No description available.

Fabry disease is an X-linked lysosomal storage disease that results from a deficiency in the enzyme α-galactosidase (α-GAL). α-GAL hydrolyzes α-galactosides, and patients with Fabry disease suffer from an accumulation of these undegraded substrates. Human α-GAL naturally occurs as a homodimer, as determined through SEC and crystallographic analysis. This means its quaternary structure consists of two identical α-GAL subunits that are associated together into a single unit. Other species, such as rice, produce a monomeric form of α-GAL, consisting of only a single subunit. If α-GAL is functional as both a homodimer and monomer, then how does homodimerization affect the activity of human α-GAL? This can be answered through two model systems. First, a monomeric form of human α-GAL can be produced, testing the activity of human α-GAL in a monomeric state. A variant of α-GAL was engineered (called α-GALF273G/W277G) that appeared promising. Secondly, another system can be produced capable of stabilizing one active site of the dimer and testing the other active site for activity. Another lysosomal enzyme, α-N-acetylgalactosaminidase (α-NAGAL), shares 46% amino acid sequence identity and share 11 of 13 active site residues. Previously, an α-GAL variant (called α-GALE203S/L206A) was produced, that maintained the antigenicity of α-GAL, but had acquired the enzymatic specificity of α-N-acetylgalactosaminidase (α-NAGAL). A heterodimeric form of α-GAL can be produced combining one subunit of α-GAL with the engineered variant. The engineered site can be stabilized, while the wild-type site can be tested for activity. SEC analysis suggests α-GALF273G/W277G is a monomer, and its kinetic properties are reported. Evidence shows monomeric α-GAL could be useful as an improved enzyme replacement therapy. Western blotting and activity assays suggest the presence of the α-GAL/ α-GALE203S/L206A heterodimer.

京都大学 / 新制・論文博士 / 博士(人間健康科学) / 乙第13562号 / 論人健博第12号 / 新制||人健||8(附属図書館) / (主査)教授 高桑 徹也, 教授 藤井 康友, 教授 長尾 美紀 / 学位規則第4条第2項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Pichia pastoris is a methylotrophic yeast that has been used in both research and industrial settings for recombinant protein expression due to the ease of genetically modifying its genome, its ability to grow to large densities in inexpensive media, and its capability to perform posttranslational modifications. Multiple tools such as the cis -acting factors MATα secretion signal and MBP fusion partners, and trans-acting modifications such as the bgs mutants have increased heterologous protein secretion. Although these techniques have already been used, their effects on the protein secretory pathway have yet to be elucidated. In this study, fluorescence microscopy was used to compare the localization of proteins expressed with the mutated MATα with the deletion of amino acids 57-70 to the wild type MATα secretion signal. Additional fluorescence microscopy was completed to visualize the localization of MBP-EGFP and EGFP-MBP fusion proteins and their spatial relativity to organelle markers. EGFP-MBP was used to further distinguish the properties of multiple bgs mutants. Additionally, secreted lipase activity levels were evaluated in bgs13 strains expressing either the wild type or the mutated MAT&agr; signal peptide. The results indicated that regardless of their differences, the MATα secretion signals and bgs mutants transported their cargo proteins through similar pathways within the cells. The results of the MBP fusion proteins suggest that the arrangement of MBP significantly influences protein secretion and localization. Lastly, the bgs13 mutant with MATα secretion signals demonstrated that lipase activity increased additively when cis- and trans -acting mutations were combined. Ultimately, these results can provide better understanding of each modified factor and the protein sorting pathway, leading to potential techniques that optimize protein secretion in P. pastoris .

Many consumers are unable to enjoy the benefits of milk, due to lactose-intolerance. Lactose-free milk is available, but at about 2 times the cost of regular milk or greater, it may be difficult for consumers to afford. The high cost of lactose-free milk is in part due to the added cost of the lactose hydrolysis process. Hydrolysis at refrigerated temperatures, possibly in the bulk tank or package, could increase the flexibility of the process, and potentially reduce the cost. A rapid β-galactosidase assay was used to determine the relative activity of commercially available lactase samples at different temperatures. Four enzymes exhibited low-temperature activity and were added to refrigerated raw and pasteurized milk at various concentrations and allowed to react for various lengths of time. The degree of lactose hydrolysis by each of the enzymes as a function of time and enzyme concentration was determined by HPLC. The two most active enzymes, as determined by the β-galactosidase assay, hydrolyzed over 98% of the lactose in 24 hours at 2°C using the supplier recommended dosage. The other two enzymes hydrolyzed over 95% of the lactose in 24 hours at two times the supplier recommended dosage at 2°C. Results were consistent in all milk types tested. The results show that it is feasible to hydrolyze lactose during refrigerated storage of milk using currently available enzymes.

The dairy industry uses enzymes to make cheese, alter product flavor, and eliminate lactose. The activities of these enzymes have been measured in clear buffered solutions, but because of the limitations of spectrophotometric methods, enzyme activities have not been measured in opaque or colored dairy products where they are used. Isothermal titration calorimetry (ITC) can be used to determine reaction kinetics in opaque and colored solutions by measuring the heat rate from enzyme-catalyzed reactions as a function of time. This study used ITC to measure β-galactosidase activity in opaque solutions of milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate with two β-galactosidase (EC 3.2.1.23) isozymes derived from A. oryzae and K. lactis. The components of the dairy fluids alter the enzyme kinetics and reaction thermodynamics, and the reactions catalyzed by the two homologs differ as shown by differing thermodynamic profiles. The study demonstrates that ITC can be used to measure enzyme activity in opaque and colored dairy fluids and identify reactions by their thermodynamic properties. To ensure that ITCs are accurately recording heat data they must be calibrated regularly. However, potential problems have been identified with standard electrical calibration procedures; primarily being that the calibration is performed outside of the sample cell. This implies that any loss of heat from the theoretically adiabatic sample cell or loss of signal through led wires would be ignored by the electrical calibration. This research describes a new means for the chemical calibration of ITCs by performing acid-base titrations into the sample cell with KHP and TRIS base. This method for reaction was shown to be accurate to theoretical values across multiple temperatures and with different models of ITCs. Measurement errors due to diffusion of substrate are described along with means for limiting this factor. The method identified provides a procedure for maintaining the accuracy of ITCs by comparing their data to well-known thermodynamic values. It is anticipated that the simplicity and low-cost for running this calibration method will further standardize ITCs, help establish the ITC as a reliable method for measuring enzyme kinetics, and will make their maintenance simple enough for their use in quality assurance and industry settings.

As a primary factor in human evolution, natural selection is an important component of genetic research. Studies of lactase persistence suggest that positive selection has played a powerful role in the adaptation to a lifelong consumption of fresh milk. Using multiple research studies of lactase persistence and suspected corresponding single nucleotide genetic polymorphisms, this study combines data sources to determine whether evidence exists for natural selection of a specific cytosine-to-thymine genetic mutation located 13,910 base pairs (T-13910) upstream from the lactase gene. This polymorphism has potential to be a causal element for lactase persistence, and data suggest that natural selection has played a role in the rising frequency and distribution of this allele, if only in some regions. European and neighboring regions appear to have the highest frequencies with little or no frequency in Asia, Africa and Indonesia; however the presence of lactase persistence in those areas suggests convergent evolution may be occurring on a phenotypic level. To examine this possibility several other identified polymorphisms in the same region as the T-13910 will be included in this study

No description available.

Yes / Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms.

Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.