**4.3. Identification of AMY and GLL1 using RP-HPLC for protein**

Nowadays, identification of proteins usually employs techniques with sophisticated and delicate instrumentation e.g. peptide-mass finger print mass spectrometry (PMF-MS) (24). Unfortunately, this technique is rather pricey and furthermore, requires convenient access to the protein database and to the amino acid sequence of the protein. Particularly in the lack of the latter, which is a very common situation in the early stage of protein works, the separation of AMY from GLL1 was confirmed by a more simple and robust technique, exploiting the use of an RP-HPLC system (25). The analysis using RP-HPLC is based on the fragmentation profile of a protein after proteolytic digestion. Proteolytic digestion of AMY was expected to result in fragmentation profile that differs to that of GLL1, as reflected from their chromatographic profiles.

276 Chromatography – The Most Versatile Method of Chemical Analysis

isolation of purified AMY and GLL1 was unanimous.

(ml)

Volume

glucoamylase.

chromatography (RP-HPLC) column. In the Spheron 300 LC column, α-amylase (strain KZ) was eluted at an ammonium sulfate concentration of 10% whilst glucoamylase was at 5%. The separation profile, however, is strikingly similar to that of AMY and GLL1 with butyl-Toyopearl column. However, separation of AMY from GLL1 in the butyl-Toyopearl column has better resolution, suggesting variations in the surface properties of *S. fibuligera* αamylase and glucoamylase from different strains, or the butyl-Toyopearl resin serves for better separation because there is no interaction between the proteins and the supporting matrix. Nevertheless, these results emphasize the power of HIC to separate α-amylase from

**4.2. Subsequent purification of AMY or GLL1 in chromatography columns** 

Total activity (Units)

**Table 1.** The final purification scheme of AMY. AS stands for ammonium sulfate.

**4.3. Identification of AMY and GLL1 using RP-HPLC for protein** 

Crude 2000 2640000 16500 160 100 25% AS 2200 2244000 13046 172 85 Butyl Toyopearl 148 1082400 753 1438 41 95% AS 35 765600 213 3595 29 DEAE-Toyopearl 60 580800 100 5808 22

Nowadays, identification of proteins usually employs techniques with sophisticated and delicate instrumentation e.g. peptide-mass finger print mass spectrometry (PMF-MS) (24). Unfortunately, this technique is rather pricey and furthermore, requires convenient access to the protein database and to the amino acid sequence of the protein. Particularly in the lack of the latter, which is a very common situation in the early stage of protein works, the separation of AMY from GLL1 was confirmed by a more simple and robust technique, exploiting the use of an RP-HPLC system (25). The analysis using RP-HPLC is based on the fragmentation profile of a protein after proteolytic digestion. Proteolytic digestion of AMY

Total protein (mg)

Specific activity (U/mg)

Yield (%)

Intended for their characterization, AMY and GLL1 were independently collected and subjected to subsequent purification with DEAE-Toyopearl 650M AEX column. An SDS PAGE analysis showed that these purification steps resulted in pure AMY (5) and GLL1 (16). The final purification scheme of AMY is summarized in Table 1. Further, whilst the presence of two and three types of α-amylase and glucoamylase, respectively, were reported upon purification of these amylolytic enzymes from strain KZ by a Mono-Q anion exchanger column (22), AMY and GLL1 appeared to be the only amylolytic enzyme species from strain R64. Nevertheless, additional analysis was performed to confirm that the The analysis of AMY and GLL1 by RP-HPLC was carried out essentially following Soedjanaatmadja and co-workers on the identification of pseudo-hevein from the latex of rubber tree (26). Firstly, purified AMY was incubated for four hours at 37oC with chymotrypsin (EC. 3.4.21.1), at an AMY to chymotrypsin mass ratio of 100:1, in 200 mM ammonium bicarbonate buffer, pH 8.0. The reaction was stopped by an addition of 10 mM hydrochloric acid, to lower the pH of the solution. Preparation of GLL1 sample was done in the same way, including the substrate to chymotrypsin mass ratio.

