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1 Trichoderma reesei Cel3A Cel1A Cel3C (BGL) BGL Cel3A Cel3C pnp-glc Cel1A BGL Cel1A BGL BGL Cel3A BGL Trichoderma reesei, which is well known to produce large amount of cellulases, expresses three β-glucosidases (BGLs), one of which is secreted Cel3A and the other are intracellular Cel1A and Cel3C, when grown on cellulose. To elucidate the physiological roles of these BGLs, we investigated the enzymatic properties of the recombinant enzymes produced by heterologous hosts. Furthermore, we constructed the deficient mutants of the corresponding BGL genes and characterized the amount of biomass and the cellulase and BGL activities of the mutants when grown on cellulose or cellobiose. The results demonstrated that Cel3A and Cel3C preferred pnp-glc to cellobiose as the substrate, whereas Cel1A had higher activity for cellobiose than that for pnp-glc. The intracellular and extracellular BGL activities of the mutants reflected the substrate specificity of the respective enzymes. We assumed that Cel1A plays the key role in cellobiose metabolism and the BGLs would not directly involve in cellulase induction. When T. reese is grown on cellobiose, the other BGL(s) besides Cel3A seems to be expressed and secreted.
2 Trichoderma reesei 10 (BGL) Cel3A (BGL I) Cel1A (BGL II) Cel3C BGL Cel3A Cel1A Cel3C Cel3A T. reesei BGL(Cel3A, Cel1A Cel3C) T. reesei T. reesei (1) Cel3A, Cel1A Cel3C Cel3A Cel1A Cel3C (Schizosaccharomyces pombe Pichia pastoris) pnp-glc pnpgase Cel3A Cel1A ph ph (2) Cel3A, Cel1A Cel3C Cel3A amds Cel1A Cel3C pyr4 0.3 CMCase pnp-lac pnplase pnpgase Cel3A, Cel1A Cel3C Cel3A bgl1 cdna pet15b Rosetta-gami B 20 Bugbuster 72kDa Cel1A bgl2 cdna pet22b Rosetta-gami B 37 Bugbuster 52kDa Cel3C S. pombe P. pastoris (2) Cel3A, Cel1A Cel3C BGL pnp-glc BGL Cel3A Cel3C pnpgase
3 Cel1A GH (3) Cel3A Cel1A Cel3A Cel1A ph ph ph Cel3A ph5 Cel1A ph6 ph Cel3A ph Cel1A ph Cel3A ph Cel1A ph Cel1A Cel3A ph (4) Cel3A, Cel1A Cel3C BGL Cel3A amds Cel1A Cel3C 5' pyr4 DNA Cel3A T. reesei QM9414 Cel1A Cel3C T. reesei QM9414 pyr4::amds BGL T. reesei (5) BGL 1 Avicel BGL Δcel3a (Δbgl1), Δcel1a (Δbgl2), Δcel3cAvicel CMCase pnplase pnpgase CMCase pnplase EG I CBH I QM9414 Δcel3a(Δbgl1) Δcel3c 48h Δcel1a(Δbgl2) 96h cel1a(bgl2) CMCase, pnplase 1% Avicel pnpgase pnpgase QM9414 Δcel3c Δcel3a(Δbgl1) Cel3A(BGL I) pnpgase Δcel1a(Δbgl2) pnpgase QM9414 Δcel3a(Δbgl1) Δcel3c Δcel1a(Δbgl2) pnpgase 1% Avicel pnpgase Δcel1a(Δbgl2) 1% Avicel
4 Δcel1a(Δbgl2) h Δcel1a(Δbgl2) pnpgase pnpgase QM9414 Δcel3c Δcel3a(Δbgl1) Δcel1a(Δbgl2) pnpgase Δcel3c BGL pnpgase BGL Δcel1a(Δbgl2) CBH EG Cel1A (BGL II) Cel3A(BGL I) Cel3C BGL (6) BGL CMCase pnpgase CMCase 1% Avicel 500 pnpgase 0.3 (7) BGL CMCase pnpgase 10 CMCase QM9414 Δcel3a(Δbgl1) Δcel3c Δcel1a(Δbgl2) CMCase 1 CMCase 0.3% pnpgase pnpgase QM9414 Δcel3c Δcel3a(Δbgl1) Δcel1a(Δbgl2) pnpgase QM9414 Δcel3a(Δbgl1) Δcel1a(Δbgl2) Δcel3c
5 pnpgase 1 10 pnpgase QM9414 Δcel3c Δcel3a(Δbgl1) Δcel1a(Δbgl2) Δcel1a(Δbgl2) Cel1A(BGL II) 10 1 T. reesei T. reesei Cel1A (BGL II) Cel3A(BGL I) Cel3C CBH T. reesei BGL 0 Trichoderma reesei Trichoderma reesei 0
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CH 2 H H H CH 2 n-1 H CH 2 n H Potato α-glucan phosphorylase H n soluble starch P i n-1 H P() 2 glucose-1-phosphate Pi H CBPase H glucose P i glucose-1-phospate cellobiose H cellooligosaccharide n+1 CDPase
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