AALYSIS OF AMPLICO OBTAIED FROM CIMAGGU METAGENOMIC DNA USING FAMILY 6 GLYCOSIL HYDROLASE DEGENERATE PRIMERS

• Main Author: Rahmah, Latri
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34740
• Institution: Institut Teknologi Bandung

Metagenomics is a useful methods to isolate novel genes from nature, a means to exploit indigenous thermophilic microorganism. This endeavour could be useful for industry since thermophilic microorganism can produce thermostabil enzymes, that could increase the effectiveness of production. One of industrial enzyme is cellulase. Cellobiohydrolase (CBH) is a cellulolytic enzyme that hydrolyzes cellulose to cellobiose. This research was proposed to isolate CBH gene from Cimanggu hotspring indigenous microorganisms. DNA was amplified using degenerate primes designed from conserved region and catalytic site of Family 6 Glycosil Hydrolase (GH). Touchdown PCR was used to obtain ~500 bp fragments. A 427 bp fragment had been amplified. However the sequencing results showned that fragment was amplified only by reverse primer, that annealed at 5’ and 3’ ends. BLASTX analized 42% to 52% sequence identity with Two- Component Response Regulator Protein. Amino acid sequence of the putative protein showed the existance of response regulator receiver domain. Isolation of CBH gene was unsuccesful and Two-Component Response Regulator Protein was unintentionally isolated. Inasmuch the result, method improvement is needed to get the final project as proposed in the beginning. Recomendation for further research is improvement of CBH primes design (a clade CBH gene of Procaryot GH family 6, Eucaryot GH or Procaryot GH from another famili).