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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.
Original language | English |
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Pages (from-to) | 354-362 |
Number of pages | 9 |
Journal | BIOCHIMICA ET BIOPHYSICA ACTA: GENERAL SUBJECTS |
Volume | 1860 |
Issue number | 2 |
DOIs | |
Publication status | Published - 1 Feb 2016 |
MoE publication type | A1 Journal article-refereed |
Keywords
- Carbohydrate binding module
- Fusion proteins
- Galactoglucomannan
- Galactomannan
- Galactose oxidase
- Galactoxyloglucan
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Projects
- 1 Active
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BHIVE: Bio-derived HIgh Value polymers through novel Enzyme function
Mollerup, F., Karppi, J., Jurak, E., Aumala, V., Master, E., Monschein, M., Parviainen, T., Dahiya, D. & Pohto, A.
01/09/2015 → 31/03/2021
Project: EU: ERC grants
Equipment
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Bioeconomy Research Infrastructure
Jukka Seppälä (Manager)
School of Chemical EngineeringFacility/equipment: Facility