Indian Journal of Biotechnology Vol 13, January 2014, pp 89-97 Medium optimization for pullulanase production from Anoxybacillus species using experimental design Ummirul Mukminin Kahar, Madihah Md. Salleh and Kian Mau Goh* Faculty of Biosciences and Medical Engineering (formerly Faculty of Biosciences and Bioengineering) Universiti Teknologi Malaysia, 81310 Skudai, Johor Darul Takzim, Malaysia Received 18 November 2012; revised 11 May 2013; accepted 22 July 2013 The aim of the present study was to improve pullulanase production from the thermophilic Anoxybacillus species strain SK3-4 by medium engineering. The strain was isolated from a Malaysian hot spring, Sungai Klah (SK). The optimization processes were performed in two stages using 2 4 full factorial design and central composite rotatable design (CCRD). Four variables were manipulated to improve pullulanase yield: initial culture pH, and concentrations of pullulan, tryptone and ammonium chloride by using a 2 4 full factorial design. Following that a quadratic model of CCRD was then used to identify the optimum yield of pullulanase by manipulating all four of these significant parameters. The optimized medium composition was a pH of 8.29, 0.79% (w/v) pullulan, 0.12% (w/v) tryptone and 0.39% (w/v) ammonium chloride, giving a final pullulanase activity of 3.86 U/mL and specific activity of 3.38 U/mg. The specific activity achieved was higher than the CCRD predicted value (3.00 U/mg). The application of two different experimental designs resulted in an improvement of pullulanase specific activity of approximately 73% from the original Thermus medium. Keywords: Anoxybacillus, central composite rotatable design, experimental design, optimization, pullulanase, response surface methodology Introduction Pullulanase (pullulan-6-glucanohydrolase, EC:3.2.1.41) is a debranching enzyme that hydrolyses the α-1,6- glycosidic bonds in pullulan and other branched polysaccharides, such as, starch 1 . This industrially important enzyme is generally used with other amylolytic enzymes, such as, α-amylase, β-amylase or glucoamylase, to increase the efficiency and yield of enzymatic reactions. For example, the quality of sugar syrup in the starch saccharification process can be enhanced by pullulanase 1 . In addition, the products of pullulan degraded by the pullulanase are frequently used in food and pharmaceutical industries 2 . Pullulanase also improves the production of cyclodextrins by pretreating the starch prior to the cyclodextrin glucanotransferase (CGTase) reaction 3 . Pullulanase also has applications in the textile, baking and detergent industries 1 . Several pullulanases were produced in vast amounts by mesophilic, thermophilic and hyperthermophilic bacteria 1 . Because pullulanase is an important industrial enzyme, several studies have been done to enhance enzyme production in cells by optimizing medium composition. The most common parameters are the types of carbon and nitrogen sources used in the fermentation medium. Pullulan 4 , maltose 5 and starch 6 are types of carbon sources previously used to improve pullulanase production. Additionally, organic nitrogen sources, including yeast extract, peptone, tryptone, trypticase, casamino acids and corn steep liquor, were found to improve the pullulanase production 7 . An optimized fermentation medium is of critical importance because the composition of medium significantly affects the production yield. The major constraints in engineering a new medium formulation are that a large number of experiments are required and it is difficult to pinpoint the true optimal point for each medium component. The conventional one-factor-at-a-time (OFAT) approach is time consuming and, therefore, statistical experimental designs provide a better platform for optimization purposes. Experimental designs, such as, two-level full factorial design and central composite rotatable design (CCRD) were previously applied successfully to optimize pullulanase production 6-8, . —————— *Author correspondence: Tel: +91-607-5557556; Fax: +91-607-5531279 E-mail: gohkianmau@utm.my