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U оvоm rаdu su ispitivаni: gајеnjе lаrvi i dоbiјаnjе rеprоduktivnо spоsоbnih јеdinki bukоvе strižibubе (Morimus funereus) u lаbоrаtоriјskim uslоvimа; аnаlizirаnjе аmilаznе аktivnоsti i izоаmilаznih prоfilа lаrvi bukоvе i vеlikе hrаstоvе strižibubе (Cerambyx cerdo) u zаvisnоsti оd hrаnjivih supstrаtа i uslоvа srеdinе; prеčišćаvаnjе i biоhеmiјskа kаrаktеrizаciја glаvnе izоfоrmе аmilаzе lаrvе bukоvе strižibubе i prеčišćаvаnjе glаvnе izоfоrmе аmilаzе lаrvi vеlikе hrаstоvе strižibubе. Оpisаnо је lаrvеnо rаzvićе bukоvе strižibubе u lаbоrаtоriјskim uslоvimа оd јаја dо rеprоduktivnо spоsоbnih јеdinki krоz dvе gеnеrаciје. Pоkаzаnо је dа sе gајеnjеm lаrvi u lаbоrаtоriјskim uslоvimа i stаlnој dоstupnоsti hrаnе skrаćuје lаrvеnо rаzvićе, i dа sе u drugој gеnеrаciјi uоčаvа vеćа uјеdnаčеnоst lаrvi pо svim pаrаmеtrimа kојimа sе оpisuје lаrvеnоrаzvićе. Nаđеnо је dа pоstоје rаzlikе u аmilаznој аktivnоsti i izоаmilаznim prоfilimа kоd lаrvi bukоvе i vеlikе hrаstоvе strižibubе u zаvisnоsti оd hrаnjivоg supstrаtа i uslоvа srеdinе gdе sе rаzviјајu, čimе је pоtvrđеnа njihоvа pоlifаgnоst i vеlikа аdаptаbilnоst. Glаvnа α-аmilаznа izоfоrmа lаrvе bukоvе strižibubе је prеčišćеnа dо hоmоgеnоsti 112 putа sа prinоsоm оd 15% i biоhеmiјski је оkаrаktеrisаnа. Оdrеđеnа јој је mоlеkulskа mаsа 33 kDа, pI vrеdnоst – 3,2, pH оptimum – 5,2 i tеmpеrаturni оptimum – 45°C. Аktivnоst α- аmilаzе lаrvе bukоvе strižibubе zаvisi оd јоnа Cа2+. Inhibirајu је inhibitоri iz pšеnicе. Pоkаzuје аktivnоst prеmа sirоvоm skrоbu. Glаvnа α-аmilаzа lаrvе vеlikе hrаstоvе strižibubе је dеlimičnо prеčišćеnа iz sirоvоg еkstrаktа, i nа izоеnzimskоm nivоu kоrišćеnjеm dvе vrstе FPLC hrоmаtоgrаfiје. Моlеkulskа mаsа јој је 34 kDа а pI mаnjа оd 3,5. This paper examined: larvae growing and getting reproductive age individuals of long – horned beetle (Morimus funereus) in laboratory conditions, analyzing the amylase activity and isoamylase profiles of M. funereus larvae and great capricorn beetle (Cerambyx cerdo) larvae depending on the nutrient substrate and environmental conditions, purification and biochemical characterization of the major amylase isoform of M. funereus larvae and purification of the major amylase isoform of C. cerdo larvae. Larval develop of M. funerus larvae in the laboratory from eggs to reproductively capable individuals in two generations were described. It is shown that growing growing larvae in laboratory conditions and constant availability of food larval development was reduced, and that in the second generation higher uniformity of larvae in all of the parameters that describe the larval development was noticed. It was found that there are differences in amylase activities and isoamylase profiles of M. funerus and C. cerdo larvae depending on the substrate and nutrient conditions in the environment where they develop, thus confirming their polyphagy and high adaptability. The main α-amylase isoform of M. funereus larvae was purified to homogeneity 112 times with a yield of 15% and was biochemically characterized. Its molecular weight is determined to be 33 kDa, pI value - 3.2, optimum pH - 5.2 and optimum temperature - 45 ° C. α-Amylase activity of M. funureus larvae is depended on concentration of Ca2 + ions. Inhibitors from wheat inhibit the M. funereus amylase. Midgut amylase of M. funereus larvae shows activity towards raw starch. The main α-amylase of C. cerdo larvae was partially purified from crude extract, and the isoenzyme pattern level using two types of FPLC chromatography. Molecular mass of midgut α-amylase of C. cerdo larvae was 34 kDa and pI was less than 3.5.

