Biologia 63/6: 10441050, 2008 Section Cellular and Molecular Biology DOI: 10.

2478/s11756-008-0167-z

Proteolysis of -amylase from Saccharomycopsis buligera: characterization of digestion products

Khomaini Hasan1,2*, Wangsa Tirta Ismaya1,3, Idar Kardi1, Yandi Andiyana1, Susanti Kusumawidjaya1, Safri Ishmayana1, Toto Subroto1 & Soetijoso Soemitro1*
1

Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jalan Singaperbangsa 2, Bandung 40133, Indonesia; e-mail: s soemitro@unpad.ac.id 2 currently on leave, present address: Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4, CZ-62500 Brno, Czech Republic; e-mail: hasan@chemi.muni.cz 3 currently on leave, present address: Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands

Abstract: -Amylase from Saccharomycopsis buligera R-64 was successfully puried by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme. However, it was found that the Km value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies showed a lower half-life time for the 39-kDa fragment. These ndings suggest that the 39-kDa fragment is the catalytic domain, while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis buligera -amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A. Key words: Saccharomycopsis buligera -amylase; tryptic digestion; thermostability; raw starch-digesting capability. Abbreviations: GluR, Saccharomycopsis buligera R-64 glucoamylase; p10, Sfamy proteolytic fragment with molecular mass of 10 kDa; p39, Sfamy proteolytic fragment with molecular mass of 39 kDa; Sfamy, Saccharomycopsis buligera -amylase.

Introduction -Amylase (1,4--D-glucan glucanohydrolase, EC 3.2.1.1) catalyzes the cleavage of 1,4--glycosidic bonds in starch. The enzyme is, together with other amylolytic enzymes, involved in the conversion of starch to -limit dextrins, oligosaccharides, maltose, and glucose. In starch-related industries, raw starch is used as the starting material, thus an energy consuming heat pre-treatment process for the gelatinization of the raw starch is required. The gelatinization results in the breakage of inaccessible starch granule complex and allows -amylase to enter the starch granules. In the recent years, the raw starch related industry has been trying to decrease the energy consumption by means of using thermostable -amylases that act on raw starches (Rodriguez-Sanoja et al. 2005; Machovic & Janecek 2006).
* Corresponding author

Only 10% of the amylase secreting organisms are capable of producing raw starch digesting -amylase (Rodriguez-Sanoja et al. 2005; Machovic & Janeek 2006), including Saccharomycopsis buligera (Hostinova 2002). This yeast has a great potential for this application because it secretes both -amylase and glucoamylase, and is a food born yeast. Hostinova et al. (2003) have reported the capability of the glucoamylase to act on raw-starch. Nevertheless, little is known about the ability of the -amylase counterpart to perform such activity. The amino acid sequence of the -amylase from S. buligera (Sfamy) derived from cDNA was reported by Itoh et al. (1987), which upon the BLAST search (Altschul et al. 1990) shares the highest identity (52%) with that of Aspergillus oryzae -amylase (Takaamylase). The X-ray structure of the latter enzyme has been elucidated (Matsuura et al. 1984) and consists of

c 2008 Institute of Molecular Biology, Slovak Academy of Sciences

Fragments of the tryptic digested S. buligera -amylase an N-terminal catalytic domain of 356 residues and a C-terminal one of 92 residues. The structure of Sfamy is yet to be elucidated; nevertheless Matsui et al. (1994) have shown that the currently available Taka-amylase structure can be employed as a structural model for Sfamy. Based on the sequence homology, the N-terminal part of the sequence of Sfamy resembles the catalytic domain of Taka-amylase (Matsuura et al. 1984). The substrate binding site and role of aromatic residues have been reported by Matsui et al. (1991, 1992a,b, 1994). Further, in some amylases the C-terminal domain is involved in starch binding (Janecek & Sevcik 1999; Sumitani et al. 2000), while in other -amylases it is responsible for thermostability and determines the enzyme kinetics (Iefuji et al. 1996). The function of the C domain in Sfamy, which exhibits only 35% sequence identity with that of Taka-amylase, remains to be concluded. This article discusses the domain organization and proposes the domain function in the Sfamy (strain R64) by means of proteolytic digestion and its action on raw starch. Two major fragments with molecular masses of 39 kDa and 10 kDa were obtained upon digestion, and these fragments were then characterized in terms of enzyme kinetics, thermostability, and the action on raw starch.
Material and methods Microorganism The S. buligera strain R-64 was obtained from the culture collection at the Center for Biotechnology, Bandung Institute of Technology, Indonesia, and was maintained at the Laboratory of Biochemistry, Padjadjaran University, Indonesia. The yeast cells were routinely grown at room temperature for 48 h on agar plates consisting of 6% sucrose, 1.5% bacto agar, and 10% tauge extract. Materials All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany) in proanalysis grade, except if specically mentioned. Toyopearl matrices (butyl- and DEAE-) were from Tosoh Ltd. (Japan), Sephadex G-25 and G-50 were from Sigma (St. Louis, MO, USA). Raw starches were purchased from a local market in Bandung, Indonesia. Production and purication The yeast S. buligera was aerobically grown in an erlenmeyer ask with a medium containing 1% sago starch and 1% yeast extract for 5 days at room temperature, on a rotary shaker (New Brunswick Scientic, Edison, NJ, USA) with a constant agitation speed of 180 rpm. The yeast cells were removed by centrifugation for 10 min at 4500g, at 4 C. As rst step in the purication procedure the crude enzyme extract was brought to 25% (w/v) saturation with ammonium sulphate in 50 mM phosphate citrate buer, pH 5.8, at 4 C for two hours. Insoluble contaminants and a protein precipitate were removed by centrifugation for 10 min at 4500g, 4 C. The supernatant obtained was loaded onto a hydrophobic interaction chromatography column butylToyopearl 650M and was step-wisely eluted with 25, 20, 15, 10 and 5% ammonium sulphate in 50 mM phosphate citrate buer, pH 5.8 (w/v). Fractions demonstrating amylolytic

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activity were pooled and were subjected to a desalting step on a Sephadex G-25 gel ltration column equilibrated with 50 mM phosphate citrate buer, pH 5.8. The nal polishing step was done on a DEAE-Toyopearl 650M anion exchanger column equilibrated with 50 mM phosphate citrate buer, pH 5.8. The enzyme was recovered from the anion exchanger column with a linear gradient of NaCl 00.5 M. The purity of the enzyme obtained was monitored by gel electrophoresis (Laemmli 1970). Amylase activity assay The amylolytic activity was routinely determined according to Fuwa (1954). The reaction mixture contained 100 L enzyme and 100 L 0.1% soluble starch (in 50 mM phosphate citrate buer, pH 5.8) and was incubated for 10 min at 50 C. The reaction was stopped by addition of 100 L of 0.1 M HCl. 100 L of an iodine solution (0.1% I2 in 0.2% KI solution) was then added, followed by 1.6 mL of water, and the absorbance of the starch-iodine complex formed was measured at 600 nm. One unit of the amylase activity is dened as 10% decrease of the color of the starch-iodine complex after 10 min of incubation at 50 C. Protein concentration determination The protein concentration was quantied by the Lowry method (Lowry et al. 1951), using bovine serum albumin as the standard. SDS PAGE analysis Samples were subjected to SDS-PAGE (12.5% of acrylamide gel with 4% of stacking gel) (Laemmli 1974) and protein bands were visualized by staining with Coomassie Brillant Blue R250 according to Blakesley & Boezi (1977). Proteolytic digestion of Sfamy Proteolysis of native Sfamy was performed with trypsin, essentially following the procedure of Gilkes et al. (1989). To a solution of pure -amylase in 25 mM Tris-HCl, pH 8.3, containing 20 mM CaCl2 , trypsin-TPCK (mol ratio of trypsin:-amylase = 1:15) was added and the mixture was incubated at 37 C for 72 h. The reaction was stopped by transferring the reaction mixture to 20 C. The fragments resulting from the digestion were detected by SDS-PAGE analysis. The fragments obtained upon proteolytic digestion were then separated by gel ltration chromatography with a Sephadex G-50 column equilibrated with 20 mM phosphate citrate buer, pH 5.8. A similar treatment was applied to the concentrated undigested enzyme. The peak fractions collected during gel ltration of fragments and undigested enzyme were used for further characterizations. Kinetic analysis Measurements of reaction kinetics of Sfamy were done at 50 C. Initial rates of soluble starch hydrolysis were measured in the concentration range of 0 to 1.8 (g/L). The kcat and Km values were obtained from the tting of the initial reaction rates as a function of substrate concentration in the Michaelis-Menten equation calculation. Because the sensitivity of the starch hydrolysis measurement according to Fuwa (1954) is not high enough for kinetic measurements, the activity of the Sfamy was measured by using the ferricyanide method as described by Walker & Harmon (1996). The procedure of amylase activity according to Walker & Harmon (1996) is described below in the section Raw starch adsorbing and digesting analysis.

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Thermostability analysis The thermostability experiment was accomplished by determining the half-life time of the enzyme, based on the decrease in enzyme activity upon thermal inactivation, essentially as described by Kim et al. (2003). The determination of the half-life time of the undigested and active fragments in 25 mM Tris-HCl, pH 8.3, was done by means of incubation at 50 C with various durations. The initial protein concentration was 1.3 103 mol/mL and remaining activity in the sample was determined according to Fuwa (1954). Raw starch adsorbing and digesting analysis The raw starch adsorbing assay was carried out by the following procedure: First, raw starch (from maize, tapioca, sago, or potato) was subsequently washed with 0.1 M HCl and water, and was dried in oven at 37 C. Prior to the assay, the raw starch (0200 mg) was suspended in 25 mM TrisHCl, pH 8.3. 1.3 103 mol/mL enzyme was added and the reaction mixture was incubated with a constant shaking in a rotary shaker (New Brunswick Scientic, Edison, NJ, USA) at room temperature for 30 min. The raw starch suspension was centrifuged for 30 min at 4500g, 4 C, and the protein concentration in the supernatant was determined according to Lowry et al. (1951). Raw starch digesting analysis of -amylase was measured based on the amount of reducing sugar which is released during enzymatic activity as described by Walker & Harmon (1996) with some modications as follows: Tubes with screw caps were used in this measurement. Prior to the assay, the raw starch (0200 mg/1.5 mL) suspended in 0.5 mL of enzyme (1.3 103 mol/mL) in 50 mM phosphate citrate buer, pH 5.8, was incubated for 24 h. The enzyme reaction was stopped by the addition of 3 mL of 10.6 mM potassium ferricyanide reagent K3 (Fe(CN))6 . Maltose standards (0120 mg/100 mL) were prepared with 1 mL of standard added to 3 mL of K3 (Fe(CN))6 reagent. Standard and sample tubes were capped, heated for 10 min in a boiling water bath, and then allowed to cool at room temperature. Distilled water was then added to each tube into total volume 20 mL, and the contents mixed by inversion. The absorbance of each solution was determined spectrophotometrically at 420 nm (model UV-160, Shimadzu, Tokyo, Japan). The baseline reducing groups present in the substrate was corrected by the blank (reagent) and negative control (the enzyme was replaced by buer). Finally, the amount of reducing sugar produced was calculated according to the standard curve using maltose as the standard.

K. Hasan et al.

Fig. 1. Hydrophobic interaction chromatography prole of Sfamy. The -amylase fraction is indicated by an arrow and is eluted by 15% (w/v) ammonium sulphate. The -amylase activity is indicated by blue color and glucoamylase activity is indicated by red color.

