Purification and characterization of a novel glutamyl aminopeptidase from chicken meat

A novel glutamyl aminopeptidase (aminopeptidase A, EC 3.4.11.7) was purified from chicken meat by ammonium sulfate fractionation, ethanol fractionation, heat treatment, and successive column chromatographies of DEAE-Sepharose CL-6B and Sephadex G-200. The purified enzyme migrated as a single band on SDS-PAGE. The molecular weight of this enzyme was found to be 55,000 and 550,000 by SDS-PAGE and Sephadex G-200 column chromatographies, respectively. This enzyme hydrolyzed Glu- and Asp-, but not Leu-, Arg-, and Ala-2-naphthylamide (-2NA) at all. The optimum pH and temperature for hydrolysis of Glu-2NA was 7.5. and 70°C, respectively. Reducing agents such as cysteine and dithiothreitol inhibited the activity of this enzyme at concentrations of 1 mM. However, the activation by Ca(2+) and the inhibition by amastatin were not observed.     

Purification and characterization of a beta-glucosidase with beta-xylosidase activity from Aspergillus sojae

A beta-glucosidase (EC 3.2.1.21) was purified as an electrophoretically homogeneous protein from a solid culture of Aspergillus sojae. The molecular mass of the purified enzyme was estimated to be 250 kDa by gel filtration chromatography and 118 kDa by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point of the enzyme was 3.80. The maximum velocity of rho-nitrophenyl beta-d-glucopyranoside degradation by the beta-glucosidase was attained at 60 degrees C and at pH 5.0. The purified enzyme was stable from pH 6.0 to 8.0, and up to 50 degrees C. The activity of the enzyme was significantly inhibited by Hg2+ and Cu2+, and stimulated by Mn2+ and Fe3+. The purified enzyme hydrolyzed beta-D-xylopyranosides as well as beta-D-glucopyranosides; the Km and Vmax values on rho-nitrophenyl beta-D-glucopyranoside were 0.14 mM and 16.7 micromol/min/mg protein, and on rho-nitrophenyl beta-D-xylopyranoside 0.51 mM and 12.2 micromol/min/mg protein, respectively.     

PURIFICATION AND CHARACTERIZATION OF A LIPASE FROM LACTOBACILLUS PLANTARUM 2739

A 65 kDa intracellular lipase from Lactobacillus plantarum 2739 was purified to homogeneity (482-fold, specific activity of 251 μmol/mg per min) and characterized. The purification procedure included chromatography with Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. The purified lipase was optimally active at pH 7.5 and 35C; it retained about 40% of the maximum activity at pH 5.0 and 15C. The enzyme was stable at 65C (D_65C= 18.6 min) and was irreversibly inactivated at 75C for 2 min. On triglycerides, the highest activity was determined on tributyrin but trilaurin and tripalmitin were hydrolyzed also. The Km on tributyrin was 2.31 mM. β-Naphthyl esters of fatty acids from C2 to C12 were hydrolyzed with a preference for β-naphthyl butyrate. After lipolysis, the fatty acid profiles in β-monoacylglycerols of milk fat showed similarities among porcine pancreatic lipase, rennet paste and lipase from Lb. plantarum 2739, but the bacterial enzyme caused a greater hydrolysis of C10 and C12 fatty acids esterified at the Sn-2 position of glycerol. The lipase was strongly inhibited by 1 mM Nethylmaleimide and iodoacetic acid, by 10 mM Hg²⁺ and Ag⁺, and was moderately stimulated by Ca²⁺ and Mg²⁺.     

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.     

The least number of phosphate groups for crosslinking of casein by colloidal calcium phosphate.

Artificial casein micelles were formed with whole human casein at 20 mM calcium, 17 mM phosphate, and 10 mM citrate. The casein micelles disaggregated by 6 M urea were separated by high performance gel chromatography on a TSK-GEL G4000SW column into crosslinked and monomeric fractions. When the crosslinked casein fraction was analyzed by high performance ionexchange chromatography on a TSK-GEL DEAE-5PW column, a small peak, representing the 3-P component of human beta-casein, and distinct peaks of the 4-P and 5-P components were found. Artificial casein micelles were formed from mixtures of each purified component of human beta-casein and bovine kappa-casein, disaggregated by urea, and separated on a TSK-GEL G4000SW column. The casein aggregates crosslinked by colloidal calcium phosphate were formed in artificial casein micelles of the 3-P and 4-P components. In contrast, no crosslinkage was formed in artificial micelles of the 1-P and 2-P components. The results indicate that at least three phosphate groups are needed for crosslinking of casein by colloidal calcium phosphate.     

