Hyper-production of a thermotolerant β-xylosidase by a deoxy-D-glucose and cycloheximide resistant mutant derivative of Kluyveromyces marxianus PPY 125

* Corresponding author Production of β-xylosidase by a cycloheximide and 2deoxy-D-glucose-resistant mutant of Kluyveromyces marxianus PPY125 was studied when cultured on growth media containing galactose, glucose, xylose, cellobiose, sucrose and lactose as carbon sources. Xylose, cellobiose, lactose and sucrose were the key substrates. Both K. marxianus PPY125 and its mutant (M 125) supported maximum β-xylosidase specific product yield (YP/X) following growth on xylose. Basal level of activity was observed in non-induced cultures grown on glucose. The mutant produced 1.5 to 2-fold more β-xylosidase than that produced by the wild cells. Synthesis of β-xylosidase was regulated by an induction mechanism in both wild and mutant cells. Addition of glucose did not inhibit the synthesis of β-xylosidase in both parental and mutant cultures in the presence of corn steep liquor. Partially purified enzyme showed good stability when incubated at 60oC and was quite stable at pH 5.0-7.0. Thermodynamic studies revealed that the enzyme derived by the mutant M125 was more thermostable as evidenced by higher midpoint inactivation temperature, lower activation energy demand for β-xyloside hydrolysis, as well as lower enthalpy and entropy demand for reversible denaturation of enzyme. β-xylosidase (EC 3.2.1.37) is one of the component enzymes of the hemicellulase complex and is widely distributed in nature. It catalyzes the hydrolysis of alkyland arylglycosides as well as xylobiose and xylooligosaccharides to xylose. In industry, it is employed for hydrolysis of bitter compounds from grape fruit during juice extraction and liberation of aroma from grapes during wine making (Manzanares et al. 1999). β-xylosidase, in synergistic action with endoβ-xylanases, (EC 3.2.1.8) and debranching enzymes namely α-glucuronidases, esterases and glycosidases have potential application in production of biofuels and in the processing of food. Cellulase-free xylanases have an important role in reducing consumption of chlorine and chlorine dioxide in paper and pulp industry (Tsujibo et al. 2001). Yeasts capable of producing glycosidases may be more suitable for the last application as they will require cloning of endo-xylanase gene only for this implication.