Biochemical Engineering Journal 33 (2007) 26–33 Production of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. TS1-1: Optimization of carbon and nitrogen concentration in the feed medium using central composite design Wan Salwanis Wan Md. Zain a , Rosli Md. Illias a,∗ , Madihah Md. Salleh b , Osman Hassan c , Roshanida A. Rahman a , Aidil Abd. Hamid c a Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia b Department of Biology, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia c School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Received 24 May 2005; received in revised form 6 March 2006; accepted 26 September 2006 Abstract Optimisation of nutrient feeding was developed to overcome the limitation in batch fermentation and to increase the CGTase production from Bacillus sp. TS1-1 in fed batch fermentation. Optimisation of the C/N ratio in the feed stream was conducted in a 5 l fermenter, where feeding was initiated at constant rate of 0.02 h -1 . In our initial screening process, the addition of nitrogen source boosted the growth of the microbes, but on the other hand reduced the CGTase production. The amount of tapioca starch and yeast extract was optimised in order to obtain a sufficient growth and thus, increased the CGTase production. Results were analysed using three-dimensional response surface plot, and the optimised values of carbon and nitrogen concentration of 3.30% (w/v) and 0.13% (w/v) were obtained, respectively. CGTase activity increased up to 80.12U/ml, which is 13.94% higher as compared to batch fermentation (70.32 U/ml). This also led to 14.54% increment of CGTase production in fed batch culture as compared to the production before the optimisation. The CGTase activity obtained was close to the predicted value, which is 78.05U/ml. © 2006 Elsevier B.V. All rights reserved. Keywords: Cyclodextrin glucanotransferase; C/N ratio; Fed batch; Central composite design 1. Introduction Cyclodextrin glucanotransferase (EC 2.4.1.19) is an extracel- lular enzyme, a member of the amylolytic glucosylase family. CGTase differs from its family members, possesses a capa- bility to catalyse multiple reactions [1]. It has both strong hydrolytic and synthetic capabilities as well as having multi- ple product specificity [2]. The ability of CGTase to convert starch into favoured industrial substance called cyclodextrin through cyclization process is of great interest to researchers [2]. Cyclodextrins are able to form an inclusion complex with various kinds of organic compounds inside the cavity of the ring structure [3]. Most production of the CGTase were carried out as batch processes [4,5]. However, CGTase production in batch pro- ∗ Corresponding author. Tel.: +60 7 5535564; fax: +60 7 5581463. E-mail address: r-rosli@utm.my (R.Md. Illias). cesses show many limitations caused by substrates suppression, catabolite repression and limiting of some essential nutrients. Therefore, applying fed batch fermentation was constructive to overcome all the limitations in batch fermentation. Unfor- tunately, limited information was obtained regarding CGTase production using fed batch culture [6–8]. The main purpose of employing fed batch culture was to remove the repressive effects of rapidly utilized carbon sources, to reduce the viscosity of the medium, to reduce the effect of toxic medium constituents or simply to extend the product formation stage of the process for as long as possible. Besides CGTase, fed batch method had been proven to increase the production of proteases by Bacil- lus sphaericus [9] and production of enzyme -1,4-endoglu- canase from recombinant Bacillus subtilis DN18859 (pCH7) [10]. Media optimisation for feed stream using statistical exper- imental design for production of novel CGTase by fed batch fermentation had not been reported before. On the other hand, the optimising of media formulation in batch fermentation was read- 1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2006.09.024 brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Universiti Teknologi Malaysia Institutional Repository