Fig. 2. Gel electrophoresis of Sfamy after purication steps. Lanes: (1) molecular weight standards, (2) crude extract, (3) after hydrophobic interaction chromatography on butyl-Toyopearl 650M, (4) after desalting on Sephadex G-25, (5) after anion exchange chromatography on DEAE-Toyopearl 650M. The black arrow indicates pure Sfamy with a molecular mass of 54 kDa.

Results Production and purication S. buligera R64 produces two amylolytic enzymes: amylase (Sfamy) and glucoamylase (GluR) (Ismaya et al. 2001), which was also demonstrated by Hostinova et al. (2003). The two enzymes demonstrate similar optimum pH for activity, molecular weight, and isoelectric point, but dier in hydrophobicity, which was exploited for separation of the two enzymes. We have successfully separated Sfamy and GluR with a butyl-Toyopearl 650 M hydrophobic interaction column (Fig. 1). The Sfamy was obtained upon elution with 15% ammonium sulphate in 50 mM phosphate citrate buer, pH 5.8 (w/v), while the GluR eluted at 5% ammonium sulphate. SDS-PAGE analysis showed that

the molecular mass of the Sfamy is 54 kDa (Fig. 2). The purication procedure resulted successfully in pure protein, as shown by the single peak from the DEAEToyopearl anion exchange chromatography that demonstrated amylolytic activity and showed a single band on SDS PAGE analysis (Fig. 2). This isolation procedure resulted in 66-fold purication and the recovery of enzyme activity was 31% (Table 1). Raw starch adsorbing and digesting analysis of Sfamy Upon raw starch adsorbing capability experiment, we observed that the amylolytic activities and the protein concentrations in the supernatant before and after the incubation of the enzyme with the raw starch substrate were similar, suggesting that no starch binding had occurred. As the positive control, starch binding by GluR was tested, whereas the enzyme adsorption onto maize, tapioca, sago, and potato starches were 90%, 80%, 25%,

Fragments of the tryptic digested S. buligera -amylase

Table 1. Purication scheme of the Saccharomycopsis buligera -amylase (Sfamy).a Step Total activity (U 103 ) 271 121 94 83 Total protein (mg) 12.200 700 121 57 Specic activity (U/mg) 22 172 777 1450 Purication (fold) 1 8 35 66 Recovery (%) 100 45 35 31

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Crude extract Butyl-Toyopearl Sephadex G-25 DEAE-Toyopearl

a

One unit -amylase activity is dened as the 10% decrease of the starch-iodine complex color after 10 minutes of incubation at 50 C.

Fig. 3. Raw starch digesting capability of Sfamy on dierent raw starches. The amount of reducing sugar was determined by the ferricyanide method (Walker & Harmon 1996), using maltose as a standard. The initial concentration of the samples was 1.3 103 mol/mL.

Fig. 4. Gel electrophoresis of Sfamy digested by trypsin. M is the molecular weight marker. Sfamy was digested for 72 hours by ratio 1 : 15 (trypsin : Sfamy). The arrow indicates trypsin.

and 20%, respectively. The result of positive control with GluR is in agreement with that of reported by Hostinova et al. (2003). Our nding indicate that the Sfamy is not a raw starch adsorbing enzyme. Nevertheless, Sfamy demonstrates digestion activity on several raw starches. After 24-h incubation with various raw starches, we found that the highest raw starch digestion activity of Sfamy was demonstrated when starch from maize was used, followed by starches from tapioca, sago, and potato, sequentially (Fig. 3). Since the character of the tested starches is various, we have shown that the dierences in shape, type, and amylose/amylopectine ratio of raw starch granules used in our experiment have a signicant inuence on the Sfamys performance upon hydrolysis. Our result is in agreement with that previously reported by Tester et al. (2006). The result indicates that Sfamy can hydrolyze dierent raw starches in dierent manner depending on the origin of the starch. Our studies on the activity of Sfamy on raw starch indicate that Sfamy is a raw starch digesting but non-adsorbing -amylase. Proteolysis of Sfamy Digestion of native Sfamy with trypsin resulted in two major fragments with apparent molecular masses of 39 kDa (p39) and 10 kDa (p10), respectively, as judged by SDS PAGE (Fig. 4). The two fragments are most likely representing the N- and C- terminal domains of the -amylase. According to Matsuura et al. (1984), the N-terminal domain of -amylase consists of the integrated A and B domains, in which the active site is

Fig. 5. Gel ltration chromatography on Sephadex G-50 column of Sfamy (dashed line, indicated by arrow) and the mixture of fragments after 72 h tryptic digestion (solid line).

located. The C-terminal domain consists of C domain, the function of which in Sfamy is not yet established. Exploiting the separation eciency of Sephadex G50 gel ltration column, the two fragments obtained from the proteolytic digestion after 72 h (Fig. 5) could be separated, as indicated in Figure 4. The undigested enzyme showed only one protein peak while the digested enzyme showed two protein peaks, of which the rst peak indicated that the larger fragment (p39) was eluted somewhat later than the undigested enzyme, while the second peak represents the smaller fragment (p10). The rst peak, which was the p39 fragment and was hypothesized as the catalytic domain, demonstrated similar amylolytic activity to that of the undi-

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K. Hasan et al. Thermostability analysis The thermostabilities of the undigested enzyme and of the p39 fragment were determined by means of measuring the remaining activity after incubation at 50 C during various time periods (Fig. 7). In the absence of p10 fragment, the remaining activity of the p39 fragment is lower than that of the undigested enzyme. This result suggests the role of the p10 fragment in the thermal stability and probably also in the integrity of the enzyme. Discussion In general, -amylases consist of two main domains, the so called catalytic domain and C-terminal domain, respectively. The catalytic domain consisting of the A and B domains is normally located at the N-terminus. These domains can be recovered as separate fragments by proteolytic digestion using either trypsin or other proteases (Desseaux et al. 1991; Kim & Kho 1993; Ferey-Roux et al. 1998; Hamilton et al. 1998; Khajeh et al. 2006). Here we have shown the two fragments obtained during tryptic digestion of Sfamy, which is in agreement with the previous studies. The amino acid sequence of the Sfamy, derived from the cDNA, diers at only 3 positions from that reported previously by Itoh et al. (1987) (data not shown). Unfortunately, the three-dimensional structure of Sfamy is yet to be elucidated. We were recently successful in obtaining Sfamy crystals suitable for preliminary X-ray studies. The crystallization and X-ray studies are currently on the way and hopefully we will be able to determine the crystal structure of the enzyme in the near future. A structural study of Sfamy was performed based on the similarity to Taka-amylase from A. niger (PDB entry code 7TAA) by Matsui et al. (1994). The amino acids at the equal position were predicted. Trypsin favors cleavage sites (lysine or arginine) that are located in a loop. Nevertheless, considering the size of p39 fragment, potential residues susceptible for cleavage are located in an -helix just before the domain C and the cleavage acquires the unfolding of the helix. Therefore a more likely primary cleavage position is a lysine residue located in a loop preceding the helix at position 382 in the translated cDNA sequence of Sfamy where in Takaamylase a threonine is located (residue 354 in the mature sequence). After this cleavage the helix may unfold bringing more sites accessible for tryptic attack. However, an amino acid sequence study has still to be done to conrm this hypothesis. The dierence in the Michaelis-Menten constant (Km ) values of native Sfamy and the p39 fragment predicted the function of the p10 fragment, which is proposed to be the domain C. The absence of the p10 fragment resulted in a decrease of enzyme anity towards soluble starch. Interestingly, the kcat values were not changed, which means that the catalytic eciency of the enzyme remains undisturbed. This result strengthens the assignment of the p10 fragment as the

Fig. 6. Michaelis-Menten plot of Sfamy with the soluble starch as a substrate. The concentration of the Sfamy was 1.3 103 mol/mL. The Km and kcat values of Sfamy are 0.54 g/L and 4.2 102 min1 , respectively. The soluble starch was used as substrate and the released amylolytic product was quantied according to Walker & Harmon (1996).

