Synthesis of raffinose by fungal α-galacotosidase from Absidia corymbifera

In order to investigate the optimal conditions for raffinose synthesis, α-galactosidase was purified from Absidia corymbifera IFO8084 with a recovery yield of approximately 8.1% (8.36 mg). The molecular weight of the wild-type α-galactosidase was about 83 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native molecular mass of the enzyme was approximately 330 kDa by gel filtration chromatography, indicating that α-galactosidase from A. corymbifera IFO8084 is a homotetrameric enzyme. The purified enzyme displayed optimal enzyme activity at pH 4.5 and 60°C. When the purified α-galactosidase was incubated in a substrate solution of sucrose and D-galactose for 48 hr at 37°C, raffinose was synthesized and was confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and ¹³C-nuclear magnetic resonance (¹³C-NMR) spectrometer analysis. Maximum rates of conversion were observed with 1.67 M galactose, 2.04 M sucrose, and 100 U α-galactosidase at pH 6.0 and 70°C. Under the optimized conditions, the overall conversion ratio was 10%(w/v), representing 2.5 times the synthesis yield that would be possible without the optimized conditions.     

Purification of properties of Saccharomyces cerevisiae cystathionine β-synthase

Cystathionine β-synthase (β-CTSase), which catalyses cystathionine synthesis from serine and homocysteine, was purified to homogeneity from Saccharomyces cerevisiae. The molecular mass of the enzyme was estimated to be 235 kDa by gel filtration and 55 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, indicating that it is a homotetramer. The N-terminal amino acid sequence of the enzyme perfectly coincided with that deduced from the nucleotide sequence of CYS4, except for the absence of initiation methionine. The purified β-CTSase catalysed cysteine synthesis from serine (or O-acetylserine) and H₂S. From this finding, we discuss the multifunctional nature and evolutionary divergence of S-metabolizing enzymes.     

Purification of angiotensin converting enzyme inhibitory peptides from sunflower protein hydrolysates by reverse-phase chromatography following affinity purification

The purification of a peptidic fraction with angiotensin converting enzyme (ACE) inhibitory activity from sunflower protein hydrolysates by affinity chromatography was recently described. We now describe that reverse-phase HPLC fractionation of this product yields several fractions with IC₅₀ one order of magnitude higher than those previously purified by reverse-phase HPLC following gel filtration chromatography, showing that affinity chromatography is much more effective than gel filtration chromatography as a first step for purification of ACE inhibitory peptides. The amino acid composition of these fractions is presented, but attempts to determine their amino acid sequence failed, showing that these fractions contained more than one peptide.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

Purification and some properties of polyphenoloxidase in eggplant (Solanum melongena)

Polyphenoloxidase (EC 1.10.3.1) in eggplant (Solatium melongena L) was purified by ammonium sulphate fractionation, DEAE-Cellulofine and DEAE-Toyopearl chromatography and Sephadex G-100 gel filtration. The enzyme was purified about 110-fold with a recovery of 5%. The purified enzyme more quickly oxidised chlorogenic acid (5-caffeoylquinic acid, IUPAC) than 10 other substrates used. The K_m value for the enzyme was found to be 0·50 mM with respect to chlorogenic acid; the optimum pH of the enzyme was about 4 with enzyme stability between pH 5 and 8. The enzyme was completely inactivated after heat treatment at 75°C for 30 min or 80°C for 5 min. Sodium metabisulphite, potassium cyanide and sodium fluoride markedly inhibited the enzyme activity.     

Identification and characterization of a novel angiotensin I-converting enzyme inhibitory peptide (ACEIP) from silkworm pupa

The angiotensin I-converting enzyme inhibitory peptide (ACEIP) was isolated and characterized from silkworm pupae and purified using Sephadex G-25 gel filtration. The structure and physicochemical properties of pupa ACEIP were analyzed. The α-P₃ fraction exhibited the most potent ACE inhibitory activity. After purification via semi-preparative reverse-phase HPLC (RP-HPLC) and HPLC, the α-P₃-6-b component was revealed to have the highest ACE inhibitory activity (IC₅₀=28.3 μg/mL). Edman degradation revealed a Val-Glu-Ile-Ser amino acid sequence in which novel active sequences were identified. Physicochemical property testing showed that purified pupa ACEIP exhibits good solubility, heat resistance, and acid resistance that all indicate ACEIP derived from silkworm pupa is an excellent food-derived ACEIP.     

