Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications.

α-Amylase from the thermophilic bacterial strain Anoxybacillus vranjensis ST4 (AVA) was cloned into the pMALc5HisEk expression vector and successfully expressed and purified from the Escherichia coli ER2523 host strain. AVA belongs to the GH13_5 subfamily of glycoside hydrolases and has 7 conserved sequence regions (CSRs) distributed in three distinct domains (A, B, C). In addition, there is a starch binding domain (SBD) from the CBM20 family of carbohydrate binding modules (CBMs). AVA is a monomer of 66 kDa that achieves maximum activity at 60-80 °C and is active and stable over a wide pH range (4.0-9.0). AVA retained 50 % of its activity after 31 h of incubation at 60 °C and was resistant to a large number of denaturing agents. It hydrolyzed starch granules very efficiently, releasing maltose, maltotriose and maltopentaose as the main products. The hydrolysis rates of raw corn, wheat, horseradish, and potato starch, at a concentration of 10 %, were 87.8, 85.9, 93.0, and 58 %, respectively, at pH 8.5 over a 3 h period. This study showed that the high level of expression as well as the properties of this highly stable and versatile enzyme show all the prerequisites for successful application in industry.

Effectiveness of game development-based learning for acquiring programming skills in lower secondary education in Croatia.

A European initiative CODING4GIRLS (C4G) promotes the acquisition of programming skills through a game development process with the aim of preparing young learners, especially girls, to enter computer science careers and raising awareness of the relationship between ICT and the real world. Using the C4G game development-based learning methodology, students develop serious games for which they need to learn specific programming concepts. This paper presents the results of a study with a mixed-gender group of both boys and girls (N = 773) carried out with the aim of examining the effectiveness of the C4G development-based learning approach in lower secondary education in Croatia. In-service and pre-service teachers organized learning activities for students based on the C4G learning scenarios, which include the development of games in the programming language Snap! with topics that are interesting for both boys and girls and which involve solving real-world problems. The results showed that students accepted the C4G methodology and were motivated to learn how to program by developing games for solving real-world problems. Teachers and experts consider this approach as a relevant and effective method for achieving learning objectives related to programming, applicable and suitable for lower secondary students (11-15 year olds).

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase.

α-Amylase from Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

Development of an efficient biocatalytic system based on bacterial laccase for the oxidation of selected 1,4-dihydropyridines.

Biocatalytic oxidations mediated by laccases are gaining importance due to their versatility and beneficial environmental effects. In this study, the oxidation of 1,4-dihydropyridines has been performed using three different types of bacterial laccase-based catalysts: purified laccase from Bacillus licheniformis ATCC 9945a (BliLacc), Escherichia coli whole cells expressing this laccase, and bacterial nanocellulose (BNC) supported BliLacc catalysts. The catalysts based on bacterial laccase were compared to the commercially available Trametes versicolor laccase (TvLacc). The oxidation product of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate was obtained within 7-24 h with good yields (70-99%) with all three biocatalysts. The substrate scope was examined with five additional 1,4-dihydropyridines, one of which was oxidized in high yield. Whole-cell biocatalyst was stable when stored for up to 1-month at 4 °C. In addition, evidence has been provided that multicopper oxidase CueO from the E. coli expression host contributed to the oxidation efficiency of the whole-cell biocatalyst. The immobilized whole-cell biocatalyst showed satisfactory activity and retained 37% of its original activity after three biotransformation cycles.

Overcoming hydrolysis of raw corn starch under industrial conditions with Bacillus licheniformis ATCC 9945a α-amylase.

α-Amylase from Bacillus licheniformis ATCC 9945a (BliAmy) was proven to be very efficient in hydrolysis of granular starch below the temperature of gelatinization. By applying two-stage feeding strategy to achieve high-cell-density cultivation of Escherichia coli and extracellular production of BliAmy, total of 250.5 U/mL (i.e. 0.7 g/L) of enzyme was obtained. Thermostability of amylase was exploited to simplify purification. The hydrolysis of concentrated raw starch was optimized using response surface methodology. Regardless of raw starch concentration tested (20, 25, 30 %), BliAmy was very effective, achieving the final hydrolysis degree of 91 % for the hydrolysis of 30 % starch suspension after 24 h. The major A-type crystalline structure and amorphous domains of the starch granule were degraded at the same rates, while amylose-lipid complexes were not degraded. BliAmy presents interesting performances on highly concentrated solid starch and could be of value for starch-consuming industries while response surface methodology (RSM) could be efficiently applied for the optimization of the hydrolysis.

