CLONING AND IN SILICO STUDY OF ENDOGLUCANASE OF THERMOPHILIC BACTERIA FROM HYDROTHERMAL VENT OF KAWIO BARAT, SANGIHE-TALAUD ISLANDS, NORTH SULAWESI

• Main Author: Arifsaputra Suherman, Edvan
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/41223
• Institution: Institut Teknologi Bandung

Endoglucanase is used in various industries, such as the bioethanol, detergents, paper, and animal feed industries. These industries apply high temperatures in processing, so thermostability is a desired property of the enzyme. Currently, most endoglucanases have very low activity at high temperatures. The aim of this study was to obtain new thermostable endoglucanase that would have potential to be used in industries. Previous research had isolated five thermophilic bacterial isolates from the hydrothermal vent of West Kawio, Sangihe-Talaud Islands, North Sulawesi. Endoglucanase activity was screened by the CMC-Congo red plate assay method. The isolate with the highest endoglucanase activity was identified using 16S rRNA gene sequence and phylogenetic analysis. Endoglucanase gene was isolated by polymerase chain reaction (PCR) and cloned into pET-32b vector using a restriction digest-ligation method. Sequences of the gene and the protein deduced from it were aligned with BLAST programs. Amino acid composition was analyzed using ProtParam ExPasy program to determine the abundance of certain amino acids and compared to two thermolabile endoglucanases from mesophilic bacteria. Bacterial isolate DSI 2 showed the highest endoglucanase activity compared to the other isolates and was identified as Bacillus safensis. Endoglucanase gene of DSI 2 was successfully cloned into pET-32b. The coding sequence is 1851 bp and has the highest homology (99.19%) with endoglucanase gene from Bacillus sp. WP8 (CP010075.1). The gene encodes a 616-amino acids protein. The amino acid sequence of DSI 2 endoglucanase has the highest identity (99.35%) with endoglucanase from B. safensis SCAL1 (KMK71141.1). The molecular mass of DSI 2 endoglucanase is predicted to be 69.41 kDa. DSI 2 endoglucanase contains a high percentage of hydrophobic amino acids, especially Ala, Leu, and Pro, and a low percentage of Gly. DSI 2 endoglucanase harbors a catalytic domain which belongs to glycosyl hydrolase family 9 (GH9) and a type 3 cellulose-binding domain (CBM3). Properties of endoglucanases with GH9-CBM3 modular structures are active in a wide pH range, high optimum temperature, and thermostable. Thus DSI 2 endoglucanase has the potential to be applied in industry.