|Li, Xin liang|
|Cotta, Michael - Mike|
Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/27/2003
Publication Date: 3/1/2004
Citation: Li, X., Ljungdahl, L.G., Ximenes, E.A., Chen, H., Felix, C.R., Cotta, M.A., Dien, B.S. 2004. Properties of a recombinant beta-glucosidase from the polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis. Journal of Applied Biochemistry and Biotechnology. 113:233-250. Interpretive Summary: Efficient conversion of lignocellulosic biomass such as rice straw and corn stalks to fermentable sugars has been recognized as the major bottleneck for the economical production of biofuels and feedstock chemicals from these and similar renewable resources. There are three major constitutes, cellulose, hemicellulose, and lignin, commonly found in lignocellulosic biomass. Biological degradation of the three constitutes requires many different enzymes working together in concert. The most needed enzymes are those which tackle cellulose and hemicelluloses. More specifically, the breakdown of cellulose requires the enzymes cellobiohydrolases, endoglucanases, and beta-glucosidases. The three types of enzymes have been found in a variety of bacteria and fungi, but only a handful of microbes can completely degrade native crystalline cellulose. Enzymes of anaerobic fungi have been demonstrated to be a source for the most active cellulases and hemicellulases. However, anaerobic fungi are hard to grow because they require strictly anaerobic cultivation techniques, and cellulases of these organisms have only recently been investigated. Beta-glucosidase has been recognized as a key enzyme and found to be limited in cellulase products currently on the market. The present paper reports a beta-glucosidase gene cloned from an anaerobic fungus, Orpinomyces PC-2, and the properties of its coded product in relation to biomass conversion. This enzyme offers many favorable features in comparison to beta-glucosidases of other organisms. Its potential for biomass saccharification is demonstrated in this paper as the recombinant enzyme produced by the baker's yeast to greatly improve the conversion of cellulose to glucose, the most commonly found fermentable sugar used for biofuel and chemical feed-stock manufacturing.
Technical Abstract: A beta-glucosidase (BglA, EC 126.96.36.199) gene, from the polycentric anaerobic fungus Orpinomyces PC-2, was cloned and sequenced. The enzyme containing 657 amino acid residues was homologous to certain animal, plant, and bacterial beta-glucosidases but lacked significant similarity to those from aerobic fungi. Neither cellulose- nor protein-binding domains were found in BglA. When expressed in Saccharomyces cerevisiae, the enzyme was secreted in two forms with masses of about 110 kDa and also found in two forms associated with the yeast cells. Km and Vmax values of the secreted BglA were 0.762 mM and 8.20 umole/min/mg, respectively, with p-nitrophenyl glucoside (pNPG) as the substrate and 0.310 mM and 6.45 umole/min/mg, respectively, for the hydrolysis of cellobiose. Glucose competitively inhibited the hydrolysis of pNPG with a Ki of 3.6 mM. The beta-glucosidase significantly enhanced the conversion of cellulosic materials into glucose by Trichoderma reesei cellulase preparations, demonstrating its potential in use for biofuel and feed-stock chemical production.