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases.

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L(-1) was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.

Production of raw-starch-digesting α-amylase isoform from Bacillus sp. under solid-state fermentation and biochemical characterization.

α-Amylase production by solid-state fermentation of different Bacillus sp. was studied previously on different fermentation media. However, no study has been reported on the influence of selected media on expression of desired amylase isoforms such as raw-starch-digesting amylase (RSDA). In this paper, the influence of different inexpensive and available agro-resources as solid media (corn, wheat and triticale) on α-amylase isoform induction from three wild-type Bacillus sp., selected among one hundred strains tested, namely 9B, 12B and 24A was investigated. For all three strains, tested amylases were detected in the multiple forms; however, number and intensity of each form differed depending on the solid media used for growth. To determine which isoform from Bacillus sp. 12B was RSDA, the suspected isoform was purified. The optimum pH for the purified α-amylase isoform was 6.0-8.0, while the optimum temperature was 60-90 °C. Isoform was considerably thermostable and Ca(2+)-independent, and actually the only α-amylase active towards raw starch. Purification and characterization of RSDA showed that not all of the solid media tested induced RSDA. From an economic point of view, it might be significant to obtain pure isoenzyme for potential use in the raw-starch hydrolysis, since it was 5 times more efficient in raw corn starch hydrolysis than the crude amylase preparation.

Bacillus amyloliquefaciens laccase--from soil bacteria to recombinant enzyme for wastewater decolorization.

One hundred wild type strains of Bacillus sp. were isolated from industrial and agricultural soil across Serbia and screened for laccase activity. Three strains showed high laccase activity temperature optimum of 65 and 80 °C towards ABTS. A new laccase gene from the strain with highest temperature optimum, namely Bacillus amyloliquefaciens 12B was cloned and expressed in Escherichia coli. Recombinant laccase degraded dye Reactive blue 52 at pH 7.0 and pH 4.0 and at elevated temperature, while fungal laccases was unable to act on this substrate at pH higher than 4.0 and was quickly inactivated at temperatures higher than 45 °C. Degradation of dye was monitored by HPLC-DAD and resulting precipitate was analyzed by FTIR spectroscopy. Single product peak without chromophore was detected in solution, while water insoluble aggregate, presumably dye polymer is formed retaining blue color.

Forensic aspects of postmortem serum carbohydrate-deficient transferrin analysis as a marker of alcohol abuse.

INTRODUCTION: Carbohydrate-deficient transferrin (CDT) has been suggested as one of alcohol abuse indicators having produced good results in forensic medicine for years. OBJECTIVE: The aim of the study was to identify correlation between present methodology of alcohol abuse diagnosis at autopsy (macroscopic and microscopic findings) and CDT examination using the method of isoelectrofocusing (IEF) in polyacrylamide gel electrophoresis (PAGE). We also analyzed if the time interval between the moment of death and blood sample collection influences CDT findings. METHODS: The method used for CDTanalysis was IEF-PAGE. Sera of 49 males and 11 females aged 14-87 years, average age 46.85+/-18.53, were used in this study. Control group consisted of five patients who died after medical treatment that lasted longer than 15 days, and five patients who started Disulfiram therapy in controlled hospital environment. RESULTS: The results obtained in CDT examination in dead bodies' sera showed sensitivity 59% and specificity 71%. A high incidence of falsely positive CDT result was noticed in liver failure and cirrhosis of non-alcoholic origin. CDT analysis is also possible to be done in samples collected postmortem up to 76 hours. CONCLUSION: In forensic medicine, the method of CDT determination is reliable for the diagnosis of alcohol abuse.

Expression and distribution of cellulase, amylase and peptidase isoforms along the midgut of Morimus funereus L. (Coleoptera: Cerambycidae) larvae is dependent on nutrient substrate composition.

