Enhancement of beta-Glucosidase Activity from a Brown Rot Fungus Fomitopsis pinicola KCTC 6208 by Medium Optimization

beta-Glucosidase, which hydrolyzes cellobiose into two glucoses, plays an important role in the process of saccharification of the lignocellulosic biomass. In this study, we optimized the activity of beta-glucosidase of brown-rot fungus Fomitopsis pinicola KCTC 6208 using the response surface methodology (RSM) with various concentrations of glucose, yeast extract and ascorbic acid, which are the most significant nutrients for activity of beta-glucosidase. The highest activity of beta-glucosidase was achieved 3.02% of glucose, 4.35% of yeast extract, and 7.41% ascorbic acid where ascorbic acid was most effective. The maximum activity of beta-glucosidase predicted by the RSM was 15.34 U/mg, which was similar to the experimental value 14.90 U/mg at the 16th day of incubation. This optimized activity of beta-glucosidase was 23.6 times higher than the preliminary activity value, 0.63 U/mg, and was also much higher than previous values reported in other fungi strains. Therefore, a simplified medium supplemented with a cheap vitamin source, such as ascorbic acid, could be a cost effective mean of increasing beta-glucosidase activity.

Biochemical Characterization of an Extracellular beta-Glucosidase from the Fungus, Penicillium italicum, Isolated from Rotten Citrus Peel

A beta-glucosidase from Penicillium italicum was purified with a specific activity of 61.8 U/mg, using a chromatography system. The native form of the enzyme was an 88.5-kDa tetramer with a molecular mass of 354 kDa. Optimum activity was observed at pH 4.5 and 60degrees C, and the half-lives were 1,737, 330, 34, and 1 hr at 50, 55, 60, and 65degrees C, respectively. Its activity was inhibited by 47% by 5 mM Ni2+. The enzyme exhibited hydrolytic activity for p-nitrophenyl-beta-D-glucopyranoside (pNP-Glu), p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-xyloside, and cellobiose, however, no activity was observed for p-nitrophenyl-beta-D-lactopyranoside, p-nitrophenyl-beta-D-galactopyranoside, carboxymetyl cellulose, xylan, and cellulose, indicating that the enzyme was a beta-glucosidase. The kcat/Km (s-1 mM-1) values for pNP-Glu and cellobiose were 15,770.4 mM and 6,361.4 mM, respectively. These values were the highest reported for beta-glucosidases. Non-competitive inhibition of the enzyme by both glucose (Ki = 8.9 mM) and glucono-delta-lactone (Ki = 11.3 mM) was observed when pNP-Glu was used as the substrate. This is the first report of non-competitive inhibition of beta-glucosidase by glucose and glucono-delta-lactone.

Proteomic Evaluation of Cellular Responses of Saccharomyces cerevisiae to Formic Acid Stress

Formic acid is a representative carboxylic acid that inhibits bacterial cell growth, and thus it is generally considered to constitute an obstacle to the reuse of renewable biomass. In this study, Saccharomyces cerevisiae was used to elucidate changes in protein levels in response to formic acid. Fifty-seven differentially expressed proteins in response to formic acid toxicity in S. cerevisiae were identified by 1D-PAGE and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analyses. Among the 28 proteins increased in expression, four were involved in the MAP kinase signal transduction pathway and one in the oxidative stress-induced pathway. A dramatic increase was observed in the number of ion transporters related to maintenance of acid-base balance. Regarding the 29 proteins decreased in expression, they were found to participate in transcription during cell division. Heat shock protein 70, glutathione reductase, and cytochrome c oxidase were measured by LC-MS/MS analysis. Taken together, the inhibitory action of formic acid on S. cerevisiae cells might disrupt the acid-base balance across the cell membrane and generate oxidative stress, leading to repressed cell division and death. S. cerevisiae also induced expression of ion transporters, which may be required to maintain the acid-base balance when yeast cells are exposed to high concentrations of formic acid in growth medium.