Isolation and Cloning of Metaloprotease Gene from Halomonas elongata BK-SM1

• Main Author: Medina, Safira
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38077
• Institution: Institut Teknologi Bandung

The enzyme with proteolytic activity (protease) has a very wide application in various industrial fields, such as for bread making, improving the quality of production of biscuits, detergents, degrading stopper (hair) in water channels, processing enzymes, etc. Utilization of microorganisms (bacteria) as a manufacturer of protease will provide high economic value since most bacteria have a fast growth rate and requires only a small area of cultivation. Halophilic bacteria of Indonesian local isolate Halomonas elongata indigenous from the Bleduk Kuwu salty mud crater has been studied previously to have a multistable extracellular metalloprotease. This study aims to isolate a gene that encode metalloprotease from halophilic bacteria using the PCR method. The first stage of this study was begun with the identification of the bacterial species by determining the sequence of a16S rRNA gene from the bacterial chromosome. Based on the 16S rRNA sequences, it was revealed that phylogenetically the species of the bacterial sample exhibited homologous relationship with Halomonas elongata. It was named in our study as Halomonas elongata BK-SM1. Next was a gene cloning stage, which was carried out by the standard procedure of PCR cloning. This stage was initiated with the identification of a metaloprotease gene that will be cloned from the genome of Halomonas elongata and the primer design based on the identified target gene. The metaloprotease gene fragment was obtained with the size of 1044 bp from the stage of cloning gene and its sequence was determined. The sequence analysis using BLASTn showed that the nucleotide sequence of Halomonas elongata BK-SM1 metaloprotease has 99% identity with a putative metalloprotease gene of Halomonas elongata DSM 2581. Halomonas elongata BK-SM1 metalloprotease composed of 347 amino acid residues with a molecular weight was about 38.25 kDa and homologous to the putative metaloprotease Halomonas elongata DSM 2581, metallopeptidase Pseudomonas stutzeri, and zinc metalloprotease of Pseudomonas sp. with respective sequence identity about 99%, 60% and 58%. The tertiary structure of H. elongata BK-SM1 metaloprotease exhibited high similarity with Serratia proteamaculans metalloproteinases (PDB.2vqxA) with a 56% sequence identity.