The reaction mixture was immediately applied to an RP-HPLC nucleosil 10 C18 column (30 x 0.45 cm) and the separation was performed for 60 minutes, at a flow rate of 1 ml/min, using 0-70% acetonitrile gradient in 0.1% trifluoroacetic acid (TFA). Elution of fragments was monitored at λ 214 nm, which is specific for detection of peptide bonds. The fragments are eluted according to their hydrophobicity, where more hydrophobic fragments are retained longer in the RP-HPLC column.

Chymotrypsin cleavage takes place on peptide bonds at the C-terminal part of preferably tyrosine, phenylalanine, tryptophan, and leucine residues, and with (much) less extent of methionine, valine, isoleucine, histidine, glycine and alanine residues (27). Due to this broad specificity, the cleavage may result in many fragments varying in size and hydrophobicity, as observed in the chromatographic profiles of proteolytically digested AMY and GLL1 (Fig. 3).

**Figure 3.** Chromatographic profile of fragments from proteolytic digestion AMY (A) or GLL1 (B) as monitored at λ 214 nm.

The fragmentation profile of AMY was significantly different from that of GLL1. Six sets of resolved peaks were recovered in both cases (Fig. 3) but their retention time, intensities, and

peak distribution were different. Intensity of the peaks suggested much less materials were recovered from GLL1 digested samples than from AMY. This situation is likely contributed from highly hydrophobic or negative charged fragments that were not eluted from the RP-HPLC column due to their poor solubility in the solvent used (28). The amino acid sequences of AMY and GLL1 (see 3.5) showed that the charged amino acid distribution in the amino acid sequence of GLL1 (100 charged amino acid residues out of 494, ~20.2%) is higher than that of AMY (86 out of 468, ~18.4%), thus AMY contains more hydrophobic and non-charged amino acids. Unfortunately, correlation between the result from RP-HPLC and hydrophobic amino acid distribution cannot firmly be established because the nature of chymotrypsin digestion was unclear. The results may indeed indicate that hydrophobic amino acids in GLL1 are likely more clustered than in AMY, resulting in highly hydrophobic peptide fragments in GLL1. This hypothesis may be related to the structure of GLL1 (Fig. 8), which consists of one globular molecule that opposes two separable domains of AMY. However, possibility for the existence of highly negative charged peptide fragments from GLL1 can also not be excluded. Nevertheless, of the eluted fragments (Fig. 3), majority of AMY fragments are localized at peaks 5 and 6 whilst GLL1 are at peaks 3 and 4, suggesting different fragmentation had occurred. Thus, the RP-HPLC profiles of AMY and GLL1 indicated successful separation of the two enzymes.

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 279

mobile phase, which was the mixture of butanol : ethanol : water (at a ratio of 5:5:3, v/v/v). The plate was retrieved from the TLC separation chamber after the mobile phase migration reached three quarters of the length of the plate and then immediately short-submersed (few seconds) in a mixture of water : methanol : sulphuric acid (at a ratio of 45:45:10, v/v/v). The plate was then heated at 120oC for 15 minutes on a hot plate for visualization of the sugars.

The TLC profile (Fig. 4) shows that the product of starch hydrolysis by GLL1 was solely monosaccharide i.e. glucose (G1). The intensity of the G1 spot was increasing from 5 to 45 minutes of incubation, indicating that glucose was produced over time. The spots at the sample application points were from the un-hydrolyzed soluble starch substrate. Apparently, most of the starch molecules were hydrolyzed within 15-45 minutes of incubation. Most importantly, no higher oligomeric sugar forms were detected, even during the first 5-10 minutes of incubation, suggesting the absence of α-amylase. This result provided the undisputed proof that GLL1 had successfully been separated from AMY despite of α-amylase activity was detected in the GLL1 fraction. Thus, the detected α-

**Figure 4.** Analysis of starch hydrolysis products of GLL1 on a TLC plate (16). Lane 1-4 is hydrolysis products after 5, 10, 15, and 45 minutes, respectively. Lane 5 is the mono- (G1), di-(G2), and oligo-

Since the purity of AMY and GLL1 was firmly established, each enzyme could now be characterized. AMY was found active at a pH range of 5.0-7.5 and a temperature range of 30-60oC, with an optimum at 5.5 and 50oC, respectively (5). GLL1 was also found active a pH range of 4.6-6.8 and a temperature range of 30-65oC, with an optimum at 5.6-6.4 and 50oC, respectively (16). These results suggest that the two enzymes are active at similar conditions.