Purification and characterization of sea squirt alpha-N-acetylgalactosaminidase

Sea squirt alpha-N-acetylgalactosaminidase was purified to homogeneity. Its molecular weight was estimated to be approximately 160,000 by gel filtration and 40,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition. The chromatographic and electrophoretic behaviors indicated that the enzyme was composed of four subunits. The optimum pH of the enzyme reaction was about 4.0 at 37 degrees C, while the enzyme was stable in the range of pH 5.0 to 6.0 during 4 h preincubation at 37 degrees C. Although the enzyme (0.1 unit) was stable at 0 degrees C for 30 min in the presence of 7.5 mM metal ions (Al3+, Ba2+, Ca2+, K+, Mn2+, Pb2+, Sr2+, and Zn2+), almost 40% of the enzyme activity was lost in the presence of Cu2+, Hg2+, monoiodoacetic acid, and EDTA. The enzyme hydrolyzed aryl N-acetyl-alpha-D-galactosaminide as well as GalNAcalpha1(-->4GalNAcalpha1-->)n 4GalNAc-p-aminobenzoic acid ethyl ester (ABEE) (n = 1-4), but GalNAcalpha1-->4GalNAc-ABEE only scarcely. Furthermore, an allergenic pentasaccharitol ABEE derivative, GalNAcalpha1-->2Fucalpha1-->3(GalNAcbeta1-->4) GlcNAcbeta1-->2(3-acetoamido-3-deoxy)L-threose-ABEE, the minimum structural unit for the sea squirt allergenicity was hydrolyzed to 95 mol% for 72 h incubation with the enzyme. The enzyme could be utilized as a powerful tool for the structural analyses of the carbohydrate epitopes of the sea squirt allergen molecules.     

Purification and characterization of a second aminopeptidase (pepC-like) from Lactobacillus delbrueckii subsp. bulgaricus B14

A second aminopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation and two steps of anion-exchange chromatography. After SDS polyacrylamide-gel electrophoresis in the presence of β-mercaptoethanol, one protein band was detected at 54 kDa. The same molecular mass was estimated by gel filtration. SDS polyacrylamide-gel electrophoresis in the absence of β-mercaptoethanol resulted in a single band at 220 kDa, indicating that the enzyme forms complexes of four molecules under non-reducing conditions. Activity was markedly increased by reducing and metal-chelating agents. Thiol-group inhibitors, such as iodoacetic acid, inhibited the enzyme strongly. In contrast to Mg²⁺ and Ca²⁺, which had slightly activating effects, other divalent cations reduced enzyme activity at a concentration of 1 mM. The aminopeptidase showed highest activity at 50°C and pH 6·5–7 and hydrolyzed a wide range of di- and tripeptides. The most suitable substrates were Leu-Gly, Leu-Gly-Gly, Ala-Ala-Ala, and Met-Gly-Gly. For Leu-Gly and Leu-Gly-Gly, K_m-values of 1·81 mM and 2·17 mM and turnover numbers of 870 s⁻¹ were calculated, with a maximal rate of hydrolysis (V_max) of 4600 and 2780 μmol/min per mg of protein, respectively. The aminopeptidase did not cleave Lys-pNA, a substrate hydrolyzed by all type-‘N’ aminopeptidases from lactic acid bacteria with high velocities. It compared well, however, with pepC found in Lactococcus.     

A novel selective nucleoside phosphorylating enzyme from Morganella morganii

A selective nucleoside phosphorylating enzyme was purified to homogeneity from Morganella morganii NCIMB10466 crude extract. The enzyme appeared to consist of six subunits identical in molecular mass (M(r) 25,000). It phosphorylated various nucleosides at the 5'-position to produce nucleoside-5'-monophosphates using pyrophosphate as the phosphate source. Energy-rich compounds, such as carbamylphosphate and acetylphosphate, were also very effective phosphate donors. The enzyme also exhibited phosphatase activity, and dephosphorylated various phosphate esters, but had a weak effect on nucleoside-3'-monophosphates. Based on the results of the kinetic study, the enzyme appeared to be an acid phosphatase. Its activity was partly inhibited by sulfhydryl reagents and heavy metal ions, but not by chelating reagents such as EDTA. Using the purified enzyme, 32.6 mM 5'-IMP was synthesized from inosine with a 41% molar yield, but the synthesized 5'-IMP was hydrolyzed back to inosine and phosphate as the reaction time was extended.     

Purification and properties of an extracellular inulinase from Rhizopus sp. strain TN-96

A filamentous fungus, Rhizopus sp. strain TN-96, was isolated from rhizosphere soil samples. An extracellular inulinase was purified from the culture filtrate of strain TN-96 grown on inulin by DEAE-Cellulofine A-500 and Sephacryl S-200 HP chromatographies. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M(r) of 83 kDa. The purified enzyme had specific activities of 17 U/mg toward inulin (I) and 0.32 U/mg toward sucrose (S) (I/S ratio, 53). Inulinase activity was optimal at pH 5.5 and 40 degrees C. The inulinase exhibited an apparent K(m) value of 9.0 mM for inulin. The enzyme also hydrolyzed raffinose, but not bacterial levan.     

