PURIFICATION AND CHARACTERIZATION OF RECOMBINANT Bacillus aquimaris MKSC 6.2 ?-AMYLASE EXPRESSED In Escherichia coli ArcticExpress (DE3)

• Main Author: Khaerunnisa Frima, Fina
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38238
• Institution: Institut Teknologi Bandung

?-Amylase (E.C 3.2.1.1) catalyzes the hydrolysis of internal ?-1,4-glycosidic linkages in starch. An ?-amylase from Bacillus aquimaris BaqA can degrade raw starch despite having no starch binding domain (SBD) which is commonly presence in the known raw starch degrading enzymes. BaqA is classified in a novel GH13 subfamily based on the presence of two consecutive tryptophans located at 201 and 202 position (W201 and W202) which are potential to play a role in starch binding. The first aim of this research was to study the function of two consecutive tryptophans W201 and W202. Secondly, this research also aimed to understand the propertises of BaqA wildtype. A recombinant plasmid pET30aEZ-BaqA was used as a template to construct BaqA mutants employing PCR based-site directed mutagenesis. Two set of primers were designed to convert a codon for tryptophan TGG into a codon for alanine GCG. The presences of mutation in the BaqA01 (pET30aEZ- BaqAW201A) and the BaqA02 (pET30aEZ-BaqAW201A-W202A) were confirmed by nucleotide sequence analysis. The BaqA was expressed in E. coli ArcticExpress (DE3) both as inclusion body and soluble protein with molecular weight of ~58 kDa based on SDS-PAGE analysis. The specific activity of BaqA was 147.6 U/mg as determined by DNS method, while the activity mutants BaqA01 and BaqA02 were 88.7 U/mg and 40.5 U/mg, respectively. The BaqA, can degrade various raw starch, such as rice, sago, potato, canna, and wheat with the amount of reducing sugar for each raw starch substrate, resepectively were 1105.2 ?mol/mg, 2431.2 ?mol/mg, 2269.4 ?mol/mg, 2246.1 ?mol/mg, and 2827.5 ?mol/mg. The BaqA01 and BaqA02 have lower activity towards raw starch compared with that of BaqA wildtype. In which the amount of reducing sugar from the degradation of rice, sago, potato, canna, and wheat by BaqA01 were 346,1 ?mol/mg, 417,3 ?mol/mg, 437,7 ?mol/mg, 484,9 ?mol/mg, and 638,2 ?mol/mg, respectively. While for BaqA02 were 579,5 ?mol/mg, 633,9 ?mol/mg, 714,9 ?mol/mg, 879,1 ?mol/mg, and 1140,0 ?mol/mg, consecutively. Taken together, these results showed that both W201 and W202 have important role in the soluble and raw starch binding. Since an attempt to purify soluble wild type BaqA has not been successful yet, therefore the inclusion body of BaqA was purified by using a Ni-NTA affinity chromatography under denaturing condition. The inclusion body of BaqA was first dissolved in 8 M urea buffer, and then was refolded with gradient urea buffer. SDS-PAGE analysis indicated that the wildtype BaqA has been purified as indicated by the appearance of a major single band at ~58 kDa. The purified BaqA has an optimum activity at the pH 7.0 and 40 oC in 1% soluble starch in the presence of 150 mM NaCl. Interestingly, BaqA retained about 69 % and 59 % activity on the presence of 500 mM and 1000 mM NaCl, respectively. And also it has more than 50 % activity in pH 4.0 to 9.0. In conclusion, these results indicated that BaqA is potential to be used in starch processing industry since its ability to degrade on raw starch in high salt concentration and broad pH range at low temperature.