Recently, the amino acid sequences of AMY (GenBank accession code HQ172905) and GLL1 (HQ415729) were successfully elucidated as derived from their chromosomal DNA (16, 33).

Moreover, their characters are similar to most other *S. fibuligera* amylases.

amylase activity was not originated from AMY.

saccharide (G3-G7) markers.

**4.5. Properties of AMY and GLL1** 

## **4.4. Analysis of starch hydrolysis products to discriminate AMY and GLL1**

Glucoamylase hydrolyzes starch at the non-reducing end of amylose or amylopectin to result in glucose therefore the enzyme activity assay is normally based on the release of reducing sugar (21, 29). Unfortunately, α-amylase random digestion of starch may also result in reducing sugar i.e. maltose (30) therefore detection of glucoamylase activity in an α-amylase preparation can be anticipated. Glucoamylase can also, at lesser extent, act on 1,6 α-glycosidic bond of amylopectin (31), although its efficiency diverse greatly depending on the source organisms. Hydrolysis of the 1,6-α- bond may result in a less integrated starch molecule, hampering the formation of iodine-starch complex, which is the basis of standard α-amylase activity assay (20). Therefore, cross-detection of the two enzyme activity assays is inevitable. This phenomenon was notorious upon separation of AMY from GLL1 at pH 5.8 (5), where α-amylase activity was detected in GLL1 fractions.

One approach to discriminate α-amylase and glucoamylase is *via* the evaluation of their hydrolysis product. α-Amylase action results in various kind of oligomeric sugars (32) whilst glucoamylase hydrolysis product is glucose (monomeric). Based on this principle, successful separation of GLL1 from AMY was assessed through their hydrolysis products, as analyzed with Thin Layer Chromatography (TLC) (16), using purified GLL1 that was obtained from the purification procedure carried out at pH 5.8.

Purified GLL1 was incubated with soluble starch substrate at 37oC and samples were taken after 5, 10, 15 and 45 minutes of incubation. Each sample was then applied onto a silica gel 60 TLC plate (20 cm x 20 cm, Merck, Darmstadt, Germany) using capillary glass tube. The plate was then placed in a TLC separation chamber that had been equilibrated with the mobile phase, which was the mixture of butanol : ethanol : water (at a ratio of 5:5:3, v/v/v). The plate was retrieved from the TLC separation chamber after the mobile phase migration reached three quarters of the length of the plate and then immediately short-submersed (few seconds) in a mixture of water : methanol : sulphuric acid (at a ratio of 45:45:10, v/v/v). The plate was then heated at 120oC for 15 minutes on a hot plate for visualization of the sugars.

The TLC profile (Fig. 4) shows that the product of starch hydrolysis by GLL1 was solely monosaccharide i.e. glucose (G1). The intensity of the G1 spot was increasing from 5 to 45 minutes of incubation, indicating that glucose was produced over time. The spots at the sample application points were from the un-hydrolyzed soluble starch substrate. Apparently, most of the starch molecules were hydrolyzed within 15-45 minutes of incubation. Most importantly, no higher oligomeric sugar forms were detected, even during the first 5-10 minutes of incubation, suggesting the absence of α-amylase. This result provided the undisputed proof that GLL1 had successfully been separated from AMY despite of α-amylase activity was detected in the GLL1 fraction. Thus, the detected αamylase activity was not originated from AMY.

**Figure 4.** Analysis of starch hydrolysis products of GLL1 on a TLC plate (16). Lane 1-4 is hydrolysis products after 5, 10, 15, and 45 minutes, respectively. Lane 5 is the mono- (G1), di-(G2), and oligosaccharide (G3-G7) markers.

## **4.5. Properties of AMY and GLL1**

278 Chromatography – The Most Versatile Method of Chemical Analysis

and GLL1 indicated successful separation of the two enzymes.

(5), where α-amylase activity was detected in GLL1 fractions.

obtained from the purification procedure carried out at pH 5.8.

