Enzyme engineering of aminotransferases for improved activity and thermostability

The production by biosynthesis of optically active amino acids and amines satisfies the pharmaceutical industry in its demand for chiral building blocks for the synthesis of various pharmaceuticals. Among several enzymatic methods that allow the synthesis of optically active aminoacids and amines, the use of minotransferase is a promising one due to its broad substrate specificity and no requirement for external cofactor regeneration. The synthesis of chiral compounds by aminotransferases can be done either by asymmetric synthesis starting from keto acids or ketones, and by kinetic resolution starting from racemic aminoacids or amines. The asymmetric synthesis of substituted (S)-aminotetralin, an active pharmaceutical ingredient (API), has shown to have two major factors that contribute to increasing the cost of production. These factors are the raw material cost of biocatalyst used to produce it and product loss during biocatalyst separation. To minimize the cost contribution of biocatalyst and to minimize the loss of product, two routes have been chosen in this research: 1. To engineer the aminotransferase biocatalyst to have greater specific activity, and 2. Improve the engineering of the process by immobilization of biocatalyst in calcium alginate and addition of cosolvents. An (S)-aminotransferase (Mutant CNB03-03) was immobilized, not as purified enzyme but as enzyme within spray dried cells, in calcium alginate beads and used to produce substituted (S)-aminotetralin at 50 °C and pH 7 in experiments where the immobilized biocatalyst was recycled. Initial rate of reaction for cycle 1 (6 hr duration) was determined to be 0.258 mM/min, for cycle 2 (20 hr duration) it decreased by ~50% compared to cycle 1, and for cycle 3 (20 hr duration) it decreased by ~90% compared to cycle 1 (immobilized preparation consisted of 50 mg of spray dried cells per gram of calcium alginate). Conversion to product for each cycle decreased as well, from 100% in cycle 1 (About 50 mM), 80% in cycle 2, and 30% after cycle 3. This mutant was determined to be deactivated at elevated temperatures during the reaction cycle and was not stable enough to allow multiple cycles in its immobilized form. A new mutant aminotransferase was isolated by applying error-prone polymerase chain reaction (PCR) on the gene coding for this enzyme and screening/selection: CNB04-01. This mutant showed a significant improvement in thermostability in comparison to CNB03-03. The new mutant was immobilized and tested under similar reaction conditions. Initial rate remained fairly constant (0.2 mM/min) over four cycles (each cycle with a duration of about 20 hours) with the mutant retaining almost 80% of initial rate in the fourth cycle. The final product concentrations after each cycle did not decrease during recycle experiments. Thermostability of CNB04-01 was much improved compared to CNB03-03. Under the same reaction conditions as stated above, the addition of co-solvents was studied in order to increase substituted tetralone solubility. Toluene and sodium dodecylsulfate (SDS) were used. SDS at 0.01% (w/v) allowed four recycles of the immobilized spray dried cells of CNB04-01, always reaching higher product concentration (80-85 mM) than the system with toluene at 3% (v/v) -70 mM-. The long term activity of immobilized CNB04-01 in a system with SDS 0.01% (w/v) at 50 °C, pH 7 was retained for three cycles (20 to 24 hours each one), reaching always final product concentration between 80-85 mM, but dropping precipitously in the fourth cycle to a final product concentration of 50 mM. Although significant improvement of immobilization on productivity and stability were observed using CNB04-01, another observation demonstrated the limitations of an immobilization strategy on reducing process costs. After analyzing the results of this experiment it was seen that a sudden drop occurred on final product concentration after the third recycle. This was due to product accumulation inside the immobilized preparation. In order to improve the economics of the process, research was focused on developing a free enzyme with an even higher activity, thus reducing raw material cost as well as improving biomass separation. A new enzyme was obtained (CNB05-01) using error-prone PCR and screening using as a template the gene derived from the previous improved enzyme. This mutant was determined to have 1.6 times the initial rate of CNB04-01 and had a higher temperature optimum (55°). This new enzyme would allow reducing enzyme loading in the reaction by five-fold compared to CNB03-03, when using it at concentration of one gram of spray dried cells per liter (completing the reaction after 20-24 hours). Also this mutant would allow reducing process time to 7-8 hours when used at a concentration of 5 grams of spray dried cells per liter compared to 24 hours for CNB03-03, assuming that the observations shown before are scalable. It could be possible to improve the economics of the process by either reducing enzyme concentration or reducing process time, since the production cost of the desired product is primarily a function of both enzyme concentration and process time.

