Catalysts are utilized in 80% of all chemical synthesis operations. The industrial catalysts primarily used in oxidation reactions are highly polluting and expensive metal catalysts. Enzymes and whole cell biocatalysts are used to a lesser extent. Nowadays, several industrial sectors are developing bio-based technologies to reduce the high costs and environmental impact of traditional chemical processes. However, these applications are limited by the challenge of developing economically competitive biologically based systems. The key for adopting these sustainable advancements is the development of novel process designs, which assure robustness, simplicity, and sustainabile operations compatible with the current development of chemical reactions. In this regard, filamentous fungi may be considered good biocatalysts due to their natural biodiversity and their broad heterogeneous enzymatic pattern. The great selectivity of fungal catalysis is now well recognized for the production of commercially valuable steroids in the pharmaceutical industry. Although this inherent capacity is mainly used for functionalization of unactivated carbons, it can be further exploited for the oxidiation of heteroatoms, such as sulfur. Focusing on the oxidation of sulfur compounds, the widely used industrial processes are produced by an organometallic catalyst. This PhD project aims to overcome low substrate conversion and enzymatic expression by proving that exposure of cells to insecticides and hydrocarbons increases cell's oxidative capacity expressed as higher substrate conversion and CYP450 content. This study is focused in the application of pest management strategies, designed to enhance the biopesticide's efficacy, to induce and improve Beauveria bassiana oxidation. B. bassiana has a very flexible metabolism and is widely used as a biocontrol agent. It can metabolize hexadecane as a sole carbon source. In addition, it shows a synergistic effect over pest control efficacy when it is applied with low pesticides (carbaryl and/or imidacloprid) concentrations.
A biocatalytic system was optimized to increase the conversion of organosulfur compounds under different fermentation conditions. Phenothiazine was used as our model substrate. Phenothiazine conversion was followed by GC-MS and HPLC. By NMR and MS fragmentation pattern product, phenothiazine metabolites were identified as (R)-hydroxyl metabolites (63% enatiomeric excess) and sulfoxide, the latter being the main metabolite. Phenothiazine conversions with growing cells resulted in 65±1.4% conversion with initial phenothiazine concentration of 500 ppm and final 325 ppm after 7 days. The highest conversion, 74±1 % was achieved with resting cells at the lowest cell concentration, 0.78 mg cell dry weight (cdw) /mL. Furthermore, the use of insecticides as inducers was an effective way to increase phenothiazine conversion from 47% to 64±3%. The major enzymes involved in catalysis of xenobiotic are heme-binding monooxygenases, in particular cytochrome P450. Heme positive proteins were identified by an SDS benzidine assay as well as the content of CYP450 by the CO difference spectrum. The P450 enzymes content was 12.3±1 pmol/µg protein for hexadecane adapted cells and 8.1± 1 pmol/µg protein for insecticides, respectively. The heme-positive proteins were characterized by MALDI-ToF and their peptide mass fingerprint compared to the available sequences on the SwissProt/Universal Protein Resource catalog of information on proteins (UniProtKB). Hemoproteins were found, including a cluster of catalase-peroxidase, alkane hydroxylase, and chloroperoxidase. The results from this project helped bridge the progress from agricultural biotechnology strain development into industrial biotechnology biocatalyst improvement. The success of this project helps us expand B. bassiana's catalysis and make it a better candidate for industrial biocatalysis.