Inhibition of myeloperoxidase by quercetin

Reaction mechanism of quercetin induced inhibition of myeloperoxidase isolated from human neutrophils was proposed by following peroxidase activity of the enzyme, using the o-dianisidine and H2O2 as substrates. The dependence of initial reaction rate vs. H2O2 concentration in the absence and presence of quercetin revealed the reaction mechanism that involved the enzyme inhibition by the excess of the substrate. The rate and equililbria constants for proposed reaction paths were determinedPhysical chemistry 2006 : 8th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 26-29 September 200

Imobilizacija invertaze preko ugljenohidratnog dela na makroporozni glicidil metakrilat

Immobilization via carbohydrate moiety is suitable for immobilization of glycoenzymes because it has little effect on enzyme active site. Macroporous glycidyl methacrylate is better support for enzyme immobilization from the much more used polystyrene because of its lesser hydrophobicity. We found optimal conditions for invertase immobilization via its carbohydrate moiety on macroporous glycidyl methacryate by varying enzyme concentration. We obtained immobilized enzyme with specific activity of 5500 lU/g, which is the highest activity reported in the literature. Immobilized enzyme has Km=43 mmol/l, temperature optimum of 60ºC, and pH optimum between 3.5 and 5.5.Imobilizacija preko šećernog dela je pogodna za glikoenzime zato što ima malo uticaja na aktivno mesto enzima. Makroporozni glicidil metakrilat je bolji nosač za imobilizaciju enzima od više korišćenog polistirena zbog svoje hidrofilnosti. Utvrđeni su optimalni uslovi za imobilizaciju invertaze preko šećernog dela na makroporozni glicidil metalkrilat variranjem koncentracije enizima. Dobijen je imobilizovani enzim specifične aktivnosti 5000 IU/g, što je najveća aktivnost do sada opisana u literaturi. Imobilizovani enzim je imao Km 43 mmol/l, temperaturni optimum na 60ºC i pH optimum između 4 i 5

Imobilizacija glukoamilaze na makroporoznim sferama

Glucoamylase was covalently immobilized through the spacer-arm of the poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) spheres by using a glutaraldehyde as a coupling agent. The influence of the enzyme load applied to the support on immobilization, yield and specific activity, has been determined. Obtained specific activity was 700 U/g with immobilization yield of 35 %. The Km value for immobilized glucoamylase was 1.28 % (w/v), pH and temperature optimum were 4.5 and 70°C, respectively. The conversion of 20 % (w/w) starch hydrolysate achieved with the immobilized glucoamylase was 97 % after 5 hours.Glukoamilaza je imobilizovana preko spejsera na sfere kopolimera glicidil metakrilata i etilen glikol dimetakrilata uz pomoć glutaraldehida. Određen je uticaj količine dodatog enzima na prinos imobilizacije kao i na specifičnu aktivnost dobijenog imobilizata. Dobijena je specifična aktivnost od 700 U/g sa prinosom imobilizacije od 35%. Km vrednost imobilizovane glukoamilaze je 1,28 % (w/v), pH i temperaturni optimumi su 4,5 i 70°S. Imobilizovani enzim je pri hidrolizi 20 % (w/w) hidrolizata skroba postigao konverziju od 97 % nakon 5 sati

Influence of nutrient substrates on the expression of cellulases in Cerambyx cerdo L. (Coleoptera: Cerambycidae) larvae

The expression and distribution of digestive cellulases along the midgut of Cerambyx cerdo larvae were analyzed for the first time and are presented in this article. Four groups of larvae were examined: larvae developed in the wild; larvae taken from the wild and successively reared on an artificial diet based on polenta; and larvae hatched in the laboratory and reared on two different artificial diets. Seven endocellulase and seven β-D-glucosidase isoforms were detected in all midgut extracts of C. cerdo with a zymogram after native PAGE. We observed that C. cerdo larvae are capable of producing cellulase isoforms with different PAGE mobilities depending on the nutrient substrate. From our findings it can be assumed that, depending on the distribution of endocellulase and β-D-glucosidase, cellulose molecules are first fragmented in the anterior and middle midgut by endo-β-1,4-glucanase; subsequently, the obtained fragments are broken down by β-D-glucosidase mostly in middle midgut.Projekat ministarstva br. 17204

Stabilizacija α-glukozidaze u organskim rastvaračima imobilizacijom na makroporoznim (poli) glicidil metakrilatima različitih površinskih karakteristika