Fabrication of nanoparticles by short-distance sputter deposition

During the past decades, tremendous research interests have been attracted to investigate nanoparticles due to their promising catalytic, magnetic, and optical properties. In this thesis, two novel methods of nanoparticle fabrication were introduced and the basic formation mechanisms were studied. Metal nanoparticles and polyurethane nanoparticles were separately fabricated by a short-distance sputter deposition technique and a reactive ion etching process. First, a sputter deposition method with a very short target-substrate distance is found to be able to generate metal nanoparticles on the glass substrate inside a RIE chamber. The distribution and morphology of nanoparticles are affected by the distance, the ion concentration and the process time. Densely-distributed nanoparticles of various compositions are deposited on the substrate surface when the target-substrate distance is smaller than 130mm. It is much less than the atoms’ mean free path, which is the threshold in previous research for nanoparticles’ formation. Island structures are formed when the distance is increased to 510mm, indicating the tendency to form continuous thin film. The trend is different from previously-reported sputtering method for nanoparticle fabrication, where longer distance between the target and the substrate facilitates the formation of nanoparticle. A mechanism based on the seeding effect of the substrate is proposed to interpret the experimental results. Secondly, in polyurethane nanoparticles’ fabrication, a mechanism is put forward based on the microphase separation phenomenon in block copolymer thin film. The synthesized polymers have formed dispersed and continuous phases because of the different properties between segments. With harder mechanical property, the dispersed phase is remained after RIE process while the continuous phase is etched away, leading to the formation of nanoparticles on the substrate. The nanoparticles distribution is found to be affected by the heating effect, the process time and the plasma power. Superhydrophilic property is found on samples with these two types of nanoparticles. The relationship between the nanostructure and the hydrophilicity is studied for further potential applications.

Dynamics of settling charged particles in turbulence : theory and experiments

It has been proposed that inertial clustering may lead to an increased collision rate of water droplets in clouds. Atmospheric clouds and electrosprays contain electrically charged particles embedded in turbulent flows, often under the influence of an externally imposed, approximately uniform gravitational or electric force. In this thesis, we present the investigation of charged inertial particles embedded in turbulence. We have developed a theoretical description for the dynamics of such systems of charged, sedimenting particles in turbulence, allowing radial distribution functions to be predicted for both monodisperse and bidisperse particle size distributions. The governing parameters are the particle Stokes number (particle inertial time scale relative to turbulence dissipation time scale), the Coulomb-turbulence parameter (ratio of Coulomb ’terminalar speed to turbulence dissipation velocity scale), and the settling parameter (the ratio of the gravitational terminal speed to turbulence dissipation velocity scale). For the monodispersion particles, The peak in the radial distribution function is well predicted by the balance between the particle terminal velocity under Coulomb repulsion and a time-averaged ’drift’ velocity obtained from the nonuniform sampling of fluid strain and rotation due to finite particle inertia. The theory is compared to measured radial distribution functions for water particles in homogeneous, isotropic air turbulence. The radial distribution functions are obtained from particle positions measured in three dimensions using digital holography. The measurements support the general theoretical expression, consisting of a power law increase in particle clustering due to particle response to dissipative turbulent eddies, modulated by an exponential electrostatic interaction term. Both terms are modified as a result of the gravitational diffusion-like term, and the role of ’gravity’ is explored by imposing a macroscopic uniform electric field to create an enhanced, effective gravity. The relation between the radial distribution functions and inward mean radial relative velocity is established for charged particles.

Study of particle movement in the nearshore region of Lake Superior with radioisotope tracers

Biogeochemical processes in the coastal region, including the coastal area of the Great Lakes, are of great importance due to the complex physical, chemical and biological characteristics that differ from those on either the adjoining land or open water systems. Particle-reactive radioisotopes, both naturally occurring (210Pb, 210Po and 7Be) and man-made (137Cs), have proven to be useful tracers for these processes in many systems. However, a systematic isotope study on the northwest coast of the Keweenaw Peninsula in Lake Superior has not yet been performed. In this dissertation research, field sampling, laboratory measurements and numerical modeling were conducted to understand the biogeochemistry of the radioisotope tracers and some particulate-related coastal processes. In the first part of the dissertation, radioisotope activities of 210Po and 210Pb in a variability of samples (dissolved, suspended particle, sediment trap materials, surficial sediment) were measured. A completed picture of the distribution and disequilibrium of this pair of isotopes was drawn. The application of a simple box model utilizing these field observations reveals short isotope residence times in the water column and a significant contribution of sediment resuspension (for both particles and isotopes). The results imply a highly dynamic coastal region. In the second part of this dissertation, this conclusion is examined further. Based on intensive sediment coring, the spatial distribution of isotope inventories (mainly 210Pb, 137Cs and 7Be) in the nearshore region was determined. Isotope-based focusing factors categorized most of the sampling sites as non- or temporary depositional zones. A twodimensional steady-state box-in-series model was developed and applied to individual transects with the 210Pb inventories as model input. The modeling framework included both water column and upper sediments down to the depth of unsupported 210Pb penetration. The model was used to predict isotope residence times and cross-margin fluxes of sediments and isotopes at different locations along each transect. The time scale for sediment focusing from the nearshore to offshore regions of the transect was on the order of 10 years. The possibility of sediment longshore movement was indicated by high inventory ratios of 137Cs: 210Pb. Local deposition of fine particles, including fresh organic carbon, may explain the observed distribution of benthic organisms such as Diporeia. In the last part of this dissertation, isotope tracers, 210Pb and 210Po, were coupled into a hydrodynamic model for Lake Superior. The model was modified from an existing 2-D finite difference physical-biological model which has previously been successfully applied on Lake Superior. Using the field results from part one of this dissertation as initial conditions, the model was used to predict the isotope distribution in the water column; reasonable results were achieved. The modeling experiments demonstrated the potential for using a hydrodynamic model to study radioisotope biogeochemistry in the lake, although further refinements are necessary.

