AHyCo: a Web-Based Adaptive Hypermedia Courseware System

Adaptive hypermedia courseware systems resolve the problem of users’ disorientation in hyperspace through the adaptive navigation and presentation support. We describe the AHyCo (Adaptive Hypermedia Courseware) - an adaptive Web-based educational system for creation and reuse of adaptive courseware with emphasis on adaptive navigation support and lessons sequencing. The proposed model consists of the domain model, the student model, and the adaptive model. The system is composed of two environments: the authoring environment and the learning environment

Hidroliza sirovog skroba - novi pravci

Improved depolymerization of grain starch to glucose is crucial for reduction of energy use in the bioconversion of glucose to industrially important compounds. In fuel ethanol production, these steps use 10-20% of the energy content of the fuel ethanol. The need to minimize energy use can be met by replacing high-temperature, liquid-phase, enzymatic digestion with low temperature, solid-phase, enzymatic digestion. Also called cold hydrolysis, the approach is a step toward a 'green' method for the production of fuel ethanol, and because of this there has been increased recent world interest in this approach.Poboljšanje procesa depolimerizacije zrna skroba do glukoze ključni je faktor u smanjenju energetske potrošnje u biokonverziji glukoze u industrijski važna jedinjenja. Kod procesa proizvodnje bioetanola ovi koraci obuhvataju 10-20% ukupne energetske potrošnje. Potreba za smanjenjem energetske potrošnje mogla bi da se zadovolji zamenom procesa enzimske hidrolize na visokoj temperaturi u tečnoj fazi, procesom enzimske hidrolize na nižim temperaturama i na čvrstoj fazi. Ovaj proces još nazvan i hladna hidroliza predstavlja korak napred u zelenoj tehnologiji za proizvodnju bioetanola, te poslednjih godina u svetu raste interes za razvojem ovakvog pristupa

Hidroliza sirovog skroba - novi pravci

Improved depolymerization of grain starch to glucose is crucial for reduction of energy use in the bioconversion of glucose to industrially important compounds. In fuel ethanol production, these steps use 10-20% of the energy content of the fuel ethanol. The need to minimize energy use can be met by replacing high-temperature, liquid-phase, enzymatic digestion with low temperature, solid-phase, enzymatic digestion. Also called cold hydrolysis, the approach is a step toward a 'green' method for the production of fuel ethanol, and because of this there has been increased recent world interest in this approach.Poboljšanje procesa depolimerizacije zrna skroba do glukoze ključni je faktor u smanjenju energetske potrošnje u biokonverziji glukoze u industrijski važna jedinjenja. Kod procesa proizvodnje bioetanola ovi koraci obuhvataju 10-20% ukupne energetske potrošnje. Potreba za smanjenjem energetske potrošnje mogla bi da se zadovolji zamenom procesa enzimske hidrolize na visokoj temperaturi u tečnoj fazi, procesom enzimske hidrolize na nižim temperaturama i na čvrstoj fazi. Ovaj proces još nazvan i hladna hidroliza predstavlja korak napred u zelenoj tehnologiji za proizvodnju bioetanola, te poslednjih godina u svetu raste interes za razvojem ovakvog pristupa

From raw starch degrading α-amylase to transglycosylase by single point mutation

Understanding the structural peculiarities and properties of both, raw starch as a substrate and raw starch digesting amylases (RSDA) as an enzymes is needed for efficient application in food and other industries. Although knowledge of these structures and properties has increased significantly in recent years, the main requirement defining whether RSDA will be efficient in raw starch hydrolysis is still a riddle. We have recently identified the surface binding site (SBS) of a potent RSDA from Bacillus paralicheniformis ATCC 9945a (BliAmy), by crystallographic study of its native form and in complexes with maltose, acarbose, maltohexaose and β-cyclodextrin. To understand role of this SBS, the two key residues identified, Phe257 and Tyr358, were mutated. Kinetic studies show that starch binding through the SBS is disrupted in the mutants and that both mutants contributed cumulatively to binding and degradation. Mutation of both sites resulted in at least 5.5 fold weaker binding and 5 fold lower efficacy with raw starch as substrate 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 granule. RSDA was further exploited for its robustness by changing its activity and converting this hydrolase into transglycosylase. The use of transglycosylases for synthetic purposes is limited since, unlike hydrolases, these enzymes are relatively rare in nature, and act on a limited substrate repertoire. To alter the activity of BliAmy, His235 was replaced with Glu. The mode of action of the mutant enzyme was tested using substrates such as starch, amylopectin, maltooligosaccharides etc. Mutant exhibited transglycosylation activity, while wild type BliAmy exhibited hydrolysis activity exclusively. Converting hyperthermostable BliAmy into transglycosylase provides potent tool in the synthesis of starch derivatives. The production of starchy foods with slow digestion properties, and thus a low glycemic index, is therefore an important goal of the modern food industry

