**5. AMY recombinant behaviour on purification in chromatography columns**

The biochemical characteristics of AMY and GLL1 are to be improved to meet specific conditions for application, such as resistance to high temperature, chemical inactivation, and proteases (42). This can be achieved by engineering at both gene and protein levels, which require convenient access to the genetic information and protein structure. Although the genes encoding for AMY or GLL1 are successfully elucidated, commencing an educated and directed genetic engineering entails the structure of the enzymes. The amino acid sequence of GLL1 is nearly identical to that of GLU (43), which its structure has been reported (in complex with amylases inhibitor acarbose, PDB accession code 2F6D). Therefore, structural study of GLL1 was performed employing the structure of GLU. The structure of *S. fibuligera* α-amylase is, on the other hands, not available. The structural study of fungal α-amylase has been employing the structure of the enzyme from *Aspergillus niger* (PDB code 7TAA) (44), which shares only 35% homology to AMY (5). Although the use of that structure is amenable, it might not be able to describe the details of characteristics of AMY. Therefore, elucidating the structure of AMY became a priority. Leading to this aim, heterelogous expression of AMY in *Escherichia coli* was attempted.

Overexpression of soluble and functional AMY in *E. coli*, with a His6-tag at its N-terminus for easy purification on a Nickel-agarose affinity column, was unsuccessful. This situation was not improved after change of the vectors that harbour the AMY gene and of *E. coli* strains, and manipulation of overproduction conditions. No amylolytic activity or protein band at the expected molecular mass upon an SDS PAGE analysis was detected in the cell lysate. Overexpression of proteins from higher organisms in bacterial system often results in the formation of inclusion bodies because of the lack of post-translational machinery (45). As α-amylase is a glycoprotein (46), which requires post-translational modification for its expression, this lack of that machinery was an obvious suspect. Actually, was AMY overexpressed successfully in *E. coli* as soluble protein, attempt leading to structure determination would be easier because post-translational modifications are reported to hamper protein crystallization (47), the initial step for structure determination by means of 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 from higher organism i.e. yeast *S. cerevisiae*.

282 Chromatography – The Most Versatile Method of Chemical Analysis

expression of AMY in *Escherichia coli* was attempted.

finding with HIC column.

**columns** 

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

**5. AMY recombinant behaviour on purification in chromatography** 

The biochemical characteristics of AMY and GLL1 are to be improved to meet specific conditions for application, such as resistance to high temperature, chemical inactivation, and proteases (42). This can be achieved by engineering at both gene and protein levels, which require convenient access to the genetic information and protein structure. Although the genes encoding for AMY or GLL1 are successfully elucidated, commencing an educated and directed genetic engineering entails the structure of the enzymes. The amino acid sequence of GLL1 is nearly identical to that of GLU (43), which its structure has been reported (in complex with amylases inhibitor acarbose, PDB accession code 2F6D). Therefore, structural study of GLL1 was performed employing the structure of GLU. The structure of *S. fibuligera* α-amylase is, on the other hands, not available. The structural study of fungal α-amylase has been employing the structure of the enzyme from *Aspergillus niger* (PDB code 7TAA) (44), which shares only 35% homology to AMY (5). Although the use of that structure is amenable, it might not be able to describe the details of characteristics of AMY. Therefore, elucidating the structure of AMY became a priority. Leading to this aim, heterelogous

Overexpression of soluble and functional AMY in *E. coli*, with a His6-tag at its N-terminus for easy purification on a Nickel-agarose affinity column, was unsuccessful. This situation was not improved after change of the vectors that harbour the AMY gene and of *E. coli* strains, and manipulation of overproduction conditions. No amylolytic activity or protein band at the expected molecular mass upon an SDS PAGE analysis was detected in the cell lysate. Overexpression of proteins from higher organisms in bacterial system often results in the formation of inclusion bodies because of the lack of post-translational machinery (45). As α-amylase is a glycoprotein (46), which requires post-translational modification for its expression, this lack of that machinery was an obvious suspect. Actually, was AMY overexpressed successfully in *E. coli* as soluble protein, attempt leading to structure determination would be easier because post-translational modifications are reported to hamper protein crystallization (47), the initial step for structure determination by means of *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 the amino acid sequence of AMY applauds this proposal.

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

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

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

glucoamylase was co-produced. Nevertheless, similarity of their elution profiles in the resource-Q AEX column suggests that AMY and AMY recombinant share similar surface charge distribution.

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 285

recombinant and AEX resin at pH 5.2 is logical, although the pH is still higher than the pI of the enzyme. However, AMY demonstrated binding to DEAE-52 cellulose matrix in that

Another approach to purify AMY recombinant is the use of α-, β-, or γ-cyclodextrin (CD) columns, which was reported previously for the purification of amylolytic enzymes (52). CD is cyclic polymer of D-glycopyranosyl that is linked by α-D-(1-4) glycosidic bond and has no non-reducing or reducing ends. The α-, β-, or γ- variant of cyclodextrin matrices differs only on the number of glucose residue that builds up the dextrin ring, being six, seven, and eight, respectively (53). The protein target is bound to the interior of the CD molecule *via* hydrophobic interactions, since this interior part is less hydrophilic than its surroundings. From this perspective, CD columns have similar function to HIC column. CD also interacts with the raw starch affinity but not active sites of α-amylases (53), unlike pullulanase or cyclodextrin glycosyltranferase. Interestingly, the binding to CD column also occurs to the raw starch degrading but not adsorbing α-amylases. These reports showed that the purification of amylolytic enzymes on a CD column is based on their affinity towards

The use of CD affinity columns is lucrative because the purification can directly be carried out after the harvesting step. After cold centrifugation, the supernatant that contained AMY recombinant was directly loaded onto α-, β-, or γ- CD affinity columns, which had already been equilibrated with 10 mM sodium acetate buffer, pH 5.5. After an extensive elution with the same buffer to remove unbound proteins, the column was eluted with 1% (w/v) α-, β-, or γ- CD in 10 mM sodium acetate buffer, pH 5.5, respective to the type of the column. Samples taken during the elution were subjected to an analysis with SDS PAGE. As the control, this

Purification of AMY on the CD columns showed an equally strong interaction with both αand β-CD matrices variant but weakly to γ-CD, as judged from the amount of AMY eluted from the respective CD column (Fig. 7). The result is in agreement with the reported use of CD column to separate α- from β-amylase from the amylolytic complex in higher plants (52). Surprisingly, AMY recombinant was not bound onto any of the CD matrices. This result set further doubt on the surface character of AMY recombinant as being different to AMY.

**Figure 7.** Elution of AMY from the α- (lane 2), β- (lane 3), and γ- (lane 4) CD columns. In lane 1 is the

purification procedure was also applied to the purified AMY.

condition, suggesting the recombinant protein has different protein surface character.

**5.2. AMY recombinant behavior upon purification in sugar affinity columns**

substrate like matrix material.

molecular mass marker.

**Figure 6.** Elution profile of AMY on a resource-Q AEX column. The blue, red, and brown lines represent absorption at λ 280 nm (protein), at λ 260 nm, and the conductivity (mS/cm).

Further, AMY recombinant was subjected to purification in DEAE-52 cellulose AEX column (Whatman Nederland BV, s'Hertogenbosch, The Netherlands) following the purification of *S. fibuligera* α-amylase strain HUT7212 (46). Although DEAE cellulose have failed to separate AMY from GLL1 in the previous attempt, the binding of AMY to this AEX resin at purification conditions similar to that of Matsui's was observed. Thus, AMY recombinant should produce a comparable elution profile, as demonstrated previously during its purification on the resource-Q AEX column.

