HYDROLYSIS OF SUCROSE BY IMMOBILIZED Saccharomyces cerevisiae INVERTASE

Invertase from Saccharomyces cerevisiae was immobilized by entrapment in carrageenan gel beads and used for sucrose hydrolysis. The immobilization efficiency was 88% when the concentration of the added enzyme was 100 unit/ml of gel and dropped to 49% upon increasing the added enzyme concentration to 500 U/ml. The immobilized enzyme remained fully active after 12 weeks of storage at 4°C. The hydrolytic activity of the enzyme was found to be influenced by temperature and pH. The preferred hydrolysis temperature was 55 °C for both the free and immobilized forms. The optimum pH was 4.5 for the free enzyme and was 4.5 – 5.5 for the immobilized enzyme. Km values for the free and immobilized invertase were 63.4 and 87.8 mM, respectively. Vmax values for free and immobilized invertase were calculated as 31.4 and 24 mM.min, respectively indicating lower affinity by the immobilized enzyme for its substrate and lower reaction velocity. In a continuous process using packed bed column, hydrolysis percentages of 84, 29 and 18% were obtained by using flow rates of 0.25, 1 and 2 bv/hr, respectively. On the contrary, the specific productivity was enhanced by increasing the flow rate in the column. INTRODUCTION The enzyme invertase (β-D-fructofuranosidase, E.C. 3.2.1.26) catalyzes sucrose hydrolysis producing an equimolar mixture of glucose and fructose named inverted sugar which represents a commercially attractive product and has been extensively used in food industries due to its sweetness and liquid form. The yeast Saccharomyces cerevisiae is used in the production of this enzyme (Mazi et al., 2006; Mahmoud, 2007; Vu and Lee, 2008). Sucrose hydrolysis can be accomplished either by hydrochloric acid or by invertase with a notorious advantage for the enzymatic over the acid process in terms of energy economy, environmental safety and low formation of by-products. However, to match the low cost of the acid process, the invertase must be used in an immobilized form (Tomotani and Vitolo, 2006). The bioprocess of invert sugar production, as any other biotechnological process, is very complex due to the number of variables involved. Hence a balanced environment with optimum temperature, pH, agitation and flow rate is necessary for a good productivity (Blanch and Clark, 1997). The enzyme immobilization technology offers technical and economical advantages such as repetitive use, easy removal of the enzyme from the reaction medium, longer half-life of the enzyme and the ability to the adaptation in the preferred continuous processes such as packed or fluidized bed reactors, in which it is possible to use higher enzyme dosage per volume of reactor than in the soluble enzyme process (Segura-Ceniceros et al., 2006). The main advantage of a continuous process over a batch process is the stabilization of operational Received 18/3/2009 accepted 4/6/2009 Mesopotamia J. of Agric. (ISSN 1815-316X) Vol.(38) No.(1) 2010 conditions, simpler quality control and ease of automation which leads to reduce the overall production costs (Arica and Bayramoglu, 2006; Emregul et al., 2006). In the present study, the immobilization of Saccharomyces cerevisiae invertase was achieved by entrapment within carrageenan gel beads. The catalytic performance of the immobilized enzyme at various temperatures, pH values, flow rates and sucrose concentrations were studied using batch and continuous processes. MATERIALS AND METHODS Materials: Saccharomyces cerevisiae invertase enzyme (46 units/mg) and carrageenan were purchased from Sigma Chemical Co. Sucrose was from Merck Chemical Co. All other chemicals were of analytical grade. Immobilization procedure: Immobilization was carried out using the method of Iborra et al. (1997). Five ml of the enzyme solution (2500 units in citratephosphate buffer, 0.1M, pH 5) were mixed with 20 ml of carrageenan solution (3%) at 4 °C. This solution was then dropped into a stirred cold solution of potassium chloride (0.3 M) using a syringe with needle. The resulting beads were kept in solution for 10 minutes after which they were filtered off, washed with sterile water and stored at 4 °C. Effect of invertase concentration on specific activity and immobilization efficiency: The immobilization procedure was carried out by using variable concentrations of the enzyme ranging from 100-500 U/ml of beads, then the activity of the immobilized enzyme was measured. Effect of temperature and pH on sucrose hydrolysis: The effect of temperature on sucrose hydrolysis by the free and immobilized invertases was investigated in the range of 30-70 °C at pH 5. The dependence of sucrose hydrolysis degree upon pH was examined in the pH range of 3-7 using citrate-phosphate buffer at 45 °C. Hydrolysis of sucrose using batch process: Sucrose solution (5%) was prepared in citrate-phosphate buffer (0.05M, pH 5). Hydrolysis was carried out at 45 °C in 100-ml conical flasks containing 20 ml of the solution and provided with a magnetic stirrer. Hydrolysis was initiated by the addition of 230 units of the enzyme as a solution or as carrageenan beads containing the same amount of the enzyme. After 15 minutes, samples were withdrawn and the total reducing sugars (TRS) produced were measured by spectrophotometric method using 3,5dinitrosalicylic acid reagent (Miller, 1959). Hydrolysis of sucrose using continuous process: The experiment was carried out in a packed bed reactor. A jacketed glass column (1.6x11 cm) was used. The carrageenan beads, containing the immobilized invertase were suspended in citratephosphate buffer solution (0.05 M, pH 5) and poured into the column. The column temperature was controlled at 45 °C by circulating water in the jacket. Sucrose solution, of 50% concentration was fed continuously into the column at flow rates of 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 and 2.0 bed volume/hour (bv/hr). After steady state had been attained, aliquot samples were collected from the outlet solution and measured for the concentration of total reducing sugars (TRS). Hydrolysis measurement: The hydrolysis percentage was calculated using the following formula: Hydrolysis (%) = TRS/So x 100 TRS is the total reducing sugar (glucose + fructose) in mmole, So is the initial sucrose concentration in mmole/liter. Mesopotamia J. of Agric. (ISSN 1815-316X) Vol.(38) No.(1) 2010 Determination of Kinetic Parameters: Km and Vmax values of the free and immobilized invertase were determined by Lineweaver-Burk plot using various substrate concentrations (0.029-0.29 M) in citrate-phosphate buffer (0.05 M, pH 5) at 45 °C. Storage stability: The free and immobilized enzymes were kept in citratephosphate buffer (0.05 M, pH 5) at 4 °C and their activities were measured at one week intervals up to 12 weeks. RESULTS AND DISCUSSION Immobilization yield: The ratio of the activity of the immobilized invertase to the activity of the free invertase was found to be 88%. The immobilized enzyme was active for 42 days without remarkable drop in activity. Approximately full activity was retained after 16 batch reactions. Effect of invertase concentration on the specific activity and immobilization efficiency: Specific activity, which is defined as the total enzyme units retained per ml of gel beads, was increased upon increasing the amount of the added enzyme in the gel matrix (Fig. 1). The relationship did not follow the first order kinetics. Total retained activities of 88, 207 and 245 U/ ml were retained when the added enzyme concentrations were 100, 300 and 500 U/ ml, respectively. Fig. (1) : Effect of enzyme concentration on specific activity of immobilized