Production and characterization of intracellular invertase from Saccharomyces cerevisiae (OL629078.1), using cassava-soybean as a cost-effective substrate.

The growing global market for industrial enzymes has led to a constant search for efficient, cost-effective methods for their production. This study reports the production of invertase using inexpensive and readily available agro-materials. Starch-digesting enzymes extracted from malted unkilned sorghum were used to hydrolyze cassava starch supplemented with 2% whole soybean. The production of intracellular invertase by Saccharomyces cerevisiae OL629078.1 in cassava-soybean and yeast sucrose broth was compared. The purification and characterization of invertase produced using the low-cost medium were also reported. The results showed that there was a 4.1-fold increase in the units of invertase produced in cassava-soybean medium (318.605 U/mg) compared to yeast sucrose broth medium (77.6 U/mg). The invertase produced was purified by chromatographic methods up to 5.53-fold with a recovery of 62.6%. Estimation of the molecular weight with gel filtration indicated a molecular weight of 118 kDa. The enzyme demonstrated its maximum activity at 50 °C and there was no decrease in its activity following a 1-h incubation at this temperature. At a pH of 5.0, the enzyme demonstrated optimal activity and it maintained over 60% of its activity in the acid range (pH 3-6). The Michalis-Menten constants Km and Vmax of intracellular invertase were 5.85 ± 1.715 mM and 6.472 ± 2.099 U/mg, respectively. These results suggest that Saccharomyces cerevisiae grown on cassava-soybean is a viable, cost-effective alternative for commercial invertase production, which can be explored for biotechnological processes.

Assessment of dynamic microbial community structure and rhizosphere interactions during bioaugmented phytoremediation of petroleum contaminated soil by a newly designed rhizobox system.

To understand the plant (Vigna unguiculata) and plant-growth promoting bacteria (PGPB) (Microcococcus luteus WN01) interactions in crude oil contaminated soil, experiments were conducted based on the newly designed rhizobox system. The rhizobox was divided into three main compartments namely the rhizosphere zone, the mid-zone, and the bulk soil zone, in accordance with the distance from the plant. Plants were grown in these three-chambered pots for 30 days under natural conditions. The plant root exudates were determined by analyzing for carbohydrates, amino acids, and phenolic compounds. The degradation of alkane, polycyclic aromatic hydrocarbons (PAHs), and total petroleum hydrocarbons (TPHs) were quantified by GC-FID. Soil catalase, dehydrogenase, and invertase activities were determined. The microbial community structure was assessed using denaturing gradient gel electrophoresis (DGGE). Results showed that the inoculation of M. luteus WN01 significantly enhanced cowpea root biomass and exudates, especially the phenolic compounds. Bioaugmented phytoremediation by cowpea and M. luteus promoted rhizodegradation of TPH. Cowpea stimulated microbial growth, soil dehydrogenase, and invertase activities and enhanced bacterial community diversity in oil contaminated soil. The rhizosphere zone of cowpea inoculated with M. luteus showed the highest removal efficiency, microbial activities, microbial population, and bacterial community diversity indicating the strong synergic interactions between M. luteus and cowpea.

This is the first study to characterize the rhizosphere effect of cowpea on microbial activities, population, and community structure in crude oil contaminated soil in the presence and absence of PGPB, M. luteus WN01. The rhizosphere of cowpea was found to be a degradation hotspot where microbial abundance and metabolic activities were most active. Cowpea-M. luteus association can be a good candidate that can be implemented in real field sites.

Low-cost production of 6G-fructofuranosidase with high value-added astaxanthin by Xanthophyllomyces dendrorhous.

The effects of medium composition and culture conditions on the production of (6)G-fructofuranosidase with value-added astaxanthin were investigated to reduce the capital cost of neo-fructooligosaccharides (neo-FOS) production by Xanthophyllomyces dendrorhous. The sucrose and corn steep liquor (CSL) were found to be the optimal carbon source and nitrogen source, respectively. CSL and initial pH were selected as the critical factors using Plackett-Burman design. Maximum (6)G-fructofuranosidase 242.57 U/mL with 5.23 mg/L value-added astaxanthin was obtained at CSL 52.5 mL/L and pH 7.89 by central composite design. Neo-FOS yield could reach 238.12 g/L under the optimized medium conditions. Cost analysis suggested 66.3% of substrate cost was reduced compared with that before optimization. These results demonstrated that the optimized medium and culture conditions could significantly enhance the production of (6)G-fructofuranosidase with value-added astaxanthin and remarkably decrease the substrate cost, which opened up possibilities to produce neo-FOS industrially.

Development of activity-based cost functions for cellulase, invertase, and other enzymes.

As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

Identification and assessment of the role of a nominal phospholipid binding region of ORP1S (oxysterol-binding-protein-related protein 1 short) in the regulation of vesicular transport.

The ORPs (oxysterol-binding-protein-related proteins) constitute an enigmatic family of intracellular lipid receptors that are related through a shared lipid binding domain. Emerging evidence suggests that ORPs relate lipid metabolism to membrane transport. Current data imply that the yeast ORP Kes1p is a negative regulator of Golgi-derived vesicular transport mediated by the essential phosphatidylinositol/phosphatidylcholine transfer protein Sec14p. Inactivation of Kes1p function allows restoration of growth and vesicular transport in cells lacking Sec14p function, and Kes1p function in this regard can be complemented by human ORP1S (ORP1 short). Recent studies have determined that Kes1p and ORP1S both bind phospholipids as ligands. To explore the function of distinct linear segments of ORP1S in phospholipid binding and vesicular transport regulation, we generated a series of 15 open reading frames coding for diagnostic regions within ORP1S. Purified versions of these ORP1S deletion proteins were characterized in vitro, and allowed the identification of a nominal phospholipid binding region. The in vitro analysis was interpreted in the context of in vivo growth and vesicle transport assays for members of the ORP1S deletion set. The results determined that the phospholipid binding domain per se was insufficient for inhibition of vesicular transport by ORP1S, and that transport of carboxypeptidase Y and invertase from the Golgi may be regulated differentially by specific regions of ORP1S/Kes1p.

Assessment of intestinal permeability: enzymatic determination of urinary mannitol, raffinose, sucrose and lactose on Hitachi analyzer.

The sugar absorption test is the usual test for measurement of intestinal permeability. After intestinal absorption of probe sugars the subsequently excreted sugars are measured in urine. We have developed four enzymatic methods for the measurement of the urinary concentration of the probe sugars mannitol, raffinose, lactose and sucrose. Mannitol, lactose and sucrose are directly measured on Hitachi 917 using mannitol dehydrogenase, beta-galactosidase and invertase, respectively, as enzyme reagents. Raffinose measurement needs a three hours preincubation with alpha-galactosidase, after which the liberated sucrose is measured. The analytical performances such as within- and between-run precision, linearity, lowest detection limit, interference of other sugars and comparison with a gas chromatographic method are described for the four methods. These methods are accurate an can easily be performed in any clinical laboratory.

Enzymes for animal feeding from Penicillium chrysogenum mycelial wastes from penicillin manufacture.

AIMS: Several enzymes from mycelial wastes of Penicillium chrysogenum fungal cultures from penicillin manufacture were studied as supplements for animal feeding. METHODS AND RESULTS: Proteins were precipitated with tannic acid, acetone, ethanol or polyethyleneglycol 6000. The levels of the enzyme activities found in the different precipitates were similar. The activities of invertase, beta-1,3-glucanase, lipase and tannase were higher and those of amylase, cellulase, pectinase, protease and xylanase were lower. Precipitation with tannic acid was selected as an easy and simple method. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: An enzymatic precipitate from these P. chrysogenum wastes, comparable to a commercial enzymatic additive used in animal feeding, was obtained.

Immobilization and stabilization of invertase on Cajanus cajan lectin support.

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.