title: Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae.
abstract: We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S-S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.
authors: VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT, Espoo, Finland.
authors: Sanni P
authors: SP
authors: Voutilainen
authors: Susanna
authors: S
authors: Nurmi-Rantala
authors: Merja
authors: M
authors: Penttilä
authors: Anu
authors: A
authors: Koivula
mesh_terms: D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D032641:Eurotiales / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D060847:Metabolic Engineering; D008969:Molecular Sequence Data; D011485:Protein Binding; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase