**3. Isolation of the amylolytic enzyme complex**

272 Chromatography – The Most Versatile Method of Chemical Analysis

starch degrading enzyme producer.

sugar.

maltotriose, or dextrin, and subsequently hydrolyzed to glucose.

Degradation of starch into sugars is performed by amylolytic enzymes, such as α-amylase, glucoamylase, β-amylase, isoamylase, pullulanase, exo(1-4)-α-D-glucanase, α-D-glycosidase, and cyclomaltodextrin-D-glucotransferase (Fig. 1) (3). *S. fibuligera* secretes amylolytic enzymes, almost exclusively α-amylase and glucoamylase. α-Amylase acts as an endoenzyme, cleaving 1,4-α-glycosidic bond at random positions to result in liquefaction of starch. Glucoamylase, on the other hands, is an exo-enzyme that cleaves 1,4-α-glycosidic bond only at the non-reducing end to result in saccharification (4). Thereby upon the combined action of α-amylase and glucoamylase, starch is degraded into maltose,

The use of amylolytic enzymes for the ethanol production in the course of renewable energy requires an ability to act on raw starch, allowing the use of biomass as the starting material. The ability of *S. fibuligera* α- and glucoamylase to degrade raw starch has been reported (4, 5). Interestingly, only 10% of amylolytic enzyme-secreting organisms are capable of producing raw starch degrading α-amylase (6, 7). Since starch degradation begins with αamylase action to produce simpler sugars, raw-starch acting α-amylase is highly desired. This situation strengthens the position of *S. fibuligera* for bioethanol production, being a raw

**Figure 1.** Starch degradation by amylolytic enzymes (3). The open and black coloured circles represent the reducing and non-reducing sugars, respectively. Note that the cleavage occurs at the reducing

α-Amylase is commonly used in food, beverage, paper industries (8), in textile industry and as additive in detergents (9, 10), for renewable energy (11, 12) and medical purpose (13). Glucoamylase has been the most important enzyme in food industry, mainly in the production of sugar or ethanol (14). Glucoamylase is normally employed in combination with amylolytic enzymes that are able to act on more complex polysaccharides, such as αThe amylolytic enzyme complex from strain R64, consisting of α-amylase (AMY) and glucoamylase (GLL1), is secreted into the production medium. Thus, the enzyme complex was harvested by simply cold-centrifugation (~4oC) at 6000 *g* for 30 minutes, to remove the yeast cells. The enzyme complex in the supernatant, which was designated as the crude extract, was subjected to a diafiltration system (Millipore Minitan II, Tangential Flow Filtration system, Merck Pte Ltd, Singapore) over a 10-kDa cut off membrane disc-plate, at a flow rate of 10-20 ml per minute, at room temperature. The enzyme complex was recovered in the retentate. Diafiltration step tremendously reduced the size of the sample, which is advantageous because the subsequent step to capture the amylolytic enzyme complex in the crude extract was precipitation with ammonium sulfate by 0-100%, on ice (~4oC). These sequential procedures demonstrate a straightforward way to isolate extra-cellular proteins that was accomplished within one-day operation, which is important for protein works due to, for example, possible degradation by proteases.
