Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use

An easily scaled-up technique has been designed to purify -mannanase from Bacillus licheniformis. Using flocculation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recovery of 47% and a specific activity of 4341 U mg^–1protein. The enzyme had maximum activity at 60 °C and pH 7.0. It was stable at 50 °C and pH 6.0 for 6 h, but lost all of its activity when held at 70 °C and pH 6.0 for 1 h.     

Short communication: Thermostability of α-amylase produced by Bacillus sp. E2—a thermophilic mutant

An -amylase from a hyper-producing strain of Bacillus (sp. E2) was stable at 70°C for 30 min but was quickly inactivated at higher temperatures. In the presence of 10mm Ca²⁺ and starch (20% w/v), however, the enzyme was stable at 90°C for 10 min and after 30 min at 100°C still retained 26% of its initial activity.     

Dependence of temperature on loss rates of rotifers,lipids, and ω3 fatty acids in starved Brachionus plicatilis cultures

Rotifer cultures of Brachionus plicatilis (SINTEF-strain, length 250 m) rich in 3 fatty acids were starved for > 5 days at variable temperature (0–18 °C). The net specific loss rate of rotifer numbers were 0.04 day^–1 (range 0–0.08 day^–1) at 5–18 °C, but reached values up to 0.25 day^–1 at 0–3 °C. The loss rate was independent on culture density (range 40–1000 ind ml^–1), but was to some extent dependent on the initial physiological state of the rotifers (i.e., egg ratio).The loss rate of lipids was 0.02–0.05 day^–1 below 10 °C, where the potential growth rate of the rotifer is low (0–0.09 day^–1). The loss rate of lipids increased rapidly for higher temperatures where the rotifer can maintain positive growth, and reached 0.19 day^–1 at 18 °C. The Q₁₀ for the lipid loss rate versus temperature was higher than the Q₁₀ for respiration found in other strains. This may suggest that other processes than respiration were involved in lipid catabolism. The content of 3 fatty acids became reduced somewhat faster than the lipids (i.e. in particular 22:6 3), but the fatty acid per cent distribution remained remarkably unaffected by the temperature during starvation.The results showed that rotifer cultures could be starved for up to 4 days at 5–8 °C without essential quantitative losses of lipids, 3 fatty acids, and rotifers. The rotifers exhausted their endogenous lipids through reproduction (anabolism) and respiration (including enhanced locomotion) at higher temperatures. At lower temperatures, the mortality rate became very high.     

Temperature dependency of induction of sexual agglutinability by α pheromone in the yeast Saccharomyces cerevisiae

When pheromone-pretreated cells of an inducible a strain of Saccharomyces cerevisiae carrying the inducible gene saa1 were incubated in a growth medium at 28°C, induction of sexual agglutinability began after a 10 min lag period. If the cells were incubated at 38°C during the lag period, no induction occurred even after incubation at 28°C. Contrary to this, if the cells were incubated at 28°C during the lag period, almost complete induction occurred, even after transfer to 38°C. Temperature shift experiments revealed that 5 min incubation at 28°C was necessary for the initiation of the temperature-sensitive period and further 5 min incubation for the completion of the period. The temperature-sensitive period was sensitive to phenylmethylsulfonyl fluoride.Non-common abbreviations PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethylsulfonyl fluoride     

Operational and storage stability of neutral β-mannanase from Bacillus licheniformis

The stability of neutral -mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l^–1 over 30 min at 25 °C. In an enzymatic membrane reactor operated at 50 °C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4 °C after one year.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

β-Glucan synthetase activity in Golgi vesicles ofPetunia hybrida

-Glucan synthetase activity has been demonstrated in a Golgi vesicle fraction isolated from pollen tubes ofPetunia hybrida. This-glucan synthetase activity differs from that of most other higher plants in its inability to incorporate [¹⁴C]glucose from GDP-[¹⁴C]glucose. UDP-[¹⁴C]glucose, however, is an appropriate glucose donor for this enzyme. The optimum conditions for this-glucan synthetase activity are: 1 mg Golgi vesicle protein/ml reaction mixture; pH=±8 and a temperature of 25°C. The newly synthesized alkali-insoluble glucan contains-1,3- as well as -1,4-glucosidic linkages.     