**4.4. Analysis of starch hydrolysis products to discriminate AMY and GLL1** 

Glucoamylase hydrolyzes starch at the non-reducing end of amylose or amylopectin to result in glucose therefore the enzyme activity assay is normally based on the release of reducing sugar (21, 29). Unfortunately, α-amylase random digestion of starch may also result in reducing sugar i.e. maltose (30) therefore detection of glucoamylase activity in an α-amylase preparation can be anticipated. Glucoamylase can also, at lesser extent, act on 1,6 α-glycosidic bond of amylopectin (31), although its efficiency diverse greatly depending on the source organisms. Hydrolysis of the 1,6-α- bond may result in a less integrated starch molecule, hampering the formation of iodine-starch complex, which is the basis of standard α-amylase activity assay (20). Therefore, cross-detection of the two enzyme activity assays is inevitable. This phenomenon was notorious upon separation of AMY from GLL1 at pH 5.8

One approach to discriminate α-amylase and glucoamylase is *via* the evaluation of their hydrolysis product. α-Amylase action results in various kind of oligomeric sugars (32) whilst glucoamylase hydrolysis product is glucose (monomeric). Based on this principle, successful separation of GLL1 from AMY was assessed through their hydrolysis products, as analyzed with Thin Layer Chromatography (TLC) (16), using purified GLL1 that was

Purified GLL1 was incubated with soluble starch substrate at 37oC and samples were taken after 5, 10, 15 and 45 minutes of incubation. Each sample was then applied onto a silica gel 60 TLC plate (20 cm x 20 cm, Merck, Darmstadt, Germany) using capillary glass tube. The plate was then placed in a TLC separation chamber that had been equilibrated with the

peak distribution were different. Intensity of the peaks suggested much less materials were recovered from GLL1 digested samples than from AMY. This situation is likely contributed from highly hydrophobic or negative charged fragments that were not eluted from the RP-HPLC column due to their poor solubility in the solvent used (28). The amino acid sequences of AMY and GLL1 (see 3.5) showed that the charged amino acid distribution in the amino acid sequence of GLL1 (100 charged amino acid residues out of 494, ~20.2%) is higher than that of AMY (86 out of 468, ~18.4%), thus AMY contains more hydrophobic and non-charged amino acids. Unfortunately, correlation between the result from RP-HPLC and hydrophobic amino acid distribution cannot firmly be established because the nature of chymotrypsin digestion was unclear. The results may indeed indicate that hydrophobic amino acids in GLL1 are likely more clustered than in AMY, resulting in highly hydrophobic peptide fragments in GLL1. This hypothesis may be related to the structure of GLL1 (Fig. 8), which consists of one globular molecule that opposes two separable domains of AMY. However, possibility for the existence of highly negative charged peptide fragments from GLL1 can also not be excluded. Nevertheless, of the eluted fragments (Fig. 3), majority of AMY fragments are localized at peaks 5 and 6 whilst GLL1 are at peaks 3 and 4, suggesting different fragmentation had occurred. Thus, the RP-HPLC profiles of AMY

> Since the purity of AMY and GLL1 was firmly established, each enzyme could now be characterized. AMY was found active at a pH range of 5.0-7.5 and a temperature range of 30-60oC, with an optimum at 5.5 and 50oC, respectively (5). GLL1 was also found active a pH range of 4.6-6.8 and a temperature range of 30-65oC, with an optimum at 5.6-6.4 and 50oC, respectively (16). These results suggest that the two enzymes are active at similar conditions. Moreover, their characters are similar to most other *S. fibuligera* amylases.

> Recently, the amino acid sequences of AMY (GenBank accession code HQ172905) and GLL1 (HQ415729) were successfully elucidated as derived from their chromosomal DNA (16, 33).

The amino acid sequence of AMY is similar to that of published earlier by Itoh and coworkers (34), having mutations at six amino acid residues (Asp>Asn153, Ile>Val159, Ser>Asn190, Ser>Xaa216, Asp>Asn239, and Ser>Thr295). The six deviating residues in AMY comprise an additional predicted glycosylation site (Asn153), which according to a structural modelling, is highly plausible because it resides in a long surface loop (33).

