Filip Mollerup1, Kirsti Parikka2, Thu V Vuong3, Maija Tenkanen2, Emma Master4. 1. Department of Biotechnology and Chemical Technology, Aalto University, 00076 Aalto, Finland. 2. Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 27, Helsinki 00014, Finland. 3. Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada. 4. Department of Biotechnology and Chemical Technology, Aalto University, 00076 Aalto, Finland; Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada. Electronic address: emma.master@utoronto.ca.
Abstract
BACKGROUND: Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO. RESULTS: Both GaO-CBM29 and CBM29-GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, kcat/Km values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO-CBM29. MAJOR CONCLUSIONS: Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO-CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs. GENERAL SIGNIFICANCE: This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time.
BACKGROUND:Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO. RESULTS: Both GaO-CBM29 and CBM29-GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, kcat/Km values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO-CBM29. MAJOR CONCLUSIONS: Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO-CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs. GENERAL SIGNIFICANCE: This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time.
Authors: Martina Andberg; Filip Mollerup; Kirsti Parikka; Sanna Koutaniemi; Harry Boer; Minna Juvonen; Emma Master; Maija Tenkanen; Kristiina Kruus Journal: Appl Environ Microbiol Date: 2017-09-29 Impact factor: 4.792
Authors: Filip Mollerup; Ville Aumala; Kirsti Parikka; Yann Mathieu; Harry Brumer; Maija Tenkanen; Emma Master Journal: PLoS One Date: 2019-05-15 Impact factor: 3.240