Cold enzyme hydrolysis of starch

With efforts to reduce global reliance on fossil fuels and lower the greenhouse gas emission, an increasing search for renewably sourced materials, which can be used as feedstock for biofuel production, is ongoing in the past few decades. At the present, ethanol is the most common alternate fuel and is already produced on a fair scale, representing a sustainable substitute for gasoline in passenger cars. Basically, in the United States ethanol is produced by fermenting starch crops that have been converted into simple sugars, and the major feedstock for this fuel is corn. In Brazil ethanol is produced through the fermentation of sugar cane molasses. Various countries have been increasing their ethanol production as well, such as India (using sugar cane), Thailand (cassava), France (sugar beet), China (corn) and Canada (wheat), among others. Improved molecular disassembly and depolymerization of grain starch to glucose are key to reducing energy use in the bioconversion of glucose to chemicals, ingredients, and fuels. In fuel ethanol production, these biorefining steps use 10-20% of the energy content of the fuel ethanol. The need to minimize energy use and to raise the net yield of energy can be met by replacing high-temperature, liquid-phase, enzymatic digestion with low temperature, solid-phase, enzymatic digestion. Also called cold hydrolysis, the approach is a step toward a “green” method for the production of fuel ethanol

Cytosolic leucyl aminopeptidase from the midgut of Morimus funereus larvae

Cilјеvi оvоg rаdа su bili: rаzviјаnjе visоkооsеtlјivоg i kvаntitаtivnоg zimоgrаmskоg еsеја zаlеucil-аminоpеptidаzе (LАP) i dеtеkciјаizоfоrmi nа оsnоvu zimоgrаmа u sirоvоm еktrаktusrеdnjеg crеvа lаrvi М. funereus; izоlоvаnjеglаvnе izоfоrmе LАP srеdnjеg crеvа lаrvi М.funereus dо hоmоgеnоsti i mоlеkulskа i еnzimskаkаrаktеrizаciја.Оpisаn је оpšti mеtоd zа dеtеkciјu lеucil-аminоpеptidаznе аktivnоsti nаkоn nаtivnеpоliаkrilаmidnе gеl еlеktrоfоrеzе in situ. Меtоdје zаsnivаn nа diаzоtоvаnju p-nitrоаnilinа,оslоbоđеnоg u gеlu dејstvоm lеucil-аminоpеptidаzе nа lеucin-p-nitrоаnilid, kојi јеzаtim kuplоvаn sа hrоmоgеnоm, 1-nаftilаminоm,dо pојаvе ružičаstе аzо-bоје nа mеstu еnzimskеаktivnоsti. Nаđеnо је dа је mеtоd rеprоduktivаn sаkоеficiјеntоm vаriјаciје mаnjim оd 15% zа 32-struki оpsеg, dоk је оbојеnа pоvršinа nа mеstuеnzimskе аktivnоsti u linеаrnој zаvisnоsti оdеnzimskе аktivnоsti.Glаvnа izоfоrmа lеucil-аminоpеptidаzе srеdnjеgcrеvа lаrvi М. funereus је prеčišćеnа dоhоmоgеnоsti i оkаrаktеrisаnа. Spеcifičnааktivnоst LАP је pоrаslа 292 putа prеčišćаvаnjеmsirоvоg еkstrаktа srеdnjеg crеvа. Prеčišćеnеnzim imа pH оptimum 7,5 i prеfеrеnciјаlnоhidrоlizuје p-nitrоаnilidе sа hidrоfоbnimаminоkisеlinаmа, sа nајvеćim Vmax/Km zа lеucin-p-nitrоаnilid. Nајеfikаsniјi inhibitоri su 1,10-fеnаntrоlin sа Ki vrеdnоšću оd 0,12 mМ icistеin sа Ki оd 0,31 mМ. ЕGТА је stimulisаоаktivnоst LАP. Zn2+, Мg2+ i Мn2+ su pоkаzаli bi-mоdаlni еfеkаt nа аktivnоst LАP. PrеčišćеniLАP (nаkоn gеl-filtrаciје nа Superose 6 kоlоni)је imао mоlеkulsku mаsu оd 400 kDа iizоеlеktričnu tаčku 6,2. SDS-PAGE-оm је pоkаzаnајеdnа prоtеinskа trаkа nа 67 kDа, štо ukаzuје dа јееnzim hеksаmеr. Šеst pеptidnih sеkvеnciја јеdоbiјеnо iz prоtеinskе trаkе ЕSI/МS-МS аnаlizоm.Pоrеđеnjеm dоbiјеnih pеptidnih sеkvеnciја ЕМBL-ЕBI prоgrаmоm zа аnаlizu sеkvеnciја sаsеkvеnciјаmа iz BLASTP bаzе pоdаtаkа pоkаzаn јеvisоk stеpеn idеntičnоsti sа drugimаminоpеptidаzаmа insеkаtа.The aims of this work were: development of sensitiveand quantitative zymogram assay for leucylaminopeptidases (LAP) and detection of LAP isoformsin the crude midgut extract of M. funereus larvae;purification of major LAP from the midgut of M.funereus larvae to homogeneity and its molecular andenzymatic characterization.A general method for detecting leucyl aminopeptidaseactivity after native polyacrylamide gel electrophoresisin situ is described. The method is based ondiazotization of p-nitroaniline, liberated in thepolyacrylamide gel by leucyl aminopeptidase action onleucine-p-nitroanilide and subsequent coupling with achromogen, 1-naphthylamine, until a pink azo dyeproduct at the position of enzyme activity is obtained.This method was found to be reproducible with thecoefficient of variation below 15% for a 32-fold range,while the colored area of enzyme activity was in lineardependence to enzyme activity.The major leucyl aminopeptidase from the midgut of M.funereus larvae was purified and characterised.Specific LAP activity was increased 292-fold bypurification of the crude midgut extract. The purifiedenzyme had a pH optimum of 7,5 and preferentiallyhydrolysed p-nitroanilides containing hydrophobicamino acids in the active site, with the highest Vmax/KMratio for leucine-p-nitroanilide. The most efficientinhibitors were 1,10-phenanthroline having a Ki valueof 0,12 mM and cysteine with Ki value of 0,31 mM.EGTA stimulated LAP activity. Zn2+, Mg2+ and Mn2+ allshowed bi-modal effects on LAP activity. The purifiedLAP (after gel-filtration on Superose 6 column) hadmolecular mass of 400 kDa with an isoelectric point of6,2. SDS-PAGE revealed one band of 67 kDa,suggesting that the enzyme is a hexamer. Six peptidesequences from protein band were obtained usingESI/MS-MS analysis. Comparison of the obtainedpeptide sequences with the EMBL-EBI sequenceanalysis toolbox and the BLASTP database showed ahigh degree of identity with other insectaminopeptidases

