**7. Conclusion**

288 Chromatography – The Most Versatile Method of Chemical Analysis

functioning of f39 and f10 were evaluated.

fair comparison.

*S. fibuligera* strain KZ (65).

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

8 (of f39 and f10, respectively) were selected for further analysis.

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

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

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

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 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.
