Isolation and characterization of a new dimeric esterase from Lactobacillus sakei 23K

The M_w of a Lactobacillus sakei intracellular esterase, determined by gel filtration, was compared to those obtained from SDS-PAGE or MALDI-TOF, pointing to a dimeric structure. Its N-terminal sequence and peptide mass fingerprint suggest that it is the putative LSA044 protein from L. sakei 23K genome.     

Expression of HIV-1 nef in yeast: The 27 kDa nef protein is myristylated and fractionates with the nucleus

The nef gene of human immunodeficiency virus type 1 (HIV-1) has been expressed in the yeast Saccharomyces cerevisiae to produce native Nef proteins. The proteins of M_r 27 kDa and 25 kDa, produced by translation from the first and second start codons of the nef gene react with human HIV-1 antisera. Under low-level steady-state expression conditions, Nef27 undergoes myristylation and is targeted to the nuclear fraction while Nef25 is not myristylated and not nuclear localized. When produced rapidly and to high levels, Nef27 is initially present in the cytoplasm as a soluble myristylated protein that later fractionates with the nucleus.     

Role of the γ component of preprotoxin in expression of the yeast K1 killer phenotype

K₁ killer strains of Saccharomyces cerevisiae secrete a polypeptide toxin to which they are themselves immune. The α and β components of toxin comprise residues 45–147 and 234–316 of the 316-residue K₁ preprotoxin. The intervening 86-residue segment is called γ. A 26-residue signal peptide is removed on entry into the endoplasmic reticulum. The Kex2 protease excises the toxin components from the 290-residue glycosylated protoxin in a late Golgi compartment. Expression of a cDNA copy of the preprotoxin gene confers the complete K₁ killer phenotype on sensitive cells. We now show that expression of immunity requires that α component and the N-terminal 31 residues of γ. An additional C-terminal extension, either eight residues of γ or three of four unrelated peptides, is also required. Expression of preprotoxin terminating at the α C-terminus, or lacking the γ N-terminal half of γ causes profound but reversible growth inhibition. Inhibition is suppressed in cis by the same 31 residues of γ required for immunity to exocellular toxin in trans, but not by the presence of β. Both immunity and growth inhibition are alleviated by insertions in α that inactivate toxin. Inhibition is not suppressed by kex2, chc1 or kre1 mutations, by growth at higher pH or temperature, or by normal K₁ immunity. Inhibition, therefore, probably does not involve processing of the α toxin component at its N-terminus or release from the cell and binding to glucan receptors. Some insertion and substitution mutations in γ severely reduce toxin secretion without affecting immunity. They are presumed to affect protoxin folding in the endoplasmic reticulum and translocation to the Golgi.     

A nuclear gene required for the expression of the linear DNA-asociated killer system in the yeast Kluyveromyces lactis

The killer system of Kluveromyces lactis is associated with two linear DNA plasmids, pGKL1 and pGKL2. The killer toxin and the immunity determinant are coded for by pGKL1. Mutations which we have named KEX1. The KEX1 gene of K. lactis has been cloned by complementation of kex1 mutations by using a recombinant plasmid pool containing the entire Kluyveromyces lactis genome, on a multicopy plasmid KEp6, which contains the Saccharomyces cerevisiae URA3 gene as a marker. Genetic analyses of strains carrying a distrupted kex1 allele demonstrated that the cloned DNA corresponded to the KEX1 gene. The cloned KEX1 gene of K. lactis has low but significant sequence homology with the KEX2 gene of Saccharomyces cerevisiae. In vivo complementation of the kex1 mutations of K. lactis by the KEX2 gene of S. cerevisiae, and complementation of the kex2 mutations of S. Cerevisiae by the KEX1 gene of K. lactis, demonstrated that KEX1 of K. Lactis is functionally related to the KEX2 gene of S. cerevisiae. K. lactis diploids homozygous for kex1 are deficient for sporulation.     

In vitro fermentation kinetics of carbohydrate fractions of fresh forage,silage and hay of Avena sativa