U оvоm rаdu su ispitivаni: gајеnjе lаrvi idоbiјаnjе rеprоduktivnо spоsоbnih јеdinki bukоvеstrižibubе (Morimus funereus) u lаbоrаtоriјskimuslоvimа; аnаlizirаnjе аmilаznе аktivnоsti iizоаmilаznih prоfilа lаrvi bukоvе i vеlikеhrаstоvе strižibubе (Cerambyx cerdo) uzаvisnоsti оd hrаnjivih supstrаtа i uslоvаsrеdinе; prеčišćаvаnjе i biоhеmiјskаkаrаktеrizаciја glаvnе izоfоrmе аmilаzе lаrvеbukоvе strižibubе i prеčišćаvаnjе glаvnеizоfоrmе аmilаzе lаrvi vеlikе hrаstоvеstrižibubе.Оpisаnо је lаrvеnо rаzvićе bukоvе strižibubе ulаbоrаtоriјskim uslоvimа оd јаја dоrеprоduktivnо spоsоbnih јеdinki krоz dvеgеnеrаciје. Pоkаzаnо је dа sе gајеnjеm lаrvi ulаbоrаtоriјskim uslоvimа i stаlnој dоstupnоstihrаnе skrаćuје lаrvеnо rаzvićе, i dа sе u drugојgеnеrаciјi uоčаvа vеćа uјеdnаčеnоst lаrvi pоsvim pаrаmеtrimа kојimа sе оpisuје lаrvеnоrаzvićе.Nаđеnо је dа pоstоје rаzlikе u аmilаznојаktivnоsti i izоаmilаznim prоfilimа kоd lаrvibukоvе i vеlikе hrаstоvе strižibubе uzаvisnоsti оd hrаnjivоg supstrаtа i uslоvаsrеdinе gdе sе rаzviјајu, čimе је pоtvrđеnаnjihоvа pоlifаgnоst i vеlikа аdаptаbilnоst.Glаvnа α-аmilаznа izоfоrmа lаrvе bukоvеstrižibubе је prеčišćеnа dо hоmоgеnоsti 112putа sа prinоsоm оd 15% i biоhеmiјski јеоkаrаktеrisаnа. Оdrеđеnа јој је mоlеkulskа mаsа33 kDа, pI vrеdnоst – 3,2, pH оptimum – 5,2 itеmpеrаturni оptimum – 45°C. Аktivnоst α-аmilаzе lаrvе bukоvе strižibubе zаvisi оd јоnаCа2+. Inhibirајu је inhibitоri iz pšеnicе.Pоkаzuје аktivnоst prеmа sirоvоm skrоbu.Glаvnа α-аmilаzа lаrvе vеlikе hrаstоvеstrižibubе је dеlimičnо prеčišćеnа iz sirоvоgеkstrаktа, i nа izоеnzimskоm nivоu kоrišćеnjеmdvе vrstе FPLC hrоmаtоgrаfiје. Моlеkulskа mаsајој је 34 kDа а pI mаnjа оd 3,5. This paper examined: larvae growing andgetting reproductive age individuals of long – hornedbeetle (Morimus funereus) in laboratory conditions,analyzing the amylase activity and isoamylase profilesof M. funereus larvae and great capricorn beetle(Cerambyx cerdo) larvae depending on the nutrientsubstrate and environmental conditions, purificationand biochemical characterization of the major amylaseisoform of M. funereus larvae and purification of themajor amylase isoform of C. cerdo larvae.Larval develop of M. funerus larvae in thelaboratory from eggs to reproductively capableindividuals in two generations were described. It isshown that growing growing larvae in laboratoryconditions and constant availability of food larvaldevelopment was reduced, and that in the secondgeneration higher uniformity of larvae in all of theparameters that describe the larval development wasnoticed.It was found that there are differences inamylase activities and isoamylase profiles of M.funerus and C. cerdo larvae depending on thesubstrate and nutrient conditions in the environmentwhere they develop, thus confirming their polyphagyand high adaptability. The main α-amylase isoform of M. funereuslarvae was purified to homogeneity 112 times with ayield of 15% and was biochemically characterized. Itsmolecular weight is determined to be 33 kDa, pI value -3.2, optimum pH - 5.2 and optimum temperature - 45 °C. α-Amylase activity of M. funureus larvae isdepended on concentration of Ca2 + ions. Inhibitorsfrom wheat inhibit the M. funereus amylase. Midgutamylase of M. funereus larvae shows activity towardsraw starch.The main α-amylase of C. cerdo larvae waspartially purified from crude extract, and the isoenzymepattern level using two types of FPLC chromatography.Molecular mass of midgut α-amylase of C. cerdo larvaewas 34 kDa and pI was less than 3.5.