alpha-Glucosidase from baker's yeast was immobilized on macroporous copolymers of ethylene glycol dimethacrylate and glycidyl methacrylate, poly(GMA-co-EGDMA), with various surface characteristics and pore sizes ranging from 44 nm to 270 nm. Immobilization was done by glutaraldehyde on the copolymer previously modified with 1,2-diaminoethane. The specific activity of the obtained immobilized enzyme varied from 27 to 81 U/g depending on the employed copolymer. The half lives of the immobilized enzyme in cosolvents were influenced by the surface characteristics of the copolymer, ranging from 60 to 150 min in 35 % methanol and from 10 to 44 min in 45 % dimethyl sulphoxide (DMSO). The best stabilities were obtained when the enzyme was immobilized onto a copolymer having a pore size of 48 rim in methanol and 270 nm in DMSO.α-Glukozidaza izolovana iz pekarskog kvasca je imobilizovana na makroporoznim glicidil-metakrilatima različitih površinskih karakteristika i veličina pora od 44 do 270 nm. Imobilizacija je izvedena glutaraldehidom na polimeru prethodno modifikovanom sa 1,2-diaminoetanom. Specifična aktivnost dobijenog imobilizovanog enzima je varirala od 27 do 81U/g u zavisnosti od vrste korišćenog polimera. Poluživoti imobilizovanog enzima u korastvaračima su zavisili od površinskih karakteristika polimera i kretali su se u opsegu od 60 do 150 min u 35%(v/v) metanolu i od 10 do 44 min u 45 % (v/v) dimetilsufoksidu. Najveća stabilnost u metanolu je dobijena imobilizacijom enzima na polimeru sa veličinom pora od 48 nm a u dimetilsulfoksidu na polimeru sa veličinom pora od 270 nm

Comparison of α-amylase isoforms from the midgut of Cerambyx cerdo L. (Coleoptera: Cerambycidae) larvae developed in the wild and on an artificial diet

α-Amylase isoforms of Cerambyx cerdo larvae from the wild (ML and SL) and reared in the laboratory (ADL) were compared. Three amylase isoforms were presented in the SL and ML extracts while two isoforms were presented in the ADL according to zymogram after isoelectric focusing (IEF). All C. cerdo amylase isoforms were acidic proteins (pI < 3.5). Seven amylase isoforms (ACC 1-7) from the midgut of C. cerdo larvae were found in the ML midgut extract, six in the SL extract, and four in the ADL extract according to native PAGE zymogram. The ADL amylase had the highest activity. All crude midgut extracts of C. cerdo larvae were fractionated on a Superose 12 HR column. The molecular mass of the ACC was estimated to be 34 kDa.Upoređene su izoforme α-amilaze larvi Cerambyx cerdo sakupljenih iz prirode (ML i SL) i gajenih na veštačkoj podlozi u laboratoriji (ADL). Zimogramskom detekcijom posle IEF-a po tri izoforme su detektovane u ML i SL ekstraktima, a u ADL dve izoforme. Sve amilazne izoforme iz C. cerdo su bile kisele (pI < 3.5). Zimogramskom detekcijom posle nativne elektroforeze sedam izoformi je detektovano u ML ekstraktu, šest u SL ekstraktu i četiri u ADL ekstraktu. Najveća amilazna aktivnost je detektovana u ADL ekstraktu. Svi ekstrakti srednjih creva larvi C. cerdo su frakcionisani na koloni Superose 12 HR. Molekulska masa ACC-a je bila 34 kDa.Projekat ministarstva br. 142026

Imobilizacija invertaze i glukoamilaze na makroporoznom kopolimeru glicidilmetakrilata i etilenglikoldimetakrilata i njihova potencijalna primena u biotehnologiji

The optimal conditions for the immobilization of invertase and glucoamylasewere found via their carbohydrate moiety on a macroporous copolymer of ehtyleneglycoldimethacry late and glycidylmethacrylate. Almost all of the added enzyme was bound to the polymer by increasing the time of incubation of the oxidized enzyme with polymer. A specific activity of 5500 U/g for invertase was obtained and 1100 U/g for glucoamylase. The specific productivity for invertase in a packed bed reactor was 3.5 kg/lh and for glucoamylase 1.9 kg/lh. During continuous use in a packed bed the reactor operational half life for invertase was 290 days, while no decrease in activity was observed for glucoamylase. In 50% (v/v) ethanol the immobilized enzymes were five to ten times more stable, and more than 200 times more stable in 25% (v/v) dioxane. The immobilized enzymes retained all activity in petroleum ether after 3 days of incubation. Because of their higher stability over native enzymes, and the large surface area of the polymer immobilized glucoamylase and invertase could be more useful for glycoside synthesis in non-aqueous solvents than native ones.Određeni su optimalni uslovi za imobilizaciju invertaze i glukoamilaze prekonjihove ugljenohidratne komponente u strukturi na makroporoznom kopolimeru glicidilmetakrilata i etilenglikoldimetakrilata. Dobijeni su imobilizati invertaze specifične aktivnosti od 5500 ILJ/g i glukoamilaze specifične aktivnosti od 1100 U/g. Specificna produktivnost cevastog protočnog reaktora sa imobilizovanom invertazom je bila 3,5 kg/dm3, a sa glukoamilazom 1,5 kg/dm3h. Operativna stabilnost imobilizovane invertaze tokom dvonedeljne upotrebe je procenjena na 290 dana, dok imobilizat glukoamilaze nije gubio aktivnost tokom 30 dana kontinuirane upotrebe. U 50% (v/v) etanolu imobilizovani enzimi su bili 10 puta stabilniji, u 25% (v/v) dioksanu preko 200 puta, dok u petrol etru nisu gubili aktivnost ni posle 3 dana

