Kinetics and regioselectivity of three GH62 α-L-arabinofuranosidases from plant pathogenic fungi.

BACKGOUND: Xylan is the second most abundant plant cell wall polysaccharide after cellulose with α-L-arabinofuranose (L-Araf) as one of the major side substituents. Capacity to degrade xylan is characteristic of many plant pathogens; and corresponding enzymes that debranch arabinoxylan provide tools to tailor xylan functionality or permit its full hydrolysis. METHOD: Three GH62_2 family α-arabinofuranosidases (Abfs) from plant pathogenic fungi, NhaAbf62A from Nectria haematococca, SreAbf62A from Sporisorium reilianum and GzeAbf62A from Gibberella zeae, were recombinantly produced in Escherichia coli. Their biochemical properties and substrate specificities were characterized in detail. Particularly with 1H NMR, the regioselectivity and debranching preference of the three Abfs were directly compared. RESULTS: The activities of selected Abfs towards arabinoxylan were all optimal at pH 6.5. Their preferred substrates were wheat arabinoxylan, followed by soluble oat spelt xylan. The Abfs displayed selectivity towards either α-(1 → 2) or α-(1 → 3)-L- Araf mono-substituents in arabinoxylan. Specifically, SreAbf62A and GzeAbf62A removed m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substituents with a similar rates, whereas NhaAbf62A released m-α-(1 → 3)-L-Araf 1.9 times faster than m-α-(1 → 2)-L-Araf. MAJOR CONCLUSIONS: Building upon the known selectivity of GH62 family α-arabinofuranosidases towards L-Araf mono-substituents in xylans, the current study uncovers enzyme-dependent preferences towards m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substitutions. Comparative sequence-structure analyses of Abfs identified an arginine residue in the xylose binding +2R subsite that was correlated to the observed enzyme-dependent L-Araf debranching preferences. GENERAL SIGNIFICANCE: This study expands the limited pool of characterized GH62 Abfs particularly those from plant pathogenic fungi, and provides biochemical details and methodology to evaluate regioselectivity within this glycoside hydrolase family.

Constructing arabinofuranosidases for dual arabinoxylan debranching activity.

Enzymatic conversion of arabinoxylan requires α-L-arabinofuranosidases able to remove α-L-arabinofuranosyl residues (α-L-Araf) from both mono- and double-substituted D-xylopyranosyl residues (Xylp) in xylan (i.e., AXH-m and AXH-d activity). Herein, SthAbf62A (a family GH62 α-L-arabinofuranosidase with AXH-m activity) and BadAbf43A (a family GH43 α-L-arabinofuranosidase with AXH-d3 activity), were fused to create SthAbf62A_BadAbf43A and BadAbf43A_SthAbf62A. Both fusion enzymes displayed dual AXH-m,d and synergistic activity toward native, highly branched wheat arabinoxylan (WAX). When using a customized arabinoxylan substrate comprising mainly α-(1 → 3)-L-Araf and α-(1 → 2)-L-Araf substituents attached to disubstituted Xylp (d-2,3-WAX), the specific activity of the fusion enzymes was twice that of enzymes added as separate proteins. Moreover, the SthAbf62A_BadAbf43A fusion removed 83% of all α-L-Araf from WAX after a 20 hr treatment. 1 H NMR analyses further revealed differences in SthAbf62A_BadAbf43 rate of removal of specific α-L-Araf substituents from WAX, where 9.4 times higher activity was observed toward d-α-(1 → 3)-L-Araf compared to m-α-(1 → 3)-L-Araf positions.

Control of protozoa contamination and lipid accumulation in Neochloris oleoabundans culture: Effects of pH and dissolved inorganic carbon.

Combined effects of pH (i.e., 7.5, 8.5, and 9.5) and bicarbonate (i.e., 0, 80 and 160mM NaHCO3) on lipid accumulation and on biological contaminant viability in a protozoa-contaminated culture of the freshwater microalga Neochloris oleoabundans were studied. Cultures grown in the media containing 160mM NaHCO3 at pH 9.5 obtained the highest biomass concentration (DCWmax=1.32g/L), lipid content (LC=327mg/g), which corresponded to a lipid productivity of 56mg/(L·d), and the culture was protozoa free one day after inoculation. Other cultures, 160mM NaHCO3 at pH 8.5 (DCWmax=1.32g/L, LC=223mg/g), and 80mM NaHCO3 at pH 9.5 (DCWmax=1.25g/L, LC=264mg/g) could delay protozoan growth, but not inhibit it completely. These results suggest 160mM NaHCO3 or slightly above at pH levels of 8.5-9.5 may be used in outdoor cultivation processes of freshwater N. oleoabundans to control protozoa contamination while maintain a high lipid content.