Regulation of β-Glucosidase in K. marxianus 75 Applied Biochemistry and Biotechnology Vol. 117, 2004 75 *Author to whom all correspondence and reprint requests should be addressed. Copyright © 2004 by Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/04/117/0075/$25.00 Influence of Carbon and Nitrogen Sources and Temperature on Hyperproduction of a Thermotolerant β-Glucosidase From Synthetic Medium by Kluyveromyces marxianus M. I. RAJOKA,* SAMIA KHAN, FAROOQ LATIF, AND RIAZ SHAHID National Institute for Biotechnology and Genetic Engineering (NIBGE), PO Box 577, Faisalabad, Pakistan, E-mail: mirajoka@nibge.org Abstract The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the indepen- dent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and pro- ductivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y P/S ) and volumetric productivity (Q P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deac- tivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes. Index Entries: β-Glucosidase; cellobiose; corn steep liquor; Kluyveromyces marxianus; shake flask. Introduction β-Glucosidase (EC 3.2.1.21) is one of the component enzymes of the cellulase complex and is widely distributed in nature. It catalyzes the