Exploration of Gene Encoded Thermostable Enzymes by Using Random PCR from Natural Sample of Domas Crater

Abstract

Thermophilic microorganisms can survive and live in extremely high-temperature habitats. This special trait made thermophilic microorganisms have heat-resistant proteins such as thermostable enzymes and have been used by the industry as biocatalysts. Some advantages of using thermostable enzymes in the industry are reducing operating costs, increasing the reaction rate, increasing productivity, and being environmentally friendly. This research aims to isolate the thermostable enzyme encoding gene and determine homology, differences in the sequence, and the closest relative of samples. The metagenomic approaches using random PCR techniques were used as a methodology in this research. The substances successfully obtained from random PCR of the metagenome samples were cloned in the cloning vector and then analyzed nucleotide sequence (sequencing). The highest protein homology in samples 23 and 24 are Short-chain Dehydrogenase Reductase (SDR) oxidoreductase [Caldivirga maquilingensis] of 58.58% and 58.02%, respectively. The 3-dehydroquinate dehydratease Type I [Metallosphaera] is the highest protein homology in sample 45, with a similarity of 79.88%. The phylogenetic tree analysis of sample 23 has the closest relationship with sample 24. Meanwhile, sample 45 has the closest relationship with 3-dehydroquinate dehydratase Type I from Metallospaera species. The three samples formed different branches with other amino acid sequences, showing that the three samples are new enzymes that are different from the previous ones.