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 281

The hydrophobicity profile of AMY and GLL1 was analyzed based on the amino acid sequence of the mature enzymes (38), using an online analysis service at ExPASy (http://web.expasy.org/protscale/) (35). Another handful online service is also available at http://www.vivo.colostate.edu/molkit/hydropathy/index.html. The window size values for the frame normalization were scanned from 3 to 21 and compared to determine the significance of the regions that represent the hydrophobic character. The profile at window frame 9 (recommended for hydrophilic protein) and 19 (for hydrophobic protein) are presented in Fig. 5. The prediction points were linearly weighted with 100% relativity at the window edges. The score for hydrophobicity is ranged from -4.5 (hydrophilic) to 4.5 (hydrophobic), where the curve above the midpoint (zero) is interpreted as regions with hydrophobic character. As shown in Fig. 5, regions with hydrophobic character in AMY are at the residues 50-75, 210-230, 310-330, 375-410, and 425-440, whilst for GLL1 are at 225-230, 375-380, 425-445, and 475-480. This *ab initio* results suggest that AMY has more fragments with hydrophobic character, which may also be due to the presence of two separable domains in AMY that each has its own hydrophobic core. However, this result may not be corresponded with RP-HPLC profile as the fragmentation of AMY and GLL1 upon digestion with chymotrypsin occurs randomly, where

more hydrophobic fragments could be produced from GLL1.

**Figure 5.** Hydrophobicity profile of AMY and GLL1 as calculated according to their amino acid sequences (38). The upper graphs are produced with window frame of 9 whilst the lower was with

that of 19.

The amino acid sequence of GLL1 is similar to glucoamylases from strain HUT 7212 (GLU) and KZ (GLA) (16). GLU and GLA share high sequence identity (4), with only seven amino acid residues different. However, these seven residues are responsible for differences in their characteristics, where GLA exhibits potential thermal stability and GLU has higher affinity towards substrate. The amino acid sequence of GLL1 also differs to both GLU and GLA at precisely those particular seven residues, which four are being identical to GLU and three to GLA. Interestingly, these mutations result in GLL1 to adopt the potential thermal stability and higher affinity towards substrate. Thus, GLL1 behaves as a hybrid of GLU and GLA.

The calculated theoretical pI values (35) of AMY and GLL1 (based on the amino acid sequences of their mature protein sequences: AMY sequence starts at residue Glu27 of the full-length protein containing signal and pre-peptides as encoded by *AMY* gene whilst GLL1 sequence starts at residue Ala1 as reported in the data base) were 4.4 and 4.3, respectively. The isoelectric point (pI) value of AMY was confirmed experimentally, being 4.6±0.2. This finding supports the early observation that AEX or CEX columns are unable to separate the two enzymes. Similarly, the calculated theoretical molecular masses of AMY and GLL1 were also similar, being 51.7 kDa and 54.6 kDa, respectively. Both values were confirmed experimentally, being 54±2 kDa for AMY and 56±2 kDa for GLL1. Additional ±2 kDa of AMY and GLL1 masses is likely contributed from glycosylation. This result also confirms the inability of SEC column to separate them.

The activity of AMY is diminished in the presence of ethylene diamine tetra acetate (EDTA), a chelating agent. Inactivation of α-amylase by chelating agent is well known, as the enzyme requires calcium ion for its activity and integrity (36). This inactivation by chelating agent was not observed with GLL1. However, the activities of both AMY and GLL1 were increased in the presence of calcium or magnesium ions (data not shown). Further, AMY demonstrated lower activity in phosphate-citrate buffer. This phenomenon may arise from the citrate, which is reported to interact with calcium ion (37). The activity of AMY decreased concomitantly with the increase of the citrate buffer concentration but was fully recovered upon back-titration with calcium chloride (data not shown). Based on this observation, citrate buffers (1, 5) should only be used with considerable reservation and the use of tris buffer is recommended. This recommendation is in line with the necessity to perform purification at basic pH for HIC. In addition, this finding may also explain higher glucoamylase activity detected in AMY fractions when the purification was carried out using phosphate citrate buffer, pH 5.8 (5). Since the buffer does not influence the glucoamylase activity as it does to AMY, the disparity between the two enzyme activities was much less pronounced in comparison to the one presented in Fig. 2.

confirms the inability of SEC column to separate them.

was much less pronounced in comparison to the one presented in Fig. 2.

GLA.

