The effectiveness of enzymatic transesterification of animal fat using the experimental enzyme catalyst NS88001 was studied. The effects of oil: alcohol molar ratio (1:1, 1:2, 1:3, 1:4 and 1:5), reaction temperature (35, 40, 45 and 50°C) and reaction time (4, 8, 12 and 16 h) on the biodiesel conversion yield were evaluated. n-Hexane was used as a solvent. The highest conversion yield of biodiesel was obtained at the1:4 oil: alcohol molar ratio, 16 h reaction time and 45°C reaction temperature. The rate of conversion of fatty acid esters increased with increases in reaction time. The reaction proceeds slowly at the beginning due to the initial mixing and dispersion of alcohol into the oil and activation of enzyme. After dispersion of alcohol, the enzyme rapidly interacted with fatty acids esters. Increasing the reaction time from 4 to 16 h increased the biodiesel conversion yield by 13.05-71.94%, depending on the reaction
temperature and oil: alcohol molar ratio. Increasing the oil: alcohol molar ratio from 1:1 to 1:4 increased the biodiesel conversion yield by 32.68-82.11% while increasing the oil: alcohol molar ratio from 1:4 to 1:5 decreased the biodiesel conversion yield by 5.43-34.27%, depending on the reaction time and reaction temperature. The increase in the oil: alcohol molar ratio promotes the reaction between the enzyme and substrate. The interactions between enzyme polymer surface and substrate appears to be dependent on reaction temperature due to hydrogen bonding and ionic interactions which play a important roles in maintaining the thermostability of lipase in the system. Increasing the reaction temperature from 40 to 45°C increased the biodiesel conversion yield by 3.64-58.78%. Higher temperature from 45 to 50°C denatured the specific structure of enzymes and resulted in a decrease in biodiesel conversion yield by 4.3-32.47%. Using n-hexane in the reaction helped to stabilize the enzyme and minimize the toxicity of alcohol. The activity of experimental enzyme catalyst (NS88001) in the presence n-hexane was slightly reduced after 10 cycles. However, the enzyme activity decreased rapidly when the number of cycles was increased above 10 and stopped completely after 50 cycles.