SYNTHETIC GENE CONSTRUCTION ENCODING RECOMBINANT METAL-DEPENDENT HYDROLASE ENZYME FROM Bacillus licheniformis F-11.4 AND CHARACTERIZATION OF THE EXPRESSION PRODUCT

• Main Author: Ahmad Ramdani, Bayu
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45791
• Institution: Institut Teknologi Bandung

Collagenase from Bacillus licheniformis F-11.4 has been succesfully used to hydrolize snakehead collagen into bioactive peptide. Based on LC-MS/MS prediction, the 26 kDa enzyme was metal-dependent hydrolase (EC 3.4.24). This research was aimed to obtain recombinant metal-dependent hydrolase from B. licheniformis F-11.4 with synthetic gene construction approach and characterize its activity. The information about metal-dependent hydrolase from B. licheniformis F-11.4 was limited in NCBI, therefore the determination of DNA sequence was done by universal primer design, then continued by Polymerase Chain Reaction (PCR) and DNA sequencing. Codon optimization of synthetic gene was done by using Escherichia coli codon preference as expression host. Synthetic gene was produced in pJ414 plasmid which has high copy number. The plasmid was transformed into E. coli BL21(DE3) by heat-shock method. The overproduction of metal-dependent hydrolase was first done in small scale with optimization of IPTG induction (0,1; 0,5; and 1 mM). The bigger scale of overproduction was done by induction of 1 mM IPTG at 37 °C for 3 hours. The protein was purified by affinity chromatography method in Ni-NTA resin column. Molecular weight characterization of metal-dependent hydrolase recombinant was performed by Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE). Activity assay was done by zymography with four different substrates, that are snakehead collagen, bovine collagen, gelatin, and casein. The result showed that metal-dependent hydrolase DNA sequence from B. licheniformis F-11.4 has been succesfully determined which has 690 base pair in size. The synthethic gene was successfully constructed with Codon Adaptation Index 0,838 and GC percentage 51,3%. Recombinant metal-dependent hydrolase was succesfully produced and has 26 kDa in size based on SDS-PAGE analysis. However, it has neither collagenolytic, gelatinolytic, nor caseinolytic activity.