PRODUCTION OF MICROBIAL ALKALINE CELLULASE AND STUDIES OF THEIR CHARACTERISTICS

In order to obtain cellulases that improve the detergency of laundry detergent products, two alkalophilic microorganims, Bacillus sp B38-2 and Streptomyces sp S36-2, were isolated from soil and compost by incubating samples in enrichment culture medium containing CMC and Na2CO3 at pH9.6. It was found that they secrete a constitutive extracellular alkaline carboxymethyl cellulase (CMCase) in high quantity. The maximum enzyme activity was observed between 48hr to 72 hr at 30-degrees-C for the Streptomyces and between 72hr to 96hr at 35-degrees-C for the Bacillus. The optimum pH and temperature of the crude enzyme activities ranged from 6.0 to 7.0 at 55-degrees-C for the Streptomyces and 7.0 to 8.0 at 60-degrees-C for the Bacillus. Two crude CMCases activities were termostable at 45-degrees-C for 1hr and the both crude enzyme activities of the Bacillus as of the Streptomyces were stable at pH 5.0 to 9.0 after pH treatments in various buffer solutions at 30-degrees-C for 24hr.

Electrochemical studies with copper-based alloys: open-circuit potential oscillations in alkaline media

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

THE PHO-2A MUTANT OF NEUROSPORA-CRASSA WHICH IS DEFICIENT IN PI-REPRESSIBLE ALKALINE-PHOSPHATASE (EC 3.1.3.1) IS ALSO DEFECTIVE IN PI-REPRESSIBLE ACID-PHOSPHATASE (EC 3.1.3.2)

1. The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively.2. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t1/2 = 4 min at 54-degrees-C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54-degrees-C, pH 5.4).3. The pho-2A mutant of N. crassa secreted a more thermolabile acid phosphatase (t1/2 = 30 min at 50-degrees-C, pH 5.4) than the wild strain (t1/2 of at least 80 min at 50-degrees-C, pH 5.4).4. The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t1/2 = 37 min at 54-degrees-C, pH 5.4) than the wild strain in high Pi medium (t1/2 = 14 min al 54-degrees-C, pH 5.4).5. The pleiotropic nature of the pho-2 locus and its possible involvement in the mechanism of phosphatase secretion by N. crassa are proposed.

Indirect polarographic and cathodic stripping voltammetric determination of cefaclor as an alkaline degradation product

Cefaclor is not reducible at a mercury electrode, but it can be determined polarographically and by cathodic stripping voltammetry as its initial alkaline degradation product which is obtained in high yield by hydrolysis of cefaclor in Britton-Robinson (B-R) buffer pH 10 at 50 degrees C for 30 min (reduction peak at pH 10, -0.70 V). Differential pulse polarographic calibration graphs are linear up to at least 1 x 10(-4) mol l(-1). Recoveries of 93% of the cefaclor (n = 3) were obtained from urine spiked with 38.6 mu g ml(-1) using this polarographic method with 1 ml urine made up to 10 ml with pH 10 buffer. Using cathodic stripping voltammetry and accumulating at a hanging mercury drop electrode at -0.2 V for 30 s, linear calibration graphs were obtained from 0.35 to 40 mu g ml(-1) cefaclor in B-R buffer pH 10. A relative standard deviation of 4.2% (eta = 5) was obtained, and the limit of detection was calculated to be 2.9 ng ml(-1). Direct determination of cefaclor in human urine (1 ml of urine was made up to 10 ml with pH 10 buffer) spiked to 0.39 mu g ml(-1) was made (recovery 98.6%). (C) 1999 Elsevier B.V. B.V. All rights reserved.