The influence of diet composition--two substrates, wheat bran and sawdust--on isoform expression of digestive enzymes (cellulase, amylase and peptidase) in the midgut of Morimus funereus larvae was examined. Their impact on larval development was demonstrated by measuring the increase of larval weight during development and by analysis of digestive enzymes zymographic profiles, where the expression of cellulase isoforms from M. funereus larvae midgut has been examined for the first time in this study. Larvae reared on wheat bran had higher body weight between day 60 and day 100 than larvae reared on sawdust; however, both groups achieved similar body weight after day 110. Wheat bran as substrate induced different cellulase and amylase isoforms. Oak sawdust in substrate acted as inducer of peptidases. The highest cellulase activity and the greatest isoform variability were detected in the midgut extracts of larvae reared on wheat bran. From our results it can be assumed that M. funereus endocellulase, amylase and peptidase are secreted in the anterior midgut, and their concentration gradually decreases towards the hindgut.

Immobilization of cell wall invertase modified with glutaraldehyde for continuous production of invert sugar.

Yeast cell wall invertase (CWI) was modified with dimethyl suberimidate, glutaraldehyde, formaldehyde, and sodium periodate. Retained activity after modification was 45% for CWI modified with formaldehyde, 77% for CWI modified with sodium periodate, 80% for CWI modified with glutaraldehyde, and 115% for CWI modified with dimethyl suberimidate. Chemically modified and native CWIs showed significantly broad pH stability (pH 3-11), whereas after incubations at 50, 60, and 70 °C, CWI modified with glutaraldehyde showed the highest thermostability. Optimum pH for CWI modified with glutaraldehyde was between 4 and 5, whereas optimum temperature was at 60 °C. Comparison to CWI modified with glutaraldehyde after immobilization within alginate beads showed broader pH optimum (4.0-5.5) as well as broader temperature optimum (55-70 °C). Column bed reactor packed with the immobilized CWI modified with glutaraldehyde was successfully used for the 95% inversion of 60% (w/w) sucrose at the flow rate of 3 bed volumes per hour, pH 4.9, and 45 °C. A 1 month productivity of 3844 kg of inverted sugar/kg of the immobilisate was obtained.

Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae.

Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5-9.5) and temperature optimum of 45 degrees C with the K(M) ratio of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (K(I) value of 0.012 mM, Ic(50) value of 0.204 mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5 kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38 kDa, suggesting that the enzyme is a monomer.

Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae.

The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V(max)/K(M) ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1,10-phenanthroline having a K(i) value of 0.12 mM and cysteine with K(i) value of 0.31 mM, while EGTA stimulated LAP activity. Zn(2+), Mg(2+) and Mn(2+) all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases.

Purification and properties of midgut alpha-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae.

Using soluble starch as a substrate five isoforms of alpha-amylase were identified in a crude extract of Morimus funereus larvae. The main alpha-amylase (termed AMF-3) was purified by gel filtration chromatography and anion exchange chromatography to obtain a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its enzymatic purity was confirmed by an in-gel activity assay after SDS-PAGE. The purity of AMF-3 was increased 112-fold with a 15.4% yield. AMF-3 had apparent molecular masses of 33 and 31 kDa when analysed using SDS-PAGE and Superdex 75 FPLC gel filtration chromatography, respectively and a calculated isoelectric point of 3.2. Purified AMF-3 showed maximal activity at pH 5.2 and had an optimum activity temperature of 45 degrees C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 degrees C. AMF-3 exhibited a high affinity towards soluble starch with a K(m) value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl(2), while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by alpha-amylase wheat inhibitor. Using a number of raw starch substrates maximum AMF-3 activity was achieved with horse-radish starch, in contrast to undetectable activity towards potato starch.

Preparation of combined extract of cell wall and cytosol antigens of Candida albicans for immunoblot analysis.

