**6. Structure function study of AMY in the absence of the structure**

The domain organization of AMY was studied by means of limited proteolysis by trypsin-TPCK, which has specific activity to cleave lysine and arginine residues. This procedure was employed to study the domain organization of bacterial cellulase (55). The fragments recovered from the limited proteolytic digestion of AMY were separated in a SEC column and subjected onto functional analysis (5).

Benefiting from the availability of amino acid sequences of AMY and GLL1, structural study *in silico* was performed (33). The structural model for AMY was prepared using the online model building program from the EMBL-EBI (56). Briefly, the amino acid sequence of AMY derived from its DNA (GenBank ID: HQ172905) was submitted to the online program SWISS-MODEL (57) using the structure of Taka-amylase from *A. niger* (PDB code 7TAA) as the template. The resulted model (7TAAmt) was evaluated using the program MolProbity (58), and manually assessed using the program *COOT* (59). The graphic representation was prepared with the program PyMOL (60), equipped with the programs PDB2PQR (61) and APBS (62) for the calculation of the surface charge distribution. As for GLL1, the structure of GLU was adopted because of near identical amino acid sequence.

The protein surface of AMY and GLL1 is mainly composed by negatively charged amino acid residues (Fig. 8). AMY does have more hydrophobic residues but they are concentrated at the interface between A/B to C domains. Discounting these domain interface hydrophobic residues in AMY, GLL1 has more hydrophobic patches on its surface. The large hydrophobic patch on the surface of AMY (Fig. 8, top right) is interrupted by positively 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 well as the low pI of the two enzymes.

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

## **6.1. Proteolytic digestion of AMY**

286 Chromatography – The Most Versatile Method of Chemical Analysis

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*

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

α-amylase from strains HUT 7212 (50) and Eksteen (51) was also not available.

**6. Structure function study of AMY in the absence of the structure** 

The domain organization of AMY was studied by means of limited proteolysis by trypsin-TPCK, which has specific activity to cleave lysine and arginine residues. This procedure was employed to study the domain organization of bacterial cellulase (55). The fragments recovered from the limited proteolytic digestion of AMY were separated in a SEC column

Benefiting from the availability of amino acid sequences of AMY and GLL1, structural study *in silico* was performed (33). The structural model for AMY was prepared using the online model building program from the EMBL-EBI (56). Briefly, the amino acid sequence of AMY derived from its DNA (GenBank ID: HQ172905) was submitted to the online program SWISS-MODEL (57) using the structure of Taka-amylase from *A. niger* (PDB code 7TAA) as the template. The resulted model (7TAAmt) was evaluated using the program MolProbity (58), and manually assessed using the program *COOT* (59). The graphic representation was prepared with the program PyMOL (60), equipped with the programs PDB2PQR (61) and APBS (62) for the calculation of the surface charge distribution. As for GLL1, the structure of

The protein surface of AMY and GLL1 is mainly composed by negatively charged amino acid residues (Fig. 8). AMY does have more hydrophobic residues but they are concentrated at the interface between A/B to C domains. Discounting these domain interface hydrophobic residues in AMY, GLL1 has more hydrophobic patches on its surface. The large hydrophobic patch on the surface of AMY (Fig. 8, top right) is interrupted by positively

use of AMY is still preferred for the structure-function study.

GLU was adopted because of near identical amino acid sequence.

and subjected onto functional analysis (5).

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 domain separation (5).

Digestion of AMY by trypsin-TPCK resulted in two fragments with molecular masses of ~39 kDa (f39) and ~10 kDa (f10) (5), as judged from an SDS PAGE analysis. Based on the size of the fragments and proteolytic cleavage prediction according to its amino acid sequence, the f39 is designated as the N-terminal domain whilst f10 as the C-terminal. α-Amylases structure comprises of an (α/β)8-TIM barrel structural motif that is built up from the Nterminal part (domain A and B) and of the C-terminal part (domain C) (63). These two major domains are linked by a long surface loop. The integrated domain A/B is assigned as the catalytic domain whilst domain C is postulated as the starch-binding domain. As the two major domains of AMY were apparently separated upon proteolytic digestion, the functioning of f39 and f10 were evaluated.

