Characterization and molecular modeling of polygalacturonase isoforms from Saccharomyces cerevisiae.

Earlier, low-temperature-active polygalacturonase isoforms from Saccharomyces cerevisiae PVK4 were isolated and purified. Substrate specificity of polygalacturonase isoforms indicated high affinity for pectins and very low enzyme activity towards non-pectic polysaccharides. To characterize the polygalacturonase isoforms, biochemical, spectral, and in silico approaches were used. The apparent Km and Vmax values for hydrolysis of pectin and galacturonic acid were 0.31 mg/ml and 3.15 mmol min/mg, respectively. Interestingly, the polygalacturonase isoforms were found to be more stable at optimal pH and temperature of 4.5 and 40 °C, respectively. These isoforms were reacted with different metal ions; Cd2+ and Ni2+ severely inhibited the enzyme activity, while Mg2+, Zn2+, Cd2+, Fe2+ Cu2+, and Ni2+ inhibited to a lesser extent, which clearly demonstrated that variations in enzyme activity were due to their differential binding affinity of metal ions. Furthermore, decrease in the viscosity of polygalacturonic acid and citrus pectin by these isoforms was approximately four and six times higher than the rate of release of reducing sugars. This indicates that polygalacturonase isoforms have an endo-mode of action. In addition to the above, thermostability of purified polygalacturonase isoforms was studied by circular dichroism and fluorescence spectroscopy. Circular dichroism showed 18% alpha helix and 57% beta sheets at pH 5, while at pH 7, 8, and 9 there was an increase of random coil. Fluorescence studies revealed small conformational changes, which were observed at 30-50 °C, while unfolding transition region was noticed between 60 and 70 °C. The purified enzyme fractions were analyzed by MALDI-TOF MS. Finally, 3D model structures for isoenzymes of polygalacturonase of S. cerevisiae were generated and validated as good quality models, which are also suitable for molecular interaction studies.

Enhanced production of xylanase by solid state fermentation using Trichoderma koeningi isolate: effect of pretreated agro-residues.

The main objective of this study was to isolate the fungal strain for enhanced production of xylanase using different agro-residues and fruit peels by solid state fermentation and its potentiality was tested on the pretreated corn cob. Fermentation was carried out with Trichoderma koeningi isolate using untreated and pretreated corn cob supplemented with pineapple peel powder showed higher production of xylanase 2,869.8 ± 0.4 (IU/g) and extracellular protein 7.6 ± 0.2 (mg/g) of corn cob, in the latter than the former yielding 1,347.2 ± 0.7 (IU/g) and 4.9 ± 0.1 (mg/g) of corn cob, respectively, at pH 6.5 and incubation period for 96 h. In the FT-IR spectrum, the bands at 1,155, 1,252 and 1,738 cm-1 had disappeared. This indicates the depolymerization of hemicellulose and the band at 1,053 cm-1 shows the presence of ß (1-4)-xylan in the pretreated corn cobs. The pretreated biomass hydrolysed with a xylanase concentration of 14 U and 6 h incubation showed mainly xylose and its oligosaccharides, which were quantified using HPLC. From the results we can conclude that pretreated energy-value and cheaply available agro-residues can be effectively used as substrates for the enhanced production of xylanase.

Very high gravity (VHG) ethanolic brewing and fermentation: a research update.

There have been numerous developments in ethanol fermentation technology since the beginning of the new millennium as ethanol has become an immediate viable alternative to fast-depleting crude reserves as well as increasing concerns over environmental pollution. Nowadays, although most research efforts are focused on the conversion of cheap cellulosic substrates to ethanol, methods that are cost-competitive with gasoline production are still lacking. At the same time, the ethanol industry has engaged in implementing potential energy-saving, productivity and efficiency-maximizing technologies in existing production methods to become more viable. Very high gravity (VHG) fermentation is an emerging, versatile one among such technologies offering great savings in process water and energy requirements through fermentation of higher concentrations of sugar substrate and, therefore, increased final ethanol concentration in the medium. The technology also allows increased fermentation efficiency, without major alterations to existing facilities, by efficient utilization of fermentor space and elimination of known losses. This comprehensive research update on VHG technology is presented in two main sections, namely VHG brewing, wherein the effects of nutrients supplementation, yeast pitching rate, flavour compound synthesis and foam stability under increased wort gravities are discussed; and VHG bioethanol fermentation studies. In the latter section, aspects related to the role of osmoprotectants and nutrients in yeast stress reduction, substrates utilized/tested so far, including saccharide (glucose, sucrose, molasses, etc.) and starchy materials (wheat, corn, barley, oats, etc.), and mash viscosity issues in VHG bioethanol production are detailed. Thereafter, topics common to both areas such as process optimization studies, mutants and gene level studies, immobilized yeast applications, temperature effect, reserve carbohydrates profile in yeast, and economic aspects are discussed and future prospects are summarized.