title: Scaled-up separation of cellobiohydrolase1 from a cellulase mixture by ion-exchange chromatography.
abstract: Enzymatic hydrolysis of cellulose often involves cellulases produced by Trichoderma reesei, of which cellobiohydrolase1 (CBH1) is the most abundant (about 60% of total cellulases) and plays an important role in the hydrolysis of crystalline cellulose. A method for separating sufficient quantities from the bulk cellulase cocktail is highly desirable for many studies, such as those that aim to characterize binding and hydrolysis kinetics of CBH1. In this work, CBH1 was separated from other Spezyme CP cellulases by ion-exchange chromatography using an efficient modification of a smaller scale process. The ion-exchange column was connected to a vacuum manifold system to provide a steady flow through parallel columns and thus achieve scale-up for enzyme separation. With five 5-mL columns running in parallel, about 55 mg of CBH1 was separated from 145 mg of Spezyme CP in a single separation. Step elution was used to replace the continuous gradient used at smaller scale. The purified CBH1 was collected in the fraction eluted with a buffer containing 0.33 M salt and showed comparable purity and activity as the enzyme purified by a fast protein liquid chromatography system. The stability of separated CBH1 was studied for up to 2 days and good thermal stability was observed. Separated CBH1 also showed both high adsorption to bacterial microcrystalline cellulose with ~4 μmol/g maximum adsorption and a K(a) of 5.55 ± 2.34 μM(-1) , and good hydrolytic activity based on atomic force microscopy observations that show a reduction in fiber height.
authors: Dept. of Chemical Engineering, University of Louisville, Louisville, KY 40292, USA.
authors: Zhuoliang
authors: Z
authors: Ye
authors: Andrew N
authors: AN
authors: Lane
authors: Gerold A
authors: GA
authors: Willing
authors: R Eric
authors: RE
authors: Berson
mesh_terms: D000327:Adsorption; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D007218:Industrial Microbiology / Q000191:economics / Q000379:methods*; D007700:Kinetics; D014242:Trichoderma / Q000737:chemistry / Q000201:enzymology*
chemical_list: D005656:Fungal Proteins; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase