Cloning and screening of the putative hexokinase genes from Rhizopus oryzae and their heterologous expression in Saccharomyces cerevisiae.

BACKGROUND: A filamentous fungus, Rhizopus oryzae (R. oryzae) is one of the ideal candidates for ethanol and lactic acid production due to its ability to grow on renewable carbon sources. METHODS AND RESULTS: In this study, the nucleotide sequence of hexokinases and glucokinase from S. cerevisiae was found on the NCBI site ( http://www.ncbi.nlm.nih.gov/blast/Blast.cgi ) were used. With these nucleotide sequences, a blast search was done on the R. oryzae genome database ( http://www.broad.mit.edu/annotation/genome/rhizopus_oryzae/Home.html ) and ten probable genes were obtained. cDNA was synthesized from the total RNA and PCR products of the seven of these putative genes were determined using the primers designed for them. CONCLUSION: The results of the sequences and the complementation studies revealed that three of these seven putative genes were expressed in R. oryzae and the growth was observed on selective media.

Single-Step Partial Purification of Intracellular ß-Galactosidase from Kluyveromyces lactis Using Microemulsion Droplets.

Partial purification of ß-galactosidase from the crude extract of Kluyveromyces lactis was carried out using water-in-isooctane microemulsions formed by the anionic surfactant, sodium di-ethylhexyl sulfosuccinate (Aerosol OT). In order to obtain the crude extract, yeast cells of K. lactis were disrupted by a cell disrupter and separated. The purification of ß-galactosidase from the extract by a recently developed one-step reversed micellar (i.e., microemulsion-based) extraction method was then tested, by measuring total protein mass and enzyme activity in the product stream and by analyzing its composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. Effects of salt concentration, protein concentration, and pH on the extraction were investigated. Using this approach, a 5.4-fold purification of ß-galactosidase was achieved with 96 % total activity recovery, using a feed containing crude extract and 50 mM K-phosphate buffer (pH 7.5) and 50 mM KCl. Gel filtration chromatography showed that the single extraction was successful at removing low molecular weight impurity proteins (molecular weight (MW) < 42 kDa) from the crude extract.

Rheological properties of lactose-free dairy desserts.

People suffering from lactose intolerance cannot digest milk or lactose-containing foods. Lactose-free diet is essential for them since they do not have the ability to produce lactase to breakdown milk sugar. Physical properties of lactose-free dairy desserts will most probably be different than that of lactose containing ones because of lactose hydrolysis. In this study, it was aimed to analyze the rheological and textural behaviors of different lactose-free dessert formulations containing different gum types and different waxy maize starch and sucrose concentrations. Waxy maize starch was used at concentrations of 0.032 g·mL-1, 0.040 g·mL-1, and 0.048 g·mL-1 In addition to waxy maize starch, guar gum, gum arabic, or κ-carrageenan at two different concentrations (1.0% w/w and 0.5% w/w) was added. Sucrose was added at concentrations of 0.14 g·mL-1 and 0.10 g·mL-1 in lactose-free desserts. Power law model was found to be suitable to explain the flow behavior of desserts. The storage and loss modulus of lactose-free desserts were higher than that of lactose-containing desserts. The κ-Carrageenan was found to be the most effective gum for structure build-up.

Cloning and expression of trehalose-6-phosphate synthase 1 from Rhizopus oryzae.

Trehalose is a reducing disaccharide acting as a protectant against environmental stresses in many organisms. In fungi, Trehalose-6-phosphate synthase 1 (TPS1) plays a key role in the biosynthesis of trehalose. In this study, a full-length cDNA from Rhizopus oryzae encoding TPS1 (designated as RoTPS1) was isolated. The RoTPS1 cDNA is composed of 2505 nucleotides and encodes a protein of 834 amino acids with a molecular mass of 97.8 kDa. The amino acid sequence of RoTPS1 has a relatively high homology with the TPS1s in several other filamentous fungi. RoTPS1 was cloned into Saccharomyces cerevisiae and secretively expressed.

One-step separation of ß-galactosidase from ß-lactoglobulin using water-in-oil microemulsions.

Solubilisation of ß-galactosidase from Kluyveromyces lactis in Aerosol-OT water-in-isooctane microemulsions was measured as a function of buffer type and concentration, pH, and protein concentration. For buffer concentrations above ∼40mM, the enzyme was largely excluded from the droplets. Based on these results, a one-step separation was developed. A protein-containing aqueous feed was injected into an AOT/isooctane solution, with the feed volume slightly in excess of the predicted water solubility. Impurity proteins were entrapped inside the microemulsion droplets that then formed in the organic phase, while the high MW target protein was excluded and entered a newly formed, excess aqueous phase. The separation of ß-galactosidase from the test protein ß-lactoglobulin was most complete at 100mM KCl salt concentration, where the droplets were large enough to carry ß-lactoglobulin but too small for ß-galactosidase. At 100mM [KCl], 92% of the total enzyme activity was recovered in a concentrated and virtually pure form.

Effect of different stresses on trehalose levels in Rhizopus oryzae.

Rhizopus oryzae accumulates and degrades trehalose in response to environmental stress conditions such as heat, osmotic stress, nitrogen starvation and pH. When heat stress was applied to R. oryzae, the trehalose content of the cells was increased from 0.9 to 4.8 mg/g dw and when the stress was relieved it decreased back to 1.1 mg/g dw. Under osmotic stress and nitrogen starvation, trehalose content was increased by 3.5 and 3 fold, respectively. The decrease in external pH increased trehalose level up to 2.6 mg/g dw from 1.1 mg/g dw. Increased trehalose level decreased back to normal after pH stress was relieved. These results indicate that trehalose accumulates and acts as a stress protectant in R. oryzae.

Lactate and ethanol productions by Rhizopus oryzae ATCC 9363 and activities of related pyruvate branch point enzymes.

In Rhizopus oryzae cultivations, lactate production decreased and ethanol and biomass productions increased as inoculated spore concentration increased. Spore concentrations of 1 x 10(3) and 1 x 10(4) spores ml(-1) induced tightly packed clumps, whereas 1 x 10(5) and 1 x 10(6) spores ml(-1) induced filamentous growth.