Dissecting the mechanism of enzymatic degradation of cellulose using low molecular weight model substrates
Kuupäev
2017-11-14
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Tselluloos on vees lahustumatu biopolümeer, mis koosneb lineaarsetest ahelatest, kus glükoosi jäägid on omavahel ühendatud glükosiidsete sidemetega. Looduses on tselluloos oluline energia- ja süsinikuallikas mitmetele baketri- ja seeneliikidele. Tselluloosi lagundamiseks kasutavad need organismid komplekti erinevatest hüdrolüütilisest ja oksüdatiivsetest ensüümidest, mida kokkuvõtvalt nimetatakse tsellulolüütiliseks süsteemiks. Kõige olulisemad komponendid tsellulolüütilistes süsteemides on tsellobiohüdrolaasid. Neid ensüüme iseloomustab protsessiivsus, see tähendab, et olles seostunud tselluloosiahelale need ensüümid hüdrolüüsivad glükosiidsidemeid järjest edasi liikudes ilma vahepeal dissotsieerumata.
Pehmemädanikseen Trichoderma reesei on üks paremini uuritud tselluloosi lagundav organism. Ehkki Trichoderma reesei tsellulaase on uuritud aastakümneid, ei mõisteta tselluloosi hüdrolüüsi lõplikult tänini. Tsellulaasidele ei ole suudetud üheselt määratleda klassikalises ensümoloogias laialdaselt kasutatavat kineetilist parameetrit katalüütiline konstant kcat. Raskused katalüütilise konstandi määramisel on tingitud ühelt poolt substraadist – tselluloos on vees lahustumatu ning sellest tulenevalt ei jaotu ruumis ühtlaselt. Teiselt poolt on see tingitud ensüümist – tsellulaasid on võimelised seostuma tselluloosiga nii produktiivselt kui ka mitteproduktiivselt. Samuti on teada, et tselluloosi hüdrolüüs tsellobiohüdrolaasidega ei järgi klassikalist Michaelis Menteni kineetikat vaid hüdrolüüsi kiirus langeb ajas järsult.
Käesolevas doktoritöös töötati välja meetod ensümaatilise tselluloosi hüdrolüüsi katalüütilise konstandi määramiseks. Meetod põhineb mudelsubstraadi hüdrolüüsil ning võimaldab eristada produktiivselt seostunud ensüümi mitteproduktiivsest. Samuti võimaldab see meetod jälgida produktiivselt seostunud ensüümi osakaalu hüdrolüüsi kulgedes ning uurida näiliste kineetiliste parameetrite muutumist ajas.
Püstitatud hüpoteesi kohaselt on tsellobiohüdrolaaside kiiruse langus tingitud ensüümimolekulide „kinni jäämisest“ tselluloosi pinnale. Protsessiivsed tsellobiohüdrolaasid liiguvad mööda tselluloosi ahelat kuni nende teele jääb takistus ning nende liikumine peatub. Kuna tsellobiohüdrolaaside dissotsiatsiooni kiirus on madal jäävad ensüümimolekulid takistuse taha pidama ning mitteproduktiivselt seostunud ensüümi oskaal suureneb. Tsellobiohüdrolaaside aktiivsust saab tõsta, kui soodustada mitteproduktiivselt seostunud ensüümide dissotsiatsiooni. Saadud tulemused annavad eelduse tootmaks paremaid ensüümisegusid tselluloosi tööstuslikuks hüdrolüüsiks.
Cellulose is a water-insoluble polysaccharide that consists of linear chains of glucose residues. Cellulose is an important energy and carbon source for many species of fungi and bacteria. These organisms secrete a set of hydrolytic and oxidative enzymes also called cellulolytic system. The major components of these cellulolytic systems are cellobiohydrolases. These enzymes are processive which means that enzyme, once bound productively to the substrate, performs several consecutive catalytic steps on a single polysaccharide chain before it dissociates. The best described cellulolytic system is that of the soft rot fungus Trichoderma reesei. While Trichoderma reesei cellulases have been subject of intensive study for decades, the mechanism of cellulase catalyzed cellulose hydrolysis is still not fully understood. One of the biggest shortcomings is the difficulty to measure the rate constant of cellulases acting on cellulose. Problems arise from the heterogeneous insoluble substrate that renders the reaction non-uniform as well as from modular structure of the enzyme that enables both productive and non-productive binding of the enzyme. Also, it is well known that the rate of enzymatic cellulose hydrolysis drops rapidly in time. The initial burst of activity is followed by a rapid decrease in the hydrolysis rate. This work introduces novel methods for determining the rate constants of cellobiohydrolase catalyzed cellulose hydrolysis. These methods are based on the hydrolysis of small soluble model substrates and enable the determination of productively bound enzyme. The methods were used to investigate the mechanism behind the decrease in activity of cellulases during the cellulose hydrolysis. Our hypothesis is that the decline of cellulose hydrolysis rate is caused by obstacles on the path of the processive movement of the enzyme. According to this model, the newly formed productive enzyme-substrate complex moves along the cellulose chain at a constant rate. Once the complex encounters an obstacle it is stalled. Since the dissociation rate constant is low, the enzyme remains “stuck” on the substrate and the proportion of non-productively bound enzyme increases. The action of cellobiohydrolases could be enhanced by promoting recruitment of the enzyme. The results provide a basis for producing better enzyme cocktails for industrial degradation of cellulose.
Cellulose is a water-insoluble polysaccharide that consists of linear chains of glucose residues. Cellulose is an important energy and carbon source for many species of fungi and bacteria. These organisms secrete a set of hydrolytic and oxidative enzymes also called cellulolytic system. The major components of these cellulolytic systems are cellobiohydrolases. These enzymes are processive which means that enzyme, once bound productively to the substrate, performs several consecutive catalytic steps on a single polysaccharide chain before it dissociates. The best described cellulolytic system is that of the soft rot fungus Trichoderma reesei. While Trichoderma reesei cellulases have been subject of intensive study for decades, the mechanism of cellulase catalyzed cellulose hydrolysis is still not fully understood. One of the biggest shortcomings is the difficulty to measure the rate constant of cellulases acting on cellulose. Problems arise from the heterogeneous insoluble substrate that renders the reaction non-uniform as well as from modular structure of the enzyme that enables both productive and non-productive binding of the enzyme. Also, it is well known that the rate of enzymatic cellulose hydrolysis drops rapidly in time. The initial burst of activity is followed by a rapid decrease in the hydrolysis rate. This work introduces novel methods for determining the rate constants of cellobiohydrolase catalyzed cellulose hydrolysis. These methods are based on the hydrolysis of small soluble model substrates and enable the determination of productively bound enzyme. The methods were used to investigate the mechanism behind the decrease in activity of cellulases during the cellulose hydrolysis. Our hypothesis is that the decline of cellulose hydrolysis rate is caused by obstacles on the path of the processive movement of the enzyme. According to this model, the newly formed productive enzyme-substrate complex moves along the cellulose chain at a constant rate. Once the complex encounters an obstacle it is stalled. Since the dissociation rate constant is low, the enzyme remains “stuck” on the substrate and the proportion of non-productively bound enzyme increases. The action of cellobiohydrolases could be enhanced by promoting recruitment of the enzyme. The results provide a basis for producing better enzyme cocktails for industrial degradation of cellulose.
Kirjeldus
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Märksõnad
tselluloos, ensümaatiline hüdrolüüs, tsellubiohüdrolaasid, cellulose, enzymatic hydrolysis, cellobiohydrolases