Toxicity of nickel and cadmium in spruce seedlings: Effect of separated and combined treatments on peroxidase and superoxide-dismutase activity

We studied the soluble peroxidase and superoxid-dismutase activity peroxidase isoenzyme pattern and metal content in the needles of 2-year-old spruce grown on soils supplemented with cadmium and nickel concentrations from 3 to 30 mg kg -1 and 50 to 500 mg kg -1 respectively. The two metals were applied both separately and simultaneously. The kinetics of metal assimilation and total accumulated quantity depended on the type of treatment. Following metal exposure, an increase in peroxidase activity in the seedlings treated with Cd and Ni/Cd, and in superoxide-dismutase activity during Ni/Cd treatment was observed. A parallel change of peroxidase isoenzyme pattern occurred. There was a good correlation between accumulated metals in needles and enzyme activities. The effect of Cd on the measured parameters was shown to be stronger comparing to the effect of Ni. The results obtained show that peroxidase and superoxide-dismutase activity and peroxidase isoenzyme pattern could be used to evaluate the capacity of one part of the defense system in spruce seedlings to with-stand metal stress.Proučavana je aktivnost slobodnih peroksidaza i superoksid-dismutaza izoenzimski profil peroksidaza i sadržaj metala u četinama dvogodišnjih smrča gajenih na zemljištu u koje su dodavani kadmijum i nikl u koncentracijama 3 do 30 mg kg -1 i 50 do 500 mg kg -1 respektivno. Metali su dodavani na dva načina: zasebno i istovremeno. Kinetika usvajanja metala i ukupna akumulirana količina zavisili su od vrste tretmana. U tretiranim izdancima došlo je do povećanja peroksidazne aktivnosti u slučaju tretmana kadmijumom i nikl/kadmijumom, kao i superoksid-dismutazne aktivnosti u toku tretmana nikl/kadmijumom, u odnosu na netretirane biljke. Istovremeno je došlo do promene izoenzimskog sastava peroksidaza. Koncentracija akumuliranih metala u četinama je bila u dobroj korelaciji sa enzimskim aktivnostima. Pokazano je da je uticaj kadmijuma na merene parametre jači u poređenju sa uticajem nikla. Dobijeni rezultati pokazuju da peroksidazna i superoksid-dismutazna aktivnost i izoenzimski profil peroksidaza mogu da se koriste za procenu kapaciteta jednog dela zaštitnog sistema u izdancima smrče u odnosu na stres izazvan metalima

Influence of nutrient substrates on the expression of cellulases in Cerambyx cerdo L. (Coleoptera: Cerambycidae) larvae

The expression and distribution of digestive cellulases along the midgut of Cerambyx cerdo larvae were analyzed for the first time and are presented in this article. Four groups of larvae were examined: larvae developed in the wild; larvae taken from the wild and successively reared on an artificial diet based on polenta; and larvae hatched in the laboratory and reared on two different artificial diets. Seven endocellulase and seven β-D-glucosidase isoforms were detected in all midgut extracts of C. cerdo with a zymogram after native PAGE. We observed that C. cerdo larvae are capable of producing cellulase isoforms with different PAGE mobilities depending on the nutrient substrate. From our findings it can be assumed that, depending on the distribution of endocellulase and β-D-glucosidase, cellulose molecules are first fragmented in the anterior and middle midgut by endo-β-1,4-glucanase; subsequently, the obtained fragments are broken down by β-D-glucosidase mostly in middle midgut.Projekat ministarstva br. 17204

Characterization of trypsin-like enzymes from the midgut of Morimus funereus (Coleoptera: Cerambycidae) larvae

The pH along the midgut of M. funereus larvae had different values, being acidic in the anterior section and basic in the middle and posterior sections. Elastase- and chymotrypsin-like activities were highest in the middle, low in the anterior, and negligible in the posterior section of the midgut. Trypsin-like activities were detected along the whole midgut, with more than 90% of activity in the anterior section. The level of elastase- and chymotrypsin-like activity was very low compared to trypsin-like activity. In the anterior section of the midgut, two isoforms of trypsin-like enzymes were found, both being basic and almost completely inhibited by benzamidine.pH vrednost duž srednjeg creva larvi M. funereus se razlikuje, kisela je u regionu prednjeg dela, dok je u srednjem i zadnjem delu bazna. Elastazama i himotripsinima slična aktivnost je najveća u srednjem delu srednjeg creva dok je u prednjem delu detektovana mala vrednost, a u zadnjem delu zanemarljiva. Tripsinima slična aktivnost je detektovana duž celog srednjeg creva, s tim da se više od 90 % aktivnosti detektuje u prednjem delu srednjeg creva. Zastupljenost elastazama i himotripsinima sličnih endopeptidaza je zanemarljivo mala u poređenju sa zastupljenošću tripsinima sličnih enzima. U prednjem delu srednjeg creva nalaze se dve izoforme tripsinima sličnih enzima, sa baznim pI vrednostima, koje su skoro u potpunosti inhibirane benzamidinom.Projekat ministarstva br. 142026