(274d) Thermostable Cellulases and Xylanases

Thermostable cellulases and xylanases offer several advantages including greater rates of hydrolysis for lignocellulosic substrates, lowered risk of contamination, and increased flexibility with respect to process design.  We have isolated several thermophilic cellulose- and xylan-degrading cultures belonging to the genera Bacillus, Clostridium, and Geobacillus using soil samples collected from the Homestake Gold Mine, SD and a local compost facility.  One of the isolates, Bacillus sp. (DUSELR13 strain) produced xylanase which had optimum pH and temperature of 7 and 75⁰C, respectively.  Other isolate Geobacillus sp. (WSUCF1 strain) secreted endoglucanase having maximum activity at pH 5 and temperature 70⁰C.  The thermal stability of enzymes secreted by DUSELR13 and WSUCF1 was tested by determining the enzyme activity remaining after incubation of the enzymes at different temperatures (50 -100⁰C) for a period of 40 days.  WSUCF1 endoglucanase showed remarkable residual activity (>50%) after prolonged incubation for 25 days at 60⁰C compared to others reported in literature.  DUSELR13 xylanase retained >50% residual activity after incubation for 35 days at 60⁰C.  The effect of various lignocellulosic feedstocks on the endoglucanase and xylanase secretion by these isolates was also examined.  K_m values for DUSEL R13 xylanase and WSUCF1 endoglucanase were calculated using Lineweaver–Burk plots.  In case of DUSELR13 xylanase, the K_m value with xylan from birchwood was 1.4 mg/ml while for WSUCF1 endoglucanase with carboxymethylcellulose this value was 1.08 mg/ml. These thermostable enzymes were overexpressed extracellularly using E.coli as a heterologous host. Comparison studies were carried out between recombinant enzymes and native enzymes. These thermostable cellulases and xylanases are currently being used to develop a novel cost-effective and more efficient form of the simultaneous saccharification and fermentation process for biorefineries.