Adsorption of a mixture of Thiobacillus thiooxidans and Thiobacillus ferrooxidans onto copper-containing furnace dust

The Langmuir adsorption parameter X(Am) of a mixture of culture of Thiobacillus ferrooxidans and Thiobacillus thiooxidans indicates that these bacteria have preferential and competitive adsorption sites on furnace dust. The constant K(A) of the mixture significantly larger than that of each component, suggesting that a synergistic effect may occur in the binding of these bacteria to the dust.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

PROPERTIES OF AMINOPEPTIDASE FROM JAPANESE CLASSIFIED BARLEY FLOUR

An aminopeptidase (AP) was purified from classified barley flour obtained from the outer layer of Japanese grain. To characterize the enzyme, the substrate specificity and pH optimum were determined with 9 aminoacyl‐p‐nitroanilide (p‐NA) and 6 aminoacyl‐β‐naphthylamide (β‐NA). Furthermore, the effects of inhibitors and the hydrolysis of natural substrates were investigated. The optimum pH was between 6.5 and 8.0 in all cases in which these artificial substrates were used. Phe‐β‐NA had the highest affinity for the enzyme and the Km value was 0.084 mM. The enzyme also showed a strong preference against leucine‐, arginine‐, and lysine‐p‐NA (Km = 0.18 mM, 0.10 mM, and 0.48 mM, respectively). Chymostatin slightly inhibited the activity, whereas AP was not affected by leupeptin and pepstatin A. The enzyme did not hydrolyze the proteins tested, but it cleaved peptide hormones.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

PURIFICATION AND CHARACTERIZATION OF α‐GALACTOSIDASE FROM PEANUTS1

Alpha‐galactosidase was characterized in two peanuts market types, Runner and Spanish. The enzyme was purified 54 fold using ammonium sulfate precipitation, anion exchange chromatography and Size Exclusion High‐performance Liquid Chromatography. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicated that the enzyme has a molecular weight of 30, 000 Da, and isoelectric focusing showed a pI of 5.2. The optimum temperature and pH were 50C and 6.0, respectively. The enzyme had a K_m of 0.221 mM when p‐nitrophenyl α‐D‐galactopyranoside (PNPG) was used as a substrate and 80.8 mM when raffmose was a substrate. Raffmose and galactose were found to be competitive inhibitors when PNPG was the substrate: K_i values were 25.4 and 189, respectively. The enzyme was very sensitive to Hg⁺⁺, Ag⁺⁺ and to a lesser extent to Cu⁺⁺. However, ethylcne diamine tetraacetic acid did not have an effect indicating no requirement for cations. The two peanut types tested showed identical enzyme activities.     

Purification and characterization of a dehydrogenase catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde to N alpha-benzyloxycarbonyl-L-aminoadipic acid from rhodococcus sp. AIU Z-35-1

The enzyme catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde (N alpha-Z-L-AASA) to N alpha-benzyloxycarbonyl-L-aminoadipic acid (N alpha-Z-L-AAA) in Rhodococcus sp. AIU Z-35-1 was identified, and its characteristics were revealed. This reaction was catalyzed by a dehydrogenase with a molecular mass of 59 kDa. The dehydrogenase exhibited enzyme activity on not only N alpha-Z-L-AASA but also N alpha-Z-D-AASA and short chain aliphatic aldehydes, but not on aromatic aldehydes and alcohols. The apparent Km values for N alpha-Z-L-AASA, N alpha-Z-D-AASA and NAD+ were estimated to be 3.8 mM, 14.1 mM and 0.16 mM, respectively. The NH2 terminal amino acid sequence of this enzyme exhibited a similarity to those of a piperidein-6-carboxylate dehydrogenase from Streptomyces clavuligerus and a putative dehydrogenase from Rhodococcus sp. RHA 1, but not to those of other microbial aldehyde dehydrogenases.     

Acid phosphatases activity in cheese and starters.

The acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5-2 and the enzyme was inhibited by F-minus,Al-3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5-2 gave a Km value for p-nitrophenyl phosphate of 1-2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with Ki values of 1-2 mM, 1-0 mM, 1-0 MM and 1-1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.     