The objective of this experiment was to evaluate the influence of preservation methods on the fermentation kinetics of carbohydrate fractions of fresh forage, hay and silage of oats, which have been harvested at the milky stage of grain ripening. Samples of unfractionated forage (WF), residue insoluble in 90% ethanol (EIR) and isolated neutral detergent fibre (NDF) were fermented in vitro and the gas production was monitored. To obtain the gas production and fermentation kinetics of the ethanol‐soluble fraction (A fraction) the gas produced from the EIR fermentation was subtracted from the WF gas at each time point. The same approach was used to obtain the gas production and fermentation kinetics of the fraction insoluble in 90% ethanol but soluble in neutral detergent solution (B₁ fraction), by subtracting the isolated NDF gas curve from the corresponding EIR curve. The fractional maximum rate of gas production (R_M) was lower for both preserved forages (p < 0.01 for silage; p < 0.05 for hay) than for fresh forage. Ensiling did not change the size of the A fraction but reduced by 40% its R_M (p < 0.01) compared with fresh forage. The potential gas production from the B₁ fraction and its R_M were reduced by 19% (p < 0.01) and 44% (p < 0.05), respectively. R_M of both A and B₁ fractions was the same for hay and fresh forage. The curve subtraction technique may be used to obtain an estimation of the rate for neutral detergent‐soluble fractions and to determine changes due to ensiling and haymaking on the rate of gas produced. Copyright © 2005 Society of Chemical Industry     

Inactivation and degradation of O Taiwan97 foot-and-mouth disease virus in pork sausage processing

This study was to investigate the levels of inactivation and degradation of a field-isolated foot-and-mouth disease virus (FMDV)-strain O Taiwan97 in pork sausage processing. FMDV antibody-free pigs were inoculated with virus suspensions and slaughtered after developing observable symptoms. Lymph nodes (LN), blood clots (BC), and muscle were collected for the evaluation of inactivation during chilling, curing, drying and steaming processes. Viable virus was assessed by the TCID₅₀ technique and RT-PCR was used to detect viral genomes. Cell culture results revealed that viruses could be detected from LN and BC, but not from muscle. Ninety percent of FMDV was inactivated after each of the chilling, curing and steaming processes as compared to <80% inactivation after the drying process. The FMDV genome was detectable in 88% of the muscle, and was found in 100% of the serum, BC and LN. After processing, a majority of the genomes were not degraded more than 60%, with the exception of the thermal-steaming process. These findings indicated that the FMDV genome was detectable after each processing step, indicating a potential infectious risk. Thus, apart from regular surveillance in the field, monitoring of meat product pH and heat treatments are essential for FMDV inactivation.     

The effects of α-amylase inhibitors on insect storage pests: Inhibition of α-amylase in vitro and effects on development in vivo

Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of M_r 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the M_r 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of M_r 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.     

Comparison between Size Exclusion Chromatography and Micro‐Batch Analyses of Corn Starches in DMSO using Light Scattering Detector

The molecular structure of corn starches different in amylose content (waxy, normal, and high‐amylose) was analyzed in 90% dimethyl sulfoxide (DMSO) solution by refractive index (RI) and multi‐angle laser light scattering (MALLS) detectors. The starch sample solutions were measured either by medium‐pressure size exclusion chromatography (MPSEC) or by the micro‐batch mode. For waxy corn starch, the average molar mass (M_w) and radius of gyration (R_g) values were similar in both methods. However, for normal and high‐amylose corn starches, M_w measured by the micro‐batch mode was 2–4 times greater than that by the chromatographic method, although R_g values obtained from both methods were not very different. The M_w difference was the greater the higher the amylose content of starch.     

A novel global hydrodynamic analysis of the molecular flexibility of the dietary fibre polysaccharide konjac glucomannan

Konjac glucomannans have been widely considered in health food products although their hydrodynamic properties have been poorly understood. The weight-average molecular weight (M_w); sedimentation coefficient (s⁰_20,w) and intrinsic viscosities ([η]) have been estimated for five different preparations. The decrease in both intrinsic viscosity and sedimentation coefficient with molecular weight enables the estimation of molecular flexibility in terms of persistence length (L_p) using the traditional Bohdanecky–Bushin and Yamakawa–Fujii analyses for intrinsic viscosity and sedimentation data respectively. However, this requires an assumption of the mass per unit length M_L. Advantage can now be taken of a recent development in data interpretation which allows the estimation of L_p from combined intrinsic viscosity and sedimentation coefficient data and also an estimate for M_L. Using this “global” procedure an estimate of (13 ± 1) nm is found for L_p and a value of (330 ± 10) g mol⁻¹ nm⁻¹ for M_L.The value for L_p suggests a molecule of considerable flexibility, comparable to galactomannans (L_p  8–10 nm) but not as flexible as pullulan (L_p  1–2 nm).     

