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

Abstract

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.