**4. Purification of AMY and GLL1 in chromatography columns**

Since the final step in the isolation procedure in the pilot experiment was precipitation with high concentration of ammonium sulfate, subsequent purification on a size-exclusion chromatography (SEC) column appears to be the most appropriate approach because it also functions as a desalting procedure. SEC column requires minimum size of sample upon application (recommended less than 3-5% of the column volume), which can easily be overcome by dissolving the protein precipitate from ammonium sulfate precipitation in a small volume. Another option is purification using an anion or cation exchanger chromatography (abbreviated as AEX and CEX, respectively) columns. However, this either type of column requires the removal of salts prior to sample application. This requirement can be countered by either diluting the sample until the conductivity of the sample solution is low or by dialysis against a buffer that contains low salt concentration.

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 275

decrement, w/v). The separation profile of AMY and GLL1 in the HIC column is presented in Fig. 2. AMY was eluted at the ammonium sulfate concentration of 15% (w/v) whilst GLL1 was at 5% (w/v). This result demonstrated that AMY and GLL1 were successfully separated based on their hydrophobicity. Moreover, the elution profile suggests that the surface

Purification of proteins in HIC column is influenced by the pH of the solution where hydrophobic interaction is stronger at lower pH (19). This means, less hydrophobic proteins are bound stronger onto the HIC matrix at lower pH value. This phenomenon may explain different separation profile of AMY and GLL1 observed upon purification in an HIC column using phosphate-citrate buffer system at pH 5.8 (5), where α-amylase activity was detected in the GLL1 fraction. Although this hypothesis is yet to be proven, the separation at neutral

**Figure 2.** Separation profile of AMY and GLL1 in the butyl-Toyopearl 650M HIC column. The black, red, and green lines represent the protein absorbance at λ 280 nm, α-amylase activity (X 500 U/ml), and glucoamylase activity (X 10 U/ml), respectively. α-Amylase activity was based on starch degradation

The use of HIC column to separate *S. fibuligera* α-amylase from glucoamylase was reported previously with an amylolytic enzyme preparation from strain KZ (22). That attempt was performed using a Spheron 300 LC HIC column, following successful separation of hog's pancreas α-amylase from its amylolytic enzyme complex (23). However, unlike butyl-Toyopearl column where proteins are captured by the butyl ligand, hydrophobic interactions in a Spheron column occur between the proteins and the hydrophobic backbone of the matrix, similar to separation of proteins in a reversed-phase high performance liquid

character of GLL1 is more hydrophobic than AMY.

(20) whilst glucoamylase was on reducing sugar (21).

to basic pH (above 7.0) is recommended for optimum operation.

The ammonium sulfate precipitate containing AMY and GLL1 was immediately dissolved in a small portion (3-5 ml) of 25 mM Tris-HCl buffer, pH 8.0. Unfortunately, AMY and GLL1 were not separated in an SEC column as suggested from the enzyme activity assays. In a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis, AMY and GLL1 was not separated and appeared as one protein band with a molecular mass of 55±2 kDa. Further, purification in a DEAE-cellulose AEX column has also failed to separate the two enzymes. This finding indicated a similarity in the distribution of charge on the surface of AMY and GLL1. In addition, there was a recommendation to avoid the use of carbohydrate- based column material for the purification of proteins with an affinity towards carbohydrate, because of the possible interaction between the protein and the supporting column matrix (17). Thus, both SEC and AEX columns were obliterated from the separation of AMY from GLL1.

As purification strategies based on either protein size or charge failed to separate AMY from GLL1, exploiting differences of their protein surface hydrophobicity profiles emerged as an alternative. This strategy was tested on a hydrophobic interaction chromatography (HIC) column, where the purification proteins is based on the hydrophobic character of the surface of the proteins (18). In an HIC column, proteins in solution are conditioned with high salt concentration, which enforces interaction between proteins and the hydrophobic resin. Separation of AMY from GLL1 in an HIC column is lucrative because it can be carried out immediately after precipitation with high concentration of ammonium sulfate. Thus, the use of the HIC column is complementary to the developed isolation procedure.

## **4.1. Separation of AMY and GLL1 in an HIC column**

The ammonium sulfate precipitated protein was dissolved in 25 mM Tris-HCl buffer, pH 8.0, containing 25% ammonium sulfate (w/v). This protein solution was applied to a butyl-Toyopearl 650M HIC column (Tosoh Bioscience Corp., Tokyo, Japan), which had previously been equilibrated with the same buffer. The column was then eluted with 25 mM Tris-HCl buffer, pH 8.0, containing a decreasing concentration of ammonium sulfate (25-0% of 5% decrement, w/v). The separation profile of AMY and GLL1 in the HIC column is presented in Fig. 2. AMY was eluted at the ammonium sulfate concentration of 15% (w/v) whilst GLL1 was at 5% (w/v). This result demonstrated that AMY and GLL1 were successfully separated based on their hydrophobicity. Moreover, the elution profile suggests that the surface character of GLL1 is more hydrophobic than AMY.

