Background. The anaerobic gut fungi (phylum Neocallimastigomycota) represent a
promising source of novel lignocellulolytic enzymes. Here, we report on the cloning,
expression, and characterization of a glycoside hydrolase family 39 (GH39) enzyme
(Bgxg1) that is highly transcribed by the anaerobic fungus Orpinomyces sp. strain C1A
under different growth conditions. This represents the first study of a GH39-family enzyme
from the anaerobic fungi. Methods. Using enzyme activity assays, we performed a
biochemical characterization of Bgxg1 on a variety of substrates over a wide range of pH
and temperature values to identify the optimal enzyme conditions and the specificity of
the enzyme. In add... More
Background. The anaerobic gut fungi (phylum Neocallimastigomycota) represent a
promising source of novel lignocellulolytic enzymes. Here, we report on the cloning,
expression, and characterization of a glycoside hydrolase family 39 (GH39) enzyme
(Bgxg1) that is highly transcribed by the anaerobic fungus Orpinomyces sp. strain C1A
under different growth conditions. This represents the first study of a GH39-family enzyme
from the anaerobic fungi. Methods. Using enzyme activity assays, we performed a
biochemical characterization of Bgxg1 on a variety of substrates over a wide range of pH
and temperature values to identify the optimal enzyme conditions and the specificity of
the enzyme. In addition, substrate competition studies and comparative modeling efforts
were completed. Results. Contrary to the narrow range of activities (β-xylosidase or α-Liduronidase)
observed in previously characterized GH39 enzymes, Bgxg1 is unique in that
it is multifunctional, exhibiting strong β-xylosidase, β-glucosidase, β-galactosidase
activities (11.5 ± 1.2, 73.4 ± 7.15, and 54.6 ± 2.26 U/mg, respectively) and a weak
xylanase activity (10.8 ± 1.25 U/mg), strength determined as compared to previously
characterized enzymes. Physiological characterization revealed that Bgxg1 is active over a
wide range of pH (3-8, optimum 6) and temperatures (25-60°C, optimum 39°C), and
possesses excellent temperature and thermal stability. Substrate competition assays
suggest that all observed activities occur at a single active site. Using comparative
modeling and bioinformatics approaches, we putatively identified ten amino acid
differences between Bgxg1 and previously biochemically characterized GH39 β-
xylosidases that we speculate could impact active site architecture, size, charge, and/or
polarity. The putative contributions of these changes to the observed relaxed specificities
in Bgxg1 are discussed. Discussion. Collectively, the unique capabilities and multifunctionality
of Bgxg1 render it an excellent candidate for inclusion in enzyme cocktails
mediating cellulose and hemicellulose saccharification from lignocellulosic biomass.
