Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

Rational design of zinc phosphide heterojunction photovoltaics

The prospect of terawatt-scale electricity generation using a photovoltaic (PV) device places strict requirements on the active semiconductor optoelectronic properties and elemental abundance. After reviewing the constraints placed on an "earth-abundant" solar absorber, we find zinc phosphide (α-Zn₃P₂) to be an ideal candidate. In addition to its near-optimal direct band gap of 1.5 eV, high visible-light absorption coefficient (>10⁴ cm^-1), and long minority-carrier diffusion length (>5 μm), Zn₃P₂ is composed of abundant Zn and P elements and has excellent physical properties for scalable thin-film deposition. However, to date, a Zn₃P₂ device of sufficient efficiency for commercial applications has not been demonstrated. Record efficiencies of 6.0% for multicrystalline and 4.3% for thin-film cells have been reported, respectively. Performance has been limited by the intrinsic p-type conductivity of Zn₃P₂ which restricts us to Schottky and heterojunction device designs. Due to our poor understanding of Zn₃P₂ interfaces, an ideal heterojunction partner has not yet been found.

The goal of this thesis is to explore the upper limit of solar conversion efficiency achievable with a Zn₃P₂ absorber through the design of an optimal heterojunction PV device. To do so, we investigate three key aspects of material growth, interface energetics, and device design. First, the growth of Zn₃P₂ on GaAs(001) is studied using compound-source molecular-beam epitaxy (MBE). We successfully demonstrate the pseudomorphic growth of Zn₃P₂ epilayers of controlled orientation and optoelectronic properties. Next, the energy-band alignments of epitaxial Zn₃P₂ and II-VI and III-V semiconductor interfaces are measured via high-resolution x-ray photoelectron spectroscopy in order to determine the most appropriate heterojunction partner. From this work, we identify ZnSe as a nearly ideal n-type emitter for a Zn₃P₂ PV device. Finally, various II-VI/Zn₃P₂ heterojunction solar cells designs are fabricated, including substrate and superstrate architectures, and evaluated based on their solar conversion efficiency.