Enhanced thermostability of a fungal alkaline protease by different additives

A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10-15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl, KHPO, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory

<span style="font-size:11.0pt;mso-bidi-font-size: 10.0pt;font-family:"Times New Roman";mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language:AR-SA" lang="EN-GB">Purification and biochemical characterization of endoglucanase from <i>Penicillium pinophilum </i>MS 20</span>

189-194<span style="mso-bidi-font-size:9.0pt;letter-spacing: -.1pt" lang="EN-GB">Cellulases find increasing prominence in sustainable production of fuel and feedstock from lignocellulosic biomass. The purification and biochemical characterization of individual components of cellulase complex is important to understand the mechanism of their action for the solubilization of crystalline cellulose. In this study, an extra-cellular endoglucanase isolated from culture filtrate of Penicillium pinophilum MS 20 was purified to homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. The purified endoglucanase (specific activity 69 U/mg) was a monomeric protein with molecular mass of 42 kDa, as determined by SDS-PAGE. The endoglucanase was active over a broad range of pH (4-7) with maximum activity at pH 5 and showed optimum temperature of 50°C. It retained 100% activity at 50°C for 6 h and half- lives of 4 h and 3 h at 60°C and 70°C, respectively. The kinetic constants for the endoglucanase determined with carboxymethyl cellulose as substrate were Vmax of 72.5 U/mg and apparent Km of 4.8 mg/ml. The enzyme also showed moderate activity towards H3PO4 swollen cellulose and p-nitrophenyl β-D-glucoside, but no activity towards filter paper, Avicel and oat spelt xylan. The activity was positively modulated by 47, 32 and 25% in the presence of Co2+, Zn2+ and Mg2+, respectively to the reaction mixture. The wide pH stability (4-7) and temperature stability up to 50°C of endoglucanase makes the enzyme suitable for use in cellulose saccharification at moderate temperature and pH. </span