Immunoblot analysis is not in wide use for diagnosis of invasive candidiasis, mostly because the procedure is not standardized and hence not reliable. This work describes a standardized method for C. albicans antigen extract preparation and immunochemical detection. The major improvement of the method is the preparation of combined antigen extract -- consisting of the cell wall and cytosol antigens. The fungal cells and lysis buffer were mixed at a 1:3 ratio and disintegrated by ultrasound for six cycles of one minute each. After centrifugation, cytosol antigens were obtained in the supernatant and cell wall antigens were in the precipitate. Precipitate was dissolved in lysis buffer with 2% sodium dodecyl sulfate (SDS) and boiled at 100 degrees C for 2 min. After centrifugation, the supernatant was combined with the previous one, so the extract of the combined antigens was obtained in the mixture. With those combined antigen extracts and with sera of three different groups of patients, immunoblot analysis showed sensitivity of 90.2%, specificity of 84.4%, and accuracy of 88.0%.

Allozyme polymorphism of Mdh and alpha-Gpdh in Ixodes ricinus populations: comparison of borreliae-infected and uninfected ticks.

Ixodes ricinus Linnaeus (Acari: Ixodidae) ticks are vectors of numerous infectious diseases in humans and animals. The allozyme variability of MDH and alpha-Gpdh was detected by native polyacrylamide gel electrophoresis in I. ricinus natural populations in three localities in Serbia. Four alleles of Mdh locus (MDH 1, MDH 2, MDH 3 and MDH X) and four alleles of alpha-Gpdh locus (VS, S, F and VF) were detected. Interpopulation differences in Mdh and alpha-Gpdh allele frequencies were statistically insignificant. Significant difference in alpha-Gpdh allele frequencies between males and females was recorded in the largest sample only. Differences in allele frequencies, detected between borreliae-infected and uninfected I. ricinus ticks, were close to the level of statistical significance, especially for alpha-Gpdh locus. Clear significant difference appeared in females when sexes were tested separatelly (P = 0.037). It is interesting that genotypes containing rarer alleles (MDH 1 and S) were infected in higher proportion in comparison to other genotypes. Our results point towards a possible role of Mdh and alpha-Gpdh loci in I. ricinus ticks in the determination of energy requirements for host seeking. Sex differences in alpha-Gpdh allele frequencies suggest that selective pressure, concerning efficiency of reserve materials utilisation, points to alpha-Gpdh rather than to Mdh locus.

Detection and quantification of leucyl aminopeptidase after native electrophoresis using leucine-p-nitroanilide.

A general method for detecting leucyl aminopeptidase activity after native polyacrylamide gel electrophoresis (PAGE) in situ is described. The method is based on diazotization of p-nitroaniline, liberated in the polyacrylamide gel by leucyl aminopeptidase action on leucine-p-nitroanilide (LpNA) and subsequent coupling with a chromogen, 1-naphthylamine, until a pink azo dye product at the position of enzyme activity is obtained. A possible use of this technique for leucyl aminopeptidase detection and quantification is indicated. This method was found to be reproducible with the coefficient of variation below 15% for a 32-fold range, while the colored area of enzyme activity was in linear dependence to enzyme activity. Applications of this method with some other aminoacyl-p-nitroanilides and for detection of kidney bean leucyl aminopeptidase isoforms are demonstrated.

Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae.

Exopeptidases of Morimus funereus larvae were partially purified and characterized. Specific leucyl aminopeptidase (LAP) activity was increased eight-fold by gel filtration of the crude midgut extract. The partially purified LAP had a molecular mass greater than 100 kDa with pH optima from 7.0-9.0 and no strict substrate specificity. M. funereus LAP preferentially hydrolyzed p-nitroanilides with hydrophobic amino acids in the active site, with a K(m) for leucine-p-nitroanilide of 0.21 mM. Zymogram analysis of an electropherogram obtained by native polyacrylamide gel electrophoresis revealed four enzymatically active proteinases using leucine-p-nitroanilide and methionine-p-nitroanilide as substrates and two enzymatically active proteinases using lysine-p-nitroanilide as a substrate. Although the optimal temperature of LAP activity was 40 degrees C, the enzyme was active over a broad temperature range from 2 to 60 degrees C. Among a number of inhibitors tested, heavy metals and 1,10-phenanthroline completely inhibited the enzyme, while methanol, ethanol and EGTA stimulated somewhat LAP activity.