PRODUCTION AND PURIFICATION OF ?-AMILASE BMAN2 RECOMBINANT

• Main Author: Jannah, Miftahul
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/47655
• Institution: Institut Teknologi Bandung

Starch is glucose polymer with ?-1,4 glucosyl linkages. Today, starch becomes one of the most important raw materials in industries. Starch prossessing in industry generally uses strong acid while some others utilize starch degrading enzymes such as ?-amylase and glucoamylase. Both of those enzymes are only able to hydrolize preheated starch (a starch gelatinization step) because of its cristalline structure. Therefore, research to find raw starch degrading enzymes has becomes important in reducing production costs (without the gelatinization step). One of the raw starch degrading enzymes known is recombinant ?-amylase from Bacillus megaterium NL3 (BmaN2). Our research group has succesfully recombined BmaN2 gene into Escherichia coli ArcticExpress (DE3), that can be expressed in cold temperature for 24 h. In order to shorten the production step, this research aims to express ?-amylase BmaN2 in Escherichia coli BL21 (DE3) system and to characterize the purified BmaN2. We choose E. coli BL21 (DE3) because of its ability to overexpress proteins in a relatively short time period at 37 oC. In order to achieve the research objective, E. coli BL21 (DE3) was transformed with pET30a(+)_bmaN2 recombinant and grown in LB broth medium containing the antibiotic kanamycin. The transformant being analyzed was induced with the addition of isopropyl ?-D- 1-thiogalactopyranoside (IPTG) 0.5 mM, 4 h, 37 oC. BmaN2 was produced after optimizing multiple parameters which were: IPTG concentration; temperature; and induction time. After that, the BmaN2 protein was purified using Ni-NTA affinity chromatography method, characterized by electrophoresis, and its activity was determined with 3,5-dinitrosalisylic acid (DNS) method. The optimum amount of BmaN2 was achieved by the addition of 0.1 mM IPTG at ±15 oC, overnight, which produced 12.05 mg/mL of total protein. BmaN2 was purified by observing a thick ~61,5 kDa band on polyacrilamid electropherogram (SDS-PAGE) gel from elution by 50 mM and 100 mM imidazole. The consentration of purified BmaN2 was determined using Bradford method, having the enzymatic activity in hydrolizing soluble starch of 16.59 U/mg. The obtained protein can be applied to determined hydrolysis patterns of raw starch.