The amino acid sequence of AMY is similar to that of published earlier by Itoh and coworkers (34), having mutations at six amino acid residues (Asp>Asn153, Ile>Val159, Ser>Asn190, Ser>Xaa216, Asp>Asn239, and Ser>Thr295). The six deviating residues in AMY comprise an additional predicted glycosylation site (Asn153), which according to a

The amino acid sequence of GLL1 is similar to glucoamylases from strain HUT 7212 (GLU) and KZ (GLA) (16). GLU and GLA share high sequence identity (4), with only seven amino acid residues different. However, these seven residues are responsible for differences in their characteristics, where GLA exhibits potential thermal stability and GLU has higher affinity towards substrate. The amino acid sequence of GLL1 also differs to both GLU and GLA at precisely those particular seven residues, which four are being identical to GLU and three to GLA. Interestingly, these mutations result in GLL1 to adopt the potential thermal stability and higher affinity towards substrate. Thus, GLL1 behaves as a hybrid of GLU and

The calculated theoretical pI values (35) of AMY and GLL1 (based on the amino acid sequences of their mature protein sequences: AMY sequence starts at residue Glu27 of the full-length protein containing signal and pre-peptides as encoded by *AMY* gene whilst GLL1 sequence starts at residue Ala1 as reported in the data base) were 4.4 and 4.3, respectively. The isoelectric point (pI) value of AMY was confirmed experimentally, being 4.6±0.2. This finding supports the early observation that AEX or CEX columns are unable to separate the two enzymes. Similarly, the calculated theoretical molecular masses of AMY and GLL1 were also similar, being 51.7 kDa and 54.6 kDa, respectively. Both values were confirmed experimentally, being 54±2 kDa for AMY and 56±2 kDa for GLL1. Additional ±2 kDa of AMY and GLL1 masses is likely contributed from glycosylation. This result also

The activity of AMY is diminished in the presence of ethylene diamine tetra acetate (EDTA), a chelating agent. Inactivation of α-amylase by chelating agent is well known, as the enzyme requires calcium ion for its activity and integrity (36). This inactivation by chelating agent was not observed with GLL1. However, the activities of both AMY and GLL1 were increased in the presence of calcium or magnesium ions (data not shown). Further, AMY demonstrated lower activity in phosphate-citrate buffer. This phenomenon may arise from the citrate, which is reported to interact with calcium ion (37). The activity of AMY decreased concomitantly with the increase of the citrate buffer concentration but was fully recovered upon back-titration with calcium chloride (data not shown). Based on this observation, citrate buffers (1, 5) should only be used with considerable reservation and the use of tris buffer is recommended. This recommendation is in line with the necessity to perform purification at basic pH for HIC. In addition, this finding may also explain higher glucoamylase activity detected in AMY fractions when the purification was carried out using phosphate citrate buffer, pH 5.8 (5). Since the buffer does not influence the glucoamylase activity as it does to AMY, the disparity between the two enzyme activities

structural modelling, is highly plausible because it resides in a long surface loop (33).

The hydrophobicity profile of AMY and GLL1 was analyzed based on the amino acid sequence of the mature enzymes (38), using an online analysis service at ExPASy (http://web.expasy.org/protscale/) (35). Another handful online service is also available at http://www.vivo.colostate.edu/molkit/hydropathy/index.html. The window size values for the frame normalization were scanned from 3 to 21 and compared to determine the significance of the regions that represent the hydrophobic character. The profile at window frame 9 (recommended for hydrophilic protein) and 19 (for hydrophobic protein) are presented in Fig. 5. The prediction points were linearly weighted with 100% relativity at the window edges. The score for hydrophobicity is ranged from -4.5 (hydrophilic) to 4.5 (hydrophobic), where the curve above the midpoint (zero) is interpreted as regions with hydrophobic character. As shown in Fig. 5, regions with hydrophobic character in AMY are at the residues 50-75, 210-230, 310-330, 375-410, and 425-440, whilst for GLL1 are at 225-230, 375-380, 425-445, and 475-480. This *ab initio* results suggest that AMY has more fragments with hydrophobic character, which may also be due to the presence of two separable domains in AMY that each has its own hydrophobic core. However, this result may not be corresponded with RP-HPLC profile as the fragmentation of AMY and GLL1 upon digestion with chymotrypsin occurs randomly, where more hydrophobic fragments could be produced from GLL1.