PURIFICATION AND CHARACTERIZATION OF INVERTASE FROM DATES (PHOENIX DACTYLIFERA L., VAR. ZAHDI)

Zahdi dates (Phoenix dactylifera) contain invertase at all development stages; the highest specific activity is present in the late yellow stage. The enzyme was purified to homogeneity, as determined by disc gel electrophoresis and isoelectric focusing, by a combination of techniques including ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sepharose 4B and Sephadex G-150 columns. A complex of invertase with a high molecular weight pectic substance of the date could not be dissociated by ammonium sulfate or DEAE-cellulose chromatography but the complex was dissociated by gel filtration on a Sepharose 4B column at pH 4.0 and ionic strength of 0.5 M. The enzyme contained 8.2% carbohydrate covalently linked probably via an amide linkage to aspartic acid. Molecular weight determination by exclusion gel chromatography and sedimentation equilibrium gave values of 130,000 and 97,100 ± 1,300, respectively. The enzyme is probably composed of two identical subunits as shown by SDS polyacrylamide gel electrophoresis. Amino acid analyses showed the enzyme to be low in sulfur-containing amino acids. Date invertase is an acid β-fructofuranosidase with a pH optimum between 3–4 and with a K_m and k_cat for sucrose of 6mM and 49 sec^-1, respectively. Activation energies for denaturation of enzyme and conversion of substrate to product were determined to be 48.7 and 17.6 kcal/mole, respectively. Chemical modification indicated that sulfhydryl groups are probably not essential for activity while carboxyl groups may be involved in the active site of the enzyme.     

Isolation and characterisation of trypsin from sardinelle (Sardinella aurita) viscera

BACKGROUND: In Tunisia, sardinelle (Sardinella aurita) catches totalled about 13 300 t in 2002. During processing, solid wastes including heads and viscera are generated, representing about 30% of the original raw material. Viscera, one of the most important by‐products of the fishing industry, are recognised as a potential source of digestive enzymes, especially proteases with high activity over a wide range of pH and temperature conditions. This paper describes the purification procedure and some biochemical characterisation of trypsin from S. aurita viscera. RESULTS: Trypsin from the viscera of sardinelle (S. aurita) was purified by fractionation with ammonium sulphate, Sephadex G‐75 gel filtration, Sepharose mono Q anion exchange chromatography, ultrafiltration and a second Sephadex G‐75 gel filtration, resulting in a 5.42‐fold increase in specific activity and 6.1% recovery. The molecular weight of the purified enzyme was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme showed esterase‐specific activity on N‐α‐benzoyl‐L ‐arginine ethyl ester (BAEE) that was four times greater than its amidase‐specific activity on N‐α‐benzoyl‐DL ‐arginine‐p‐nitroanilide (BAPNA). The optimal pH and temperature for enzyme activity were pH 8 and 55 °C respectively using BAEE as a substrate. The trypsin kinetic constants K_m and k_cat on BAPNA were 1.67 mmol L^?1 and 3.87 s^?1 respectively, while the catalytic efficiency k_cat/K_m was 2.31 s^?1 L mmol^?1. CONCLUSION: Trypsin was purified from sardinelle (S. aurita) viscera. Biochemical characterisation of S. aurita trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish‐processing and food industries. Copyright © 2008 Society of Chemical Industry     

Multiplicity of Glucoamylase of Aspergillus oryzae Part 1. Separation and Purification of Three Forms of Glucoamylase

The glucoamylase system of Aspergillus oryzae cultured on wheat bran was separated into 3 active fractions by (NH₄)₂SO₄, rivanol and ethanol precipitation followed by ion exchange chromatography on DEAE-Sephadex A-25. One of these fractions, referred to as glucoamylase I was purified by further chromatography on hydroxyapatite gel and Sephadex G-200. The other two fractions referred to as glucoamylase II and III were purified by further gel filtration on Sephadex G-200. The purified glucoamylases were found to be homogeneous on 7.5% polyacrylamide gel electrophoresis and isoelectric focusing by carrier ampholites.     

Partial purification,heat stability and kinetic characterization of the pectinmethylesterase from Brazilian guava,Paluma cultivars

Pectinmethylesterase (PME) was extracted from guava fruit (Psidium guajava L.), cultivar Paluma, by 70% ammonium sulphate saturation and partially purified by gel filtration on Sephadex G100. Gel filtration showed PME isoenzymes with different values of molecular mass. Two samples were examined: concPME (70% saturation by ammonium sulphate) and Iso4 PME (one of the isoforms from gel filtration with the greatest specific activity). Optimum pH of the enzyme (for both samples) was 8.5 and optimum temperature ranged from 75 and 85 °C. The optimum sodium chloride concentration was 0.15 M. The K_M and V_max ranged from 0.32 to 0.23 mg ml⁻¹ and 244 to 53.2 μmol/min, respectively, for concPME and Iso4PME. The activation energies (E_a) were 64.5 and 103 kJ/mol, respectively, for concPME and Iso4PME. Guava PME, cv Paluma, is a very thermostable enzyme, showing great heat stability at all temperatures studied.     

Purification of β-carotene 15,15′-monooxygenase from pig intestine and its enzymatic hydrolysis of pigment in soybean oil

β-Carotene 15,15′-monooxygenase was isolated and purified from the intestinal mucosa of pigs, and then, it was applied to hydrolyse the pigment in soybean oil, and thus, vitamin A fortified soybean oil was obtained. The pig intestinal mucosal protein solution was purified to a specific activity of 2.487 × 10⁻⁴ U mg⁻¹, maximum reaction velocity (V_max) of 8.42 × 10⁻⁹ mol/h and Michaelis constant (K_m) of 2.03 × 10⁻⁵ m . The protein solution had a molecular mass of 156 kDa by gel filtration. The sodium deoxycholate concentrations, optimum pH, Tween 40 amount and enzyme amount for vitamin A production in soybean oil were determined to be 6.0 mm , 8.0, 3.0% (w/v) and 0.2 U/mL enzyme, respectively. Under these conditions, β-carotene 15,15′-monooxygenase produced 14.65 mg/L vitamin A after 20 h, with a conversion yield of β-carotene of 33.29% (w/w). Therefore, the nutrients in soybean oil were improved.     

Purification and Characterization of β-Glucosidase from Aspergillus niger

Aspergillus niger, an isolate of soil contaminated with effluents from cotton ginning mill was grown in Czapek-Dox medium containing sawdust, Triton-X 100 and urea for production of an extracellular β-glucosidase. β-Glucosidase enzyme was purified (86-fold) from culture filtrate of A. niger by employing ammonium sulphate precipitation and gel filtration on sephadex G-75. The molecular mass of the purified enzyme was estimated to be 95 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme had an optimal activity on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5.0. The K_m and V_max of the enzyme on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5 were 8.0 mM and 166 µmol/min/mg of protein, respectively. The enzyme could hydrolyze cellobiose and lactose but not sucrose. Heavy metals like Hg²⁺, Al³⁺, and Ag⁺ inhibited the activity, whereas Zn²⁺ and detergents such as Triton-X 100 and Tween-80 increased the activity at 0.01%. The enzyme activity increased in the presence of methanol and ethanol.     

Purification and characterization of aminopeptidase yspI from Schizosaccharomyces pombe

Aminopeptidase yspI was purified to apparent homogeneity from the fission yeast Schizosaccharomyces pombe. The molecular mass of the native enzyme was estimated to be 184 kDa by gel filtration chromatography. A value of 92 kDa was calculated after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme is thus a dimer with two identical subunits. Optimum pH for cleavage of synthetic aminoacyl-4-nitroanilides is 7·0. Mercury ions, EDTA and chloroquine were found to be potent inhibitors of aminopeptidase yspI activity. Substrate specificity studies indicate that the purified enzyme cleaves L-lysine-4-nitroanilide with high efficiency.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

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.     

Purification of a lipase from the hepatopancreas of oil sardine (Sardinella longiceps linnaeus) and its characteristics and properties

Lipase has been purified from the hepatopancreas of oil sardine (Sardinella longiceps) by defatting, water extraction, ammonium sulphate fractionation and chromatography on DEAE Sephadex and Sephadex G-100. The preparation was homogeneous on polyacrylamide disc gel electrophoresis and on gel filtration through Sephacryl S-200. The enzyme showed a molecular weight of 54000±57000 with 6.1% of carbohydrate. The pH and temperature optima of purified sardine lipase were 8 and 37°C respectively. Sardine lipase remained stable up to 45°C (15 min) and in the pH range 5 to 9.5. The K_m values obtained for the substrates tributyrin and triacetin were 4 × 10^?2 and 30 × 10^?2, respectively. The effect of halogens and various metal ions on sardine lipase activity, substrate specificity, amino acid and carbohydrate composition are also reported.     

PURIFICATION AND PHYSICOCHEMICAL PROPERTIES OF AN EXTRA CELLULAR AMYLASE FROM A STRAIN OF BACILLUS AMYLOLIQUEFACIENS ISOLATED FROM DRY ONION POWDER

An extracellular α-amylase from Bacillus amyloliquefaciens, isolated from dry onion powder, has been purified to homogeneity by ammonium sulfate fractionation, adsorption on starch, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100 column. The enzyme consisted of one polypeptide chain with a molecular weight of 60,000. The isoelectric point was pH 5.2, the pH optimum 5.5 and the temperature optimum ranging from 50°-70°C. Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 6.9 mg/ml. The enzyme was quite stable at 50°C, but lost about 85% of its activity at 60° after 30 min (pH 6.0).     

Purification and Characterization of a Pullulan-Hydrolyzing Glucoamylase from Sclerotium rolfsii

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50°C between pH 4.0–4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0–4.5 and at 65°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml⁻¹ (V_max 1.0U · mg⁻¹) and for soluble starch, it was 0.6mg · ml⁻¹ (V_max 8.33 U · mg⁻¹). The enzyme was observed to be a glycoprotein (12–13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed α-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 μM). The enzyme has been characterized as a glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.     

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

Background: γ‐Aminobutyric acid (GABA) is a non‐protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. Results: The molecular mass of the enzyme estimated by Sephadex G‐100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg²⁺, Cu²⁺, Fe³⁺, aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA‐Na₂), L ‐cysteine and β‐mercaptoethanol. The K_m value of DAO was 0.23 mmol L^?1 for putrescine and 0.96 mmol L^?1 for spermidine. However, the enzyme did not degrade spermine. Conclusion: DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine. Copyright © 2011 Society of Chemical Industry