Date of Award

Winter 2014

Project Type

Thesis

Program or Major

Chemical Engineering

Degree Name

Master of Science

First Advisor

Kang Wu

Second Advisor

Palligarnai T Vasudevan

Third Advisor

Harish Vashisth

Abstract

The use of enzymes as industrial oxidants has become popular due to their high substrate specificity and mild reaction conditions. Specifically, laccases are multi-copper oxidases that can oxidize a disparate range of organic substrates using oxygen and producing water as a byproduct without requirement for additional reactive compounds. Currently, all laccases used in industrial processes are fungal in origin. Although fungal laccases have high activities under near-ambient conditions, their use is limited at higher temperatures. Also, expression of fungal laccases in heterologous hosts is limited due to incorrect glycosylation. Bacterial laccases are much easier to express heterologously and are more active and stable at high temperatures, pH and salt concentrations.

Geobacillus is a genus of gram-positive thermophilic bacteria, many of which have been found to naturally secrete proteins at high levels. A novel laccase has been predicted to be present in multiple Geobacillus strains using comparative genomics. This laccase is approximately half the size of those found in other gram-positives or fungi, making it a better candidate for lignocellulosic biomass degradation because of easier access to the substrate. In this work, we seek to isolate and characterize this laccase, and determine the types of substrates it can oxidize. We then want to compare the activity of our laccase with that of a fungal laccase at different temperatures.

A plasmid was successfully constructed for the overexpression of laccase in Geobacillus thermoglucosidasius 95A1 and Escherichia coli DH5á. The novel laccase was isolated and purified from E. coli. The laccase was characterized by determining the activity for 5 substrates at a range of pHs and temperatures. Finally, the thermal stability of our laccase was compared with that from a fungal source, Trametes versicolor. Laccase from G. thermoglucosidasius demonstrated a 20-fold higher initial activity than Trametes laccase at 80°C, and was superior to the latter in terms of thermal stability and activity at high temperatures.

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