ROLE OF LYS202, GLU231, AND HIS294 RESIDUES IN THE HYDROLYSIS OF STARCH ?-AMYLASE BMAN1?C

• Main Author: Nafisah, Zahrotun
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/55376
• Institution: Institut Teknologi Bandung

?-Amylase is an enzyme that hydrolyzes ?-(1,4)-glycosidic bonds of starch. ?- Amylase is used in various industries, such as food, pharmaceutical, textile, paper and detergent industries. BmaN1 is one of the starch degrading enzymes produced by Bacillus megaterium NL3 in Kakaban Lake, East Kalimantan, Indonesia. BmaN1 belongs to the GH13 family. A common feature of the GH13 family is the presence of a catalytic residue in the form of two aspartates and one glutamate. There’s only glutamate found in conserved regions of BmaN1, while one aspartate was replaced by His294 and another aspartate (Asp203) was shifted one residue i + 1 by Lys202. Expression of BmaN1 in Escherichia coli produces low solubility protein because of transmembrane region at the C end. This study aims to construct the bmaN1 gene without 22 hydrophobic residues at the C end (bmaN1?C) and characterize functions of Lys202, Glu231, and His294 residues of BmaN1?C on starch hydrolysis. Amplified bmaN1?C gene fragments were ligated into pGEMT cloned plasmids. Sub-cloning into pET30a(+) expression plasmid was performed by cutting the recombinant plasmid pGEMT_bmaN1?C using XhoI and NdeI. To characterize function residues in conserves region, recombinant plasmid pET30a_bmaN1?C was used as a mutation template using Site Directed Mutgenesis (SDM) method. Three recombinant plasmids containing bmaN1?C mutant with a codon change resulted substitution of K202D, E231Q, and H294D. Expression BmaN1 and BmaN1?C yielded proteins with masses of ~55 kDa and ~49 kDa, respectively. The best level expression of BmaN1?C was obtained in the host E. coli ArcticExpress (DE3), followed by E. coli BL21 (DE3) RIPL and E. coli BL21 (DE3). Specific activities for BmaN1, BmaN1?C, E231Q, and H294D lysates were 0,43 U/mg, 1,26 U/mg, 0,25 U/mg, and 0,59 U/mg, respectively, while K202D had no activity. Based on the research results, it was estimated that the K202D substitution made repulsion and changed Asp203 orientation, while Glu231 acted as donor proton, and His294 stabilezed state transition on starch hydrolysis of ?- amylase Bman1?C.