Development of a Refolding Method for Recombinant ?-Amylase BaqA

• Main Author: Musnaini, Bibit
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32213
• Institution: Institut Teknologi Bandung

Indonesia has an abundant sources of starch but processing into high economic value products has not been optimal. This is because the starch processing need ?-amylase enzymes that can degrade starch grains. Our group has successfully produced recombinant ?-amylase BaqA from Bacillus aquimaris in Escherichia coli BL21(DE3) cells, that can degrade starch grains. However, BaqA formed an inclusion body. This study aims to develop a protein refolding method to obtain soluble and active BaqA. To achieve these objectives, BaqA refolding was optimized using urea concentration gradient with two methods. The first method, inclusion body was dissolved in buffer A (20 mM Tris-Cl pH 8.0, 10 mM imidazole; 300 mM NaCl) containing 8 M urea and incorporated in Ni-NTA (Nickel-Nitrilotriacetic Acid) matrix. BaqA was refolded in matrix with a buffer stream containing urea concentration gradient (8 M, 4 M, 2 M, 1 M, 0.5 M, and 0 M). BaqA was released from matrix by elution buffer (20 mM Tris-Cl pH 8.0; 200 mM imidazole; 300 mM NaCl). The second method, inclusion body solution in 8 M urea was dialyzed gradually to free urea. The results of these two methods was characterized by SDS-PAGE (Sodium Dedocyl Sulphate Poly-Acrylamide Gel Electrophoresis). SDS-PAGE results for both methods showed band at molecular weight of about 72 kDa corresponding to the molecular weight of ?-amylase BaqA. BaqA activity was determined by DNS (Dinitrosalicylic Acid) method using two kinds of starch grains, such as rice and corn. The activity of refolded BaqA from urea concentration gradient method on rice substrate was 42,00 U/mL and on corn substrate was 9,98 U/mL. While the activity of refolded BaqA from dialysis method on rice substrate was 28,28 U/mL and on corn was 7,95 U/mL.