Purification and Characterization of Cellobiose Dehydrogenase from the Plant PathogenSclerotium(Athelia)rolfsii

Abstract

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochrome c, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, ␤-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

“…2a and Supplementary Tables 5-7). As 17 expected, the genes encoding enzymes used for modification of fungal cell walls (e.g., GH16,18 GH17 and GH72 proteins) are expressed at similar levels during growth on glucose and plant 19 straws for the two organisms. In contrast, transcripts encoding enzymes that deconstruct plant 20 cell walls are upregulated only during growth on barley or alfalfa straws.…”

“…The major difference occurs in 15 chromosome (Ch)1 of T. terrestris, which harbors most of the genes located on Ch2 and Ch4 of 16 M. thermophila. In addition, extensive translocation is observed between Ch1/Ch6 of M. 17 thermophila and Ch2/Ch5 of T. terrestris. The protein coding fractions of the genomes include 18 9,110 genes in M. thermophila and 9,813 genes in T. terrestris ( Table 1); both are smaller than 19 average proteomes of other fungi in the class Sordariomycetes, and substantially smaller than the 20 closely related mesophile Chaetomium globosum 7 , which has 11,124 predicted genes in a 34.…”

“…The best studied and most widely used cellulases and 15 hemicellulases are produced by Trichoderma, Aspergillus and Penicillium species, and they are 16 most effective over a temperature range from 40 °C to ~50 °C. At these temperatures, complete 17 saccharification of biomass polysaccharides (>90% conversion to fermentable sugars) requires 18 long reaction times, during which hydrolysis reactors are susceptible to contamination. One way 19 to overcome these obstacles is to raise the reaction temperature, thereby increasing hydrolytic 20 rates and reducing contamination risks.…”

“…The protein coding fractions of the genomes include 18 9,110 genes in M. thermophila and 9,813 genes in T. terrestris ( Table 1); both are smaller than 19 average proteomes of other fungi in the class Sordariomycetes, and substantially smaller than the 20 closely related mesophile Chaetomium globosum 7 , which has 11,124 predicted genes in a 34. [9][10][11][12][13][14][15][16][17][18][19][20][21] Mbp genome. These three species within the family Chaetomiaceae share 6,279 three-way 22 orthologs and extensive synteny with >6,000 genes in syntenic blocks between each pair, 23 including four blocks of >400 genes between M. thermophila and T. terrestris (Supplementary Table 1).…”

“…4). The two thermophiles can be 16 considered all-purpose decomposers with respect to their CAZymes and their ability to degrade 17 plant polysaccharides (Supplementary Fig. 5).…”