**Figure 5.** Hydrophobicity profile of AMY and GLL1 as calculated according to their amino acid sequences (38). The upper graphs are produced with window frame of 9 whilst the lower was with that of 19.

Another computational study was performed using the on-line hydrophobic cluster analysis (HCA) program (39). This approach has previously been done to compare the hydrophobic clusters in α-amylases (40). The sequence of AMY and GLL1 were submitted to the drawcha server (http://bioserv.impmc.jussieu.fr/hca-form.html) and the resulted profiles were analyzed manually following Gaboriaud *et al.* (41). The profiles were manually mismatched with the structures of AMY (*in silico*, generated from its homolog, see section 5) and GLU, correspondingly. AMY was predicted to have 34 hydrophobic clusters whilst GLL1 was 17. However, only 3 hydrophobic clusters of AMY are exposed on the protein surface as oppose to 7 of GLL1. The analysis suggests the presence of more hydrophobic patches on the surface of GLL1 than on that of AMY. Thus the result corresponded with the experimental finding with HIC column.

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 283

X-ray crystallography. Nevertheless, based on the result of overexpression, the attempt for heterelogous expression of AMY in *E. coli* was withdrawn for the use of expression system

*S. cerevisiae* has been a popular choice for heterelogous expression of proteins (48) and tested to express α-amylase and glucoamylase from *S. fibuligera* strain HUT 7212 (49, 50). *S. cerevisiae* expression system for expression of *S. fibuligera* α-amylase and glucoamylase has also been improved, i.e. the enzymes expression was drastically increased under the control of the *S. cerevisiae* constitutive phosphoglycerate kinase (PGK) promoter (51). Motivated from that success, AMY was overexpressed in *S. cerevisiae* INVSc1, using yEP-secretex vector and galactose as the inducer. However, although AMY recombinant demonstrated similar activity to AMY, its molecular mass is substantially higher, being 67±2 kDa. Unfortunately, no such information was provided from the expression of *S. fibuligera* α-amylase from strains HUT 7212 (50) and Eksteen (51). Since no polypeptide chain or protein tag was added, different glycosylation profile was likely the reason for the increase in the molecular mass of the recombinant protein. The finding of a plausible additional glycosylation site in

from higher organism i.e. yeast *S. cerevisiae*.

the amino acid sequence of AMY applauds this proposal.

respective buffer in the cold room (~4oC).

**5.1. AMY recombinant behavior upon purification in AEX columns**

Likewise AMY, AMY recombinant was secreted into overproduction medium therefore it was harvested by cold centrifugation at 6000 *g* for 30 minutes to remove the yeast cells. The enzyme was captured from the medium by fractionation with ammonium sulfate at a saturation degree of 0-100%. The ammonium sulfate protein precipitate was dissolved in 50 mM Tris-HCl buffer, pH 8.0 (~5 ml) and then dialyzed overnight against one litre of that

The dialyzed AMY recombinant was then loaded onto a resource-Q AEX column (GE Healthcare Europe GmbH, Diegem, Belgium), which had been equilibrated with that respective buffer. Purification was performed in a cold cabinet (~7oC) using a fast protein liquid chromatography (FPLC) Äkta system (GE Healthcare Europe GmbH, Diegem, Belgium) and monitored on-line with the Unicorn program. The enzyme was recovered from the column upon an elution with an increasing gradient of sodium chloride 0-1 M. As

Purification of AMY in the resource-Q column showed that minor contaminants were still present in the purified sample (Fig. 6). The major contaminant has, however, higher absorbance at λ 260 nm, suggesting that it may not be protein. This contaminant appeared yellowish in colour, which may be originated from the overproduction medium component that was co-purified in HIC, AEX, and SEC columns. Further, the elution profile of AMY recombinant was very similar to that of AMY, except for an additional large protein peak upon sample application and washing step prior to the sodium chloride salt gradient. These additional peaks unmistakably are originated from other proteins and components of overproduction medium because the AMY recombinant sample applied was not purified prior to this column. AMY recombinant was not pre-purified with the HIC because no

the control, purified AMY was also subjected to purification with the same column.
