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.

Economic viability of shale gas production in the Marcellus Shale; indicated by production rates, costs and current natural gas prices

The U.S. natural gas industry has changed because of the recent ability to produce natural gas from unconventional shale deposits. One of the largest and most important deposits is the Marcellus Shale. Hydraulic fracturing and horizontal drilling have allowed for the technical feasibility of production, but concerns exist regarding the economics of shale gas production. These concerns are related to limited production and economic data for shale gas wells, declines in the rates of production, falling natural gas prices, oversupply issues coupled with slow growth in U.S. natural gas demand, and rising production costs. An attempt to determine profitability was done through the economic analysis of an average shale gas well using data that is representative of natural gas production from 2009 to 2011 in the Marcellus Shale. Despite the adverse conditions facing the shale gas industry it is concluded from the results of this analysis that a shale gas well in the Marcellus Shale is profitable based on NPV, IRR and breakeven price calculations.

Spatial and temporal analysis of land cover and landscape structure change in Zagros Forests

The Zagros oak forests in Western Iran are critically important to the sustainability of the region. These forests have undergone dramatic declines in recent decades. We evaluated the utility of the non-parametric Random Forest classification algorithm for land cover classification of Zagros landscapes, and selected the best spatial and spectral predictive variables. The algorithm resulted in high overall classification accuracies (>85%) and also equivalent classification accuracies for the datasets from the three different sensors. We evaluated the associations between trends in forest area and structure with trends in socioeconomic and climatic conditions, to identify the most likely driving forces creating deforestation and landscape structure change. We used available socioeconomic (urban and rural population, and rural income), and climatic (mean annual rainfall and mean annual temperature) data for two provinces in northern Zagros. The most correlated driving force of forest area loss was urban population, and climatic variables to a lesser extent. Landscape structure changes were more closely associated with rural population. We examined the effects of scale changes on the results from spatial pattern analysis. We assessed the impacts of eight years of protection in a protected area in northern Zagros at two different scales (both grain and extent). The effects of protection on the amount and structure of forests was scale dependent. We evaluated the nature and magnitude of changes in forest area and structure over the entire Zagros region from 1972 to 2009. We divided the Zagros region in 167 Landscape Units and developed two measures— Deforestation Sensitivity (DS) and Connectivity Sensitivity (CS) — for each landscape unit as the percent of the time steps that forest area and ECA experienced a decrease of greater than 10% in either measure. A considerable loss in forest area and connectivity was detected, but no sudden (nonlinear) changes were detected at the spatial and temporal scale of the study. Connectivity loss occurred more rapidly than forest loss due to the loss of connecting patches. More connectivity was lost in southern Zagros due to climatic differences and different forms of traditional land use.

SIZE OPTIMIZATION OF A BIOMASS TO LIQUID FUEL CONVERSION FACILITY WITHIN A BIOMASS SUPPLY CHAIN

Bioenergy and biobased products offer new opportunities for strengthening rural economies, enhancing environmental health, and providing a secure energy future. Realizing these benefits will require the development of many different biobased products and biobased production systems. The biomass feedstocks that will enable such development must be sustainable, widely available across many different regions, and compatible with industry requirements. The purpose of this research is to develop an economic model that will help decision makers identify the optimal size of a forest resource based biofuel production facility. The model must be applicable to decision makers anywhere, though the modeled case analysis will focus on a specific region; the Upper Peninsula (U.P.) of Michigan. This work will illustrate that several factors influence the optimal facility size. Further, this effort will reveal that the location of the facility does affect size. The results of the research show that an optimal facility size can be determined for a given location and are based on variables including forest biomass availability, transportation cost rate, and economy of scale factors. These variables acting alone and interacting together can influence the optimal size and the decision of where to locate the biofuel production facility. Further, adjustments to model variables like biomass resource and storage costs have no effect on facility size, but do affect the unit cost of the biofuel produced.

INVESTIGATING THE IMPACTS OF STREAMBED HABITAT HETEROGENEITY ON ECOSYSTEM STRUCTURE AND PROCESSES USING BASIC AND APPLIED PERSPECTIVES

Stream restoration often focuses on increasing habitat heterogeneity to reverse ecosystem degradation. However, the connection between heterogeneity and ecosystem structure and processes is poorly understood. We looked to investigate this interaction from both applied and basic science perspectives. For the applied study, we examined two culvert replacements designed to mimic natural stream channels, to see if they were better at maintaining ecosystem processes within as well as upstream and downstream of culverts compared to non-replaced culverts. We measured three ecosystem processes (nutrient uptake, hydrologic characteristics, and coarse particulate organic matter retention) and found that stream simulation culvert restoration improved organic matter retention within culverts, and that there were no differences in processes measured upstream and downstream of both restoration designs. Our results suggest that measurements of ecosystem processes are more likely to show a response to restoration if they match the scale of the restoration activity. For the basic science study, we quantified the longitudinal spatial heterogeneity of physical and biofilm characteristics at microhabitat to segment scales on streams with different streambed variability. We found that all physical characteristics and biofilm characteristics were spatially independent at the macro-habitat scale and greater. Together, these studies demonstrate the importance of scale in ecological interactions and the value of incorporating considerations of scale into restoration activities.