Production and Characterization of a Novel Yeast Extracellular Invertase Activity Towards Improved Dibenzothiophene Biodesulfurization

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml^?1) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5–6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5–6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l^?1 sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l^?1 sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μ_max from 0.035 to 0.070 h^?1 and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.     

Characterization of invertase entrapped into calcium alginate beads

A solution of 10 g/L of sodium alginate (Satialgine® types used [Sanofi trademark]: SG800® and S1100® with manuronic/guluronic ratio of 0.5 and 1.2, respectively) containing invertase (0.08 g of protein/L) was dropped into 0.1 M CaCl₂ solution buffered at pH 4.0, 7.0, or 8.0. The beads were left to harden in CaCl₂ solution for 24 h. The high immobilization yield of 60% occurred with SG800 at pH8.0. The activity of soluble and insoluble invertase was measured against pH (2.8–8.0), sucrose concentration (4.5–45 mM), and temperature (30–60°C). Both forms presented an optimum pH of 4.6. However, the soluble invertase was stable at the overall pH interval studied, whereas insoluble invertase lost 30% of its original activity at pH > 5.0. At temperatures above 40°C, the insoluble form was more stable than the soluble one. The kinetic constants and activation energies (E _a ) for free invertase were K _M =41.2 mM, V _max=0.10 mg of TRS/(min · mL), and E _a 28 kJ/mol for entrapped invertase they were (K _M ) _ap =7.2 mM, (V _max) _ap =0.060 mg of TRS/(min · mL), and (E _a )_ap=24 kJ/mol.     

Screening of Dowex® anion-exchange resins for invertase immobilization

Commercial yeast invertase (Bioinvert^®) was immobilized by adsorption on anion-exchange resins, collectively named Dowex^® (1×8:50–400, 1×4:50–400, and 1×2:100–400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1×4–200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/invertase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The K _m and V _max for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL.     

Comparative effects of different triazole compounds on growth,photosynthetic pigments and carbohydrate metabolism of Solenostemon rotundifolius

The effects of two triazole compounds, triadimefon and hexaconazole, on the growth and carbohydrate metabolism were studied in Solenostemon rotundifolius Poir., Morton plants under pot culture. Plants were treated with triadimefon at 15 mg l⁻¹ and hexaconazole at 10 mg l⁻¹ separately by soil drenching on 80, 110 and 140 days after planting (DAP). The plants were harvested randomly and growth parameters were studied on 90, 120 and 150 DAP for determining the effect of both the triazole on growth and chlorophyll pigments. These triazole compounds increased the chlorophyll pigments. However, both the treatments decreased the fresh and dry weights of shoot and leaf area. Both these triazole resulted in a marginal increase in starch content and decreased the sugar contents. The carbohydrate metabolizing enzymes α- and β-amylase activities were reduced and invertase activity increased in S. rotundifolius under triadimefon and hexaconazole treatments.     

Invertase production is related to the nitrogen source in Hansenula anomala

Differences in invertase accumulation of Hansenula anomala cultivated in ammonium and nitrate are reported. Media supplemented with sucrose and ammonium sulfateas the solecarbon and nitrogen source, respectively, show strong media acidification (pH 3.0 or lower), and vigorous cell growth. Invertase activity was not detected under such conditions. A cell-free imitation experiment suggests that, under such low pH, extensive chemical break-down of sucrose (>22%) occurs. Thus, H. anomala is able togrow under strong acidic conditions that permit sucrose metabolism by the uptake of monosaccharides generated from chemical hydrolysis. In addition, invertase activity is not present in cells grown in nitrate as nitrogen source at pH 5.0, but at pH 7.0 activity is detected. If ammonium is supplied instead of nitrate, cells grown at pH 5.0 show invertase activity and at pH 7.0 high levels of activity are detected. These results indicate a specific physiological response of the sucrose metabolism to the presence of alternate nitrogen source.     

Entrapment of plant invertase within novel composite of agarose-guar gum biopolymer membrane

Invertase or β-d-Fructofuranosidase (E.C.3.2.1.26) was extracted from Cucumis melo. L. fruit (Family-Cucurbitaceae). Soluble, plant invertase enzyme was immobilized in novel composite of agarose-guar gum biopolymer matrix in the form of hydrophilic, porous membranes. The immobilized invertase was characterized for sucrose hydrolytic activity and leakage from the matrix support. The efficiency of immobilization was found to be 91% with negligible leaching. The kinetic parameters K_m and V_max for free and immobilized invertase were also determined. Immobilized invertase was optimally active in the wide pH range of 4.5-6.5. The immobilization process also enhanced the thermal stability of enzyme up to 65 °C. Immobilized invertase membranes showed excellent storage stability with shelf life of 110 days. Entrapped invertase showed better operational stability and reusability up to 12 cycles. The fluorescence spectra of the composite membranes were studied and compared with that of soluble enzyme. All these characteristics of the immobilized invertase membranes make them suitable for the fabrication of biosensors.     