PURIFICATION AND PROPERTIES OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE FROM CORIANDER (CORIANDRUM SATIVUM) LEAVES

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP⁺ oxidoreductase, EC 1.1.1.49; G6PD) was purified from coriander (Coriandrum sativum) leaves; the kinetic behavior and some properties of the enzyme were also investigated. The purification was done at 4C and involved two steps: ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 26.4% and had a specific activity of 1.826 U/mg protein. Optimum pH, stable pH, optimum temperature, molecular weight, K_M and V_max values for NADP⁺ and glucose 6-phosphate (G6-P) were also determined.
The overall purification was about 74-fold. SDS-PAGE of the purified enzyme showed a single band. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm.
The molecular mass was estimated to be 74.4 kDa by SDS-PAGE and 73.2 kDa by Sephadex G-200 gel filtration column chromatography. The enzyme had an optimum pH at 8.5 and was stable at pH 8.0 in 0.1 M Tris-HCl buffer. The optimum temperature was at 30C. The K_M values for NADP⁺ and G6-P were 0.026 mM and 0.116 mM, respectively. The V_max values for these substrates were 0.035 EU/mL and 0.038 EU/mL, respectively.     

Characterization of Polyphenol Oxidase from Airen Grapes

Polyphenol oxidase (PPO) was isolated from grapes grown in Spain and its characteristics were studied. The partially purified enzyme had both cresolase and catecholase activities. Catecholase activity had a pH optimum in a range 3.5–4.5 and was characterized by a relatively high stability to heat. The apparent K_M for 4-methylcatechol was 9.5 mM. Cresolase activity presents a lag period which is modulated by different factors: enzyme concentration, substrate concentration, temperature or pH. The presence of o-diphenols in the reaction medium abolishes the lag period, these acting as co-substrates. The apparent K_M towards p-cresol and the activation constant for o-diphenol for cresolase activity were 0.35 mM and 1.75 μM, respectively.     

THE β-N-ACETYLGLUCOSAMINIDASE ACTIVITY OF EGG WHITE. 1. Kinetics of the Reaction and Determination of the Factors Affecting the Stability of the Enzyme in Egg White

SUMMARY– Enzymatic activity of β-N-acetylglucosaminidase, which occurs naturally in chicken egg white, was characterized to establish conditions suitable for routine assay for this enzyme in egg products. A variation in enzyme content of approximately 3-fold was observed in individual fresh eggs. The enzyme has a pH optimum between 3.0 and 3.4, and a K_m of approximately 0.6 mM for the substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide. Activation energy for hydrolysis of this substrate is 10.7±0.8 kcal/mole. The enzyme is stable for at least several hours at ambient temperature from pH 6.8 to approximately 8.8. Above pH 8.8, inactivation is first order with respect to time. Enzyme activity in shell eggs decreases fairly rapidly at ambient temperature; loss of activity probably results from increase in pH of egg white, which occurs normally upon loss of carbon dioxide. Eggs held at 4°C retain activity much longer.     

Biological deodorization of hydrogen sulfide using porous lava as a carrier of Thiobacillus thiooxidans

Biological deodorization of hydrogen sulfide (H2S) was studied using porous lava as a carrier of Thiobacillus thiooxidans in a laboratory-scale biofilter. Three different samples of porous lava, A, B, and C, which were obtained from Cheju Island in Korea, were used. The water-holding capacities of samples A, B and C were 0.38, 0.25, and 0.47 g-H2O/g-lava, respectively. The pHs and densities of the lava samples ranged from 8.25-9.24 and 920-1190 kg/m3, respectively. The buffering capacities, expressed as the amount of sulfate added to lower the pH to 4, were 60 g-SO4(2-)/kg-lava for sample A, 50 g-SO4(2-)/kg-lava for B, and 90 g-SO4(2-)/kg-lava for C. To investigate the removal characteristics of H2S by the lava biofilters, T. thiooxidans was immobilized on the lava samples. Biofilters A and C showed a removal capacity of 428 g-S.m(-3).h(-1) when H2S was supplied with 428 g-S.m(-3).h(-1) of inlet load at a space velocity (SV) of 300 h(-1). At the same inlet load and SV, the removal capacity of biofilter B was 396 g-S.m(-3).h(-1). The H2S critical loads of biofilters A, B and C at a SV of 400 h(-1) were 396, 157 and 342 g-S.m(-3).h(-1), respectively. It is suggested that natural, porous lava is a promising candidate as a carrier of microorganisms in biofiltration.     