“In vitro” Antifungal activity of protease inhibitors

In the last five years, as HAART has become standard therapy in HIV seropositive or AIDS patients, changes have been noted in the numbers and types of opportunistic fungal infections in these cohorts of patients. Particularly, oropharyngeal candidiasis have become rare in HIV infected patients since the introduction of new anti-HIV drugs of the protease inhibitors type. At the Immunology Institute of the Universidad Central de Venezuela the most frequent protease inhibitors (PIs) used for the treatment of these patients have been: Nelfinavir (Viracept^TM, Roche),Indinavir (Crixivan^® Merck),Ritonavir (Norvir^®, Abbott),Saquinavir (Fortovase^®, Roche).Recently, we observed that recurrent candidiasis was less frequent and no Candidacould be isolated in our patients. A direct relation to the PIs was suspected. In order to assess the in vitro antifungal activity of the afore mentioned protease inhibitors on Candida sp., we used both the well diffusion test and the NCCLS broth microdilution test to assay 100 Candida sp. isolates from HIV seropositive or AIDS patients with syntomatic oropharyngeal Candida infection. In general, the data obtained with the well diffusion test were in agreement with those obtained by the broth microdilution test. All 100 isolates were susceptible to Saquinavir and 32 were susceptible to Indinavir using the NCCLS microdilution test,while 97 were susceptible to Saquinavir and 52 to Indinavir by the well diffusion test. From 17 C. albicans resistant to fluconazole, all were susceptible to Saquinavir by the NCCLS micro method and 16 by the well diffusion test. Our results showed anticandidal activity in vitro of PIs, mainly Saquinavir.This revised version was published online in October 2005 with corrections to the Cover Date.     

Cellodextrin utilization and β-glucosidase production by Bacteroides polypragmatus

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a -glucosidase with a pI value of 4.2 and a molecular weight of approximately 100000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The K _m values obtained with p-nitrophenyl--d-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55°C. It was also stable during long-term storage at either 4°C or-20°C, provided that 10% (v/v) glycerol was added to preparations maintained at-20°C.Abbreviations HPLC high-performance liquid chromatography - IEF isoelectric focusing - pNPG p-nitrophenyl--d-glucoside NRCC No. 25676     

Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts

An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg^–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg²⁺ up to 168% of the original activity, while Zn²⁺, Mn²⁺ and Cu²⁺ ions, chelating agent (EDTA) and NaN₃ (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C.     

Effect of temperature on some characteristics of the thermostable α-amylase from Bacillus licheniformis

The V _max of an extracellular, thermostable -amylase from Bacillus licheniformis 44MB82 were 5.70×10^-3 and 9.70×10^-3 mM s^-1 at 30 and 90°C, respectively, whereas the K _m values were similar (0.9 mg ml^-1) at both temperatures. Excluding dextrins, the dominant products from soluble starch and amylopectin hydrolysis contained less than six glucose residues. The enzyme hydrolysed amylopectin better than soluble starch. Increasing the temperature from 30 to 90°C was accompanied by an increase in the production of malto-oligosaccharides, especially maltotetrose, and this was related to the secondary hydrolysis of maltopentose and maltohexose.The authors are with the Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113. 26 Academician G. Bonchev, Bulgaria     

The responsiveness of Avena fatua aleurone protoplasts to gibberellic acid

The sensitivity to gibberellic acid (GA₃) of aleurone protoplasts isolated from a single harvest of an inbred line of Avena fatua seed that had been after-ripened over anhydrous CaCl₂ at 25±2°C and 4±2°C for three years was assessed. Protoplasts isolated from aleurones of seed stored at 25°C produced substantially more -amylase in response to 10^–7 M GA₃ than those isolated from aleurones of seed stored at 4°C. The apparent difference in responsiveness does not appear to be due to a change in the duration of the lag phase between addition of GA₃ and the production of -amylase. The dose response of aleurone protoplasts to GA₃, measured as -amylase production, is complex and appears to have three phases. Protoplasts from seed stored at both temperatures respond appreciably to 10^–14 M GA₃. With increasing concentrations of GA₃, up to 10^–9 M, -amylase production increases similarly in protoplasts from both lots of seed, reaching a level approximately 2.7–3.8 times greater than when no GA₃ is applied. GA₃-induced -amylase production increases markedly as the concentration is raised from 10^–9 M to 10^–6 M, and the response then appears to be saturated. Over this part of the response curve protoplasts from the two seed lots differ markedly in their responsiveness to GA₃. Those from seed stored at 25°C produce considerably more -amylase, >130-fold higher than the minus GA₃ control, than those from seed stored at 4°C, <35-fold higher than the minus GA₃ control. This apparent difference in the responsiveness of aleurone protoplasts to GA₃ could be correlated with the loss of embryo dormancy in seed stored at 25°C. Seed stored at 4°C retained the dormancy characteristics present immediately after harvesting.     