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. 142026B

Application of specific chromogenic substrates, use of class-specific inhibitors, and zymogram analysis enabled us to identify several peptidase classes in extracts of the midgut of Morimus funereus larvae. Zymogram analysis with gelatin as a peptidase substrate and phenylmethylsulfonyl fluoride as an inhibitor showed that serine peptidases were the most abundant endopeptidases in the midgut of M. funereus larvae. By zymogram analysis with gelatin as a peptidase substrate and 1,10-phenanthroline as an inhibitor, metallopeptidases were also detected. Analyses of serine peptidases with specific chromogenic substrates revealed dominance of elastase-like peptidases in extracts of the midgut of M. funereus larvae, with less pronounced chymotrypsin- and trypsin-like activities. Primenom specifičnih hromogenih supstrata, klasno-specifičnih inhibitora i zimogramske analize identifikovano je nekoliko klasa peptidaza u sirovom ekstraktu srednjeg creva larvi koleoptere Morimus funereus. Zimogramskom analizom sa želatinom kao supstratom i fenilmetilsulfonil-fluoridom kao inhibitorom utvrđeno je da su serin-peptidaze najzastupljenije peptidaze u ekstraktu srednjeg creva larvi M. funereus. Zimogramskom analizom sa želatinom kao supstratom i 1,10-fenantrolinom kaoinhibitorom takođe su detektovane metalopeptidaze. Analizom serin-peptidaza, upotrebom specifičnih hromogenih supstrata, dokazano je da su dominantni elastazi-slični enzimi u sirovom ekstraktu srednjeg creva larvi M. funereus, dok su himotripsinima- i tripsinima-slični enzimi manje zastupljeni. Projekat ministarstva br. 142026B

Ova disertacija se bavi ispitivanjem mogućnosti primene izolata gljive roda Trichoderma harzianum za produkciju enzima celulaznog kompleksa, optimizacijom uslova za njihovu produkciju upotrebom otpadnih lignoceluloznih materijala, ispitivanjem mehanizma kontorole produkcije i karakterizacijom produkovanih celulaznih enzima. Razvijen je brz i pouzdan test za selekciju endocelulaznih hiperprodukujućih izolata Trichoderma spp., koji je korišćen za analizu sto Trichoderma spp. izolata. Obećavajući produceri su identifikovani do nivoa vrste. Najbolji producer endocelulaza je identifikovan kao Trichoderma guizhouensis (UB483FTH2) iz grupe T. harzianum i korišćen je za produkciju celulaznih enzima. Optimizovani su uslovi za produkciju celulaznog kompleksa enzima u uslovima tečne fermentacije upotrebom slame kao inducibilnog supstrata. Maksimalna produkcija endocelulaza (28,32 U/g) je dobijena u 3 danu fermentacije, egzocelulaza (0,049 U/g) nakon 5 dana fermentacije i β-glukozidaza (14,1 