Storage stability of the natural colourant from Justicia spicigera microencapsulated in protective colloids blends by spray‐drying

Muitle (Justicia spicigera), a Mexican native plant, produces a purple aqueous extract (MAE) because of its anthocyanin content. The aim of this work was to microencapsulate MAE by spray‐drying in two different protective colloids blends in a 1:1 weight ratio: gum Arabic‐mesquite gum (GA50‐MD50) and mesquite gum‐maltodextrin DE10 (MG50‐MD50), yielding the microcapsules M_GA50‐MD50 and M_MG50‐MD50. The minimum integral entropy of the microcapsules was determined at 20, 35 and 40 °C, and the resulting water activities (a_W) were 0.555, 0.592, 0.627 for M_GA50‐MD50 and 0.581, 0.587, 0.648 for M_MG50‐MD50, respectively. These a_W temperature sets were considered as the most adequate conditions for achieving maximum storage stability of the microcapsules. Total anthocyanin content (TAC) and total colour change (ΔE) suffered considerable degradation at all storage conditions, but that degradation was significantly inhibited by encapsulating MAE in the biopolymers blends especially that made up by MG50‐MD50.     

FERMENTATION PROPERTIES OF BREWING YEAST WITH KILLER CHARACTER

The cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae has been transferred to three different commercially-used brewing yeasts; two ale strains and one lager strain. The ease with which the character can be transferred is very strain dependent. In addition to killer character, mitochondria from the brewing strain have been transferred into the new ‘killer’ brewing strains. Fermentations carried out with the manipulated strains produced beers which were very similar to those produced by the control brewing strains. The beers produced by killer brewing strains containing brewing yeast mitochondria were most like the control beers and could not be distinguished from them in three glass taste tests. In addition to producing good beers the genetically manipulated yeasts killed a range of contaminant yeasts and were themselves immune to the action of Kil-k₁ killer yeasts.     

Investigation of molecular weight distribution of LBG galactomannan for flours prepared from individual seeds, mixtures, and commercial samples

The molecular weight distribution has been determined for the galactomannan solubilized from three types of locust bean gum (LBG) flours: single carob seeds, mixtures, and a range of commercial products. To prepare crude endosperm flours from carob seeds with minimal galactomannan degradation, a new extraction and milling method was developed. The method consists of applying a brief thermal shock to the seeds, followed by an extended 3-day swelling period, and manual separation of endosperms; particle size reduction to a flour is accomplished on hydrated endosperms using a centrifugal mill. This method was optimized so that redissolved LBG flours produced solutions with the highest possible viscosity and the least amount of galactomannan degradation as determined by SEC. For the three samples types, the molecular weight distribution, w(M), was found to be unimodal, appearing as a sharply defined main peak (M_p≈1.1×10⁶ g/mol) with a small high molecular weight tail (up to 2.0×10⁶ g/mol) and broad low molecular weight tail (down to 0.01×10⁶ g/mol); polydispersities (M_w/M_n) were estimated to be 1.5–1.8. Variations in M_w and [η] for galactomannans extracted from individual seeds originating from the same carob tree were minimal and nearly indistinguishable from a bulk mixture (6 seeds, M_w=0.96–1.1×10⁶ g/mol, [η]=14.2–15.1 dl/g). There was a higher variability in these molecular parameters for galactomannans solubilized from commercial LBG flours, which generally exhibited lower M_w and [η], broader distributions, and reduced solubilities (M_w=0.86–1.0×10⁶ g/mol, [η]=12.4–13.6 dl/g). These side-effects were attributed to damage caused by industrial scale seed processing. The near constancy of M_p for the three sample types suggests that the average molecular size of LBG galactomannan varies only slightly due to natural or biological causes.     

Thermal and rheological properties of the supersaturated sucrose solution in the presence of different molecular weight fractions and concentrations of dextran

In our current research work, we investigated the effects of molecular weight (M _w) and the concentration of dextran presence during cane sugar manufacturing on the rheological and glass transition properties of supersaturated sucrose solution. Three dextrans of various M _w, namely 100,000 g/mol (T ₁₀₀), 500,000 g/mol (T ₅₀₀) and 2,000,000 g/mol (T ₂₀₀₀), were admixed in concentrations between 1,000 and 10,000 ppm with 65 and 75% w/w sucrose solution. The results indicated that both the apparent viscosity and dynamic modulus increased with an increase in dextran concentrations and they demonstrated strong dependence on its M _w. Glass transition temperature (T _g) of the samples was measured by differential scanning calorimetry, and their dependence on dextran M _w and concentration was analyzed by the Fox and expanded Gordon–Taylor mathematical models. It was found that the higher the M _w and concentration of the dextran, the greater the increase in T _g. The expanded Gordon–Taylor equation has proved useful in predicting the T _g of the sucrose solution in the presence of polymer.     