Hemijske modifikacije β-laktoglobulina hinonima

The avarone/avarol quinone/hydroquinone couple. as well as their derivatives show considerable antitumor activity. In this work, covalent modifications of beta-lactogglobulin. isolated from cow milk by avarone, its model compound 2-tert-butyl-1,4-benzoquinone. and several of their alkylthio derivatives were studied. The techniques applied for as-saying the modifications were: UV/VIS spectrophotometry, SDS PAGE and isoelectrofocusing. The results of the SDS PAGE suggest that polymerisation of the protein occurs. The shift of the pI of the protein upon modification toward lower values indicates that lysine amino groups are the principal site of die reaction of beta-lactoglobulin with the quinones.Hinonsko/hidrohinonski par avaron/avarol i njihovi derivati pokazuju značajnu antitumorsku aktivnost. U ovom radu proučavane su kovalentne modifikacije β-laktoglobulina, izolovanog iz kravljeg mleka, avaronom, njegovim model-jedinjenjem 2-tert-butil-1,4-benzohinonom i njihovim alkiltio-derivatima. Za ispitivanje modifikacija korišćene su UV/VIS spektrofotometrija, SDS PAGE i izoelektrofokusiranje. Rezultat SDS PAGE ukazuje da se protein polimerizuje. Pomeranje pI vrednosti proteina nakon modifikacije ka nižim vrednostima pokazuje da su amino grupe lizina glavna mesta reakcije β-laktoglobulina sa hinonima

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

Bacillus licheniformis 9945a α-amylase (BliAmy) has been described as potent enzyme for raw starch hydrolysis. Starch represents an inexpensive source for production of glucose, maltose syrups and fructose which are widely used in food industries. Regarding energy costs, effective utilization of natural resources and viscosity problems, direct hydrolysis of raw starch below the gelatinization temperature by using raw-starch-digesting enzymes, such as α-amylase is desirable. In spite of the extensive studies concerning the structure and thermal properties of B. licheniformis amylase and the numerous reports in the literature referring to the molecular mechanism of irreversible thermoinactivation, little attention has been paid to its enzymological characterisation. Detailed knowledge about subsite architecture of B. licheniformis amylase is scarce. No report on kinetics and mode of action of this industrially important enzyme can be found in the literature especially when raw starch is used as a substrate. For mechanistic studies enzyme preparations of high purity are required and improving downstream processing is very beneficial. BliAmy was produced using optimized fed-batch approach in defined media and significant overexpression of 1.2 g L-1 was achieved. These amylases have exposed tyrosine and tryptophan residues as part of their surface binding sites. Mixed mode Nuvia cPrime™ resin is tested as improvement of the downstream processing of raw starch digesting amylases aiming at exploiting hydrophobic patches at their surface. This resin combines hydrophobic interactions with cation exchange groups. Presence of salt facilitates hydrophobic interactions while ionexchange groups enable proper selectivity. Surface response methodology was used to optimize binding and eluting conditions of BliAmy. This single step procedure enables simultaneous concentration, pigments removal and purification of amylase with a yield of 96% directly from fermentation broth

