The acquisition of cellulose chain by a processive cellobiohydrolase
Failid
Kuupäev
2017-07-07
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Taimsesse biomassi talletatud taastuvat süsinikuvaru on võimalik kasutada biokütuste ja keemiatööstuse toorainena. Kõige suuremal hulgal esindatud polüsahhariid taime rakukestas on tselluloos. Tselluloosiahelad koosnevad omavahel järjestikku liitunud glükoosjääkidest ja tänu ahelate korrapärasele paigutusele esineb tselluloos rakukestas peamiselt kristallilisel kujul. Tööstuslikuks kasutamiseks on esmalt vaja kristalle moodustavad ahelstruktuurid lagundada. Looduses toodavad mikroorganismid selleks erinevaid ensüüme nagu tselluloosi hüdrolüüsi eest vastutavaid tsellulaase. Tselluloosi efektiivseks hüdrolüüsiks leidub tsellulaaside hulgas erineva toimemehhanismiga ensüüme. Kõige rohkem on protsessiivseid tsellulaase. Need ensüümid viivad tselluloosile seostudes läbi mitmeid katalüüsiakte järjest. Tselluloosi lagundajana tuntud mudelorgansim – pehmemädanik seen Trichoderma reesei – toodab kõige rohkem protsessivset tsellulaasi TrCel7A.
Minu töö põhifookuses oli TrCel7A katalüütilise tsükli molekulaarsete mehhanismide väljaselgitamine. Töö põhitulemus on TrCel7A aktiivtsentris paikneva aminohappejäägi Trp38 rolli väljaselgitamine ensüümi protsessiivses tsüklis. Näitasime, et Trp38 on oluline tselluloosiahela seondumiseks ensüümi aktiivtsentri tunnelisse. Lisaks kiirendab Trp38 puudumine ensüümi lahkumist tselluloosilt, mistõttu väheneb ka ensüümi protsessiivsus. Veel uurisime, miks vabaneb TrCel7A protsessivses hüdrolüüsis esimese produktina ülekaalukalt glükoos. Leidsime, et see fenomen on vähemalt osaliselt seletatav glükoosijäägi anomeeri spetsiifilise seondumisega ühes seondumiskohas TrCel7A aktiivtsentris. Pakume välja hüpoteesi, et see anomeeri spetsiifiline interaktsioon võib olla oluline ensüümi protsessiivsuse tagamisel. Kokkuvõttes loodan oma uurimistööga anda panuse tselluloosi lagundamise mõistmisele ja seeläbi biomassi rafineerimistehnoloogiate arendamisele
Deposits of carbon in plant biomass has a huge potential to contribute to the worldwide chemical and biofuel production. The major component of plant cell walls is cellulose. In cellulose, glucose residues are joined into long chains, which, in turn, have a regular arrangement and form a crystalline lattice in plant cell walls. For industrial use, it is often essential to degrade polysaccarides into its soluble monomeric compounds. The main biomass degraders in nature are microorganisms who secrete a myriad of different enzymes for deconstruction of biomass. The enzymes secreted to deconstruct cellulose are cellulases. The key enzymes for cellulose deconstruction use processive hydrolytic mechanism. Once associated with cellulose chain, these enzymes perform several consecutive catalytic acts before dissociation. The industrially relevant producers of cellulases are the cellulase hyper-producing strains of soft rot fungus Trichoderma reesei. The key cellulase of T.reesei is a processive cellulase TrCel7A. The primary targets of my research were the molecular mechanisms of the processive catalytic cycle of TrCel7A. In particular, we focused on the role of aromatic amino acid Trp38 at the entrance region of the active site of TrCel7A in processive cycle. Trp38 was found to be important for the cellulose chain feeding into the active site. Additionally, its absence increases the dissotiation of the enzyme from the substrate and, thereby, decreases the processivity. Regarding the acquisition of cellulose chain by TrCel7A, we also found that the binding of glucose residues in the subsite +2 in the active site of the enzyme was anomer-selective. We propose that this anomer-specific interaction with the substrate contributes to the processivity of TrCel7A. I hope that the main findings of my research will contribute to the understanding of the deconstruction mechanisms of cellulose and, thereby, to the development of technologies of biorefinement.
Deposits of carbon in plant biomass has a huge potential to contribute to the worldwide chemical and biofuel production. The major component of plant cell walls is cellulose. In cellulose, glucose residues are joined into long chains, which, in turn, have a regular arrangement and form a crystalline lattice in plant cell walls. For industrial use, it is often essential to degrade polysaccarides into its soluble monomeric compounds. The main biomass degraders in nature are microorganisms who secrete a myriad of different enzymes for deconstruction of biomass. The enzymes secreted to deconstruct cellulose are cellulases. The key enzymes for cellulose deconstruction use processive hydrolytic mechanism. Once associated with cellulose chain, these enzymes perform several consecutive catalytic acts before dissociation. The industrially relevant producers of cellulases are the cellulase hyper-producing strains of soft rot fungus Trichoderma reesei. The key cellulase of T.reesei is a processive cellulase TrCel7A. The primary targets of my research were the molecular mechanisms of the processive catalytic cycle of TrCel7A. In particular, we focused on the role of aromatic amino acid Trp38 at the entrance region of the active site of TrCel7A in processive cycle. Trp38 was found to be important for the cellulose chain feeding into the active site. Additionally, its absence increases the dissotiation of the enzyme from the substrate and, thereby, decreases the processivity. Regarding the acquisition of cellulose chain by TrCel7A, we also found that the binding of glucose residues in the subsite +2 in the active site of the enzyme was anomer-selective. We propose that this anomer-specific interaction with the substrate contributes to the processivity of TrCel7A. I hope that the main findings of my research will contribute to the understanding of the deconstruction mechanisms of cellulose and, thereby, to the development of technologies of biorefinement.
Kirjeldus
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Märksõnad
tselluloos, tsellubiohüdrolaasid, hüdrolüüs, fluorestsentsanalüüs, cellulose, cellobiohydrolases, hydrolysis, fluorescence analysis