Pacific Cod Muscle 5'-Nucleotidase

SUMMARY: A 5'-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability.
The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl₂ (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl₂ (10 to 100 mM). It was strongly activated by Mn⁺⁺ and weakly activated by Mg⁺⁺. The optimum pH was 7.6, and the Km was 5 × 10⁻⁴M with UMP and 8 ⁻⁴M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2',3'-nucleotides.     

Hydrolysis of the potato glycoalkaloid alpha-chaconine by filamentous fungi

Three strains of filamentous fungi have been isolated from potato sprouts to obtain an enzyme degrading the glycoalkaloids. All of the strains hydrolyzed alpha-chaconine and not alpha-solanine when grown on the sprouts. From strain HP341, identified as Plectosphaerella cucumerina, the enzyme hydrolyzing alpha-chaconine was purified on columns of DEAE-Toyopearl and Phenyl-Toyopearl. The partially purified enzyme hydrolyzed alpha-chaconine to beta1-chaconine but not to beta2- or gamma-chaconine, suggesting that the enzyme is a rhamnosidase specific for the hydrolysis of the rhamnose (C1-C4) glucose linkage in alpha-chaconine. Conversion of alpha-chaconine to beta1-chaconine may be the first step of detoxification for filamentous fungi to grow on potato sprouts that accumulated antifungal alpha-chaconine.     

Purification and Characterization of Cathepsin B from the Skeletal Muscle of Fresh Water Fish, Tilapia mossambica

Cathepsin B from the skeletal muscle of a fresh water fish Tilapia mossambica was purified 4280-fold with 9% recovery. The electrophoretic homogeneity of the preparation was established both under native and denatured conditions. The molecular weight of cathepsin B on the basis of its gel filtration profile was 23,500 daltons. The enzyme, an endopeptidase, hydrolysed Z-arg-arg-NNap and Bz-arg-NNap, with Km values of 0.57 and 3.23 mM, respectively. Cathepsin B did not display aminopeptidase activity, but cleaved Bz-arg-NH₂, exhibiting the specificity of a carboxypeptidase. Among protein substrates tested, only azocoll was hydrolyzed at lower pH values. Leu-peptin, antipain and thiol blockers abolished the enzyme activity completely. The Kcat set^-1 value of fish cathepsin B seemed to be lower than that of mammalian enzyme.     

Cloning and expression of chitin deacetylase gene from a Deuteromycete, Colletotrichum lindemuthianum

The chitin deacetylase gene was cloned from cDNA of Colletotrichum lindemuthianum ATCC 56676, and the open reading frame consisted of a possible prepro-sequence of 27 amino acids at the N-terminus and a mature chitin deacetylase. The deduced amino acid sequence of the mature enzyme revealed 26% identity and 46% similarity with a chitin deacetylase from Mucor rouxii. The molecular mass of the protein estimated from the amino acid sequence data was 24.3 kDa, which was in good agreement with the MALDI-TOF MS analysis data of the purified protein (24.17-24.36 kDa). The gene product was overexpressed in Escherichia coli cells as a fusion protein with six histidine residues at its C-terminus. The fusion protein formed inclusion bodies, but chitin deacetylase activity was restored from the inclusion bodies by a simple renaturation step with 8 M urea treatment. The recombinant enzyme was purified by affinity chromatography and gel filtration steps, and had a final specific activity of 4.22 units mg(-1) of protein. Trypsin digestion of the recombinant enzyme resulted in 2.1-fold increase in activity, suggesting that the removal of the prepro-domain from the recombinant enzyme resulted in an increase in its activity.     

Antioxidative activity of whey protein hydrolysates in a liposomal system

Whey protein isolate (WPI) with or without preheating (90 degrees C for 5 min) was hydrolyzed for 0.5 to 6 h using four pure enzymes (pepsin, papain, trypsin, and chymotrypsin) and three commercial crude proteases. After determining the degree of hydrolysis, the hydrolysates were incubated (37 degrees C, 1 h) with a liposome oxidizing system (50 mM FeCl3/0.1 mM ascorbate, pH 7.0). Lipid oxidation was measured by determining the concentrations of TBA-reactive substances (TBARS). The degree of hydrolysis of WPI ranged from 4 to 37% depending on the enzymes used and whether the substrate was heated or not. WPI hydrolysates prepared by pure enzyme treatments did not prevent TBARS formation in the oxidative model system, but WPI hydrolyzed by the commercial crude enzymes, especially protease F, exhibited antioxidant activity. The antioxidative potential of hydrolyzed WPI was not affected by the degree of hydrolysis, and it was improved by preheat treatment in only some samples.     

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.     