Effect of<Emphasis Type="Italic"> p</Emphasis>CO<Subscript>2</Subscript> and temperature on the boron isotopic composition of the zooxanthellate coral<Emphasis Type="Italic"> Acropora</Emphasis> sp.

Culture experiments were carried out with Acropora sp. (a branching scleractinian coral) in seawater at two pCO₂ conditions (438 and 725 µatm) and two temperatures (25 and 28 °C) in order to establish the pH and temperature dependence of the boron isotopic composition of the skeleton. A clear pCO₂ effect, but no temperature effect, on the coral boron isotope composition is seen. For corals cultured at normal pCO₂ (438 µatm), the ¹¹B of the skeleton was 24.0±0.2 at 25 °C, and 23.9±0.3 at 28 °C. The values of ¹¹B measured for corals cultured at higher pCO₂ (725 µatm) were lower: 22.5±0.1, and 22.8±0.1 at 25 and 28 °C, respectively. The ¹¹B of corals cultivated at both high and normal pCO₂ conditions are consistent with a dominant pH control, and are very close to that calculated from theoretical considerations. Thus, the corals do not seem to significantly alter ambient seawater for calcification with respect to pH. Co-variation between boron and carbon isotope values is explored.Communicated by: Guest Editor A. Grottoli     

Influence de la température sur la cinétique,de croissance et le coefficient de maintenance de Candida lipolytica cultivé sur n-alcane

The effect of growth temperature on the evolution of kinetic parameters and yields was determined for Candida lipolytica cultures with ntetradecane as substrate, in a temperature range of 18°C to 30°C, which is below the critical growth temperature in order to work only in the activation zone of these parameters.In such a culture limited by substrate transfer, growth rate depends on biological rates, related to microorganisms characteristics, and diffusional rates, related to mass transfer. The effect of temperature thus depends on the limiting step. The activation energy, calculated from exponential growth rate determinations is .When the activation energy is calculated from the maximal rate of cell production (determined at the growth curve's inflexion point), it's found to be E _X=71,200 J/mole in the 18°C–24°C range, and E _X=28,000 J/mole in the 24°C–30°C range. The latter one is characteristic of a diffusion-limited process. Above 24°C, growth is controlled by substrate-transfer, as physiological potentialities are preferentially increased with temperature than diffusional ones: 24°C is thus the transition temperature T _t from physiological to diffusional limitation.The apparent yield is almost constant, over the 18°C to 30°C temperature range, although maintenance coefficients are very dependent on temperature. The activation energies related to maintenance coefficients for alkane and oxygen respectively are and .The m _s/m_{O 2} ratio is about 3 (g/g), whereas that, for a strict oxidation reaction of n-tetradecane ought to be 3.47 (g/g). A satisfactory correlation, relating maintenance coefficients to the maximal growth rate of yeast, is given.

---

Liste des symboles A constante de saturation de modèle de croissance(1) - B vitesse spécifique considérée - C substrat carboné ou oxygène (g/l) - E energie d'activation (J/mole) - S _m quantité de substrat consommée par maintenance au cours d'une fermentation discontinue (g) - O₂ quantité d'oxygène transférée au milieu de culture (g/l) - R rendement global de la fermentation - R rendement global de la fermentation - constante des gaz parfaits (J/mole K) - S concentration en substrat carboné (g/l) - T température de croissance (°K) - X concentration en biomasse (g/l) - Y rendement limite - m coefficient de maintenance (h^-1) - t duree de fermentation (h) - tømpérature de croissance (^o Celsius) - taux de croissance (h^-1) Indices 1 relatif à la température 1. - 2 relatif à la température 2 - c relatif au substrat carboné ou à l'oxygène - f relatif au temps final - i relatif au point d'inflexion - m maximum - mO₂ relatif au coefficient de maintenance sur l'oxygène - m _s relatif au coefficient de maintenance sur le substrat carboné - o relatif au temps initial - O₂ relatif à l'oxygène - s relatif au substrat carboné - t de transition - T relatif à la température de croissance T - U _m relatif au taux de croissance maximal - X relatif à la productivité maximale en biomasse     