274 Chromatography – The Most Versatile Method of Chemical Analysis

separation of AMY from GLL1.

**4. Purification of AMY and GLL1 in chromatography columns** 

is low or by dialysis against a buffer that contains low salt concentration.

Since the final step in the isolation procedure in the pilot experiment was precipitation with high concentration of ammonium sulfate, subsequent purification on a size-exclusion chromatography (SEC) column appears to be the most appropriate approach because it also functions as a desalting procedure. SEC column requires minimum size of sample upon application (recommended less than 3-5% of the column volume), which can easily be overcome by dissolving the protein precipitate from ammonium sulfate precipitation in a small volume. Another option is purification using an anion or cation exchanger chromatography (abbreviated as AEX and CEX, respectively) columns. However, this either type of column requires the removal of salts prior to sample application. This requirement can be countered by either diluting the sample until the conductivity of the sample solution

The ammonium sulfate precipitate containing AMY and GLL1 was immediately dissolved in a small portion (3-5 ml) of 25 mM Tris-HCl buffer, pH 8.0. Unfortunately, AMY and GLL1 were not separated in an SEC column as suggested from the enzyme activity assays. In a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis, AMY and GLL1 was not separated and appeared as one protein band with a molecular mass of 55±2 kDa. Further, purification in a DEAE-cellulose AEX column has also failed to separate the two enzymes. This finding indicated a similarity in the distribution of charge on the surface of AMY and GLL1. In addition, there was a recommendation to avoid the use of carbohydrate- based column material for the purification of proteins with an affinity towards carbohydrate, because of the possible interaction between the protein and the supporting column matrix (17). Thus, both SEC and AEX columns were obliterated from the

As purification strategies based on either protein size or charge failed to separate AMY from GLL1, exploiting differences of their protein surface hydrophobicity profiles emerged as an alternative. This strategy was tested on a hydrophobic interaction chromatography (HIC) column, where the purification proteins is based on the hydrophobic character of the surface of the proteins (18). In an HIC column, proteins in solution are conditioned with high salt concentration, which enforces interaction between proteins and the hydrophobic resin. Separation of AMY from GLL1 in an HIC column is lucrative because it can be carried out immediately after precipitation with high concentration of ammonium sulfate. Thus, the use

The ammonium sulfate precipitated protein was dissolved in 25 mM Tris-HCl buffer, pH 8.0, containing 25% ammonium sulfate (w/v). This protein solution was applied to a butyl-Toyopearl 650M HIC column (Tosoh Bioscience Corp., Tokyo, Japan), which had previously been equilibrated with the same buffer. The column was then eluted with 25 mM Tris-HCl buffer, pH 8.0, containing a decreasing concentration of ammonium sulfate (25-0% of 5%

of the HIC column is complementary to the developed isolation procedure.

**4.1. Separation of AMY and GLL1 in an HIC column** 

Purification of proteins in HIC column is influenced by the pH of the solution where hydrophobic interaction is stronger at lower pH (19). This means, less hydrophobic proteins are bound stronger onto the HIC matrix at lower pH value. This phenomenon may explain different separation profile of AMY and GLL1 observed upon purification in an HIC column using phosphate-citrate buffer system at pH 5.8 (5), where α-amylase activity was detected in the GLL1 fraction. Although this hypothesis is yet to be proven, the separation at neutral to basic pH (above 7.0) is recommended for optimum operation.

**Figure 2.** Separation profile of AMY and GLL1 in the butyl-Toyopearl 650M HIC column. The black, red, and green lines represent the protein absorbance at λ 280 nm, α-amylase activity (X 500 U/ml), and glucoamylase activity (X 10 U/ml), respectively. α-Amylase activity was based on starch degradation (20) whilst glucoamylase was on reducing sugar (21).

The use of HIC column to separate *S. fibuligera* α-amylase from glucoamylase was reported previously with an amylolytic enzyme preparation from strain KZ (22). That attempt was performed using a Spheron 300 LC HIC column, following successful separation of hog's pancreas α-amylase from its amylolytic enzyme complex (23). However, unlike butyl-Toyopearl column where proteins are captured by the butyl ligand, hydrophobic interactions in a Spheron column occur between the proteins and the hydrophobic backbone of the matrix, similar to separation of proteins in a reversed-phase high performance liquid 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 glucoamylase.

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 277

was expected to result in fragmentation profile that differs to that of GLL1, as reflected from

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 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

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

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

the same way, including the substrate to chymotrypsin mass ratio.

their chromatographic profiles.

retained longer in the RP-HPLC column.

monitored at λ 214 nm.
