Mahiran Basri

The effect of carbon sources on the expression level of thermostable L2 lipase in Pichia pastoris

Thermostable lipases are enzymes that are particularly pleasing for industrial purposes such as in the production of detergents, animal skin-based industry and food processing endeavours. Thermostable L2 lipase gene fished out from Bacillus sp. L2 was cloned into Pichia pastoris strain GS115 under constitutive expression system of pGAPZαA. Study performed on various carbon sources revealed that glucose and glycerol support the growth of Pichia pastoris and expression of L2 lipase. In addition, the by-product of sugar refinery processes (molasses) was found to be a potential carbon source for the growth of P. pastoris and L2 lipase expression. Since the thermostable L2 lipase will be extracellularly secreted into the medium, the use of P. pastoris expression system is seen as an alternative to the conventional expression system of Escherichia coli that applies intracellular expression of the L2 lipase gene encoded. Key words : Thermostable lipase, Pichia pastoris, constitutive expression, carbon source.

Biomolecular Design and Receptor-Ligand Interaction of a Potential Industrial Biocatalsyt: A Thermostable Thermolysin-Phosphoethanolamine-Ca2+ Protein Complex

Protein structures are prone to modification based on the fundamental rules of design and function. Calculations of free binding energies ( G) of chemical molecules (effectors) that bind to proteins are important in molecular signaling processes and catalytic mechanisms of certain key enzymes. These calculations can be obtained via in silico and theoretical approaches. A series of 48 pockets were identified in thermolysin (KEI) and the four biggest pockets were selected for their suitable sites for modification. Application of molecular docking on phosphoethanolamine (PSE) and 1,10-phenanthroline (PHN) that act as intermediate ligands in the designated protein complex showed favorable final docked energy at different pockets (-8.49 to -4.80 kcal/mol). Analysis on docking of a divalent metal ion (Ca) to ligand (PSE) produced a final docked energy of -4.15 kcal/mol within acceptable distance (1.5 Å M 3.0 Å). It was found that the thermolysin-phosphoetanolamine-Ca represented the putative protein complex of semisynthetic metalloprotease. Combinatorial modeling methods were applied in order to determine the best metalloenzyme complex. The identification of the potential protein pocket was conducted using CASTp. Selected ligands and metal ions were docked into each pocket using AutoDock 3.05. Analyses on their docking energy, non-covalent interaction as well as their geometry were conducted in order to determine the best metalloenzyme complex. This complex displayed the lowest docking energy with the additional Ca suitably docked. It was hypothesized that metal ions can add new functionality to proteins and catalyze some of the challenging biological reactions, particularly in the pharmaceutical and fine chemicals industries.

Structure improvement and analysis of F16L lipase crystallized under microgravity environment

C791 and oxidation of three residues (Gln65-Tyr66-Gly67) located at the center of internal α-helix. The imidazolinone moiety of chromophore was exposed to the outside of the β-barrel through the characteristic water-filled channel. It is considered that oxygen molecules are converted to ROS with light induced energy transfer at chromophore, followed by ROS diffuse to outside of β-barrel through this channel.