Biochemical Engineering Journal 31 (2006) 8–13 Activity and stability of caffeine demethylases found in Pseudomonas putida IF-3 Juan G. Beltr´ an, Richard L. Leask ∗ , Wayne A. Brown Mc Gill University, Department of Chemical Engineering, 3610 University Street, Montreal, Que., Canada Received 6 July 2005; received in revised form 9 January 2006; accepted 12 May 2006 Abstract Resting cell suspensions and cell-free extracts of Pseudomonas putida IF-3 were tested to assess their ability to degrade caffeine, and to determine their capacity to retain activity at different temperatures. A method to quantify cell lysis using optical density was developed in order to allow the comparison of cell free extract and resting cell caffeine degradation rates on the same basis. Caffeine degradation rates for cell free extracts were found to be 2.4 mol g cells -1 min -1 ; this rate is 55 times faster than previously reported P. putida data. Resting cells degraded caffeine 12 times faster than cell free extracts, at 22 ◦ C. However, both systems were equivalently active at 50 ◦ C. Resting cells were significantly more stable than cell free extracts, retaining their ability to degrade caffeine even at elevated temperatures. Cell free extracts lost all activity after 15min at 55 ◦ C. © 2006 Elsevier B.V. All rights reserved. Keywords: Caffeine; Decaffeination; Enzymes; Enzyme activity; Enzyme deactivation; Cell disruption 1. Introduction The worldwide coffee market is estimated at 70 billion dol- lars per annum [1]. In the United States, decaffeinated coffee accounts for more than 12% of the coffee market [2]. Currently, almost all of the commercial decaffeination methods use sol- vents to remove caffeine from whole green coffee beans, prior to roasting. Extraction agents include methyl chloride, ethylene acetate, supercritical CO 2 , and hot water. Up to now, a process that targets freshly brewed coffee for decaffeination has not been commercialized, although there are many incentives for such an approach. For example, current decaffeination methods require dedicated facilities for decaffeination, which could be eliminated by targeting brewed coffee. A cup-by-cup decaffeination method would increase the variety accessible to the consumer, making all types of coffee available with or without caffeine. Such a process could turn decaffeination into a domestic operation, vir- tually eliminating the need to buy decaffeinated coffee beans. A decaffeination method that targets hot beverages should be highly selective for caffeine, in order to reduce caffeine content without affecting flavor. To this end, processes involving the use of enzymes are attractive, with both bacterial and fungal sources having been studied [3–12]. Of interest is the bacterium Pseu- ∗ Corresponding author. E-mail address: richard.leask@mcgill.ca (R.L. Leask). domonas putida [6,13,14] as a number of researchers have shown that selective caffeine removal by this organism is possible in aqueous solutions [3,5,15]. Caffeine degradation by P. putida has been proposed to start via three successive demethylation steps, followed by oxidation of xanthine to uric acid [12]. These steps are known to require oxygen and NADH as cofactors [3]. A number of factors must be considered in developing a decaffeination method applicable to brewed coffee. Amongst them, temperature is a key factor if a biological method to decaf- feinate hot beverages is to be developed. However, from the studies reported to date, it is difficult to assess the potential application of biological enzymes to single cup decaffeination as the effect of temperature on resting cells and cell free extract to caffeine degradation rates has not been reported. This paper investigates the effect of temperature on caffeine degradation rates by resting cells and cell free extracts of P. putida IF-3 in buffered aqueous solutions. 2. Materials and methods 2.1. Culture and culture conditions The strain of P. putida used in this study was P. putida IF- 3 [16]. The bacterium was stored at -70 ◦ C (Revco Model ULT1386) in 1.5 mL mini-centrifuge vials, containing a 1:1 (v/v) mixture of growth medium and 40% glycerol. The microorgan- isms were grown in shake flasks holding 150 mL of aqueous 1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2006.05.006