THE ROLE OF GLYCOSYLATION RESIDUES ASN153 AND ASN224 ON ACTIVITY AND STABILITY OF SACCHAROMYCOPSIS FIBULIGERA R64 ?-AMYLASE

• Main Author: Dewi Rosahdi, Tina
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/36072
• Institution: Institut Teknologi Bandung

?-Amylase (1,4-?-D-glucanglucanohydrolase, EC 3.2.1.1) is an enzyme that catalyzes the hydrolysis of ?-1,4-glycosidic bonds in starch to produce ?-limit dextrin, oligosaccharide, and maltose. The enzyme is widely used in the field of biotechnology, especially in the food, pharmaceutical, textile, detergent, paper and bioethanol industries. ?-Amylases are ubiquitous enzymes which are produced by prokaryotes, eukaryotes, and archaea. One potential source of ?-amylase for industry is yeast Saccharomycopsis fibuligera. ?-Amylase (Sfamy) from S. fibuligera strains HUT 7212, KZ, and R64 have been isolated and characterized. Bioinformatics analysis shows that the three Sfamys share one conserved N-linked glycosylation residue at position 224 (Asn224). Interestingly, SfamR64 has another N-linked glycosylation residue at position 153 (Asn153). The role of both residues is unknown and has not been studied. Therefore, the aims of the research were to investigated the roles of glycosylation residues Asn153 and Asn224 on the SfamyR64 activity and stability. Asn224 and Asn153 SfamyR64 were substituted into Gln224 and Gln153 (glutamine) to remove the glycosylation sites of SfamyR64 by site directed mutagenesis with Polymerase Chain Reaction. Three types of variant SfamyR64, namely SfamyR64Asn153Gln, SfamyR64Asn224Gln, and SfamyR64Asn153Gln/Asn224Gln, had been generated. SfamyR64 wild type and the three SfamyR64 variants were produced in Pichia pastoris KM71, and purified using DEAE-Toyopearl anionic chromatography, and were analyzed in term of physico-chemical parameters, kinetics parameters, dan structural modelling. Determination of physico-chemical parameters covered optimum temperature, raw starch hydrolysis, the effect of 2 mM CaCl2, and enzyme half- life. The kinetics parameters determined include the maximum rate (Vmax), Michaelis-Menten constant (KM), catalytic constant (kcat), and catalytic efficiency (kcat/KM). Structure prediction analysis was performed using several programs, including I-TASSER, Phyre2, ModRefiner, MolProbity, and ProBis. Wild type SfamyR64, SfamyR64Asn153Gln, SfamyR64Asn224Gln, and SfamyR64Asn153Gln/Asn224Gln were produced as extracellular enzymes in P. pastoris KM71 and were successfully purified by DEAE anion exchange chromatography. Wild type SfamyR64, SfamyR64Asn153Gln, SfamyR64Asn224Gln, and SfamyR64Asn153Gln/Asn224Gln have specific activities of 117.2; 607.8; 20.1; and 6.6 U/mg; respectively. The activity of SfamyR64Asn153Gln towards soluble starch increased five-fold compared to that of wild type SfamyR64. Meanwhile, SfamyR64Asn224Gln and SfamyR64Asn153Gln/Asn224Gln activities decreased significantly. Regarding raw starch (rice, wheat, canna, corn, and sago) degrading activity, SfamyR64Asn153Gln, SfamyR64Asn224Gln, and SfamyR64Asn153Gln/Asn224Gln showed higher activity than that of wild type SfamyR64. The presence of 2 mM calcium metal ion decreased the activity of wild type SfamyR64, SfamyR64Asn153Gln, SfamyR64Asn153Gln/Asn224Gln, and SfamyR64Asn224Gln towards soluble starch. Based on the kinetic parameters, wild type SfamyR64, SfamyR64Asn153Gln, SfamyR64Asn224Gln, and SfamyR64Asn153Gln/Asn224Gln have kcat/KM values of 0.06; 0.45; 0.01; and 0.005, respectively. The half-life of wild type SfamyR64, SfamyR64Asn153Gln, SfamyR64Asn224Gln, and SfamyR64Asn153Gln/Asn224Gln were 8.4; 8.8; 5.4; and 5.8 minutes, respectively at 50 °C. SfamyR64Asn224Gln lost its activity towards soluble starch after 10 minutes, while the activities of wild type SfamyR64, SfamyR64Asn153Gln, and SfamyR64Asn153Gln/Asn224Gln disappear after 30 minutes. The results of soluble starch activity indicated that both glycosylation residues of SfamyR64 play a role in the hydrolysis soluble and raw starches. It is likely that the secondary binding of calcium ions might affect the stability of the local structure, especially in the catalytic site. The results of kinetics parameter indicated that SfamyR64N153Q has the highest catalytic efficiencies, while SfamyR64Asn153Gln/Asn224Gln showed very low catalytic efficiencies towards soluble starch. Temperature stability results sugested that the conserved glycosylation residue (Asn224) plays an important role in maintaining the stability of SfamyR64. Based on model structure, SfamyR64Asn153Gln is predicted to have more open catalytic cavity which in turn will facilitate substrate binding, while the conserved glycosylation site (Asn224) located in the domain A might act as a structural stabilizer in which SfamyR64Asn224Gln has high structural dynamics especially in its catalytic region. Taken together, this study sheds light in the important function of glycosylation in the activity and stability of SfamyR64. The glycosylation site of Asn153 plays a role in SfamyR64 activity, whereas Asn224 plays a role in the stability of SfamyR64.