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Effect of calcium ions on rat osseous plate alkaline phosphatase activity

Rat osseous plate alkaline phosphatase is a metalloenzyme with two binding sites for Zn2+ (sites I and III) and one for Mg2+ (site II). This enzyme is stimulated synergistically by Zn2+ and Mg2+ (Ciancaglini et al., 1992) and also by Mn2+ (Leone et al., 1995) and Co2+ (Ciancaglini et al., 1995). This study was aimed to investigate the modulation of enzyme activity by Ca2+. In the absence of Zn2+ and Mg2+, Ca2+ had no effects on the activity of Chelex-treated, Polidocanol-solubilized enzyme. However, in the presence of 10 mu M MgCl2, increasing concentration of Ca2+ were inhibitory, suggesting the displacement of Mg2+ from the magnesium-reconstituted enzyme. For calcium-reconstituted enzyme, Zn2+ concentrations Zip to 0.1 mu M were stimulatory, increasing specific activity from 130 U/mg to about 240 U/mg with a K-0.5 = 8.5 nM. Above 0.1 mu M Zn2+ exerted a strong inhibitory effect and concentrations of Ca2+ up to I mM were not enough to counteract this inhibition, indicating that Ca2+ was easily displaced by Zn2+. At fixed concentrations of Ca2+, increasing concentrations of Mg2+ increased the enzyme specific activity from 472 U/mg to about 547 U/mg, but K-0.5 values were significantly affected (from 4.4 mu M to 38.0 mu M). The synergistic effects observed for the activity of Ca2+ plus magnesium-reconstituted enzyme, suggested that these two ions bind to the different sites. A model to explain the effect of Ca2+ on the activity of the enzyme is presented. (C) 1997 Elsevier B.V.

OSSEOUS PLATE ALKALINE-PHOSPHATASE IS ANCHORED BY GPI

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The Mr of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mu mol min(-1) mg(-1)), ATP (42.0 mu mol min(-1) mg(-1)) and pyrophosphate (28.4 mu mol min(-1) mg(-1)). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited ''Michaelian'' kinetics with K-0.5 = 70 and 979 mu M, respectively. For pyrophosphate, K-0.5 was 128 mu M and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K-d = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (K-d = 6.2 mu M) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K-0.5 = 10.1 mu M), magnesium (K-0.5 = 29.5 mu M) and manganese ions (K-0.5 = 5 mu M) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K-0.5 = 653 mu M) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.

Production of extracellular alkaline proteases by Aspergillus clavatus

The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37degreesC. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25degreesC for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH4NO3 showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40degreesC and the half-lives at 40, 45 and 50degreesC were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.

KINETIC-PROPERTIES OF OSSEOUS PLATE ALKALINE-PHOSPHATASE FROM DIABETIC RATS

1. Increased levels of bone alkaline phosphatase activity were observed in diabetic rats. These animals exhibited impaired bone development without concomitant alterations of the sequence of cellular transformations.2. Alkaline phosphatase activity was delayed in diabetic rats but the kinetic parameters for the hydrolysis of p-Nitrophenylphosphate (PNPP) were virtually the same observed for controls (N = 1.2 and K0.5 = 43 muM).3. Alkaline phosphatase from diabetic rats had a better affinity (K0.5 = 38 muM) for magnesium ions than controls (K0.5 = 9 1 muM).4. Zinc ions affected alkaline phosphatase activity from control and diabetic rats in the same way (K0.5 = 10 muM).

SURFACE EFFECTS IN THE HYDROGEN EVOLUTION REACTION ON NI-ZN ALLOY ELECTRODES IN ALKALINE-SOLUTIONS

Hydrogen evolution reaction was studied on Ni-Zn (25% of Ni before leaching) in 1 M NaOH at 25 degrees C. These electrodes were characterized by very low Tafel slopes of 67 mV dec(-1). Other techniques used included potential and current pulse, potential relaxation in an open circuit, and ac impedance spectroscopy. Analysis of the experimental results led to the conclusion that hydrogen adsorption in the surface layers was responsible for the observed behavior. Influence of the oxidation of the electrode surface and the addition of poisons, thiourea and cyanides, were also studied. These processes inhibit the hydrogen absorption and restore ''normal'' Tafel slopes. Kinetic parameters of the hydrogen evolution reaction were determined.