Primary structure of 2S albumin from seeds of Lupinus cosentinii

 The 2S albumin from seeds of Lupinus cosentinii Guss. was purified, and the complete amino acid sequences of the dominating small and large subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin consists of 35 amino acid residues resulting in a molecular mass (M _r) of 4233. The large subunit contains 73 amino acid residues (M _r = 8627). The two polypeptide chains are linked by two interchain disulphide bonds. In addition, the large polypeptide contains two intrachain disulphide bridges and one free sulphydryl group. A high degree of homology (88–89%) exists between the primary structure of the 2S albumin from L. cosentinii and those from other Lupinus species. The positions of the cysteines and of some other amino acids are conserved not only in most of the Dicotyledoneae 2S albumins sequenced so far but also in other storage proteins. Received: 26 May 1997     

Primary structure of 2S albumin from seeds of Lupinus cosentinii

 The 2S albumin from seeds of Lupinus cosentinii Guss. was purified, and the complete amino acid sequences of the dominating small and large subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin consists of 35 amino acid residues resulting in a molecular mass (M _r) of 4233. The large subunit contains 73 amino acid residues (M _r = 8627). The two polypeptide chains are linked by two interchain disulphide bonds. In addition, the large polypeptide contains two intrachain disulphide bridges and one free sulphydryl group. A high degree of homology (88–89%) exists between the primary structure of the 2S albumin from L. cosentinii and those from other Lupinus species. The positions of the cysteines and of some other amino acids are conserved not only in most of the Dicotyledoneae 2S albumins sequenced so far but also in other storage proteins. Received: 26 May 1997     

Size and charge properties of the pectic and cellulolytic enzymes in a commercial enzyme preparation

Summary The size and charge properties of the pectic and cellulolytic enzymes in a commercial preparation were studied using with isoelectric focusing, nondenaturating polyacrylamide gradient gel electrophoresis, titration curve analysis and a two-dimensional technique. Enzyme activity was detected with the zymogram technique. Five major isoenzymes of endo-polygalacturonase were detected having isoelectric points (pI) of 3.2, 3.6, 3.7, 4.4, and 4.5. These separate into two major size species after PAGGE (M _r 28000 and 48000). — Endo-pectinlyase was detected at pI 3.6/M _r 38000 after two-dimensional electrophoresis. — Pectin esterase was found to have a pI of 3.5 and aM _r of 27000. — Endo-Cx-cellulases were found at pI 3.1–3.2/M _r 38000; pI 3.5/M _r 27000; pI 3.6–3.8/M _r 80000; pI 5.6/M _r 70000.

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Molekülgröße und Ladungseigenschaften von pectolytischen und cellulolytischen Enzymen in einem Handelsenzympräparat

Zusammenfassung Die Molekülgröße und Ladungseigenschaften von pectolytischen und cellulolytischen Enzymen in einem Handelspräparat wurden mittels isoelektrischer Focussierung (IEF), nichtdenaturierender Polyacrylamid-Gradienten-Gelelektrophorese (PAGGE), Titrationskurvenanalysen und zweidimensionaler Technik untersucht. Die Enzymaktivität wurde mit der Zymogramm-Technik bestimmt. Fünf Haupt-Isoenzyme der endo-Polygalakturonase (PG) wurden aufgrund ihrer isoelektrischen Punkte (pI) gefunden (PI 3,2; 3,6; 3,7; 4,4 und 4,5). Diese werden durch die PAGGE in zwei Hauptbanden mit den MolekülgrößenM _r 28000 und 48000 aufgetrennt. — Endo-Pectinlyase (PL) wurde bei pI 3,6/M _r 38 000 nach 2-D-Elektrophorese gefunden. — Für Pectinesterase (PE) wurde ein pI von 3,5 und ein Molekulargewicht von 27 000 bestimmt. — Endo-Cx-Cellulasen wurden bei pI 3,1–3,2/M _r 38 000; pI/M _r 27 000; pI 3,6–3,8/M _r 80 000; pI 5,6/70000 gefunden.

     

Diffusion of homologous model migrants in rubbery polystyrene: molar mass dependence and activation energy of diffusion

Published diffusion prediction models for the diffusion of additives in food packaging simplify reality by having a small number of parameters only. Therefore, extrapolation of such models to barrier polymers, larger ranges of temperature and/or additive molecular weight (M_W ) is questionable. Extra data is still required to generalize these existing prediction models. In this paper, diffusion of a specifically designed homologous set of model additives (from 236 to 1120 g mol^?1) was monitored in two polystyrenes in the rubbery state (from 100 to 180°C): syndiotactic semi-crystalline polystyrene and its amorphous equivalent. Variations in associated diffusion coefficient D and activation energy Ea with migrant M_W and temperature were surprisingly low. Comparison of experimental behaviour with model predictions was performed. In their actual form, none of the models is capable of describing all experimental data, but there is evidence of convergence of the different approaches.     