A novel selective nucleoside phosphorylating enzyme from Morganella morganii

A selective nucleoside phosphorylating enzyme was purified to homogeneity from Morganella morganii NCIMB10466 crude extract. The enzyme appeared to consist of six subunits identical in molecular mass (M_r 25,000). It phosphorylated various nucleosides at the 5′-position to produce nucleoside-5′-monophosphates using pyrophosphate as the phosphate source. Energy-rich compounds, such as carbamylphosphate and acetylphosphate, were also very effective phosphate donors. The enzyme also exhibited phosphatase activity, and dephosphorylated various phosphate esters, but had a weak effect on nucleoside-3′-monophosphates. Based on the results of the kinetic study, the enzyme appeared to be an acid phosphatase. Its activity was partly inhibited by sulfhydryl reagents and heavy metal ions, but not by chelating reagents such as EDTA. Using the purified enzyme, 32.6 mM 5′-IMP was synthesized from inosine with a 41% molar yield, but the synthesized 5′-IMP was hydrolyzed back to inosine and phosphate as the reaction time was extended.     

Plate assay for endo-beta-N-acetylglucosaminidase activity using a chromogenic substrate synthesized by transglycosylation with Arthrobacter endo-beta-N-acetylglucosaminidase

The transglycosylation activity of Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A) was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-5-bromo-4-chloro-3-indolyl-beta-glucoside (Man6GlcNAc-Glc-beta-X). Various endo-beta-N-acetylglucosaminidases hydrolyzed this oligosaccharide, producing Man6GlcNAc and Glc-beta-X. The E. coli strains coexpressing Endo-A and beta-glucosidase formed blue colonies in the presence of Man6GlcNAc-Glc-beta-X. Therefore, endo-beta-N-acetylglucosaminidase activity could be directly detected by the plate assay. This simple plate assay is useful for screening microorganisms for endo-beta-N-acetylglucosaminidase activity.     

Purification and characterization of a novel extracellular beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from ezo abalone Haliotis discus hannai

A novel extracellular alkaline stable beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from the ezo abalone Haliotis discus hannai was purified by ammonium sulfate precipitation, DEAE-Sepharose FF ion exchange chromatography and Sephacryl S-200HR gel filtration. The molecular weight of the purified enzyme was estimated to be 71 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was very stable at pH 5.3 to 11.5 but unstable at pH 4.0 to 4.5. The optimum temperature and thermostability of the enzyme increased in the presence of CaC1, The enzyme hydrolyzed R-1,3-glucan from marine organisms, but did not show activity against any other beta-1,3-glucans. The major hydrolysis products of beta-1,3-glucan from Laminaria digitata and Eisenia bicyclis were laminaritriose and laminaritetraose, respectively. The N-terminal amino acid sequence of the purified enzyme was similar to that of several beta-1,3-glucanases in the glycoside hydrolase family 16.     

Purification and Characterization of a β-Amylase of Hendersonula toruloidea

β-Amylase produced by Hendersonula toruloidea was purified to homogeneity by salting out with ammonium sulphate, ion-exchange chromatography on DEAE-cellulose and gel-filtration on Sephadex G-75. The relative molecular mass of the enzyme was estimated to be 60,000 by gel filtration. The enzyme was optimally active at pH 6.0 and 60°C, stable between pH 6 and 8 (24 h) and retained 74% activity at 70°C (30 min). It was strongly activated by Na⁺ but inhibited by Hg²⁺, Zn²⁺ and Cu²⁺. The enzyme hydrolyzed amylopectin (Km 0.42 mg/ml) forming maltose, maltotetraose and unidentified maltooligosaccharide, and hydrolyzed soluble starch (Km 0.3 mg/ml) and glycogen (Km 0.5 mg/ml) forming maltose and unidentified maltooligosaccharide.     

Isolation and characterization of proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614

A dimeric, 90 kDa subunit intracellular proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614 was purified to homogeneity by chromatography on hydroxyapatite, Sephacryl 200, Phenyl Superose and Mono Q. The enzyme was specific on Pro-p-nitroanilide and Pro-X dipeptides. It hydrolyzed 2 fragments of hormone oligopeptides with an N-terminal proline: bradykinin, f2-7 and substance P, f4-11. A number of oligopeptides containing 5-11 amino acids residues and proline at the penultimate position from N-terminus or other internal position were not hydrolyzed. The enzyme was most active at pH 7-7.5 and at 37-40 degrees C but it retained 9% of maximal activity at pH 5.5 and >12% of maximal activity at 10 or 60 degrees C. The enzyme was inhibited strongly by the serine protease inhibitor 3,4-dichloroisocoumarin, and stimulated markedly by 1 mol/l of NaCl. The results indicate that the enzyme may lead to the accumulation of proline from dipeptides and oligopeptides during the ripening of cheese.