U/g) u 7 danu fermentacije. Razvijena je zimogramska metoda za istovremenu detekciju različitih klasa celulaznih izoformi, nakon izoelekrofokusiranja. β-glukozidaze su detektovane nakon printovanja na nitroceluloznoj membrani upotrebom eskulina. Endocelulaze su detektovane na poliakrilamidnom gelu sa koopolimerizovanom karboski metil celulozom, dok su egzocelulaze detektovane upotrebom 4-metilumberiferil-β-D-celobiozida kao supstrata. Ispitana je i upotreba iskorišćenog komposta šampinjona kao novog supstrata za produkciju celulaznih enzima gajenjem 6 izolata Trichoderma spp. Produkovana je značajna količina endocelulaza i β-glukozidaza, uz istovremeno smanjenje početnog supstrata za 30%. Mehanizam regulacije produkcije celulaznih proteina izolata UB483FTH2 je analiziran korelisanjem nivoa transkribovane iRNA za gene xyr1 i lae1, regulatora produkcije celulaza, sa produkcijom svih celulaznih enzima. Pronađeno je da eksprimirani geni, xyr1 i lae1, imaju pozitivan uticaj na produkciju celulaznih enzima i proteina. This thesis examines application of isolates of Trichoderma harzianum for production of cellulase enzyme complex, optimization of their production using lignocellulose waste materials, studying of control mechanisms for production and characterization of the produced cellulases enzymes. Fast and reliable test for the selection of endocellulase hyper-producing isolates of Trichoderma spp. has been developed and used for screening one hundred isolates of Trichoderma spp. The promising isolates have been isolated to species level. The best endocellulase producer is identified as Trichoderma guizhouensis (UB483FTH) which belongs to the Trichoderma harzianum species complex. This strains was used for production of cellulase enzyme complex. Cellulase production was optimized using wheat straw as a substrate in conditions of submerged fermentation. Maximum of endocelullase production (28.32 U/g) was obtained in 3rd days of fermentation, exocellulase (0.049 U/g) after 5th days and β- glucosidase (14.1 U/g) in 7th days of fermentation. Reliable zymographic method for simultaneous characterization of cellulolytic complex enzymes after isoelectric focusing has been developed. β-glucosidase is detected after printing on nitrocellulose membrane using esculin as substrate. Endocellulase was detected on polyacrilamide gel with copolymerized carboxymethyl-cellulose and exocellulase were detected using 4-metillumberiferil-β-D-celobioside as a substrate. Reuse of spent mushroom compost as a new substrate for the production of cellulase enzymes by 6 isolates Trichoderma spp. was examined here too. Significant amount of endocellulase and β-glucosidase were produced, while the amount of initial substrate decreased by 30%. The regulation mechanism of protein production of isolate UB483FTH2 analysed as a correlation of transcribed level of mRNA for genes xyr1 and lae1 as a regulators of production of cellulase enzymes. It was found that expressed genes xyr1 and lae1 have a positive effect on the production of cellulases proteins and total proteins.