Enzymatic synthesis of ethyl hexanoate by transesterification

Summary Enzymatic synthesis of ethyl hexanoate by means of an acyl transfer reaction has been studied by using an immobilized Rhizomucor miehei lipase (RML). The effect of reaction parameters on ester synthesis has been investigated. Rhizomucor miehei lipase showed more specificity than other lipases when ethyl hexanoate was synthesized in n‐hexane. Maximum ester synthesis was obtained by using a 0.5 m substrate concentration (equimolar ratio). Temperatures in the range of 45–55 °C were found to be optimum and at higher temperatures (>60 °C) deactivation of enzyme was observed. Higher molar concentrations of hexanoic acid inhibited RML, but no inhibitory effect of ethyl caprate, even at higher molar concentrations, was observed. Apparent kinetic parameters have been determined. The values are as follows: K_M (ester), 0.0135 m ; K_M (acid), 0.08466; K_i (ester), 3.07 m ; K_i (acid), 0.550 m ; V_max, 1.861 µmol min^?1 mg^?1 enzyme.     

Characterization of Physical Properties of Flour and Starch Obtained from Gamma‐Irradiated White Rice

Physical and structural characteristics of rice flour and starch obtained from gamma‐irradiated white rice were determined. Pasting viscosities of the rice flour and starch, analyzed by using a Rapid Visco Analyser, decreased continuously with the increase in irradiation dosage. Differential scanning calorimetry showed that gelatinization onset, peak and conclusion temperatures of rice flour and starch changed slightly but the enthalpy change decreased significantly with increase of irradiation dosage. All irradiated starch displayed an A‐type X‐ray diffraction pattern like the native starch. Gel permeation chromatography showed that the blue value ratio of the first peak (amylopectin) to the second one (amylose) decreased with the increase of the irradiation dosage. The weight‐average molecular weight (M_w) and gyration radius (R_z) of amylopectin analyzed by using HPSEC‐MALLS‐RI (high‐performance size‐exclusion chromatography equipped with multiangle laser‐light scattering and refractive index detector) decreased gradually from 1.48×10⁹ (M_w) and 384.1 nm (R_z) of native rice starch to 2.36×10⁸ (M_w) and 236.8 nm of 9 kGy‐irradiated starch. The branch chain‐length distribution of amylopectins determined by HPAEC‐ENZ‐PAD (high‐performance anion‐exchange chromatography with amyloglucosidase post‐column on‐line reactor and pulsed amperometric detector) showed that gamma irradiation had no significant effect on the amylopectin branch chains with 13≤DP≤24 and 37≤DP, but produced more branch chains with 6≤DP≤12 when the irradiation dosage was less than 9 kGy. It might be deduced that gamma irradiation caused the breakage of the amylopectin chains at the amorphous regions, but had little effects on the crystalline regions of starch granules, especially at low dosage irradiation.     

Water Sorption of Amaranthus cruentus L. Seeds Modelled by GAB Equation

Abstract

The GAB (Guggenheim, Andersen, and de Boer) equation was adjusted to literature data of sorption of Amaranthus cruentus L. (M _e vs. a _w for adsorption and desorption) determined at 25, 30, 35, 40, 45, 50, 55, 65, 70, and 90°C, in the range of water activity from 0.029 to 0.979. To quantify the goodness of fit, the correlation coefficient (R ²), the sum of squares (RSS), the standard error of the estimate (S _y ), the mean relative deviation (MRD) and the plots of residuals were analysed. The three theoretical parameters of the GAB model (M _o , C, and K) gave a good correlation (R ² > 0.9817, RSS < 0.0297, MRD < 0.138, S _y  < 0.0143, and random residuals‐plots) in the range of a _w from 0.029 to 0.979, of interest in seed storage and processing. However this correlation does not consider the effect of temperature (T) on coefficient values. In a second stage, parameters M _o and K were adjusted at each temperature. Very low variances were obtained in the range 25–65°C for desorption and in the range 25–55°C for adsorption. These results suggested that M _o and K remain almost constant and a correlation with T is not justified. On the contrary sense, parameter C showed stronger variation with T. This was explained by the analysis of sensitivity for the influence of C on moisture content. On this basis, the relation C–T was proposed by an Arrhenius‐type relation [C = A.exp(B/T)] and this function was incorporated to the original GAB model to re‐estimate the parameters A, B, M _o , and K. The developed modification provides a generalised and precise expression of GAB model for Amaranth.