Comparative effects of different triazole compounds on growth, photosynthetic pigments and carbohydrate metabolism of Solenostemon rotundifolius

The effects of two triazole compounds, triadimefon and hexaconazole, on the growth and carbohydrate metabolism were studied in Solenostemon rotundifolius Poir., Morton plants under pot culture. Plants were treated with triadimefon at 15mg l(-1) and hexaconazole at 10mg l(-1) separately by soil drenching on 80, 110 and 140 days after planting (DAP). The plants were harvested randomly and growth parameters were studied on 90, 120 and 150 DAP for determining the effect of both the triazole on growth and chlorophyll pigments. These triazole compounds increased the chlorophyll pigments. However, both the treatments decreased the fresh and dry weights of shoot and leaf area. Both these triazole resulted in a marginal increase in starch content and decreased the sugar contents. The carbohydrate metabolizing enzymes alpha- and beta-amylase activities were reduced and invertase activity increased in S. rotundifolius under triadimefon and hexaconazole treatments.     

Immobilization of invertase through its carbohydrate moiety onOcimum basilicum seed

Yeast invertase, a glycoprotein, was covalently coupled toOcimum basilicum seeds either through its protein or carbohydrate moiety. Of the various methods investigated, binding of the enzyme through its carbohydrate moiety resulted in the retention of considerably higher amounts of enzyme activity. Immobilized invertase showed a shift in the pH optimum toward the alkaline side without appreciable change in temperature optimum. However, the immobilized preparation was more thermostable than the free enzyme. Invertase bound to the seeds could be used repeatedly for the hydrolysis of sucrose syrups in a batch process without appreciable loss in activity. The seeds could serve as an inexpensive, ready-to-use, natural pellicular polysaccharide support for immobilizing enzymes.     

Genome Classification of Musa cultivars from Northeast India as Revealed by ITS and IRAP Markers

Genome classification of 38 banana cultivars found in northeast India was successfully carried out using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer (ITS) region and inter-retrotransposon amplified polymorphism (IRAP) techniques. The RsaI digestion of the ITS region revealed the composition of A genome in 32 cultivars and B genome in 29 cultivars. With the gypsy-IRAP marker, 33 cultivars were identified to be composed of B genome. The AluI digestion of the 420-bp PCR amplification product using copia-IRAP primer resulted in the identification of the ABB genome in 17 cultivars. Overall, the data obtained from 36 cultivars using the molecular markers were in accordance with the initial classification based on morphological characters except in two cultivars. The present findings provide the reliable information on the genome classification and the status of the existing banana genetic resources from the northeastern Indian region, which could be utilized in improvement and conservation programs.     

Purification,Characterization and N‐terminal Sequence Analysis of Betel Leaf (Piper betle) Invertase

The present study is the first report describing the purification, enzymatic properties and N‐terminal amino acid sequence of a native invertase in betel leaf. The invertase was purified as a monomeric glycoprotein of molecular mass (Mr) 68 kDa. The enzyme was capable to attack β‐fructofuranoside linkages from the fructose end of sucrose, raffinose and stachyose indicating it as an authentic β‐D‐fructofuranosidase with high specificity for sucrose (Km 4.83 mM). The maximum activity was detected at pH 5.2 and 37 °C. Glucose and fructose showed typical inhibitory effect on the enzyme activity where as lectin was found to be effective activators of the enzyme. Significant inhibition by heavy metal ion Hg²⁺ and sulfhydryl group modifying agents suggesting that free sulfhydryl group containing amino acid, cysteine is necessary for the catalytic activity of the invertase. A BLAST search of the N‐terminal amino acid sequence of betel leaf invertase showed significant homology with the homologous invertases in database.     

Analytical method development using FTIR-ATR and FT-Raman spectroscopy to assay fructose,sucrose, glucose and dihydroxyacetone,in Leptospermum scoparium nectar

The carbohydrate dihydroxyacteone (DHA) occurs in significant levels in Leptospermum scoparium (mānuka) nectar and is the precursor of methylglyoxyl (MGO), the unique non-peroxide antibacterial activity (NPA) component in mānuka honey. The nectar of ten different cultivars of L. scoparium was assayed quantitatively for fructose, glucose, sucrose, and DHA with high pressure liquid chromatography (HPLC) for comparison with FT-Raman and FTIR-ATR spectroscopic methods. FT-Raman spectroscopy and ATR-FTIR spectroscopy, alongside chemometric methods using principal component analysis (PCA) and partial least squares (PLS) prediction were shown to be useful techniques to quantify and compare the nectar composition in a range of cultivars of L. scoparium.     

