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Title:	Protein engineering of cota laccase by using bacillus subtilis spore display
Author:	Sheng, Silu
View Online:	njit-etd2017-057 (xiii, 130 pages ~ 9.3 MB pdf)
Department:	Department of Chemistry and Environmental Science
Degree:	Doctor of Philosophy
Program:	Chemistry
Document Type:	Dissertation
Advisory Committee:	Farinas, Edgardo Tabion (Committee chair) Mitra, S. (Committee member) Gund, Tamara M. (Committee member) Li, Mengyan (Committee member) Dias, Cristiano L. (Committee member)
Date:	2017-05
Keywords:	Directed evolution Laccase Spore display Biocatalysis Protein engineering
Availability:	Unrestricted
Abstract:	Spore display offers advantages over more commonly utilized microbe cell-surface display systems. For instance, protein-folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes. The aim is to improve pH stability using spore display. The maximum activity of CotA is between pH 4 and 5 for the substrate ABTS (ABTS = diammonium 2, 2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate)). However, the activity dramatically decreases at pH 4. The activity is not significantly altered at pH 5. CotA is used as a model to prove that enzymes could be improved for pH resistance by using Bacillus subtilis spore display. First, CotA is evolved for increased half-life (t1/2) at pH 4. Next, a double mutant is constructed. This variant combines the amino acid substitutions from the improved t1/2 variant (E498G) and organic solvent tolerant mutant (T480A). The t1/2 and kinetic parameters are evaluated for the double mutant. Consequently, T480A/E498G-CotA is constructed and the t1/2 is 62.1 times greater than wt-CotA. Finally, T480A/E498G-CotA yields 5.3-fold more product than does wt-CotA after recycling the biocatalyst seven times over 42 h. Also, the mutant and wild-type are overexpressed in E. coli and purified. The enzymes immobilized in the spore coat are compared with the purified free protein. The t1/2 and catalytic efficiency follow the same trends for spore or E. coli expressed wt-CotA and E498G-CotA, although the kinetic parameters are different. In a previous investigation, a laccase (CotA), which is found on the spore coat of Bacillus subtilis, was engineered by directed evolution for improved activity in organic solvents. A CotA variant was identified with a Thr480Ala (T480A-CotA) amino acid substitution after only one round of evolution. The screen was performed at 60 % DMSO and it was 2.38-fold more active than the wild-type CotA (wt-CotA) with substrate ABTS. T480A-CotA was more active from a range of 0 - 70 % DMSO. In addition, the variant was more active in ethanol, methanol and acetonitrile. In this study, the catalysis of T480A-CotA and wt-CotA in the spore coat is determined with natural phenolic compounds, such as (+)-catechin, (-)-epicatechin and sinapic acid in aqueous-organic media. In general, the catalytic efficiency (Vmax/Km (δA/OD580)/mM) of T480A-CotA is higher than wt-CotA for all the substrates. Then, the Vmax for T480A-CotA is greater than the wt-CotA in all organic solvents used in this study. The Vmax for T480A-CotA is up to 3.4-fold, 7.9-fold and 6.4-fold greater than wt-CotA for substrate (+)-catechin, (-)-epicatechin and sinapic acid, respectively. In addition, the catalyst can be easily removed from the reaction solution and reused. This allows for simpler recovery of the product from the enzyme. This investigation indicates that enzymes expressed on the spore coat can be utilized for industrial applications.