. Verification of the proposed method was carried out on the experimental model. Recorded output voltage variation is within 5% over the wide coupling range, implying a quite well characteristic of the coupling independent voltage output. Experimental results match well with the calculations, validating the rightness of the proposed variable inductor tuning method.     

Miscibility in blends of sulfonylated poly(2,6-dimethyl-1,4-phenylene oxide) and poly(p-bromostyrene-co-o-bromostyrene)

Miscibility in blends of random copolymers of p-bromostyrene (pBrSt) and o-bromostyrene (oBrSt) [P(pBrSt-co-oBrSt)] with partially sulfonylated poly(2,6-dimethyl-1,4-phenylene oxide) (SPPO) have been studied by differential scanning calorimetry (DSC). For an SPPO of given degree of sulfonylation, a miscibility window in terms of the isomeric composition of the brominated copolymer was seen; the location and width of the window was a function of sulfonylation. In general, copolymers with a higher content of pBrSt exhibit miscibility with SPPOs with higher degrees of sulfonylation. Upon annealing to temperatures of 280° and 320°C, only small changes in the miscibility regime were observed. The miscibility behavior was analyzed on the basis of the mean-field theory in terms of the individual segmental interaction parameters. © 1994 John Wiley & Sons, Inc.     

Structural analysis of Aleksinac oil shale kerogen by high-resolution solid-state C n.m.r. spectroscopy

A sample of kerogen from Aleksinac oil shale was examined by high-resolution solid-state ¹³C n.m.r. spectroscopy. The presence and relative proportions of kerogen structural units were estimated using a combination of NQS and T_1C methods with a peak-synthesis technique applied to the ¹³C CP—MAS spectrum. Relaxation parameters from these experiments were used to estimate differences in relative ‘mobility’ of various structural units. The kerogen was found to be highly aliphatic and to contain 79% long-chain aliphatic plus alicyclic structures, as well as 9% aromatic structures. These findings are in good agreement with the characterization of the same kerogen from its oxidation products.     

Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense

The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0–11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80 % of the initial activity after 1 h incubation at 45 °C, and 65 % of the initial activity after 24 h incubation in 30 % dimethyl sulfoxide.     

Epidemiology of end‐stage renal disease and hemodialysis treatment in Serbia at the turn of the millennium

The study presents the epidemiological features of patients treated with renal replacement therapy (RRT) in Serbia from 1997 to 2009 and compares the results of hemodialysis treatment in 1999 and 2009. Epidemiological data were obtained from the National Registry of RRT patients and data on hemodialysis treatment from special surveys conducted in 1999 and 2009. Within the period 1997–2009 the incidence of patients on RRT increased from 108 to 179 per million population (pmp), prevalence rose from 435 to 699 pmp, while mortality rate fell from 20.7% to 16.7%. The frequency of patients with glomerulonephritis decreased, while that of patients with diabetes and hypertensive nephropathy increased. In late 2009 there were 5208 patients receiving RRT in Serbia. Within the examined period new hemodialysis and reverse osmosis equipment were purchased, high‐flux dialyzers with synthetic membranes were increasingly used and the number of patients receiving hemodiafiltration increased to 17.6%. Kt/V greater than 1.2 was recorded in 16% of the patients in 1999 but 52% in 2009. Options for correction of anemia and mineral disorders have also improved. The percentage of patients with HbsAg (13.8% vs. 4.8%) as well as anti‐hepatitis C virus antibodies positive patients (23.2% vs. 12.7%) was significantly lower in 2009 than in 1999. Both the incidence and prevalence of RRT patients in Serbia are rising continuously, while the mortality rate is falling. More favorable conditions for dialysis treatment have brought about significant improvement in the results over the last 10 years.     

Actively recharged single photon counting avalanche photodiode integrated in an industrial CMOS process

An actively recharged single photon counting avalanche photodiode (SPAD) is integrated in a conventional CMOS process. A fast recharge through a low impedance path leads to a dead time lower than 10 ns. This outstanding feature allows one to work with pulse repetition rate up to 100 MHz in time correlated single photon counting based experiments. Biased 2.5 V above its breakdown voltage, the 30 μm² sensitive area photodiode has a maximum detection probability of about 20% at λ=440 nm and up to 5% in the visible part of the spectrum. At this bias condition, the dark count rate is as low as 60 Hz at room temperature, making a cooling of the microsystem unnecessary. The AR-SPAD exhibits no afterpulsing phenomenon revealing the maturity of the CMOS process used. The timing resolution of the AR-SPAD is less than 50 ps. For applications where the photons can be focused on the detector with an objective, the AR-SPAD is highly competitive with commercially available single photon counter. Furthermore, CMOS integration opens the way to arrays fabrication as well as co-integration of additional functions to develop smart optical sensors.     

Small Capacity Mobile Dryers for Drying Biological Materials

Creation of the universal technology and solution for drying of biological materials is almost impossible since various materials are concerned and various parts of plants are dried (root, stem, flower, bloom, seed, and fruit). Three original designs of devices for drying of biological materials have been developed. First, there are two types of mobile solar dryers for drying various biological materials by direct and combined use of solar energy, a design with the direct use of solar energy, solar air collector, and photovoltage system, and a design with combined use of solar energy, solar air collector, and wind motor. The procedure for dimensioning some basic elements of the dryer has been presented, and the analysis is given showing how the parameters of materials being dried, the collector construction, and climatic, and weather conditions influence the efficiency of the collector active surface. Having considered the advantages of radiation drying as well as the reasons for its limited application, a design concept of a mobile radiation dryer for drying biological materials at atmospheric pressure is given. Furthermore, optimization of operation modes and construction parameters of the proposed mobile dryer design is provided, with respect to the analysis showing how radiation source and characteristics of material being dried influence the drying rate and dried material quality.