ROLE OF HIS141 RESIDUE RECOMBINANT BACILLUS MEGATERIUM NL3 ????-AMYLASE

• Main Author: Eka Putri, Liza
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/65275
• Institution: Institut Teknologi Bandung

?-Amylase from recombinant Bacillus megaterium NL3 (BmaN2) obtained from Kakaban Lake, East Kalimantan, Indonesia is one of the enzymes for degrading raw starch without gelatinization process. BmaN2 is believed to have residues that play a role in substrate binding called Surface Binding Site (SBS) namely Tyr101, His141, Trp179 and Trp198. In a previous study, a profile of the hydrolysis activity of starch by the BmaN2/H141K mutant was carried out on Escherichia coli BL21 (DE3) host cells, but aggregation formation occurred. So it is necessary to replace the host cell for the BmaN2/H141K mutant. The purpose of this study was to determine the expression of the gene encoding BmaN2 H141K in Escherichia coli BL21 PlysS and Escherichia coli BL21 CodonPlus-Ripl cells and to determine the hydrolysis activity profile of the BmaN2 H141K variant against raw and soluble starch. In this study, Escherichia coli BL21 PlysS and Escherichia coli BL21 CodonPlus-Ripl cells were transformed with recombinant pET-30a(+)_bmaN2 and grown on Luria Bertani (LB) solid media containing the antibiotics kanamycin and chloromphenicol. The grown transformants were analyzed by adding 0.1 mM isopropyl-D-1-thiogalactopyranoside (IPTG) inducer for 4 hours at 37°C. BmaN2 was further purified by Ni-NTA affinity chromatography, characterized by electrophoresis and determined for activity by the 3,5-dinitrosalicylate (DNS) method. These proteins have been successfully produced in Escherichia coli BL21 PlysS and Escherichia coli BL21 CodonPlus-Ripl for 16 ? 18 hours with 0.1 mM isopropyl -D-1-thiogalactopyranoside (IPTG) inducer and incubation temperature of ±10°C. The molecular weight of these proteins is ~61.5 kDa which is characterized by sodium dodecyl sulfate-polyacramid gel electrophoresis (SDS-PAGE). These proteins are soluble and insoluble (inclusion bodies). In the His141Lys mutant, inclusion bodies were more dominant than the dissolved fraction. The specific activity of wildtype BmaN2 soluble protein was 21.08; His141K mutant in Escherichia coli BL21 CodonPlus-Ripl 4.28; and His141K mutant in Escherichia coli BL21 PlysS did not have enzymatic activity. The results showed that the gene encoding ?-amylase BmaN2 with altered His141Lys residue was successfully expressed in Escherichia coli BL21 (DE3) CodonPlus-Ripl cells. In E. coli BL21 PlysS, the gene encoding BmaN2/His141Lys could not be expressed. The mass of the pellets of E. coli BL21 (DE3) CodonPlus-Ripl cells was 0.84 grams and E. coli BL21 PlysS 0.82 grams with total soluble protein concentrations of 34.12 mg/mL and 14.34 mg/mL, respectively. In addition, the results of the solubility test of BmaN2 in Escherichia coli BL21 (DE3) CodonPlus–Ripl showed that there was BmaN2 in the debris phase and lysate was more dominant in the debris phase. In the results of BmaN2 purification in Escherichia coli BL21 (DE3) CodonPlus-Ripl, BmaN2 eluted at 75 mM imidazole wash fraction which is in accordance with BmaN2 under wildtype conditions. With the qualitative test using the Fuwa method, it can be seen that before and before purification there was activity of the enzyme which was marked by the fading of the purple color to slightly clear. The resulting BmaN2 can be applied to determine the hydrolysis pattern of raw starch.