Purification of xylanases from Aureobasidium pullulans CCT 1261 and its application in the production of xylooligosaccharides

Xylanolytic enzymes are involved in xylan hydrolysis, the main ones being endo-beta-1,4-xylanases (xylanases). This can be applied in the bioconversion of lignocellulosic materials into value-added products such as xylooligosaccharides (XOS). This study aimed to establish a protocol for the purification of xylanases, as well as to characterize and apply the purified enzyme extract in the production of XOS. The enzyme purification techniques studied were ammonium sulfate ((NH4)(2)SO4) and ethanol precipitation. Purification of xylanase by fractional precipitation (20-60%) with (NH4)(2)SO4 was more efficient than with ethanol because the salt precipitation reached a purification factor of 10.27-fold and an enzymatic recovery of 48.6% The purified xylanase exhibited optimum temperature and pH of 50 degrees C and 4.5, respectively. The Michaelis-Menten constant using beechwood xylan for the enzyme was 74.9 mg/mL. The addition of salts such as CaCl2, ZnCl2, and FeCl3 in the reaction medium increased the xylanase activity. Xylanase showed greater thermal stability (half-life = 169 h) at 45 degrees C and pH 4.5. Under these conditions and in the presence of Ca2+ (10 mmol/L) the enzyme was even more stable (half-life = 231 h). Total XOS contents (6.7 mg/mL) and the conversion of xylan to XOS (22.3%) between 2 and 24 h were statistically equal. The hydrolysates showed the majority composition of xylobiose, xylotriose, and xylose. The addition of Ca2+ ions did not contribute to an increase in the total XOS content or to a greater conversion of xylan into XOS, but the hydrolysate was richer in xylobiose and had a lower xylose content.

Automated summary

This study successfully established a method for the purification of xylanases and investigated their application in the production of xylooligosaccharides (XOS). Fractional precipitation with ammonium sulfate was found to be more efficient for xylanase purification compared to ethanol precipitation. The purified xylanase showed optimal activity at a temperature of 50°C and a pH of 4.5. Addition of salts such as CaCl2, ZnCl2, and FeCl3 increased the xylanase activity. Xylanase exhibited good thermal stability at 45°C and pH 4.5, and the stability was enhanced in the presence of Ca2+ ions. XOS production from xylan was achieved with a conversion rate of 22.3%. The hydrolysates mainly contained xylobiose, xylotriose, and xylose. The addition of Ca2+ ions did not significantly affect the XOS content, but altered the composition of the hydrolysate.