**2.** *S. fibuligera* **R64 secretes amylolytic enzyme**

*S. fibuligera* is a food-borne yeast that is widely used in the production of rice- or cassavabased fermented food, i.e. *Tape* in Indonesia and other Southeast Asia countries (1). The yeast, in combination with *Saccharomyces cerevisiae* or *Zymomonas mobilis*, has been employed in the production of ethanol using cassava starch as the starting material (2), where the starch is firstly degraded into simple sugars prior to (bio-) ethanol. Bioethanol has been promoted as a renewable energy replacing fossil fuels and at the moment is already used as an additive. As the demand for renewable energy grows, *S. fibuligera* emerges as an attractive workhorse for the bio-ethanol production.

© 2012 Ismaya et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ismaya et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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, maltotriose, or dextrin, and subsequently hydrolyzed to glucose.

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 273

amylase and pullulanase (14). Recently, amylolytic enzymes are employed in the production of lactic acid and ethanol by-product by lactic acid bacteria (LAB) (15), demonstrating that

Interestingly, numbers and characteristics of amylolytic enzymes secreted by *S. fibuligera* vary from one strain to another. For example, strain IFO 0111 secretes only glucoamylase whilst strain KZ secretes α-glucosidase along with α-amylase and glucoamylase (4). Further, glucoamylases from strain IFO 0111 and HUT 7212 demonstrate raw starch degradation whereas that from the strain KZ does not. Glucoamylase from strain KZ, however, demonstrates better thermal stability (4). Fascinating to find these variations in their properties despite of their near identical acid sequences (16). Strain KJ-1, an *S. fibuligera* strain from Indonesia, is also reported to secrete only glucoamylase and its partial amino acid sequence (residues 28-47) is identical to that of glucoamylase from strain KZ (1). This observation indicates differences in expression and secretion of amylolytic enzymes between *S. fibuligera* strains. Nevertheless, the practicality of *S. fibuligera* in producing

amylolytic enzymes applauds the proposal to sequence its whole genomic DNA (2).

Of 136 isolates screened from various places in Indonesia, *S. fibuligera* strain R64 was selected for demonstrating the highest amylolytic activities (α-amylase and glucoamylase). Strain R64 is able to degrade raw starch and its amylolytic enzymes demonstrate potent thermal stability. This finding makes the amylolytic enzymes from strain R64 attractive.

Extra-cellular amylolytic enzyme production by strain R64 is relatively simple, using a medium that contains of 1% sago starch and 1% yeast extract. The enzyme was harvested after 4-5 days of cultivation in a one-litre bioreactor, with constant aeration rate 1 vvm, volumetric oxygen transfer coefficient (*kLa*) 1.53 per hour, agitation speed 100 rpm, 30oC, and pH 7.0. Under this condition, enzyme activity observed on starch degradation was 1320 U/ml (Table 1). The production was also easily reproduced at a laboratory scale by means of Erlenmeyer flask (5). Thus, strain R64 offers a simple but convenient production scheme.

The 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

the application of amylolytic enzymes continues to expand.

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

to, for example, possible degradation by proteases.

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 starch degrading enzyme producer.

**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 sugar.

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 αamylase and pullulanase (14). Recently, amylolytic enzymes are employed in the production of lactic acid and ethanol by-product by lactic acid bacteria (LAB) (15), demonstrating that the application of amylolytic enzymes continues to expand.

Interestingly, numbers and characteristics of amylolytic enzymes secreted by *S. fibuligera* vary from one strain to another. For example, strain IFO 0111 secretes only glucoamylase whilst strain KZ secretes α-glucosidase along with α-amylase and glucoamylase (4). Further, glucoamylases from strain IFO 0111 and HUT 7212 demonstrate raw starch degradation whereas that from the strain KZ does not. Glucoamylase from strain KZ, however, demonstrates better thermal stability (4). Fascinating to find these variations in their properties despite of their near identical acid sequences (16). Strain KJ-1, an *S. fibuligera* strain from Indonesia, is also reported to secrete only glucoamylase and its partial amino acid sequence (residues 28-47) is identical to that of glucoamylase from strain KZ (1). This observation indicates differences in expression and secretion of amylolytic enzymes between *S. fibuligera* strains. Nevertheless, the practicality of *S. fibuligera* in producing amylolytic enzymes applauds the proposal to sequence its whole genomic DNA (2).

Of 136 isolates screened from various places in Indonesia, *S. fibuligera* strain R64 was selected for demonstrating the highest amylolytic activities (α-amylase and glucoamylase). Strain R64 is able to degrade raw starch and its amylolytic enzymes demonstrate potent thermal stability. This finding makes the amylolytic enzymes from strain R64 attractive.

Extra-cellular amylolytic enzyme production by strain R64 is relatively simple, using a medium that contains of 1% sago starch and 1% yeast extract. The enzyme was harvested after 4-5 days of cultivation in a one-litre bioreactor, with constant aeration rate 1 vvm, volumetric oxygen transfer coefficient (*kLa*) 1.53 per hour, agitation speed 100 rpm, 30oC, and pH 7.0. Under this condition, enzyme activity observed on starch degradation was 1320 U/ml (Table 1). The production was also easily reproduced at a laboratory scale by means of Erlenmeyer flask (5). Thus, strain R64 offers a simple but convenient production scheme.