ALPHA-GALACTOSIDASE OF GERMINATING SEEDS OF CASSIA SERICEA SW.

Germinating seeds of Cassia sericea Sw. contain two molecular forms of α-galactosidase which were partially purified and characterized. Both enzyme forms had an optimum pH of 5.0 and an optimum temperature of 50 °C. K_m values for the substrate p-nitrophenyl-α-D-galactoside (PNPG) were 0.91 mM and 1.05 mM for the two forms, and substrate inhibition was observed at high concentrations of PNPG. The two forms of the enzyme had molecular weights of 46,000 and 33,000 by gel filtration. The enzyme forms were inhibited completely by Ag⁺, Cu²⁺ and Hg²⁺ ions and by p-chloromercuribenzoate showing that thiol groups are probably involved in catalysis. Both α-galactosidases hydrolyzed melibiose and raffinose, although hydrolysis of stachyose could not be detected. The enzyme may find potential use in the food industry to hydrolyze flatulence-causing sugars in processed foods and in production of sucrose from high-raffinose sugar beets. That the source (C. sericea seeds) of the enzyme is inexpensive provides an added advantage over use of cultivated legumes as a source of α-galactosidase.     

Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini

An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.     

Properties of wheat bran polyphenol oxidase

Polyphenol oxidase (PPO) obtained from wheat bran catalyzed the oxidation of 4-methyl catechol. Phenolic compounds found naturally in crude extract played role as an endogeneous substrate and activity of crude extract needed correction. Activity versus enzyme concentration gave a linear plot at high substrate concentration whereas a nonlinear plot was obtained at low substrate concentration which proved the presence of endogeneous substrate. Adsorption on celite and extraction with polyvinylpyrrolidone (PVPP) caused the removal of phenols. Adsorption of PPO on celite yielded a 4-fold increase in specific activity whereas extraction with PVPP yielded a 2.5-fold increase in specific activity compared to the crude extract. The kinetics of PPO catalyzed oxidation obeyed Michaelis-Menten model; Km and Vmax values were found as 218 mM and 99 microM/min, respectively. The enzyme was inhibited by ethyl alcohol, dithiothreitol (DTT) and isoproterenol and exhibited heat stability up to a temperature of 90 degrees C. The optimum pH of the enzyme was found to be 5.0.     

MICROSOMAL-ASSOCIATED GLYCEROPHOSPHATE ACYLTRANSFERASE ACTIVITY IN GERMINATING SOYBEANS

Acyl CoA:sn-glycero-3-phosphate O-acyltransferase (glycerophosphate acyltransferase, EC 2.3.1.15) catalyzes the acylation of glycero-3-phosphate to form lysophosphatidic acid. This enzyme catalyzes the first committed step in the biosynthesis of phospholipids and acylglycerols in soybeans. Glycerophosphate acyltransferase was predominatly associated with the microsomal fraction of germinating soybeans. The pH optimum for the reaction was 7.0. The enzyme exhibited saturation kinetics for glycero-3-phosphate (K_m of 0.2 mM) and palmitoyl CoA (K_m of 6.4 μM). A variety of acyl CoA derivatives served as substrates for the enzyme. Palmitoyl CoA was the most effective acyl CoA substrate. The addition of the nonionic detergent Triton X-100 inhibited glycerophosphate acyltransferase activity. Thioreactive agents inhibited the enzyme indicating that a sulphydryl group is essential for activity. The activation energy for the reaction was 8.8 Kcal per mole. The microsomal enzyme was reasonably stable to temperatures up to 70° C.     

Purification and Properties of Beta-galactosidase from Streptococcus thermophilus

A β-galactosidase from Streptococcus thermophilus was purified to homogeneity by ammonium sulfate and acetone fractionation, gel filtration on Sephadex G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme preparation exhibited an optimum pH at 6.6–7.0 and an optimum temperature of 57°C. The enzyme was stable at pH 6.8–7.0. Km and V_max for the enzyme, using ortho-nitrophenyl β-D-galactopyranoside as the substrate, were 0.25 mM and 83 μmoles/mg protein/min, respectively. It was strongly inhibited by Hg⁺⁺, Ag⁺, and Cu⁺⁺ as well as pchloro-mercuri benzoate. The enzyme had a molecular weight of about 6 × 10⁵ and was highly specific for β-galactoside bonds.     