Electrochemical decomposition of cyanides on tin dioxide electrodes in alkaline media

The electrochemical oxidation of cyanide in alkaline media was studied at different pH levels on SnO2 doped with Sb supported on titanium, at 25 degrees C, the electrooxidation of CN- at constant current follows a first-order rate law with a half life of t(1/2) = 35 min on SnO2-SbOx electrodes and t(1/2) = 69 min on SnO2-SbOx-RuO2 electrodes, in K2SO4(aq), pH 12, the reaction rate increases with the applied current and tends to reach a plateau when j > 20 mA cm(-2), In the pH range 10-13.5 the reaction rate diminishes as pH is increased owing to an increasing competition between CN- and OH- ions for the electrode surface. Addition of chloride to the solution does not alter the rate law but increases the reaction rate, A mechanism is proposed to explain the observed behaviour.

Study of the alkaline earth metals with 8-hydroxyquinolinate derivatives - Calcium complexes

Metal complexes of calcium with 5,7-dibromo, 7-iodo and 5-chloro-7-iodo-8-hydroxyquinolate were precipitated in aqueous ammonia and acetone medium, except for the solid state compound with 5,7-dichloro-8-hydroxyquinoline which hasn't been obtained under these conditions. The complexes obtained through the mentioned precipitation are Ca[(C9H4ONBr2)(2)](3).H2O, Ca[(C9H5ONI)(2)].2H(2)O and Ca[(C9H4ONICl)(2)].2.5H(2)O. Their intermediate from the thermal decomposition found through TG/DTA curves in air indicated the presence of different kinds of calcium carbonates related to the reversibility and crystalline structure, depending on the original compounds. The initial compounds and the intermediate from the thermal decomposition were also characterized through IR spectra and X-ray diffraction.

Electrochemical behavior of cobalt oxide coatings on cold-rolled steel in alkaline sodium sulfate

The electrochemical behavior of a coating of cobalt oxide on cold-rolled steel in alkaline sodium sulfate was Studied using the electrochemical techniques of open-circuit potential measurements and electrochemical impedance spectroscopy. The coating was prepared at different annealing temperatures ranging from 350 to 750 degreesC and characterized by SEM, EDX and XRD. Below 550 degreesC the composition of the coating was basically of Co3O4. At 750 degreesC CoO was formed and big cracks appeared on the film exposing an inner layer of iron oxides. Analysis of the EIS data is very difficult because of the complexity of the interface structure. It can be inferred that the charge transfer resistance of the coatings prepared at 350 and 450 C were higher than those for the coatings prepared at temperatures above 550 degreesC. (C) 2002 Published by Elsevier B.V. Ltd.

Alkaline ribonuclease activity in maternal serum and in serum of newborns from birth up to thirty days of age. A study made with full-term appropriate-, full-term small- and preterm appropriate-for-gestational-age infants

We have studied the alkaline ribonuclease (RNase) activity in maternal serum and serum of full-term small- (T-SGA), full-term appropriate- (T-AGA) and preterm appropriate-for-gestational age (PT-AGA) newborns. A significantly lower level of RNase was observed in T-AGA and T-SGA newborns on the 30th day of age and in PT-AGA newborns on the 15th and 30th days of age, as compared to other T-AGA, T-SGA and PT-AGA groups of infants at birth. RNase activity was significantly higher in cord blood than in the maternal blood in all categories studied. Moreover, in preterm newborns, RNase activity in cord blood was significantly higher in those presenting a lower gestational age. We did not observe any significant difference in RNase levels in the cord blood of newborns from the 3 categories studied. The same results were observed concerning maternal blood. We, therefore, conclude that RNase activity in cord blood or in maternal blood is not a very statisfactory indicator of fetal malnutrition.