Properties of a thermostable nonspecific fructofuranosidase produced by cladosporium cladosporioides cells for hydrolysis of Jerusalem artichoke extract

Thermostable invertase (E.C. 3.2.1.26) and inulinase 2,1-beta-D-fructan fructanohydrolase (E.C. 3.2.1.7) activities were produced by Cladosporium cladosporioides grown on sucrose, inulin, yam extract, or Jerusalem artichoke. The ratio I (inulinase)/S(invertase) activity was between 0.31 and 0.36. Both activities had high temperature optima (60 degrees C) and were stable during pretreatment for 4.5 h at this temperature. Whole cells of C. cladosporioides were used for batch fructose production from Jerusalem artichoke extract at several concentrations. With the highest extract concentration used (260 g total sugars/L), total hydrolysis was achieved in 150 min at 60 degrees C. Thin-layer chromatography of the enzymatic hydrolysis of inulin and Jerusalem artichoke extract showed that from the beginning of the reaction, fructose was the only product released. This suggests an exoaction mechanism, beta-D-fructofuranoside fructohydrolase [E.C. 3.2.1.2.6].     

Simultaneous determination of glucose and sucrose by a glucose-sensing enzyme electrode combined with an invertase-attached cell

Glucose and sucrose are simultaneously determined by using a glucose-sensing enzyme electrode combined with a cell that contains immobilized invertase. The electrode current changes linearly with time for several minutes from ca. 1 min after the addition of a glucose-sucrose mixture. The concentration of sucrose (60 μM-6 mM) is determined from the rate of current change in the linear region, and that of glucose (5 μM-1 mM) is determined by extrapolating the straight current-time line to t=0.45 min and by measuring the intercept on the vertical (current) axis at t=0.45 min. The relative standard deviations are 1.8% for glucose and 3.7% for sucrose (n=10). More than 20 food samples can be analysed in 1 h.     

The optimization of the liquid affinity chromatography conditions of the extracellular invertase isolation from theCandida utilis cultures

Summary TheCandida utilis yeast strain was used as a source for the extracellular invertase (E.C.3.2.1.26). The conditions for the highest extracellular invertase activity production were established using three liquid media. The enzyme was purified using one-step affinity chromatography method in the optimized conditions. Three sacharides: sucrose, maltose and unilin were comparatively used as ligands in the affinity technique. The partially purified invertase was afterwards immobilized on the newly described solid matrix with keratin as an activator. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Cool temperature hinders flux from glucose to sucrose during cellulose synthesis in secondary wall stage cotton fibers

Current knowledge about the integration of cellulose synthesis into cellular carbon metabolism and the cool temperature sensitivity of cellulose synthesis is reviewed briefly. Roles for sucrose synthase (to channel UDP-glucose to the cellulose synthase) and sucrose phosphate synthase (to recycle the fructose released by sucrose synthase to more sucrose) in secondary wall cellulose synthesis are described. Data are presented that implicate sucrose synthesis within cotton fibers as a particularly cool temperature-sensitive step in the partitioning of carbon to cellulose. Sugar metabolism during fiber secondary wall deposition was analyzed in in vitro cultures of ovules from two cultivars of Gossypium hirsutum L. (cv. Acala SJ-1 and cv. Paymaster HS 200), which had different levels of cool temperature sensitivity. The sizes of the sucrose, glucose, and fructose pools within fibers at 4 and 7 h after a temperature shift to 15 or 34 °C did not change in either cultivar. Feeding exogenous U-¹⁴C-glucose in pulse and pulse/chase experiments showed that uptake of glucose and transport through the ovule into fibers occurred at the same rate at 34 and 15 °C. In contrast, the flux from glucose to sucrose within fibers was greatly hindered at 15 °C in both cultivars. Since sucrose is the preferred donor of UDP-Glc to the cellulose synthase during secondary wall deposition, this sensitivity in sucrose synthesis is likely to at least partially explain the cool temperature sensitivity of cotton fiber cellulose synthesis that is observed in the field.     

