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.

Evaluation of surface defect detection in reinforced concrete bridge decks using terrestrial LiDAR

Routine bridge inspections require labor intensive and highly subjective visual interpretation to determine bridge deck surface condition. Light Detection and Ranging (LiDAR) a relatively new class of survey instrument has become a popular and increasingly used technology for providing as-built and inventory data in civil applications. While an increasing number of private and governmental agencies possess terrestrial and mobile LiDAR systems, an understanding of the technology’s capabilities and potential applications continues to evolve. LiDAR is a line-of-sight instrument and as such, care must be taken when establishing scan locations and resolution to allow the capture of data at an adequate resolution for defining features that contribute to the analysis of bridge deck surface condition. Information such as the location, area, and volume of spalling on deck surfaces, undersides, and support columns can be derived from properly collected LiDAR point clouds. The LiDAR point clouds contain information that can provide quantitative surface condition information, resulting in more accurate structural health monitoring. LiDAR scans were collected at three study bridges, each of which displayed a varying degree of degradation. A variety of commercially available analysis tools and an independently developed algorithm written in ArcGIS Python (ArcPy) were used to locate and quantify surface defects such as location, volume, and area of spalls. The results were visual and numerically displayed in a user-friendly web-based decision support tool integrating prior bridge condition metrics for comparison. LiDAR data processing procedures along with strengths and limitations of point clouds for defining features useful for assessing bridge deck condition are discussed. Point cloud density and incidence angle are two attributes that must be managed carefully to ensure data collected are of high quality and useful for bridge condition evaluation. When collected properly to ensure effective evaluation of bridge surface condition, LiDAR data can be analyzed to provide a useful data set from which to derive bridge deck condition information.

Determining bridge deck deterioration through the use of 3D photogrammetry

The bridge inspection industry has yet to utilize a rapidly growing technology that shows promise to help improve the inspection process. This thesis investigates the abilities that 3D photogrammetry is capable of providing to the bridge inspector for a number of deterioration mechanisms. The technology can provide information about the surface condition of some bridge components, primarily focusing on the surface defects of a concrete bridge which include cracking, spalling and scaling. Testing was completed using a Canon EOS 7D camera which then processed photos using AgiSoft PhotoScan to align the photos and develop models. Further processing of the models was done using ArcMap in the ArcGIS 10 program to view the digital elevation models of the concrete surface. Several experiments were completed to determine the ability of the technique for the detection of the different defects. The cracks that were able to be resolved in this study were a 1/8 inch crack at a distance of two feet above the surface. 3D photogrammetry was able to be detect a depression of 1 inch wide with 3/16 inch depth which would be sufficient to measure any scaling or spalling that would be required be the inspector. The percentage scaled or spalled was also able to be calculated from the digital elevation models in ArcMap. Different camera factors including the distance from the defects, number of photos and angle, were also investigated to see how each factor affected the capabilities. 3D photogrammetry showed great promise in the detection of scaling or spalling of the concrete bridge surface.

Erupted magma volume estimates at Santiaguito and Pacaya Volcanoes, Guatemala using digital elevation models

High resolution digital elevation models (DEMs) of Santiaguito and Pacaya volcanoes, Guatemala, were used to estimate volume changes and eruption rates between 1954 and 2001. The DEMs were generated from contour maps and aerial photography, which were analyzed in ArcGIS 9.0®. Because both volcanoes were growing substantially over the five decade period, they provide a good data set for exploring effective methodology for estimating volume changes. The analysis shows that the Santiaguito dome complex grew by 0.78 ± 0.07 km3 (0.52 ± 0.05 m3 s-1) over the 1954-2001 period with nearly all the growth occurring on the El Brujo (1958-75) and Caliente domes (1971-2001). Adding information from field data prior to 1954, the total volume extruded from Santiaguito since 1922 is estimated at 1.48 ± 0.19 km3. Santiaguito’s growth rate is lower than most other volcanic domes, but it has been sustained over a much longer period and has undergone a change toward more exogenous and progressively slower extrusion with time. At Santiaguito some of the material being added at the dome is subsequently transported downstream by block and ash flows, mudflows and floods, creating channel shifting and areas of aggradation and erosion. At Pacaya volcano a total volume of 0.21 ± 0.05 km3 was erupted between 1961 and 2001 for an average extrusion rate of 0.17 ± 0.04 m3 s-1. Both the Santiaguito and Pacaya eruption rate estimates reported here are minima, because they do not include estimates of materials which are transported downslope after eruption and data on ashfall which may result in significant volumes of material spread over broad areas. Regular analysis of high resolution DEMs using the methods outlined here, would help quantify the effects of fluvial changes to downstream populated areas, as well as assist in tracking hazards related to dome collapse and eruption.

Comparison of three methods for driven pile capacity

A significant cost for foundations is the design and installation of piles when they are required due to poor ground conditions. Not only is it important that piles be designed properly, but also that the installation equipment and total cost be evaluated. To assist in the evaluation of piles a number of methods have been developed. In this research three of these methods were investigated, which were developed by the Federal Highway Administration, the US Corps of Engineers and the American Petroleum Institute (API). The results from these methods were entered into the program GRLWEAPTM to assess the pile drivability and to provide a standard base for comparing the three methods. An additional element of this research was to develop EXCEL spreadsheets to implement these three methods. Currently the Army Corps and API methods do not have publicly available software and must be performed manually, which requires that data is taken off of figures and tables, which can introduce error in the prediction of pile capacities. Following development of the EXCEL spreadsheet, they were validated with both manual calculations and existing data sets to ensure that the data output is correct. To evaluate the three pile capacity methods data was utilized from four project sites from North America. The data included site geotechnical data along with field determined pile capacities. In order to achieve a standard comparison of the data, the pile capacities and geotechnical data from the three methods were entered into GRLWEAPTM. The sites consisted of both cohesive and cohesionless soils; where one site was primarily cohesive, one was primarily cohesionless, and the other two consisted of inter-bedded cohesive and cohesionless soils. Based on this limited set of data the results indicated that the US Corps of Engineers method more closely compared with the field test data, followed by the API method to a lesser degree. The DRIVEN program compared favorably in cohesive soils, but over predicted in cohesionless material.