Enhancing Performance of Lipase for Biodiesel Production by Cell Surface Displaying and Increased Thermostability

Dr. Suvit Loprasert
Chulabhorn Graduate Institute and Chulabhorn Research Institute

The organic solvent resistant lipase (Lipase A) producing Proteus sp. SW1 was isolated from the beer factory waste in Thailand. The enzyme was found to have characteristics suitable for biodiesel production. The gene encoding this Lipase A (lipA) was successfully cloned from the chromosome of Proteus sp. SW1. The attempt to improve Lipase A to express on the cell surface of E. coli and Pseudomonas putida has been made by creating the lipase fusion proteins with three different surface display carrier proteins which are Antigen 43, EstA and OprF.

All the fusion clones expressed lipase A as seen by the lypolytic activities on tributyrin agar. Unfortunately, all protein fusion constructs did not well translocate to the cell surface as demonstrated by fluorescence microscope and the protein detection western technique could not reveal the presence of lipase A and carriers proteins fusions. Moreover, we used error prone technique and in vivo mutator E. coli strain to genetically improve lipase A to become more thermostable resulting in clone C5 and M1 which resist heat treatment at 70 oC for 30 minutes compared to undetectable activity of the wild type control lipase A. The gene lip A from both clones (C5 and M1) were sequenced and found amino acid residue alterations as R147H, S148C in C5 and S148N,G206C in M1. To confirm the thermo stable phenotypes and amino acid residue changes, enzyme mutants were created by sited-directed mutagenesis and purified before characterization. The purified mutant enzymes did not demonstrate different thermostability when compared to wild type enzyme.

แหล่งข้อมูล:

  • โครงการวิจัยชื่อ “Enhancing Performance of Lipase for Biodiesel Production by Cell Surface Displaying and Increased Thermostability” – ระยะเวลาโครงการ: September 2011- August 2013