MODELING DYNAMICS OF OZONE AND NITROGEN OXIDES AT SUMMIT, GREENLAND WITH A 1-D PROCESS-SCALE MODEL

This work presents a 1-D process scale model used to investigate the chemical dynamics and temporal variability of nitrogen oxides (NOx) and ozone (O3) within and above snowpack at Summit, Greenland for March-May 2009 and estimates surface exchange of NOx between the snowpack and surface layer in April-May 2009. The model assumes the surface of snowflakes have a Liquid Like Layer (LLL) where aqueous chemistry occurs and interacts with the interstitial air of the snowpack. Model parameters and initialization are physically and chemically representative of snowpack at Summit, Greenland and model results are compared to measurements of NOx and O3 collected by our group at Summit, Greenland from 2008-2010. The model paired with measurements confirmed the main hypothesis in literature that photolysis of nitrate on the surface of snowflakes is responsible for nitrogen dioxide (NO2) production in the top ~50 cm of the snowpack at solar noon for March – May time periods in 2009. Nighttime peaks of NO2 in the snowpack for April and May were reproduced with aqueous formation of peroxynitric acid (HNO4) in the top ~50 cm of the snowpack with subsequent mass transfer to the gas phase, decomposition to form NO2 at nighttime, and transportation of the NO2 to depths of 2 meters. Modeled production of HNO4 was hindered in March 2009 due to the low production of its precursor, hydroperoxy radical, resulting in underestimation of nighttime NO2 in the snowpack for March 2009. The aqueous reaction of O3 with formic acid was the major sync of O3 in the snowpack for March-May, 2009. Nitrogen monoxide (NO) production in the top ~50 cm of the snowpack is related to the photolysis of NO2, which underrepresents NO in May of 2009. Modeled surface exchange of NOx in April and May are on the order of 1011 molecules m-2 s-1. Removal of measured downward fluxes of NO and NO2 in measured fluxes resulted in agreement between measured NOx fluxes and modeled surface exchange in April and an order of magnitude deviation in May. Modeled transport of NOx above the snowpack in May shows an order of magnitude increase of NOx fluxes in the first 50 cm of the snowpack and is attributed to the production of NO2 during the day from the thermal decomposition and photolysis of peroxynitric acid with minor contributions of NO from HONO photolysis in the early morning.

Design of a lab scale direct-filtration system and its application in process lab

The Environmental Process and Simulation Center (EPSC) at Michigan Technological University started accommodating laboratories for an Environmental Engineering senior level class CEE 4509 Environmental Process and Simulation Laboratory since 2004. Even though the five units that exist in EPSC provide the students opportunities to have hands-on experiences with a wide range of water/wastewater treatment technologies, a key module was still missing for the student to experience a full cycle of treatment. This project fabricated a direct-filtration pilot system in EPSC and generated a laboratory manual for education purpose. Engineering applications such as clean bed head loss calculation, backwash flowrate determination, multimedia density calculation and run length prediction are included in the laboratory manual. The system was tested for one semester and modifications have been made both to the direct filtration unit and the laboratory manual. Future work is also proposed to further refine the module.

Effect of high order interpolation in the stability and efficiency of the time-integration process in vorticity-velocity CFD algorithms

The numerical solution of the incompressible Navier-Stokes Equations offers an effective alternative to the experimental analysis of Fluid-Structure interaction i.e. dynamical coupling between a fluid and a solid which otherwise is very complex, time consuming and very expensive. To have a method which can accurately model these types of mechanical systems by numerical solutions becomes a great option, since these advantages are even more obvious when considering huge structures like bridges, high rise buildings, or even wind turbine blades with diameters as large as 200 meters. The modeling of such processes, however, involves complex multiphysics problems along with complex geometries. This thesis focuses on a novel vorticity-velocity formulation called the KLE to solve the incompressible Navier-stokes equations for such FSI problems. This scheme allows for the implementation of robust adaptive ODE time integration schemes and thus allows us to tackle the various multiphysics problems as separate modules. The current algorithm for KLE employs a structured or unstructured mesh for spatial discretization and it allows the use of a self-adaptive or fixed time step ODE solver while dealing with unsteady problems. This research deals with the analysis of the effects of the Courant-Friedrichs-Lewy (CFL) condition for KLE when applied to unsteady Stoke’s problem. The objective is to conduct a numerical analysis for stability and, hence, for convergence. Our results confirmthat the time step ∆t is constrained by the CFL-like condition ∆t ≤ const. hα, where h denotes the variable that represents spatial discretization.