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 289

**Figure 9.** Separation of the f39 and f10 using a Sephadex G50 SEC column (5). The open circle represents absorption profile of AMY (54 kDa) whilst the closed circle is f39 and f10. The arrow marks

Further, AMY was pre-treated under various conditions that resulted in denatured and partially denatured enzymes prior to proteolysis. Similar experiments were also carried out using a chemically modified AMY (33). The results were employed to assess the domain organization and assignment of AMY as well as to predict the precise location of trypsin cleavage and the nature of the catalytic domain. These results are being reported

Amylolytic enzymes from *S. fibuligera* R64 (AMY and GLL1) were successfully separated and identified using chromatography as the key tool. The two enzymes have a different protein surface hydrophobicity profile and their fragmentation profiles provided undisputed proof for their separation. Their assignment was confirmed by the analysis of the products from enzymatic hydrolysis. Further, the domain organization and functioning of AMY has been explored, which led to the structural study of AMY in the absence of its structure. Thereby, this chapter demonstrates how the results from the chromatographic analysis of AMY and GLL1 are complementary to the structural study of the enzymes.

*Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,* 

*Institute for Bioengineering and Bioscience, School of Chemistry and Biochemistry, Georgia Institute of Technology, IBB Petit Building, Atlanta, Georgia, USA* 

the peak of AMY.

elsewhere (33).

**7. Conclusion** 

**Author details** 

Wangsa T. Ismaya

*Jatinangor, Sumedang, Indonesia* 

## **6.2. Separation of f39 and f10 in an SEC column**

The separation of f39 and f10 was performed in a Sephadex G50 SEC column (∅1.3 x 50 cm, bed volume ~48 ml) with gravity flow, in 20 mM phosphate citrate buffer, pH 5.8. Fractions of 5 ml were collected and the protein elution profile was measured by absorbance at λ 280 nm (UV-160, Shimadzu Corp., Tokyo, Japan). Only the collected protein absorption peak fractions were used for further analysis. As the control, purified AMY was also applied to the column and eluted with the same conditions for the proteolytically digested sample. The amount of AMY applied was also kept similar to that of used in the proteolytic reaction for fair comparison.

Two distinct protein peaks were recovered from the proteolytic digestion reaction mixture (Fig. 9) as oppose to one peak from the purified AMY. The use of SEC column at a 50 kDa cut off allowed a clear separation because AMY was eluted right at the end of the void volume retention whilst digested AMY was eluted after the void volume. Trypsin (~23 kDa) was not detected because its amount was very small (out numbered by f39 and f10, having an AMY to trypsin mass ratio of 34:1). Should trypsin be detected, it may contribute to a small increase of absorbance at fraction 7 of the digested AMY. Fractions 3 (of AMY), 4 and 8 (of f39 and f10, respectively) were selected for further analysis.

Only AMY and f39 demonstrated α-amylase activity, confirming the assignment of f39 function as the catalytic domain. However, the KM value of f39 suggested lower affinity towards starch substrate. Interestingly, lower f39 KM value was not followed by the decrease of the *kcat* value of the reaction, suggesting that the catalytic efficiency was not disturbed (5). These observations further supported the assignment of f39 as the catalytic domain as well as suggested the function of f10 in substrate binding. Furthermore, the half-life time (IC50) value of f39 upon incubation at 50oC was also lower than that of AMY (5). This finding suggested that the absence of f10 also resulted in lower stability of f39. In conclusion, these findings served as an evidence for the proposed function of f10 to house the substrate recognition site (63) and to maintain thermostability (64) of α-amylase. This finding assigned AMY as a raw starch degrading but not-adsorbing enzyme. Ultimately, these results were confirmed by the independent group working on AMY homolog from *S. fibuligera* strain KZ (65).

Chromatography as the Major Tool in the Identification and the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 289

**Figure 9.** Separation of the f39 and f10 using a Sephadex G50 SEC column (5). The open circle represents absorption profile of AMY (54 kDa) whilst the closed circle is f39 and f10. The arrow marks the peak of AMY.

Further, AMY was pre-treated under various conditions that resulted in denatured and partially denatured enzymes prior to proteolysis. Similar experiments were also carried out using a chemically modified AMY (33). The results were employed to assess the domain organization and assignment of AMY as well as to predict the precise location of trypsin cleavage and the nature of the catalytic domain. These results are being reported elsewhere (33).
