Characterization of the Type 2 L-Asparaginase Gene in Thermohalophilic Bacterial from Wawolesea Hot Springs, Southeast Sulawesi, Indonesia.

<b>Background and Objective:</b> Type 2 L-asparaginase enzyme can be used as a cancer therapy agent and prevent acrylamide formation in food products. Enzymes produced by thermohalophilic bacteria can provide high activity at high temperatures so they are needed on an industrial scale. Hence, this study aims to determine the characteristics of the gene encoding type 2 L-asparaginase enzyme in the thermohalophilic bacterial isolate CAT3.4. <b>Materials and Methods:</b> This research is a type of exploratory research. The characteristics of the gene encoding type 2 L-asparaginase were determined using the PCR technique using the primer pairs AsnBac2-F2 (5'-CTCACGGGAATCTCCATAACTC-3') and AsnBac2-R2 (5'CAGCGATGTAACAGACAGCATC-3'). The characterization process was carried out in stages: Isolation of genomic DNA using a modified alkali-lysis method, nucleotide and protein similarity analysis using BLASTn analysis on the NCBI website, construction of a phylogenetic tree using the MEGAX program, restriction enzyme mapping and amino acid analysis using the Bioedit program. <b>Results:</b> The characterization results showed that the PCR product has a size of 1594 bp with a CDS of 1128 bp, has a similarity value of 100% with <i>Bacillus subtilis</i>, has seven restriction enzymes as molecular markers for the type 2 L-asparaginase gene at the species level: <i>Bsr</i>GI, <i>Dra</i>I, <i>Eco</i>RV, <i>Hind</i>III, <i>Hpy</i>CH4IV , <i>Ssp</i>I and <i>Tai</i>I, have dominant hydrophilic regions and are in the same subclass as <i>Bacillus subtilis</i> strain GOT9. <b>Conclusion:</b> The target gene was similar to the gene encoding type 2 L-asparaginase from <i>Bacillus subtilis</i> with a max identity of 98.85%, query coverage value of 100% and E-value of 0.