Comparative characterization of extracellular enzymes secreted by Phanerochaete chrysosporium during solid-state and submerged fermentation.

Influence of water content on the expression of lignocellulolytic enzymes by Phanerochaete chrysosporium remains unclear. This work compares the enzyme production profiles of P. chrysosporium during solid-state and submerged fermentation. There were 110 and 64 extracellular carbohydrate-active enzymes identified in solid-state and submerged fermentation respectively, among which 57 enzymes were common to both of the secretomes. P. chrysosporium secreted more cellulases (especially lytic polysaccharide monooxygenase) and hemicellulases during solid-state fermentation while the proportion of enzyme containing carbohydrate-binding module was higher for submerged fermentation. Although its activities were weaker, the enzyme cocktail from submerged fermentation was surprisingly more effective in hydrolysis at low substrate loading. This advantage of enzymes from submerged fermentation was mainly attributed to carbohydrate-binding module because more xylanases bound with substrate at the beginning of hydrolysis. These results reveal the influence of fermentation conditions on enzyme produced by P. chrysosporium for the first time and show the importance of carbohydrate-binding module in the hydrolysis process of lignocellulose.

Efficient ferulic acid and xylo-oligosaccharides production by a novel multi-modular bifunctional xylanase/feruloyl esterase using agricultural residues as substrates.

Liberating high value-added compounds ferulic acid (FA) and xylo-oligosaccharides (XOSs) from agricultural residues is a promising strategy for the utilization of lignocellulose. In this study, a bifunctional xylanase/feruloyl esterase from bacterial consortium EMSD5 was heterogeneously expressed in Escherichia coli. Depending on the inter-domain synergism of the recombinant enzyme rXyn10A/Fae1A, high yields of FA (2.78, 1.82, 1.15 and 7.31 mg/g substrate, respectively) were obtained from 20 mg in-soluble wheat arabinoxylan, de-starched wheat bran, ultrafine-grinding corn stover and steam-exploded corncob. Meanwhile, 3.210, 1.235, 1.215 and 0.823 mg xylose/XOSs were also released. For cost-saving enzyme production, we firstly constructed a recombinant E. coli, which could secrete the bifunctional xylanase/feruloyl esterase out of cells. When the recombinant E. coli was cultured in medium containing 200 mg de-starched wheat bran, 474 µg FA and 18.2 mg xylose/XOSs were also detected. Hence, rXyn10A/Fae1A and the recombinant strain showed great applied potential for FA and XOSs production.

Characterization of a high performance α-galactosidase from Irpex lacteus and its usage in removal of raffinose family oligosaccharides from soymilk.

Raffinose family oligosaccharides (RFOs) negatively affect nutritional value of legume-derived food and feed. It has been challenging to develop a high performance α-galactosidase excelled on catalytic efficiency, thermostability, pH stability and protease-resistance that could efficiently hydrolyze RFOs. In this study, the first GH family 27 α-galactosidase gene from Irpex lacteus was cloned. The gene had an open reading frame of 1314 bp interrupted by 12 introns. The recombinant α-galactosidase expressed in Pichia pastoris (rILgalA) had an apparent molecular mass of 64 kDa and was highly N-glycosylated. rILgalA was maximally active at pH 4.8 and 70 °C. It was stable over a broad pH range of 3-11, retained 90% of its activity after incubation at 60 °C for 10 h and exhibited strong resistance to digestive proteases. Unlike many other α-galactosidases, rILgalA was hyperactive on RFOs. Its specific activities toward melibiose, raffinose and stachyose were 644, 755 and 833 U mg-1, respectively. The corresponding Kcat/Km values were 120, 130 and 180 mM-1 s-1, which were the highest among reported α-galactosidases. rILgalA almost completely hydrolyzed raffinose and stachyose in soymilk at 60 °C in 30 min. These superior properties would make rILgalA an ideal remover of RFOs in food and feed industries.