Properties of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1

A NAD+-dependent medium-chain alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1 was expressed in Escherichia coli and purified. The recombinant enzyme was a homotetramer of molecular mass 1.6 x 10(2) kDa. The optimum pH for the oxidative reaction was around 10.5 and that for the reductive reaction was around 8.0. The enzyme had a broad substrate specificity including aliphatic and aromatic alcohols, aliphatic and aromatic ketones, and benzylaldehyde. This enzyme produced (S)-alcohols from the corresponding ketones. The enzyme was thermophilic and the catalytic activity increased up to 95 degrees C. It maintained 24% of the original catalytic activity after incubation for 30 min at 98 degrees C, indicating that this enzyme is highly thermostable.     

PURIFICATION OF AMINOPEPTIDASE FROM THE JAPANESE CLASSIFIED BARLEY FLOUR

In a search for biologically active materials from the classified barley flour produced in Japan an aminopeptidase activity was identified. In this paper, the purification of aminopeptidase is described. The activity of the enzyme was monitored using L-leucine-p-nitroanilide as the substrate. After extraction using 20 mM sodium acetate buffer, pH 5.5, from the 95–75% classification flour, ammonium sulfate fractionation was performed between 30 and 50% saturation. The aminopeptidase was then purified about 160-fold to homogeneity as assessed by HPLC using the following sequential chromatography steps: hydrophobic interaction chromatography, Sephacryl S-200HR gel chromatography, DEAM-ion exchange chromatography, hydroxylapatite chromatography, Sephacryl S-100HR gel chromatography. The molecular weight of this enzyme was estimated as 62 kDα by size exclusion HPLC. The enzyme had a K_m value of 0.22 mM and α pH optimum of 7.0.     

STABILITY AND PROPERTIES OF A THERMOSTABLE β-GALACTOSIDASE IMMOBILIZED ON CHTTIN

Thermostable β‐galactosidase from an E. coli transformant containing the enzyme gene from P. woesei was immobilized at pH 4.0 and a glutaraldehyde concentration of 10 mM on chitin isolated from shrimp Crangon crangon shells. These preparations had a specific activity of 43,000 U/g of chitin at 85C using ONPG as substrate. The optimum pH and temperature for immobilized β‐galactosidase activity were 5.2 and 93C. Immobilization shifts the optimum pH for the activity of the enzyme by 0.2 units towards the acid side. The immobilized enzyme is stable at temperatures close to the optimal value, and the residual activity for ONPG hydrolysis of the preparations incubated 5 h in 0.1 M phosphate citrate buffer (pH 5.4) at 90C and 100C was 70% and 40% of the initial value, respectively.     

Characterization of Ypr1p from Saccharomyces cerevisiae as a 2-methylbutyraldehyde reductase

The metabolism of aldehydes and ketones in yeast is important for biosynthetic, catabolic and detoxication processes. Aldo-keto reductases are a family of enzymes that are able to reduce aldehydes and ketones. The roles of individual aldo-keto reductases in yeast has been difficult to determine because of overlapping substrate specificities of these enzymes. In this study, we have cloned, expressed and characterized the aldo-keto reductase Ypr1p from the yeast Saccharomyces cerevisiae and we describe its substrate specificity. The enzyme displays high specific activity towards 2-methylbutyraldehyde, as well as other aldehydes such as hexanal. It exhibits extremely low activity as a glycerol dehydrogenase. The enzyme functions over a wide pH range and uses NADPH as co-factor. In comparison to other mammalian and yeast aldo-keto reductases, Ypr1p has relatively high affinity for D,L-glyceraldehyde (1.08 mM) and hexanal (0.39 mM), but relatively low affinity for 4-nitrobenzaldehyde (1.07 mM). It displays higher specific activity for 2-methylbutyraldehyde than does yeast alcohol dehydrogenase and has a K(m) for 2-methyl butyraldehyde of 1.09 mM. The enzyme is expressed during growth on glucose, but its levels are rapidly induced by osmotic and oxidative stress. Yeast in which the YPR1 gene has been deleted possess 50% lower 2-methylbutyraldehyde reductase activity than the wild-type strain. This suggests that the enzyme may contribute to 2-methyl butyraldehyde reduction in vivo. It may therefore play a role in isoleucine catabolism and fusel alcohol formation and may influence flavour formation by strains of brewing yeast.