Optimizacija rasta i produkcije α-amilaze Bacillus subtilis IP 5832 u šaržnim kulturama u erlenmajeru i laboratorijskom fermentoru

Cell growth and the level of alpha-amylase in response to the carbon and nitrogen sources used for the growth of the strain Bacillus subtilis IP 5832 were examined. Based on the amylase productivity level in shake flask cultures after 24 hours of growth, the growth medium containing starch and peptone was selected as the best medium. Amylase production was greatly reduced when glutamate or citrate as sources of carbon were used. Experiments performed at different initial concentrations of starch showed that although the strain grew well with all the starch concentration used, 0.5 % starch was necessary for maximum alpha-amylase production, inducing 1.55 IU mL(-1) of amylase to be secreted after 8 h of cultivation in shaking flasks. During the batch fermentation of B. subtilis IP 5832 strain in 2 L laboratory fermenter, a 60 % higher activity (2.5 IU mL(-1)) was obtained. The production of the enzyme was directly related to the growth of the strain. Maximum enzyme activity was obtained at the beginning of the stationary growth phase.Ćelijski rast i nivoi α-amilaze nakon gajenja Bacillus subtilis IP 5832 ispitivani su u zavisnosti od različitih izvora ugljenika i azota korišćenih u podlozi. Na osnovu produktivnosti amilaze nakon dvadesetčetvoročasovnog gajenja kulture u erlenmajerima sa mešanjem pokazano je da je najbolja hranljiva podloga ona koja sadrži skrob i pepton. Kada su glutamat ili citrat korišćeni kao izvori ugljenika produkcija enzima je bila značajno redukovana. Eksperimentima u kojima je varirana inicijalna koncentracija skroba pokazano je da je, iako soj dobro raste na svim korišćenim koncentracijama, 0,5 % skrob neophodan za maksimalnu produkciju enzima, indukujući sekreciju 1,55 IU mL-1 amilaze nakon 8 sati gajenja kulture u erlenmajerima sa mešanjem. Tokom šaržne fermentacije B. subtilis IP 5832 soja u laboratorijskom fermentoru od 2 L dobijeno je 60 % više aktivnosti enzima (2,5 IU mL -1). Produkcija enzima je bila direktno vezana za rast mikroorganizma. Maksimalna enzimska aktivnost dobijena je na početku stacionarne faze rasta

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

Bacillus licheniformis 9945a α-amylase (BliAmy) has been described as potent enzyme for raw starch hydrolysis. Starch represents an inexpensive source for production of glucose, maltose syrups and fructose which are widely used in food industries. Regarding energy costs, effective utilization of natural resources and viscosity problems, direct hydrolysis of raw starch below the gelatinization temperature by using raw-starch-digesting enzymes, such as α-amylase is desirable. In spite of the extensive studies concerning the structure and thermal properties of B. licheniformis amylase and the numerous reports in the literature referring to the molecular mechanism of irreversible thermoinactivation, little attention has been paid to its enzymological characterisation. Detailed knowledge about subsite architecture of B. licheniformis amylase is scarce. No report on kinetics and mode of action of this industrially important enzyme can be found in the literature especially when raw starch is used as a substrate. For mechanistic studies enzyme preparations of high purity are required and improving downstream processing is very beneficial. BliAmy was produced using optimized fed-batch approach in defined media and significant overexpression of 1.2 g L-1 was achieved. These amylases have exposed tyrosine and tryptophan residues as part of their surface binding sites. Mixed mode Nuvia cPrime™ resin is tested as improvement of the downstream processing of raw starch digesting amylases aiming at exploiting hydrophobic patches at their surface. This resin combines hydrophobic interactions with cation exchange groups. Presence of salt facilitates hydrophobic interactions while ionexchange groups enable proper selectivity. Surface response methodology was used to optimize binding and eluting conditions of BliAmy. This single step procedure enables simultaneous concentration, pigments removal and purification of amylase with a yield of 96% directly from fermentation broth