Ova disertacija se bavi ispitivanjem mogućnosti primene izolata gljive rodaTrichoderma harzianum za produkciju enzima celulaznog kompleksa, optimizacijomuslova za njihovu produkciju upotrebom otpadnih lignoceluloznih materijala, ispitivanjemmehanizma kontorole produkcije i karakterizacijom produkovanih celulaznih enzima.Razvijen je brz i pouzdan test za selekciju endocelulaznih hiperprodukujućihizolata Trichoderma spp., koji je korišćen za analizu sto Trichoderma spp. izolata.Obećavajući produceri su identifikovani do nivoa vrste. Najbolji producer endocelulaza jeidentifikovan kao Trichoderma guizhouensis (UB483FTH2) iz grupe T. harzianum ikorišćen je za produkciju celulaznih enzima.Optimizovani su uslovi za produkciju celulaznog kompleksa enzima u uslovimatečne fermentacije upotrebom slame kao inducibilnog supstrata. Maksimalna produkcijaendocelulaza (28,32 U/g) je dobijena u 3 danu fermentacije, egzocelulaza (0,049 U/g)nakon 5 dana fermentacije i β-glukozidaza (14,1 U/g) u 7 danu fermentacije.Razvijena je zimogramska metoda za istovremenu detekciju različitih klasacelulaznih izoformi, nakon izoelekrofokusiranja. β-glukozidaze su detektovane nakonprintovanja na nitroceluloznoj membrani upotrebom eskulina. Endocelulaze sudetektovane na poliakrilamidnom gelu sa koopolimerizovanom karboski metil celulozom,dok su egzocelulaze detektovane upotrebom 4-metilumberiferil-β-D-celobiozida kaosupstrata.Ispitana je i upotreba iskorišćenog komposta šampinjona kao novog supstrata zaprodukciju celulaznih enzima gajenjem 6 izolata Trichoderma spp. Produkovana jeznačajna količina endocelulaza i β-glukozidaza, uz istovremeno smanjenje početnogsupstrata za 30%.Mehanizam regulacije produkcije celulaznih proteina izolata UB483FTH2 jeanaliziran korelisanjem nivoa transkribovane iRNA za gene xyr1 i lae1, regulatoraprodukcije celulaza, sa produkcijom svih celulaznih enzima. Pronađeno je da eksprimiranigeni, xyr1 i lae1, imaju pozitivan uticaj na produkciju celulaznih enzima i proteina. This thesis examines application of isolates of Trichoderma harzianum forproduction of cellulase enzyme complex, optimization of their production usinglignocellulose waste materials, studying of control mechanisms for production andcharacterization of the produced cellulases enzymes.Fast and reliable test for the selection of endocellulase hyper-producing isolates ofTrichoderma spp. has been developed and used for screening one hundred isolates ofTrichoderma spp. The promising isolates have been isolated to species level. The bestendocellulase producer is identified as Trichoderma guizhouensis (UB483FTH) whichbelongs to the Trichoderma harzianum species complex. This strains was used forproduction of cellulase enzyme complex.Cellulase production was optimized using wheat straw as a substrate in conditionsof submerged fermentation. Maximum of endocelullase production (28.32 U/g) wasobtained in 3rd days of fermentation, exocellulase (0.049 U/g) after 5th days and β-glucosidase (14.1 U/g) in 7th days of fermentation.Reliable zymographic method for simultaneous characterization of cellulolyticcomplex enzymes after isoelectric focusing has been developed. β-glucosidase is detectedafter printing on nitrocellulose membrane using esculin as substrate. Endocellulase wasdetected on polyacrilamide gel with copolymerized carboxymethyl-cellulose andexocellulase were detected using 4-metillumberiferil-β-D-celobioside as a substrate.Reuse of spent mushroom compost as a new substrate for the production ofcellulase enzymes by 6 isolates Trichoderma spp. was examined here too. Significantamount of endocellulase and β-glucosidase were produced, while the amount of initialsubstrate decreased by 30%.The regulation mechanism of protein production of isolate UB483FTH2 analysedas a correlation of transcribed level of mRNA for genes xyr1 and lae1 as a regulators ofproduction of cellulase enzymes. It was found that expressed genes xyr1 and lae1 have apositive effect on the production of cellulases proteins and total proteins.