Kinetic phenomena in the enzymatic hydrolysis of wheat arabinoxylan
Special Symposium - Biotechnology
Invited Session on Biotechnology: Honoring Prof. John Villadsen (S-3)
Keywords: arabinoxylan, enzyme kinetics, b-xylosidase, a-L-arabinofuranosidase
A.S. Meyer oral presentation at John Villadsen symposium ECCE-6
Kinetic phenomena in the enzymatic hydrolysis of wheat arabinoxylan
A.S. Meyer1a, A. Arnous1b, M. K. Nielsen2
1 Dept. of Chemical Engineering, Technical University of Denmark, 2800 Lyngby, Denmark
1a am@kt.dtu.dk; 1b aar@kt.dtu.dk
2 BioCentrum-DTU, Technical University of Denmark, 2800 Lyngby, Denmark
mkn@biocentrum.dtu.dk
Arabinoxylans constitute one of the major types of structural polysaccharides in cell walls of cereal grains. Arabinoxylans consist of a linear backbone of b-1,4-linked D-xylopyranosyl units which are partially substituted with arabinofuranosyls that may be attached as (1→2) or (1→3) to xylose, i.e. singly substituted xyloses, or as (1→2) and (1→3) to doubly substituted xyloses. The enzymatic degradation of arabinoxylans is of increasing importance in e.g. bioethanol processes, high-fibre bread baking, and beer brewing. The study of enzymatic hydrolysis of arabinoxylans is also a study case for enzyme kinetics on branched heteropolysaccharides that require different side-group cleaving and depolymerizing enzyme activities. The kinetics of enzyme catalyzed degradation of wheat arabinoxylan were assessed by quantifying the rates of enzyme catalyzed release of arabinose and xylose by HPAEC at different substrate concentrations during individual and combined treatments with: a) b-xylosidase and endo-b1,4-endoxylanase from a commercial enzyme preparation, Celluclast 1.5 L, produced by Trichoderma reesei, b) a-L-arabinofuranosidase (a-Ara 1) from Aspergillus niger attacking only a-1,3-linked arabinoses on singly substituted xyloses, and
c) a newly discovered a-L-arabinofuranosidase (a-Ara 2) produced by Bifidobacterium adolescentis attacking only a-1,3-linked arabinoses on doubly substituted xylose in wheat arabinoxylan. The effective substrate concentration (S0) for b-xylosidase attack was estimated via a statistical probability calculation of available non-reducing ends in the substrate. For purified soluble wheat arabinoxylan (Megazyme, Bray, Ireland) the averageDP of the xylan backbone was estimated to be ~1310 and the effective [S0] for b-xylosidase attack at 1.865 mg arabinoxylan/mL was 5.63mM. On this basis the effective [S0] was equal to [2.5 – (0.60 + 0.61)] × [Sreducing ends]. Vmax and Km values were calculated from Hanes plots of product release during initial reaction rate modes at pH 5, 50 °C, at initial arabinoxylan levels 0.0625-1.865 mg/mL. During extended reactions with T. reesei b-xylosidase (Celluclast 1.5 L) + a-Ara 1 (A. niger), the rates of xylose release increased. This rate increase was apparently due to the continuously increasing availability of new unsubstituted xyloses at the non-reducings ends allowing increased depolymerization of the xylan backbone plus the continued endo-b1,4-xylanase catalyzed release of new reducing ends for b-xylosidase attack. These findings as well as a further understanding of enzyme kinetics of complex polysaccharide degradation are important for designing optimal enzyme based reactions for arabinoxylan and other branched polymer modifications and for predicting optimal enzyme reaction times at different substrate concentrations and scales of reactions.
Presented Wednesday 19, 16:00 to 16:30, in session Invited Session on Biotechnology: Honoring Prof. John Villadsen (S-3).
