Production, purification and immobilization of inulinases from Aspergillius species

Inulinases are enzymes catalysing hydrolysis of polyfructosans to produce fructose or fructooligosaccharides; these properties have attracted interest of many researchers towards exploring various plant sources (agro food waste) as substrates. According to the literature, various microorganisms, such as fungi, yeast, bacteria, and actinomycetes, can synthesize inulinase. Producer microorganisms can be the micromycetes Aspergillus, Penicillium, Rhizopus and Fusarium, the yeast Kluyveromyces and the bacteria Clostridium thermosuccinogenes and Bacillus subtilis, using inulin, sucrose, fructose, lactose, raffinose, xylose as sources of carbon. The present study mainly envisages inulinase produced by Aspergillius species grown on a fermentation medium based on different carbon sources (inulin and agro-alimentary by-products). The crude extract was purified by fractional precipitation with ammonium sulphate. The dissolved and dialyzed precipitate was further purified by ion exchange chromatography on DEAE-Sephadex. The experiments led to the obtaining of an inulinase with a specific activity of 43.34 U/mg proteins. Inulinase immobilization tests on chitosan beads have led to a stable biocatalyst at temperature and pH variations that could be useful for obtaining fructose syrup.

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

Investigating the Central Metabolism ofClostridium thermosuccinogenes

ABSTRACTClostridium thermosuccinogenesis a thermophilic anaerobic bacterium able to convert various carbohydrates to succinate and acetate as main fermentation products. Genomes of the four publicly available strains have been sequenced, and the genome of the type strain has been closed. The annotated genomes were used to reconstruct the central metabolism, and enzyme assays were used to validate annotations and to determine cofactor specificity. The genes were identified for the pathways to all fermentation products, as well as for the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway. Notably, a candidate transaldolase was lacking, and transcriptomics during growth on glucose versus that on xylose did not provide any leads to potential transaldolase genes or alternative pathways connecting the C5with the C3/C6metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related thermophilic species of industrial relevance. Furthermore, the gene responsible for malate dehydrogenase was identified via heterologous expression inEscherichia coliand subsequent assays with the cell extract, which has proven to be a simple and powerful method for the basal characterization of thermophilic enzymes.IMPORTANCERunning industrial fermentation processes at elevated temperatures has several advantages, including reduced cooling requirements, increased reaction rates and solubilities, and a possibility to perform simultaneous saccharification and fermentation of a pretreated biomass. Most studies with thermophiles so far have focused on bioethanol production.Clostridium thermosuccinogenesseems an attractive production organism for organic acids, succinic acid in particular, from lignocellulosic biomass-derived sugars. This study provides valuable insights into its central metabolism and GTP and PPicofactor utilization.

Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin

ABSTRACT Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin byClostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70°C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58°C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70°C. No acetate kinase activity was observed at 70°C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58°C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70°C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70°C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.