CLONING OF PROTEASE GENE FROM Pseudomonas stutzeri BK-AB12 BLEDUG KUWU MUD CRATER ISOLATE

• Main Author: Retno Gumilar, Winda
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32365
• Institution: Institut Teknologi Bandung

Protease is an enzyme that act as catalyst in hydrolisis reaction of peptide bond protein molecules. It is also called as proteolytic enzyme. Protease is used mostly in food and non-food industries. In food industry, protease is used in cheese, beer, bread and meal industry while for non-food industry, it is mostly used in pharmacy, photography, textiles and leather industry. Advanced in biotechnology allows the utilization of protease as catalyst in industry to produced commercial product. One of them is in detergent industry. Protease is needed in large amount because of the increase demand for industry. One way to anticipate this by production of recombinant protease. Microorganism as source of protease have the some advantages compare to other source such as plant or animal. That is it can produced in large scale, its productivity easily to increase, low price, microorganism could be grown fast, its growth is easily to manage, enzyme easily to isolate and environment friendly. Microorganism used in this study derives from Bledug Kuwu Mud Crater, Central Java. Bledug Kuwu Mud Crater has unique environment like high salinity, pH of mud 7.5, 8% water salinity, 5 - 6% mud salinity. Halophilic microorganism that able to live at this environment was predicted having the potential enzymes to be developed. One of it is protease production of protease in large number could be performed by isolating the protease coding gene from potential microbes and expressing it in host cell that produce recombinant halostable enzymes. Recombinant microorganism could produce halostable enzyme in higher number by lower price if it is compare with the original microorganism. In this study, halophilic protease coding gene was isolated from local isolate Pseudomonas stutzeri BK-AB12. Based on literature study, it have been reported that Pseudomonas stutzeri has subtilisin-like serin protease gene. Subtilisin-like serin protease have been used in large number for detergent industry. From the result of proteolytic activity selection by formation of clear zone, it is known that local isolate Pseudomonas stutzeri BK-AB12 is an alkaline protease. In this study, the protease gene was isolated from local isolate halophilic microorganism with polymerase chain reaction (PCR) method. A pair of primers have been designed for amplification of whole protease gene XFPPst and XRPPst. Nucleotide sequence of forward primer are 5’-CAA CGG CGT TGA CCC ATG ACG-3’ and the sequence for the reverse primer are 5’-ATG GAG TGA CGT GGC CTA GCG-3’. DNA band which corresponde to fragment DNA with size of ± 1800 bp in agarose gel from electrophoresis analysis shows that protease gene has successfully amplified. The PCR product was ligated to cloning vector pGEM-T easy to give recombinant plasmid pGT-PPS. The recombinant plasmid which contain protease gene as insert was selected based on restriction analysis using EcoRI enzymes result and PCR analysis. The sequencing shows that recombinant plasmid contain protease gene with size 1800 bp. This gene encoding 589 amino acids and the protease has moleculer weight 64.8 kDa. BLAST analysis shows that protease gene from Pseudomonas stutzeri BK-AB12 isolate have 99% homology with the sequence of subtilisin like-serine protease Pseudomonas stutzeri A1501. Three dimensional structure was done by analysis method of comparative model in 3D structure swiss-model server. The analysis shows that structure of local isolate protease was similar to extracellular serine protease from Aeromonas sobria (PDB ID: 3HJR) by 21.25%.