Comprehensive analysis of a metabolic model for lipid production in Rhodosporidium toruloides.

The yeast Rhodosporidium toruloides has been extensively studied for its application in biolipid production. The knowledge of its metabolism capabilities and the application of constraint-based flux analysis methodology provide useful information for process prediction and optimization. The accuracy of the resulting predictions is highly dependent on metabolic models. A metabolic reconstruction for R. toruloides metabolism has been recently published. On the basis of this model, we developed a curated version that unblocks the central nitrogen metabolism and, in addition, completes charge and mass balances in some reactions neglected in the former model. Then, a comprehensive analysis of network capability was performed with the curated model and compared with the published metabolic reconstruction. The flux distribution obtained by lipid optimization with flux balance analysis was able to replicate the internal biochemical changes that lead to lipogenesis in oleaginous microorganisms. These results motivate the development of a genome-scale model for complete elucidation of R. toruloides metabolism.

α-L-rhamnosidase and ß-D-glucosidase activities in fungal strains isolated from alkaline soils and their potential in naringin hydrolysis.

α-L-Rhamnosidases (EC 3.2.1.40) and ß-D-glucosidases (EC 3.2.1.21) obtained from several microbial sources are potential catalysts in food, beverage, and pharmaceutical industries. However, the enzyme preparations currently used have limitations related to the stability and activity of the enzyme as well to their reuse. A microtiter screening was carried out in 55 fungal strains isolated from alkaline soils, to obtain active α-L-rhamnosidases and ß-D-glucosidases at pH 9.0. While α-L-rhamnosidase activity was detected in 45% of the strains tested, ß-D-glucosidase activity was found only in 27%. Based on the fungal ability to produce α -L-rhamnosidase activity, cultures were supplemented with naringin to study the activities of the enzymes and the potential of the fungal strains on naringin hydrolysis. About 70% of the fungal strains tested increased the activities of both enzymes in the naringin-supplemented cultures as compared to non-supplemented ones. This effect was higher in Acrostalagmus luteo-albus LPSC 427 (15.3 fold) for α-L-rhamnosidase activity and Metarrhizium anisopliae LPSC 996 (51.1 fold) for ß-D-glucosidase activity. All the enzyme preparations tested hydrolyzed naringin at pH 9.0, being that obtained from Acremonium murorun LPSC 927 cultures the one which showed highest hydrolysis. Here, different fungal species are reported for the first time for their ability to produce α-L-rhamnosidase and ß-D-glucosidase activity at alkaline pH.

Purification and characterization of a novel alkaline α-L-rhamnosidase produced by Acrostalagmus luteo albus.

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal nonreducing L-rhamnose for the bioconversion of natural or synthetic rhamnosides. They are of great significance in the current biotechnological area, with applications in food and pharmaceutical industrial processes. In this study we isolated and characterized a novel alkaline rhamnosidase from Acrostalagmus luteo albus, an alkali-tolerant soil fungus from Argentina. We also present an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In the presence of rhamnose as the sole carbon source, this fungus produces a rhamnosidase with a molecular weight of 109 kDa and a pI value of 4.6, as determined by SDS-PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electrophoretic techniques. Using p-nitrofenil-α-L-rhamnopiranoside as substrate, the enzyme activity showed pH and temperature optima of 8.0 and 55°C, respectively. The enzyme exhibited Michaelis-Menten kinetics, with K (M) and V (max) values of 3.38 mmol l(-1) and 68.5 mmol l(-1) min(-1), respectively. Neither divalent cations such as Ca(2+), Mg(2+), Mn(2+), and Co(2+) nor reducing agents such as ß-mercaptoethanol and dithiothreitol showed any effect on enzyme activity, whereas this activity was completely inhibited by Zn(2+) at a concentration of 0.2 mM. This enzyme showed the capacity to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. Based on these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered a potential new biocatalyst for industrial applications.

One-step concentration and partial purification of Aspergillus kawachii non-acidic polygalacturonases by adsorption to glass fiber microfilters.

The non-acidic polygalacturonases produced by Aspergillus kawachii in a glucose/tryptone medium were adsorbed to a glass fiber microfilter that was used to clarify the fermentation broth. Maximum adsorption occurred at pH 3 under low ionic strength conditions. The adsorbed activity could be readily released with a buffer solution at pH 5. Based upon these observations, a separation process was developed which enabled the broth to be clarified and, at the same time, the non-acidic polygalacturonases to be concentrated 20-fold and purified 100-fold in a unique filtration step. The practical advantage of recovering polygalacturonases by a filtration process lies in the simplicity and efficiency of the operation involved.