Protein engineering of rhizopus oryzae lipase for improved thermostability

Official URL: http://192.168.1.20/record=b1301595 (Table of Contents)

Protein engineering provides tools for understanding the structure function relationship and deduces the rules governing this relationship. Enzymes catalyze several types of reactions and have wide range of applications ranging from the textile industry to the pharmaceutical industry. Today one of the most important topics in biotechnology industry focus on replacing chemicals with enzymes accordingly turning all the industrial processes into green industry. Especially lipases have great importance in such processes. Unfortunately currently available active lipases do not function well under the harsh industrial conditions such as high temperature and extreme pH. In this thesis I have focused on the thermostability of Rhizopus oryzae lipase and tried to deduce the rules that make it stable at high temperatures while preserving their activity. I have developed a protocol involving the computational analysis predicting beneficial point mutations, and analyzed the effects of predicted mutations in silico. I have cloned, expressed and purified the mutant proteins to verify the predictions. I showed that the P195G mutation in Rhizopus oryzae lipase sustained 80.5 percent of the activity at high temperatures in comparison to the native lipase.