#TITLE_ALTERNATIVE#

• Main Author: Suci Mulyani, Nies
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/8812
• Institution: Institut Teknologi Bandung

ABSTRACT: <br /> <br /> <br /> Penicillin acylase (EC 3.5.1.11) is an enzyme that catalyses the hydrolisis of penicillin molecule into 6-aminopenicillanic acid (6-APA) and carboxylic acid. Some microorganisms produced the enzyme intracellularly or extracellularly. The main part of a penicillin molecule is 6-APA, which can be used as a precusor of semisynthetic penicillin. <br /> <br /> <br /> In this research, penicillin acylase was isolated from Escherichia soli B-130 cultivated in the medium of Morita and Iwata using 0,2% phenylacetic acid as an inducer. Since E.coli B-130 produces penicillin G acylase intracellularly, it is necersary to use Potter Elvehjem Homogenizer and Ultrasonicator to disrupt the cell wall. The crude enzyme was further purified by (NH4)2SO4 precipitation technique followed by ion exchange chromatography on DEAE-sephacel column. <br /> <br /> <br /> The activity of penicillin G acylase was determined by the method based upon the derivate of 2,4 pentanadion and 6-APA formed followed by second reaction using p-dimethylaminobenzaldehyde (Erlichs reagent). As a result of this reaction is a red product which absorbs at X. 538 nm The incubation time was 30 minutes, and the benzylpenicilin concentration was 10 mg/mL. <br /> <br /> <br /> The optimum pH of penicillin G acylase was found to be 7,5 and the optimum temperature was C 55 degree. The Michaelis-Menten constant and the maximum rate of 6-APA formation were 0,86 pM and 147 pmol 6 APA/mL enzyme /30 minute, respectively. <br /> <br /> <br /> The (NH4)2504 purified enzyme had a specific activity of 3,88 unit per mg. Further purification of this partially purified enzyme with DEAE-sephacel at pH 7.8 yielded higher purity enzyme which can be determined by electrophoretic analyses. Its specific activity was 70.10 unit per mg protein. <br /> <br /> <br /> Polyacrylamide gel eletrophoretic analysis showed that the crude extract of enzyme consists of 14 protein bands. After the enzyme was purified by (NH4)2SO4 precipitation and followed by ion exchange chromatography on DEAE-sephacel column, the amounts of protein bands decreased into 12 bands and 4 bands, respectively. The yield of the DEAE-sephacel fraction is 39.10 X.