Fig. 7. The inuence of p10-removal on thermostability. The remaining activity of native Sfamy (open circles) and a p39 sample (closed circles) after incubation at 50 C at dierent times were determined by the Fuwa (1954) method. The initial concentration of the samples was 1.3103 mol/mL. Initial activities: 60 U/mL (Sfamy); 50 U/mL (p39).

gested Sfamy. The second peak, which was the p10 and was hypothesized as C-domain, demonstrated no amylolytic activity. Kinetic analysis The Michaelis-Menten curve of Sfamy is presented in Figure 6. From these data a Km value of 0.54 g/L starch and a kcat value of 4.2102 min1 could be calculated. Although the preliminary kinetic experiments showed that the undigested enzyme and the p39 fragment have similar kcat values, it was found that the substrate anity (Km ) of the later one is approximately two-times lower than that of the undigested enzyme. This result suggests that the p39 fragment has a lower anity towards the substrate and the C-terminal p10 fragment may have a role related to the enzyme-substrate interaction.

Fragments of the tryptic digested S. buligera -amylase C-terminal domain in Sfamy and the importance of the p10 fragment for recognition of substrate by the enzyme. The p10 fragment plays also a role to maintain the thermal stability of the enzyme, which is in agreement to the report of Iefuji et al. (1996). Removal of the p10 fragment caused destabilization of the catalytic domain during incubation at 50 C. The result shows that the C domain is important for both the enzyme activity and stability. The p39 fragment demonstrated amylolytic activity in the absence of the p10 fragment, which supports the assignment of this fragment as the main catalytic domain. Further, we also observed that the p39 and p10 fragments do not have any anity to raw starch, as previously demonstrated by the undigested enzyme. Interestingly, the p39 fragment is still able to digest several raw starches (data not shown) although at a lower extent than the undigested enzyme. The catalytic domain of an -amylase is formed by a (/)8 -barrel (TIM barrel) which has a rigid conformation. According to Matsui et al. (1994), this rigidity is maintained by hydrophobic interactions in the core, which is built mainly by aromatic residues, and is strengthened by three disulde bridges (Matsui et al. 1994), and may be responsible for the resistance of the p39 fragment against proteolytic digestion. Although Sfamy has the capability to digest raw starch, the enzyme intriguingly does not adsorb onto raw starch. The amino acid sequence of Sfamy indicates the absence of tryptophan residues in the C domain, which are known to be responsible for raw starch binding (Machovic & Janecek 2006). Instead of the tryptophans, tyrosine residues occupy identical positions in Sfamy. Therefore it is our interest to study whether the tyrosine is responsible for the enzyme weak interaction with starch. The elucidation of the enzyme structure as well as the ongoing site-directed mutagenesis study will hopefully provide a more clear picture on this subject. Conclusions We have successfully puried, separated, and characterized the p39 and p10 fragments obtained from tryptic digestion of Sfamy that resemble the catalytic and C-terminal domains of the homologous Taka-amylase. Although the amylolytic activity of the catalytic domain is independent from presence of the domain C, the later one most likely plays a role in the anity of the enzyme towards substrate and in the enzyme thermostability. Moreover, Sfamy is capable of degrading raw starch, but shows no adsorption to this substrate, suggesting that Sfamy is a raw starch degrading but not adsorbing enzyme.
Acknowledgements This work was nancially supported by Competitive Grant batch IX from the Directorate for Higher Education, the Ministry of National Education (20012004), and the Indonesia Toray Science Foundation (ITSF, 2005). We thank Prof. J. J. Beintema (University of Groningen, The Nether-

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lands) for reviewing the manuscript and his full support, and Prof. B.W. Dijkstra (University of Groningen, The Netherlands) and Prof. M.J.E.C van der Maarel (TNO-Groningen, The Netherlands) for discussion on -amylases and starchbinding domains. References
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