Biochemical characterization of selected carbohydrases from Beauveria bassiana and their potential applications

Abstract

Different filamentous fungi have continued to attract scientific interests as novel sources of enzymes and other important bioproducts. Beauveria bassiana, a well-known entomopathogenic fungi has long been valued for its biotechnological application as a biocontrol agent in its entomopathogenic state and as a plant-growth promoter in its endophytic state. The fungus has also been proven to be safe for human health, as studies have shown B. bassiana strains to be non-pathogenic to humans, other animals and plants. Furthermore, its ability to utilize various agro-residues for its growth and the concomitant production of important bioproducts have been well demonstrated. However, despite all of these, there has been no appreciable attempt at exploring this remarkable fungus for the production of industrially important enzymes, especially in its saprophytic state. Recently, a filamentous fungus was isolated in its endophytic state from onion leaves, in our laboratory. It was confirmed by rDNA ITS sequencing to be a B. bassiana strain and was subsequently designated as B. bassiana SAN01. Preliminary experiments revealed the remarkable ability of this novel strain to utilize lignocellulosic biomass for its metabolism while secreting various biomass-degrading enzymes in the process. Hence, carbohydrases from B. bassiana SAN01 were considered worthy of investigation because of the established safety of the source organism, as well as the probable low production cost of the enzymes using readily available plant biomass. Besides, it was also observed that there has been no significant investigation into the biochemical properties of lignocellulolytic enzymes from B. bassiana, which has probably hindered their industrial applicability. Hence, this Ph.D. research was focused on investigating the production, the biochemical properties, as well as the potential applicability of selected biomass-degrading enzymes, viz., amylase, cellulase (endoglucanase), pectinase (polygalacturonase) and xylanase from B. bassiana SAN01. To achieve these, the phylogenetic relationship of the fungal strain was established, and its carbon utilization profile was annotated using phenotypic microarray technology. Furthermore, to understand the dynamics surrounding its lignocellulosic biomass utilization and its carbohydrase-production capabilities, comparative transcriptomics analysis was carried out B. bassiana SAN01 under three different simulated conditions i.e., endophytic, fermentation and lab control conditions. In addition, to fully explore the carbohydrase production potential of the fungus, the production of the selected carbohydrases was optimized using response surface methodology; subsequently, all the selected enzymes were purified to enhance the evaluation of their biochemical properties as well as their potential industrial applications. The proclivity of B. bassiana SAN01 for polyols, pentoses, N-acetyl-D-glucosamine and some other carbon sources was demonstrated by the phenotype microarray profiling. While the comparative genome-wide transcriptome analyses revealed a clear distinction between the fungus under the different trophic conditions investigated. It was observed that 4-5% of the 10,365 B. bassiana SAN01 genes were differentially expressed between these conditions, and a significant proportion of the genes were found to be involved in lignocellulose deconstruction. The annotation of CAZymes from the B. bassiana SAN01 transcriptome under fermentation (saprophytic) conditions confirmed the upregulation of biomass-degrading enzymes such as amylases, cellulases, chitinases, glucanases, laccases, lignases, pectinases and xylanases. The subsequent optimization of the production parameters of B. bassiana SAN01 amylase, endoglucanase, polygalacturonase and xylanase led to heightened yields of 34.82 UmL-1, 23.03 UmL-1, 51.05 UmL-1, and 1061 UmL-1, respectively. These were estimated to be 1.79-, 1.35-, 1.87- and 3.44- folds higher than unoptimized production levels and are also the highest ever production levels recorded for these enzymes from any B. bassiana strain. Further in the study, the xylanase from B. bassiana SAN01 was purified to homogeneity while the other three enzymes were partially purified. The purified xylanase was demonstrated to have a molecular mass of ~37 kDa and performed optimally at pH 6.0 and 45oC. However, the optimum pH of the partially purified amylase, endoglucanase, and polygalacturonase were found to be pHs 6.0, 6.0 and 7.0, while the optimum temperatures were observed to be 35oC, 35oC and 45oC, respectively. Consequently, the purified B. bassiana SAN01 xylanase was demonstrated to be effective in deinking wastepaper with an optimized deinking rate of 106.72% relative to the control. In addition, the partially purified amylase-polygalacturonase from B. bassiana SAN01 was demonstrated to adequately clarify pear juice with a 1.37-fold improvement in clarity recorded under optimal conditions. Furthermore, results also showed that the enzymatic- assisted juice clarification was without any detrimental effect on some quality parameters of the juice. In the same vein, crude endoglucanase-xylanase from the fungus was shown to significantly hydrolyze sugarcane bagasse, releasing ~20% reducing sugars under optimal conditions. Finally, to gain insights into the structure-function relationship of B. bassiana carbohydrases, the structural properties of B. bassiana chitinases and xylanases were elucidated for the first time using computational techniques. The in silico prediction revealed that the enzymes were generally hydrophilic, thermostable, negatively charged and extracellularly secreted. The modelled tertiary structures of B. bassiana chitinase and xylanase were validated by the presence of ~ 90% of their amino acid residues in the Ramachandran plot’s favoured region. The findings from this study have thus created a strong framework for the prospective utilization of B. bassiana and its carbohydrases in alternative biotechnological processes.