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The early-lineage, aerobic, zoosporic fungi from the Chytridiomycota constitute less than 1% of the described fungi and can use diverse sources of nutrition from plant or animal products. One of the ancestral sources of fungal nutrition could be products following enzymatic degradation of plant material. However, carbohydrate-active enzymes from these ancient fungi have been less studied. A GH11 xylanase (RrXyn11A) (EC 3.2.1.8) and a GH43 xylosidase (RrXyl43A) (EC 3.2.1.37) were identified from an early-lineage aerobic zoosporic fungus, Rhizophlyctis rosea NBRC 105426. Both genes were heterologously expressed in Pichia pastoris and the recombinant enzymes were purified and characterized. The optimal pH for recombinant RrXyn11A and RrXyl43A was pH 7. RrXyn11A had high stability over a wide range of pH (4-8) and temperature (25-70 °C). RrXyn11A also showed high substrate specificity on both azurine-cross-linked (AZCL) arabinoxylan and AZCL xylan. RrXyl43A had β-xylosidase and minor α-L-arabinofuranosidase activity. This enzyme showed low product inhibition and retained 51% activity in the presence of 100 mM xylose. A combination of RrXyn11A and RrXyl43A exhibited significantly higher hydrolytic and polymer degradation capability and xylose release on wheat bran and beechwood xylan compared to treatment with commercial enzymes. This study was the first to heterologously express and characterize the GH11 xylanase (RrXyn11A) and GH43 xylosidase (RrXyl43A) from the ancient fungus, R. rosea. Meanwhile, this study also demonstrated that the enzymes from the ancient fungus R. rosea can be easily handled and heterologously expressed in Pichia, which presents a promising path to a new source of enzymes for biomass degradation. [ABSTRACT FROM AUTHOR]

Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β- d-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose ( N, N′-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β- d-glucosaminide (1 → 4)-β-linkages and are thus ' exo-chitobiose hydrolases.' In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose. [ABSTRACT FROM AUTHOR]