ISOLATION AND EXPRESSION OF OXALATE DECARBOXYLASE GENE FROM Flammulina Velutipes (Curtis) Singer

• Main Author: KRISHNA ADIANTO PUTRA (NIM: 21115001), MOKHAMAD
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29031
• Institution: Institut Teknologi Bandung

<p align="justify">Brassica oleracea L. or kale is one of the plant with measurable content of oxalic acid. Oxalic acid (C2H2O4) itself is a common plant metabolite that widely known for its toxicity effects upon humans consumption. Oxalate decarboxylase is an enzyme that capable of degrading oxalic acid to other non-hazardous compounds. Isolation and expression study of the gene coding for oxalic acid degrading enzyme (OXDC) was conducted by using a globally known edible mushroom, Flammulina velutipes. The mushroom used in this research was the cultivar that was available for commercial purpose and widely sold in the market. The research involved growing mushrooms in culture media and proceed to gene induction by oxalic acid treatment. Optimization of cultivation time revealed that OXDC gene could be isolated from mRNA following one week of cultivation. Amplification of the gene by using a pair of specific primers on genomic DNA and cDNA template, resulted in PCR products of 2252 bp and 1344 bp respectively. The gene fragments were then sequenced and analyzed by using the BLASTN program from NCBI. The result showed identity values of 98% and 99%. Sequence alignment of both genomic DNA and cDNA showed 18 regions of exon and 17 regions of intron. Protein prediction analysis by using ExPASy-Translate tool program showed an open reading frame with 447 amino acid and identity value of 100% with BLASTP analysis. Structural analysis of prediction protein by using CDD and CDART program indicated two major domains belonged to cupin superfamily and was assumed to have enzymatic activity. A signal peptide motif was also discovered at the N-terminus which indicated its function in secretory pathway. It is concluded that the commercial product of F. velutipes mushroom that had been cultured was indeed carried oxalate decarboxylase gene (OXDC) on its genome and the gene could still be expressed by using appropriate culture and induction method. <p align="justify">