Expression and functional and structural characterization of genomic sequences encoding lipolytic enzymes.

Author(s)

Thais Carvalho Maester

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2015-08-27

Examining board members

Eliana Gertrudes Macedo Lemos; Welington Luiz de Araujo; Tsai Siu Mui; Elen Aquino Perpetuo; Jackson Antonio Marcondes de Souza

• Advisor: Eliana Gertrudes Macedo Lemos

Abstract

Two enzymes of PL14.H10 clone from a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation, EST3, from family IV, and EST5, from the V of lipolytic enzymes were characterized. Structural models showed cap-domain and large internal cavity. EST3 hydrolyzed pNP-esters of up to 12 carbons, with Kcat/Km in pNP-acetate of 1533.27 s-1.mM-1. EST5 hydrolyzed pNP-esters from 2 to 14 carbon atoms, with greater activity on pNP-valerate, and Kcat/Km of 1732.3 s-1.mM-1. Both showed the thermal activation phenomenon. EST5 activity was optimal at 45°C and pH 7.5. EST3 exhibited maximum activity at pH 6.0, 41°C. The esterases activity increased in the presence of almost all ions tested, and EST5 was not influenced by detergents. They were relatively stable in organic solvents. One of the crystals of EST5 protein diffracted at 2.311 Å and is expected to solve the structure of this protein. The results of this study revealed interesting features of the EST3 and EST5 proteins for possible biotechnological applications. (AU)

• FAPESP's process: 11/09064-6 - Expression and structural and functional characterization of genomic sequences encoding lipolytic enzymes
• Grantee: Thaís Carvalho Maester Casanova
• Support Opportunities: Scholarships in Brazil - Doctorate