Influence of temperature on the properties of the xylanolytic enzymes of the thermotolerant fungus<Emphasis Type="Italic"> Aspergillus phoenicis</Emphasis>

This study reports on the effects of growth temperature on the secretion and some properties of the xylanase and -xylosidase activities produced by a thermotolerant Aspergillus phoenicis. Marked differences were observed when the organism was grown on xylan-supplemented medium at 25 °C or 42 °C. Production of xylanolytic enzymes reached maximum levels after 72 h of growth at 42 °C; and levels were three- to five-fold higher than at 25 °C. Secretion of xylanase and -xylosidase was also strongly stimulated at the higher temperature. The optimal temperature was 85 °C for extracellular and 90 °C for intracellular -xylosidase activity, independent of the growth temperature. The optimum temperature for extracellular xylanase increased from 50 °C to 55 °C when the fungus was cultivated at 42 °C. At the higher temperature, the xylanolytic enzymes produced by A. phoenicis showed increased thermostability, with changes in the profiles of pH optima. The chromatographic profiles were distinct when samples obtained from cultures grown at different temperatures were eluted from DEAE–cellulose and Biogel P-60 columns.     

Purification and biochemical properties of a thermostable xylose-tolerant β-<Emphasis Type="SmallCaps">D</Emphasis>-xylosidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic -D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level of -D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial -xylosidase induced by xylan was purified using a procedure that included heating at 50°C, ammonium sulfate fractioning (30–75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified -D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60°C and 5.0, respectively. -D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for 1 h at 60°C and has half-lives of 11 and 30 min at 65°C in the absence or presence of calcium, respectively. The purified -D-xylosidase hydrolyzed p-nitrophenol--D-xylopyranoside and p-nitrophenol--D-glucopyranoside, exhibiting apparent K_m and V_max values of 1.3 mM, 88 mol min^–1 protein^–1 and 0.5 mM, 20 mol min^–1 protein^–1, respectively. The purified enzyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true -D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most -xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum -xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.     

Alpha-N-acetylgalactosaminidase from marine bacterium Arenibacter latericius KMM 426T removing blood type specificity of A-erythrocytes

An -N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426^T. The -N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes. The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50°C and for a week at 20°C. The enzyme can be stored under the sterile conditions for any length of time at 4°C, but it does not withstand freezing. The -N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl--N-acetyl-D-galactosaminide, the K _m is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.     

Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Two intracellular -glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for -glucosidase A and -glucosidase B, respectively. -Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (-glucosidase A, 29.2%; -glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for -glucosidases A and B, respectively. Both purified -glucosidases. especially -glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both -glucosidases were highly activated by CuCl₂, and inhibited by SnCl₂ and KMnO₄. Hg²⁺ and Ag⁺ also inhibited severely -glucosidase B. The K _m and V _max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2mol min^–1 mg^–1 for -glucosidase A and 2.76 mM and 143.5 mol min^–1 mg^–1 for -glucosidase B.     

Effects of the plant alkaloid castanospermine as an antimetabolite of storage pests

The secondary plant compound castanospermine is toxic to the larvae of the bruchid beetle Callosobruchus maculatus F. and the flour beetle Tribolium confusum J. & V. when incorporated into the diet. The larval alimentary tract -D-glucosidase and -D-glucosidase activities of C. maculatus were strongly inhibited by castanospermine in a non-competitive and competitive manner respectively. The larval alimentary tract -D-glucosidase activity of T. confusum was strongly inhibited in a competitive manner, but the -D-glucosidase activity was not markedly inhibited; however, the -D-galactosidase activity exhibited strong uncompetitive inhibition.

---

Résumé La castanospermine, substance secondaire végétale, incorporée à l'aliment est toxique pour les larves de Callosobruchus maculatus F. et de Tribolium confusum J. & V. Les activités -D-glucosidase et -D-glucosidase du tube digestif de C. maculatus ont été fortement inhibées par la castanospermine respectivement de façon non-compétitive et compétitive. L'activité -D-glucosidase du tube digestif de T. confusum était, fortement inhibée d'une façon compétitive, mais l'activité -D-glucosidase n'était pas nettement inhibée; cependant, l'activité -D-galactodase ne présentait pas une forte inhibition compétitive.