Structure And Directed Evolution Of Alcohol Dehydrogenase And Enzymatic Preparation Of (S)-Naproxen

As the increasing of energy, resource, and environmental crisis, development of bio-energy and bio-resources has become a hot topic of the modern biochemical industry. Enzyme-mediated biotransformation is one of the key technologies due to its high efficiency, high regio-and stereoselectivity, and mild conditions. A non-steroidal anti-inflammantory drug, naproxen ((+)-6-methoxy-a-methyl-2-naphthaleneacetic acid), has been causing increasing concern due to its wide use and large market demand. Isobutanol (2-methylpropan-l-ol), while the next-generation biofuel, has also gained more and more attention with advantages over bio-ethanol, such as high energy density, low vapor pressure and low hygroscopicity.Based on accurate and reliable high-performance liquid chromatography (HPLC) for chiral compound analysis, high-throughput screening platform for directed enzyme evolution and advanced X-ray diffraction technique, this dissertation is mainly focused on the enzymatic resolution process for chiral drugs and directed evolution of a key enzyme in the isobutanol production pathway. This dissertation involves a variety of techniques such as gene cloning and expression, directed molecular evolution, enzyme fermentation, purification, characterization, and crystallization, as well as bio-product isolation and refinement. It is composed of three main parts:(1) Cloning and expression of a carboxylesterase and its application in enzymatic preparation of naproxen;(2) directed evolution of an alcohol dehydrogenase for improved isobutanol production; and (3) structural basis of the increased isobutyraldehyde (2-methylpropanal) activity generated by directed evolution.The first part of the dissertation is targeted for the preparation of optically pure (S)-naproxen through enzymatic resolution of naproxen methyl ester (NME). A carboxylesterase from Bacillus subtilis ECU0554, namely BSE-NP01, was cloned and over-expressed in Escherichia coli BL21, which showed98%enantiomeric excess(ee) for (S)-naproxen in the kinetic resolution of NME. BsE-NP01was shown to be a carboxylesterase with a molecular mass of～32kDa, and the optimal temperature and pH at50℃and8.5, respectively. A mechanic-grinding approach to substrate dispersion, which is considered to be a replacement for deleterious surfactants such as Tween-80, was also applied, resulting in improved performance of the hydrolysis reaction. Batch production of (S)-naproxen was repeatedly carried out in a solid-water biphasic system at2-L scale, achieving an average total isolation yield of about85%and hundred-gram (S)-naproxen (>99%ee) after ten runs with recycling of (R)-substrate and recrystallization of final product.The second part of the dissertation is aimed to develop an efficient NADH-dependent isobutanol production pathway through directed evolution of an alcohol dehydrogenase for increased activity toward isobutyraldehyde. An NADH-dependent alcohol dehydrogenase from Lactococcus lactis, namely L1AdhA, was chosen to be the starting point of directed evolution. After one round of random mutagenesis, followed by a recombination of beneficial mutations, mutant LlAdhARE1harboring three amino acid mutations (Y50F,1212T and L264V) was identified with7-fold decrease in KM value and-30-fold increase in catalytic efficiency(kcat/KM), compared to wild-type LlAdhA. Based on the structures of LlAdhA and L1AdhARE1, site-saturation mutagenesis targeting residues close to the active site yielded a quadruple mutant Y50F/N110S/1212T/L264V, denoted LlAdhA29C8, with-17-fold lower KM and～160-fold higher catalytic efficiency toward isobutyraldehyde than wild-type LlAdhA.The third part of the dissertation utilizes the crystal structures of LlAdhA and its laboratory-evolved variant LlAdhAREI to elucidate the structural basis of the improved isobutyraldehyde activity in LlAdhARE1created by directed evolution. The X-ray crystal structures of LlAdhA and LlAdhARE1were determined at1.9A and2.5A resolution, respectively. Structural comparison of LlAdhA and LlAdhARE1indicates no large structural difference between wide-type LlAdhA and LlAdhARE1except for three amino acids changes (Y50F, I212T and L264V), and the enhanced isobutyraldehyde activity in LlAdhARE1stems from increases in the protein’s active site size, hydrophobicity, and substrate access due to mutations Y50F and L264V, which facilitate the catalysis of bulky substrates.