Celulaze gljive Trichoderma harzianum: produkcija, kontrola produkcije i karakterizacija eksprimiranih enzima

Abstract

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.