Lipolytic bacteria attract great attention to various biotechnology industries because of their enzymatic potential. This study aims to isolate and identify lipolytic bacteria from cashew nutshell waste using the 16S rRNA gene as a molecular marker. Lipolytic bacteria were isolated using serial dilutions and inoculated on lipolytic media. A total of 3 isolates of lipolytic bacteria were obtained from cashew nutshell waste based on screening in LA Rhodamine B. The partial sequence of 16S rRNA gene from LB15 amplified using a pair of primers 63F and 1387R having a size of 1238 bp, while BL6 and BK6 were 1283 bp, respectively. Based on genetic distance analysis and phylogenetic reconstruction, we proposed that LB15 be identified as Burkholderia sp. with 99.92% similarity. In addition, because the 16S rRNA gene sequence similarity of BL6 was 99.87% with Paraburkholderia kururiensis strain 979, BL6 was classified as Paraburkholderia kururiensis. Then, isolate BK6 was identified as Ralstonia sp. with a similarity of 99.53%. The similarity value can be used as a reference in determining the identity of bacteria. A bacterium can be categorized as the same species if it has a similarity value of more than 99%.

This study aimed to obtain a functional lipase (LipRM) from Ralstonia pickettii BK6 through a co-expression involving its foldase. The ORF of the LipRM was 999 bp while the gene encoding of lipase-specific foldase (LifRM) was 1,030 bp. LipRM and LifRM genes were cloned into a plasmid and were successfully co-expressed in Escherichia coli strains to produce functional LipRM. Enzyme activity from partially purified enzymes showed quite surprising results, LipRM activity in E. coli BL21 (DE3) was 25.84 U/ml, while the other strains (DH5α, HB101, S17-1λpir) were 628.98 U/ml, 761 U/ml, and 1206.46 U/ml, respectively. The highest relative activity of LipRM was found at 50-55°C and pH 7-8 with pNP-laurate (C12) as the preferred substrate specificity. LipRM activity was enhanced sharply in the presence of 30% organic solvents (methanol and ethanol) but decreased by more than 50% in the presence of detergents. This study was the first to report heterologous expression of Ralstonia pickettii lipase employing its native foldase resulting in functional lipase from subfamily I.2.