A fragment of the ß-glucosidase gene from the rumen fungus Neocallimastix patriciarum J11 encodes a recombinant protein that exhibits activities in ß-glucosidase and ß-glucanase.

Lignocellulose, the most abundant organic waste on Earth, is of economic value because it can be converted into biofuels like ethanol by enzymes such as ß-glucosidase. This study involved cloning a ß-glucosidase gene named JBG from the rumen fungus Neocallimastix patriciarum J11. When expressed recombinantly in Escherichia coli, the rJBG enzyme exhibited significant activity, hydrolyzing 4-nitrophenyl-ß-d-glucopyranoside and cellobiose to release glucose. Surprisingly, the rJBG enzyme also showed hydrolytic activity against ß-glucan, breaking it down into glucose, indicating that the rJBG enzyme possesses both ß-glucosidase and ß-glucanase activities, a characteristic rarely found in ß-glucosidases. When the JBG gene was expressed in Saccharomyces cerevisiae and the transformants were inoculated into a medium containing ß-glucan as the sole carbon source, the ethanol concentration in the culture medium increased from 0.17 g/L on the first day to 0.77 g/L on the third day, reaching 1.3 g/L on the fifth day, whereas no ethanol was detected in the yeast transformants containing the recombinant plasmid pYES-Sur under the same conditions. These results demonstrate that yeast transformants carrying the JBG gene can directly saccharify ß-glucan and ferment it to produce ethanol. This gene, with its dual ß-glucosidase and ß-glucanase activities, simplifies and reduces the cost of the typical process of converting lignocellulose into bioethanol using enzymes and yeast.

Catalytic efficiency diversification of duplicate ß-1,3-1,4-glucanases from Neocallimastix patriciarum J11.

Four types of ß-1,3-1,4 glucanase (ß-glucanase, EC 3.2.1.73) genes, designated bglA13, bglA16, bglA51, and bglM2, were found in the cDNA library of Neocallimastix patriciarum J11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias to Streptococcus equinus. The presence of expansion and several predicted secondary structures in the 3' untranslated regions (3'UTRs) of bglA16 and bglM2 suggest that these two genes were duplicated recently, whereas bglA13 and bglA16, which contain very short 3'UTRs, were replicated earlier. These findings indicate that the ß-glucanase genes from N. patriciarum J11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. ß-Glucanase genes of Streptococcus equinus, Ruminococcus albus 7, and N. patriciarum J11 were cloned and expressed by Escherichia coli. The recombinant ß-glucanases cloned from S. equinus, R. albus 7, and N. patriciarum J11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial ß-glucanases were also significantly lower than those of the fungal ß-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal ß-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived ß-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated ß-glucanases, showed much higher k(cat) values than others. These results support the notion that duplicated ß-glucanase genes, namely, bglA16 and bglM2, increase the reaction efficiency of ß-glucanases and suggest that the catalytic efficiency of ß-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms in N. patriciarum J11.

A highly active beta-glucanase from a new strain of rumen fungus Orpinomyces sp.Y102 exhibits cellobiohydrolase and cellotriohydrolase activities.

A new strain of rumen fungus was isolated from Bos taurus, identified and designated Orpinomyces sp.Y102. A clone, celC7, isolated from the cDNA library of Orpinomyces sp.Y102, was predicted to encode a protein containing a signal peptide (Residues 1-17), an N-terminal dockerin-containing domain, and a C-terminal cellobiohydrolase catalytic domain of glycoside hydrolase family 6. CelC7 was insoluble when expressed in Escherichia coli. Deletion of 17 or 105 residues from the N-terminus significantly improved its solubility. The resulting enzymes, CelC7(-17) and CelC7(-105), were highly active to ß-glucan substrates and were stable between pH 5.0 and 11.0. CelC7(-105) worked as an exocellulase releasing cellobiose and cellotriose from acid-swollen Avicel and cellooligosaccharides, and displayed a Vmax of 6321.64µmole/min/mg and a Km of 2.18mg/ml to barley ß-glucan. Further, the crude extract of CelC7(-105) facilitated ethanol fermentation from cellulose. Thus, CelC7(-105) is a good candidate for industrial applications such as biofuel production.