CLONING AND EXPRESSION OF GENE ENCODING ?-AMYLASE RAW STARCH-DEGRADING FROM Bacillus megaterium NL3 IN Escherichia coli

• Main Author: Yondi, Violeta
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32324
• Institution: Institut Teknologi Bandung

Starch is a polysaccharide consists of amylose and amylopectin. Needs of starch as food resource increases along with the growth of population. A side from being food resource, many industries which food industry, detergents, paper and bioethanol production use starches as raw materials. Enzymatic starch hydrolysis process in starch processing industry involves two stages, namely liquefaction and saccharification. In the liquefaction stage, starch is gelatinized at high temperature, thus requires high energy and in turn would increase the production cost. ?-Amylases which capable of hydrolyzing starch at lower temperature has been widely studied. In the previous study, a novel raw starch degrading ?-amylase Bacillus megaterium NL3, BmaN1, has been discovered. BmaN1 has one conserve catalytic residue, namely glutamate, has no starch-binding domain, and the activity is not dependent on the presence of Ca2+ ions. The purposes of this study ware to clone the encoding gene of ?-amylase B. megaterium NL3 in the E. coli expression vector, to express BmaN1 recombinant in E. coli ArticExpress (DE3) as host cells, and to characterize the biochemical properties of BmaN1 recombinant. bmaN1 gene was amplified by PCR using primers containing the BamHI and EagI restriction. BmaN1 fragment amplification resulted in a fragment with a size of ~1.5 kb. BmaN1 fragment was ligated to the pGEM-T cloning vector. Recombinant plasmid pGEM-bmaN1 subsequently cut with BamHI and EagI restriction enzymes, as well as the expression vector pET-30a. Both fragments were ligated and transformed to E. coli TOP 10F’. Recombinant plasmid pET30a-bmaN1 was expressed in E. coli ArcticExpress (DE3) with the addition of IPTG as inducer. Nucleotide sequencing analysis showed that bmaN1 has 98% similarity with the ?-amylase B. megaterium QM B1551 and B. megaterium DSM319, and 95% similarity with ?-amylase B. megaterium WSH-002. bmaN1 encoding 538 amino acids without the peptide signal and has polihistidin sequence at the N and C terminal end. Amino acid alignment results showed that BmaN1 belong to the Glycoside Hydrolase (GH) family 13. BmaN1 known to only has one conserve catalytic residues, namely glutamate (Glu259), which located in the region II. BmaN1 also known to have two consecutive tryptophan residues namely Trp217 and Trp218. Those residues may contribute to its raw starch-degrading ability, that is to act as binding residues enabling the stacking with glucose molecule because their side chain are positioned outside. BmaN1 was expressed as soluble and insoluble protein in the host cell. SDS PAGE results showed that the molecular mass of BmaN1 approximately 59 kDa. The recombinant BmaN1 has the ability to degrade soluble starch, amylopectin, pullulan, and ?-cyclodextrin. BmaN1 relative hydrolysis activity to soluble substrates are as follows: amylopectin ? soluble starch ? ?-cyclodextrin ? pullulan. BmaN1 is also capable to hydrolyze starch from cassava, canna, rice, potato, and corn. In which rice is the most difficult substrate to be hydrolyzed by BmaN1.