BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF Aspergillus oryzae Lipase

• Main Author: KORRY NOVITRIANI (NIM : 20510045); Pembimbing I : Rukman Hertadi. D.Sc; Pembimbing II : Dr. Sant
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/16238
• Institution: Institut Teknologi Bandung

Lipase is one of the enzymes that have high economical value. Some applications of lipase are to hydrolysis of fats and oils, as a flavor enhancer in food processing and to synthesis of organic compounds. Based on the above applications, lipase is widely used in the biodiesel industry, detergent formulation, food, processing of organic chemicals and pharmaceuticals synthesis. Versatility of this enzyme in the industrialized field requires lipase with special properties such as stereospecific, and stable to high temperatures. One of the commercial lipases that has been widely used in the industry is the one isolated from Aspergillus oryzae fungal. Relevant information regarding the biochemical and biophysical property of this commercial enzyme is still limited. Therefore, the aim of this work is to study biochemical and biophysical properties of lipase from Aspergillus oryzae. The properties being studied from this commercial enzyme will be used as a reference in comparing the isolated lipase from other microorganisms or from genetic engineering. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> In this study, the commercial lipase from Aspergillus oryzae was obtained in the form of immobilized one. Therefore, initially we released it from the matrix immersing them in glycine -NaOH buffer solution with pH 9.4. The purity of lipase that has been released from its immobilized matrix was then checked with SDS-PAGE. There are three bands appeared in the electrophoregram of SDS-PAGE indicating that the protein need to be further purified. One of the bands has been confirmed to be lipase by virtue of zymogram test with the molecular weight <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> approximately about 37 kDa.Gel filtration method has been used to separate lipase from its protein mixture based on its molecular weight. The fraction containing lipase was confirmed for its purity as a single band of SDS-PAGE appeared in its electrophoregram. The purified enzyme was then subjected for biochemical and biophysical characterization. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> Biochemical characterizations of the enzyme covers the determination of optimum pH and temperature, study the effect of effectors in the forms of metallic ions (Mg2+, Hg2+, Fe2+, Cu2+, Ba2+, Zn2+, Mn2+ and Ca2+) and organic compounds (SDS, PMSF dan EDTA). The enzyme showed its optimum activity at pH 9 and 45 oC. The activity of the enzyme was enhanced significantly in the addition of Mg2+ dan Zn2+ ions to the solution. On the contrary, the activity prominently declined when Hg2+ ion was added. is The activity of the enzyme relatively less affected by the addition of PMSF and EDTA suggesting that this lipase is strong likely not a serine hydrolase-like enzyme nor metaloenzyme. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> In order to seek understanding about the structure-function relationship of the enzyme in various pH and in the presence of effectors, we evaluated the conformational dynamics and compactness of the protein with fluoresesence spectroscopy method.This measurement showed the difference in emission spectra when the protein was dissolved in the buffer of pH 7, 9, and 10. The compactness analysis employing Stern-Volmer method revealed the increment of compactness level in the order of pH 7, pH 9 and pH 10, respectively. The compactness level of the protein was also affected as Mg2+ and Hg2+ ions added to the solution when compared to its metal free form. The addition of both metal ions increased the compactness level of the protein in the order of Mg2+ and Hg2+, respectively. Above all analysis revealed that the optimum activity of lipase may be achieved when the protein conformation has similar level of compactness to the state in pH 9 and in the presence of Mg2+.