Comparative studies on performance of radiation-induced and thermal cross-linked ion-exchange membrane for water electrolysis

Radiation-induced and thermal cross-linked sulfonated poly(ether sulfone) (SPS)–sulfonated poly(ether ether ketone) (SPK) composite ion-exchange membranes (SPS/SPK(γ) and SPS/SPK(T), respectively) were prepared. Their performances for water electrolysis were comparatively assessed. Thermal cross-linked membrane (SPS/SPK(T)) showed cross-linking of part functional groups (–SO₃H) and thus deterioration in membrane conductivity. While, radiation-induced cross-linked membrane (SPS/SPK(γ)) avoided any cross-linking between functional groups and thus conductivity. Electrolysis performances of these membranes were evaluated in comparison with Nafion117 membrane. Relatively low current efficiency (CE) for SPS/SPK and SPS/SPK(T) membranes was due to their high mass transfer (water) via electro-osmotic drag, which was negligible for SPS/SPK(γ) membrane. SPS/SPK(γ) membrane exhibited comparable stabilities and water splitting performance with Nafion117 membrane, which revealed its suitability as substitute for electrochemical applications.     

Antioxidant activities and bioactive compounds of five Jalopeno peppers (Capsicum annuum) cultivars

The present study was designed to evaluate the contents of different antioxidants compounds and their antioxidant activities in Jalopeno peppers (Capsicum annuum) cultivars (El Dorido, Grande, Tula, Sayula and El Rey) extracts. Free radical scavenging activity of Grande was recorded as high as 87% followed by El Dorido (83%). Results of reducing power (Fe³⁺ to Fe²⁺) showed that Grande (0.85%) and El Dorido (0.81%) fruit extract absorbance value were close to synthetic antioxidant BHT (0. 97%) obtained at100 μg/mL. The results showed that total phenolic content of El Dorido and Grande were significantly higher compared to other Jalapeno pepper. Results indicated strong and positive correlation between antioxidant activity and carotenoids content (r = 0.75), vitamin C (r = 0.78) and total capsaicinoids (r = 0.84), respectively. The results of the antioxidant activity assays showed that the El Dorido and Grande had strongest antioxidant activity compared to other peppers cultivars in this study.     

Nitrogen regulation of Saccharomyces cerevisiae invertase

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE 2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure 2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure 2 mutant cells from both phases. When these cells were trans-formed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cells was not observed. These results suggested that the URE2 protein plays a role in invertase activity.     

Normal and second excimers in macromolecules—I: Poly(1-methoxy-4-vinylnaphthalene) in fluid solution

The emission properties of poly(1-methoxy-4-vinylnaphthalene) (PMVN) in 2-methyltetrahydrofuran solution have been investigated over the range 77–350 K. It exhibits, in addition to the monomer fluorescence (348 and 360 nm), two structureless emissions derived from two different types of excimer, viz. the normal excimer (420 nm) and the second excimer (380 nm), the latter having a partially overlapping structure of aromatic rings. The intensity of second excimer emission of PMVN increases with decreasing temperature, while the normal excimer emission shows a maximum at 215 K. Kinetic analysis of transient decay curves for the fluorescence of PMVN gave results consistent with the previously reported kinetic scheme for the dimer model, 1,3-bis(4-methoxy-1-naphthyl)propane, showing that the second excimer is not formed from or converted to the normal excimer and that two types of excimer are formed independently from the excited monomer.     

Effects of Holdfast of Laminaria japonica on Listeria Invasion on Enterocyte-Like Caco-2 Cells and NO Production of Macrophage RAW 264.7 Cells

Listeria monocytogenes (Lm) causes food poisoning in humans mainly through consumption of ready-to-eat foods. Immunocompromised persons are at the highest risk for infection. We investigated effects of crude soluble polysaccharides (SPS) and ethanolic extract (EE) fractions of frond (kombu) and holdfast (ganiashi) parts of Laminaria japonica on Lm invasion into human enterocyte-like Caco-2 cells and immune and/or inflammatory reactions of murine macrophage RAW 264.7 cells. Recovery and viscosity were high in kombu SPS. Total phenolic content and antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity and Fe-reducing power) were higher in ganiashi EE. EE of ganiashi, rather than kombu, suppressed the Lm invasion into the differentiated Caco-2 cells, though the inhibitory effect of SPS was not significant. Ganiashi SPS increased the nitric oxide (NO) production of intact RAW 264.7 cells. On the other hand, the NO production from Escherichia coli O111 lipopolysaccharide-activated cells was suppressed by kombu SPS and ganiashi EE. These results suggest that L. japonica, particularly ganiashi, might suppress the invasion and infection of Lm and also the inflammation.