Laboratory Evoluon of CotA Laccase for Increased Half-life at pH 4 Sunandan Dhar 1 , Silu Sheng 2 , and Edgardo T. Farinas 2 1 Department of Biotechnology, Heritage Instute of Technology, Kolkata, India 700107 2 Department of Chemistry and Environmental Science, New Jersey Instute of Technology, Newark NJ 07102 Engineer proteins to optimize properties DNA sequence is randomly mutated and expressed (1-2 mutations per gene) Screening of library of mutants for desired parameters Iterative process Protein surface display is a technique used to screen protein li- braries. Recently, bacterial endospores have been demonstrated to be an effective microbial display tool [1]: No protein folding problems Preimmobilized Stable in extreme conditions Easy to engineer CotA protein on spore coat of B. subtilis: Laccase enzyme activity [2] Oxidizes variety of sub- strates (copper dependent) Maximum catalytic activity in pH 4, but highly unstable After screening 3000 clones, a triple mutant (E498G, E29V, L343S) was found. However, only E498G is responsible for increased half-life. This mutant was further characterized. Wild-type B. subtilis and mutant cells are grown on LB plate, then transferred to Schaeffer plate. Sporulation is induced by incuba- tion is 2SG media (nutrient starvation) for 72 hours at 37⁰C. Purification of spores Washing with salt solution & water Centrifuge at 17000 R.P.M. Dark brown spores obtained Study of Kinetics Activity: oxidation of ABTS substrate Acid inactivation: spores suspended in pH 4 buffer V max = 13.88 μM/ min/ OD unit K m = 63.54 μM V max = 32.77 μM/ min/ OD unit K m = 473.65 μM Objective: Increase stability in pH 4 using Directed Evolution Wild-type: t½ = 21 min E498G : t½ = 869 min 41 fold Improvement of Stability Kinetic parameters of mutant slightly hindered Significant improvement of stability in acidic conditions Industrial & biotechnological applications Further research to improve catalytic activity 0 2 4 6 8 10 12 14 0 200 400 600 800 1000 1200 Activity (μM /min /OD unit) ABTS Concentration (μM) Wild-type Kinetics 0 5 10 15 20 25 30 35 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 Activity (μM /min /OD unit) ABTS Concentration (μM) E498G Kinetics Directed Evolution Procedure Results Conclusion References: [1] Gupta, Nirupama, et al. “Directed evolution of CotA laccase for increased substrate specificity using Bacillus subtilis spores.” Protein Engineering, Design & Selection 23.8 (2010): pp 679-682. [2] Martin, Ligia, et al. “Molecular and Biochemical Characterization of a Highly Stable Bacterial Laccase That Occurs as a Structural Component of the Bacillus subtilis Endospore Coat.” The Journal of Biological Chemistry 227.21 (2002): pp18849-18859. Acknowledgements: National Science Foundation Closed circle: t = 0 min Open circle: t = 120 min 0% 20% 40% 60% 80% 100% 120% 0 20 40 60 80 100 120 140 160 180 200 Relative Activity (%) Time (minutes) Acid Inactivation Wild-type E498G