HETEROLOGOUS EXPRESSION, PURIFICATION, AND IMMOBILIZATION OF THERMOSTABLE LIPASE FROM GEOBACILLUS THERMOLEOVORANS PPD2 ISOLATE

• Main Author: HERAWATI PERMANA NIM: 30512014, ANITA
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/25638
• Institution: Institut Teknologi Bandung

Lipase is widely used in industrial processes; such as fat and fatty acids, organic synthesis, polymer, and bioenergy industries. The enzyme catalyzes both of hydrolytic and transesterification reactions, depends on the aqueous and organic composition in the solution. Beside of high catalytic activity, biocatalyst in industrial process required high resilience and stability during the catalysis process. There are several methods to improve enzyme performance as biocatalyst, such as screening for enzyme with high activity and stability, genetic engineering, and enzyme immobilization. <br /> <br /> <br /> <br /> <br /> <br /> Geobacillus thermoloeovorans PPD2 is a thermophilic microorganism which was isolated from Indonesian hot spring. The isolate is a potential source for thermostable lipase, and its lipase gene had been cloned into Escherichia coli in the previous research. Heterologous expression, purification, and immobilization of recombinant lipase from G. thermoleovorans PPD2 were carried out in this research. Immobilization of thermostable lipase from PPD2 isolate is important to improve the enzyme performance as a biocatalyst. <br /> <br /> <br /> <br /> <br /> <br /> Two active bands were obtained in the heterologous expression, in which both bands showed lipolytic activity with the size of 51 (LipA) and 43 (LipB) kDa. Both of the protein bands did not appeared on crude extract of the control E. coli BL21(DE3)-pET-30a(+). LipA was highly expressed, however it formed an inclusion body which was precipitated in the debris; while LipB was soluble in the supernatant. The solubility of LipA was increased by adding detergen solution in the cell lysis process. Purification of crude extract using chromatography affinity Ni-NTA resulted one dominant band of LipA, meanwhile LipB could not bind to the resin. Another purification for LipB was carried out by acetone fractionation, and LipB was precipitated at 60–95 % acetone fraction. Purification results suggested that LipB had lost its His-tag which was designed at the N-terminal. <br /> <br /> <br /> <br /> <br /> <br /> Both of LipA and LipB showed high activity toward medium chain length substrates, with optimum activity at 50 oC and pH 8.5. LipA and LipB showed tolerance toward short chain alcohols, such as methanol, ethanol, n-propanol, and isopropanol. The characterization results showed no significant differences between LipA and Lip B, which suggested that LipB is the result of post translational modification of LipA. LipA still contains a putative signal peptide, when the region was cleaved resulting 388 amino acid residues with molecular weight of 43 kDa (LipB). The post-translational modification process, which was occured to soluble LipA, was resulted in LipB with reduced molecular weight and His-tag loss. <br /> <br /> <br /> <br /> <br /> <br /> Immobilization system on Ni-NTA agarose matrix and carboxymethyl chitosan was succesfully immobilized LipA. Hydrolytic activity was decreased to 10.5% and 9.08%; while transesterification activity was retained at 304.5% and 80.6% for LipA immobilized on Ni-NTA agarose and carboxymethyl chitosan matrix, respectively. Ni-NTA/His-tag system gave better result since the binding site at the end of N-terminal did not interfere with the active site and lid region. <br /> <br /> <br /> <br /> <br /> <br /> Purified thermostable lipase of G. thermoleovorans PPD2 has adequate hydrolytic and transesterification activity. The transesterification activity increased after immobilization on the Ni-NTA agarose matrix. Application of G. thermoleovorans PPD2 lipase as a catalyst in hydrolytic and transesterification reactions could be carried out by considering the appropriate conditions for the enzyme. <br />