Highly Thermostable Xylanase Production from A Thermophilic Geobacillus sp. Strain WSUCF1 Utilizing Lignocellulosic Biomass

Abstract

Efficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylooligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail) when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70°C, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70°C, respectively. At 70°C, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, Cellic-HTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70°C). High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.

Keywords: biofuels; corn stover; prairie cord grass; thermostable; untreated lignocellulose; xylanase.

Figure 1

Effect of (A) growth pH and (B) growth temperature on production of WSUCF1 xylanase. Enzyme production at optimum pH and optimum temperature was defined as 100% (19.3 and 18.7 U/ml, respectively). Values shown were the mean of duplicate experiments, and the variation about the mean was below 5%.

Figure 2

Effects of xylan and various inexpensive lignocellulosics (prairie cordgrass – PCG, corn stover – CS, pretreated prairie cordgrass – PPCG, and pretreated corn stover – PCS) on xylanase production by WSUCF1.

Figure 3

SDS-PAGE (12%) and zymogram of WSUCF1 (A) endoxylanase activity, (B) β-xylosidase activity. Lane S, precision plus protein standards (BioRad); Lane CS, corn stover; Lane PCS, pretreated corn stover; Lane PCG, prairie cord grass; Lane PPCG; pretreated prairie cord grass.

Figure 4

Impact of (A) pH and (B) temperature on the xylanase activity of WSUCF1 isolate. The enzyme activity was expressed as percentages of the maximum activity. The points are the averages of triplicates, and error bars indicate ±SDs of the means (n = 3). Error bars smaller than the symbols are not shown.

Figure 5

Thermal stability of xylanase activity produced by WSUCF1 isolate. The enzyme activities were expressed as percentages of the initial activity. The points are the averages of triplicates, and error bars indicate ±SDs of the means (n = 3). Error bars smaller than the symbols are not shown.

Figure 6

Hydrolysis of Xylan from Birchwood – comparison of hydrolytic activity of WSUCF1 xylanase, Cellic HTec2, and Genencor Accellerase XY. The points are the averages of duplicates, and error bars indicate ±SDs of the means (n = 2). Error bars smaller than the symbols are not shown.