Supernatant from cold centrifugation was mixed with DEAE-52 cellulose AEX resin that had been equilibrated with 50 mM sodium acetate buffer, pH 5.2. After 1.5 hours of incubation at 4oC, the resin was allowed to settle and the unbound proteins were carefully decanted. The resin was washed three times with and then suspended in the respective buffer. The DEAE-52 cellulose AEX suspension was poured into an empty chromatographic column and eluted with the respective buffer. The proteins were recovered from the column by a sodium chloride salt gradient 0-1 M.

Surprisingly, AMY recombinant was not bound to DEAE-52 cellulose AEX resin, as judged from both SDS PAGE analysis and enzymatic assay. Reflecting back to purification on the resource-Q AEX column at pH 8.0, AMY and AMY recombinant were eluted at 30-35% of B solution (1 M sodium chloride, thus 300-350 mM), suggesting that the enzymes were not strongly bound to the AEX matrix. Thus, to observe no interaction between AMY recombinant and AEX resin at pH 5.2 is logical, although the pH is still higher than the pI of the enzyme. However, AMY demonstrated binding to DEAE-52 cellulose matrix in that condition, suggesting the recombinant protein has different protein surface character.

## **5.2. AMY recombinant behavior upon purification in sugar affinity columns**

284 Chromatography – The Most Versatile Method of Chemical Analysis

purification on the resource-Q AEX column.

chloride salt gradient 0-1 M.

charge distribution.

glucoamylase was co-produced. Nevertheless, similarity of their elution profiles in the resource-Q AEX column suggests that AMY and AMY recombinant share similar surface

**Figure 6.** Elution profile of AMY on a resource-Q AEX column. The blue, red, and brown lines represent absorption at λ 280 nm (protein), at λ 260 nm, and the conductivity (mS/cm).

Further, AMY recombinant was subjected to purification in DEAE-52 cellulose AEX column (Whatman Nederland BV, s'Hertogenbosch, The Netherlands) following the purification of *S. fibuligera* α-amylase strain HUT7212 (46). Although DEAE cellulose have failed to separate AMY from GLL1 in the previous attempt, the binding of AMY to this AEX resin at purification conditions similar to that of Matsui's was observed. Thus, AMY recombinant should produce a comparable elution profile, as demonstrated previously during its

Supernatant from cold centrifugation was mixed with DEAE-52 cellulose AEX resin that had been equilibrated with 50 mM sodium acetate buffer, pH 5.2. After 1.5 hours of incubation at 4oC, the resin was allowed to settle and the unbound proteins were carefully decanted. The resin was washed three times with and then suspended in the respective buffer. The DEAE-52 cellulose AEX suspension was poured into an empty chromatographic column and eluted with the respective buffer. The proteins were recovered from the column by a sodium

Surprisingly, AMY recombinant was not bound to DEAE-52 cellulose AEX resin, as judged from both SDS PAGE analysis and enzymatic assay. Reflecting back to purification on the resource-Q AEX column at pH 8.0, AMY and AMY recombinant were eluted at 30-35% of B solution (1 M sodium chloride, thus 300-350 mM), suggesting that the enzymes were not strongly bound to the AEX matrix. Thus, to observe no interaction between AMY

Another approach to purify AMY recombinant is the use of α-, β-, or γ-cyclodextrin (CD) columns, which was reported previously for the purification of amylolytic enzymes (52). CD is cyclic polymer of D-glycopyranosyl that is linked by α-D-(1-4) glycosidic bond and has no non-reducing or reducing ends. The α-, β-, or γ- variant of cyclodextrin matrices differs only on the number of glucose residue that builds up the dextrin ring, being six, seven, and eight, respectively (53). The protein target is bound to the interior of the CD molecule *via* hydrophobic interactions, since this interior part is less hydrophilic than its surroundings. From this perspective, CD columns have similar function to HIC column. CD also interacts with the raw starch affinity but not active sites of α-amylases (53), unlike pullulanase or cyclodextrin glycosyltranferase. Interestingly, the binding to CD column also occurs to the raw starch degrading but not adsorbing α-amylases. These reports showed that the purification of amylolytic enzymes on a CD column is based on their affinity towards substrate like matrix material.

