José Cardoso Duarte

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

Abstract A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml−1 was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l−1 h−1, and in reactor, 104,000 U l−1 h−1 was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l−1 h−1, compared to shake-flask fermentations, 12,000 U l−1 h−1. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.

Electroelution as a simple and fast protein purification method : isolation of an extracellular xylanase from Bacillus sp. CCMI 966

An efficient and simple modified method of electroelution is described that can be used as a time-saving method for eluting multiple protein bands. Provided that the proteins are highly expressed, they can be purified rapidly and without requiring any prior knowledge of the protein characteristics. A xylanase excreted by Bacillus sp. CCMI 966 was purified directly from the polyacrylamide gel. Some of the properties of this enzyme are presented. It had an unusually apparent high molecular mass of 340kDa, as determined by native PAGE. The specific activity of the purified xylanase was 137 U/mg.

On the relationship between cellobiose dehydrogenase and cellobiose:quinone oxidoreductase under conditions where [14C]DHP is mineralized by whole cultures of Phanerochate chrysosporium

Phanerochaete chrysosporium ME-446 cultivated in celluose-based media degraded synthetic lignin to about the same extent in high-nitrogen (24 mM N as asparagine) and low-nitrogen (2.4 mM) media when Tween 80 was employed at 0.15%, and 0.075%, respectively. Under these specified conditions of high 14C-DHP itmineralization which occurred after 6 days of cultivation, the amount of lignin peroxidase (113 U l−1) in high-N media was much higher than that in low N (17 U l−1 LiP). In contrast, the Mn-dependent peroxidase activity was not affected by the N concentration. Likewise, the total (extracellular plus bound) cellobiose:quinone oxidoreductase activity was of the same order (about 15 to 25 U l−1) in both high- and low-N/cellulose-based media. These data suggest that the amount of cellobiose:quinone oxidoreductase/cellobiose dehydrogenase formed is critical for optimal ligninolytic activity and that too high levels of these enzymes may be inhibitory. The use of the cellulose-based media has made it possible to observe a wide spectrum of enzymatic activities, including protease activity. The relationship among these enzymes is analyzed in the light of the synthetic lignin degradation.