The use of CD affinity columns is lucrative because the purification can directly be carried out after the harvesting step. After cold centrifugation, the supernatant that contained AMY recombinant was directly loaded onto α-, β-, or γ- CD affinity columns, which had already been equilibrated with 10 mM sodium acetate buffer, pH 5.5. After an extensive elution with the same buffer to remove unbound proteins, the column was eluted with 1% (w/v) α-, β-, or γ- CD in 10 mM sodium acetate buffer, pH 5.5, respective to the type of the column. Samples taken during the elution were subjected to an analysis with SDS PAGE. As the control, this purification procedure was also applied to the purified AMY.

Purification of AMY on the CD columns showed an equally strong interaction with both αand β-CD matrices variant but weakly to γ-CD, as judged from the amount of AMY eluted from the respective CD column (Fig. 7). The result is in agreement with the reported use of CD column to separate α- from β-amylase from the amylolytic complex in higher plants (52). Surprisingly, AMY recombinant was not bound onto any of the CD matrices. This result set further doubt on the surface character of AMY recombinant as being different to AMY.

**Figure 7.** Elution of AMY from the α- (lane 2), β- (lane 3), and γ- (lane 4) CD columns. In lane 1 is the molecular mass marker.

Excessive amount of contaminants from the production medium was one of the suspects for this altered AMY recombinant behaviour upon purification with AEX DEAE-52 cellulose or CD columns, but the result from the latter challenged that possibility. Instead, different glycosylation pattern emerged as the prime suspect because glycosylation has indeed been reported as the main drawback for heterelogous expression of protein in *S. cerevisiae* (48). Hypermannosylation was detected upon the overexpression of *A. niger* glucoamylase in *S. cerevisiae* (54). Change of glycosylation profile may have negative impact as it can demolish enzyme activity or even change protein function (48). Furthermore, different glycosylation pattern may explain the observed higher molecular mass of AMY recombinant. Unfortunately, comparable information from similar works on the expression of *S. fibuligera* α-amylase from strains HUT 7212 (50) and Eksteen (51) was also not available.

Chromatography as the Major Tool in the Identification and

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charged residues (lysine) and putative additional glycosylation site, which increases the overall hydrophilicity of that hydrophobic patch. The surface representation suggests that the surface profile of GLL1 is more hydrophobic, thus the *in silico* study supports results from the purification in HIC column. The overall negative charged residues on the protein surface might also explain the need for basic pH for the buffers used in the purification as

**Figure 8.** Surface charge distribution representation of AMY and GLL1. The red and blue colour represents negatively and positively charged amino acid residues, whilst the whitish are neutral of

Purified AMY was incubated with trypsin treated with tosyl phenylalanyl chloromethyl ketone (TPCK) for 72 hours at 37oC at a substrate to protease mol ratio of 15:1. The reaction was carried out in 25 mM Tris-HCl buffer, pH 8.3 containing 20 mM calcium chloride. The reaction mixture was then transferred to -20oC for storage prior to further analysis in SDS PAGE analysis, or immediately applied to a sephadex G50 SEC column for the enzyme's

hydrophobic amino acids. The C domain of AMY is shifted away for clarity.

**6.1. Proteolytic digestion of AMY** 

domain separation (5).

well as the low pI of the two enzymes.

Overexpression of amylolytic enzymes in *S. cerevisiae* is very attractive because it leads to direct conversion of starch to ethanol (11), for generation of renewable energy. Heterelogous expression of AMY in yeast *S. cerevisiae* has been successful to produce AMY recombinant, in term of enzyme activity. This AMY recombinant could be employed in further work leading to production of bio-ethanol. However, the purification profile of AMY recombinant differs substantially to AMY and the cause for this problem is yet to disclose. Therefore, the use of AMY is still preferred for the structure-function study.
