Purification and characterization of a novel beta-agarase from an alkalophilic bacterium, Alteromonas sp. E-1

A novel beta-agarase (EC 3.2.1.81) was purified from an agar-degrading alkalophilic bacterium, Alteromonas sp. E-1 isolated from the soil. This enzyme was obtained from a cell-free extract after sonication and purified 40.9-fold through treatment with streptomycin, ammonium sulfate fractionation and successive chromatography on anion-exchange and gel filtration columns. The molecular weight was estimated to be 82 kDa by SDS-polyacrylamide gel electrophoresis and 180 kDa by Superdex 200 gel filtration. The enzyme was inhibited by Mn2+, Cu2+, Fe2+, Zn2+ and Hg2+, and activated by K+, Na+ and EDTA, and its optimum pH and temperature for agarose degradation were 7.5 and 40 degrees C, respectively. This beta-agarase hydrolyzed agarose with rapid reduction of viscosity, and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose] was detected from the early stage of the reaction. Neoagarobiose as the final product was selectively released from agarose, neoagarohexaose and neoagarotetraose by the reaction with this beta-agarase. This observation was different from that of other beta-agarases which produced mixtures of neoagarobiose and neoagarotetraose as the final hydrolysis products. The N-terminal amino acid sequence of this beta-agarase shows no homology to those of other beta-agarases that were so far reported.     

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.     

Production of cyclic tetrasaccharide from starch using a novel enzyme system from Bacillus globisporus C11

Production of cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS, cyclic tetrasaccharide) from starch was attempted using 1,6-alpha-glucosyltransferase (6GT) and 1,3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus C11. The optimal conditions for production from partially hydrolyzed starch were as follows: substrate concentration, 3%; pH 6-7; temperature, 30 degrees C; 6GT, 1 unit/g-dry solid (DS); IMT, 10 units/g-DS. The production of CTS was demonstrated and 544 g of CTS hydrate crystal powders were obtained from 3500 g of partially hydrolyzed starch. Two major by-products were also isolated from the reaction mixture and identified as the branched derivatives of CTSs, 4-O-alpha-D-glucopyranosyl-CTS and 3-O-alpha-isomaltosyl-CTS.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.     

Purification and characterization of cis-aconitic acid decarboxylase from Aspergillus terreus TN484-M1

cis-Aconitic acid decarboxylase (CAD) was assumed to be a key enzyme in the production of itaconic acid by comparing the activity of CAD from Aspergillus terreus TN484-M1 with that of CAD from the low-itaconate yielding strain Aspergillus terreus CM85J. The constitutive CAD was purified to homogeneity from A. terreus TN484-M1 by ammonium sulfate fractionation, and column chromatography on DEAE-toyopearl, Butyl-toyopearl, and Sephacryl S200HR, and then characterized. A molecular mass of 55 kDa for the native enzyme was determined by SDS-PAGE. The enzymic activity was optimal at a pH of 6.2 and temperature of 45 degrees C. The K(m) value for cis-aconitic acid was determined as 2.45 mM (pH 6.2, 37 degrees C). The enzyme was completely inactivated by Hg+, Cu2+, Zn2+, p-chloromercuribenzoate, and 5,5'-dithio-bis(2-nitrobenzoate).     

Purification and characterization of an alkaline manganese peroxidase from Aspergillus terreus LD-1

We report that Aspergillus terreus LD-1 produces an extracellular ligninolytic enzyme, manganese peroxidase (MnP), that reacts under alkaline conditions. This MnP was purified 13.1-fold from the culture supernatant to elicit a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of this MnP was estimated as either 43 kDa by SDS-PAGE or 44 kDa by gel permeation chromatography, suggesting a monomeric structure. The optimum pH and temperature of this MnP are 12.5 and 37 degrees C, respectively. This MnP is stable in the pH range 11.0 to 12.5 and also up to 40 degrees C. The K(m) values of this MnP for hydrogen peroxide, 2,6-dimethoxyphenol (2,6-DMP) and Mn2+ were 320 microM, 20 microM and 33 microM at pH 12.5, respectively. The activity of the MnP is completely inhibited by Hg2+, Pb2+, Ag+ and lactate. On the other hand, the MnP is activated by oxalate, maleate and fumarate. Maleate at 5 mM increased the MnP activity 5-fold. EDTA at 1 mM inhibited the MnP activity completely, but this inhibition was not observed in the presence of 1 mM Fe2+.     

Purification and properties of hydrogen sulfide oxidase from Bacillus sp. BN53-1

A hydrogen sulfide oxidase was purified to homogeneity from the heterotroph Bacillus sp. BN53-1 isolated from pig feces compost. The enzyme was found to be a monomer with a M(r) value of approximately 37 kDa. It required FAD for its activity, which was not replaced by FMN. The optimum reaction pH and temperature were 7.5 and 40 degrees C, respectively. The enzyme was stable between pH 6.0 and 7.0 and up to 30 degrees C. Its activity was stimulated by Ca2+ and Mn2+ and inhibited by Al3+, dithiothreitol, and 2-mercaptoethanol. The main product was elemental sulfur, and H2O2 was not detected. The N-terminal sequence of the enzyme showed similarity to other FAD-requiring enzymes.     

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.     

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.     

Purification and characterization of N-Acetylglucosamine 6-phosphate deacetylase from Thermus caldophilus

N-Acetylglucosamine 6-phosphate deacetylase [EC 3.5.1.25] was purified and biochemically characterized from an extreme thermophile, Thermus caldophilus GK24. The optimum temperature and pH of the enzyme were 80 degrees C and 7.5, respectively. The enzyme is a tetramer composed of identical 45 kDa subunits. The N-terminal amino acid sequence of the purified enzyme was determined to be MSVDLKTLHRRHVLTP. It hydrolyzed GlcNAc-6-P, but not GlcNAc-1-P or chitin oligosaccharides. The deacetylase activity was completely inhibited by the addition of 1 mM Cu2+, but moderately activated by that of 1 mM Mn2+ and Co2+. Within 2 h of reaction, 2 mM GlcNAc-6-P was completely hydrolyzed to GlcN-6-P and acetate by the action of the deacetylase.     

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.     

Purification and properties of a phytase from Candida krusei WZ-001

A phytase from Candida krusei WZ-001 isolated from soil was purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The phytase is composed of two different subunits with molecular masses of 116 kDa and 31 kDa on SDS-PAGE (or 120 kDa and 30 kDa on gel chromatography), with the larger subunit having a glycosylation rate of around 35%. The phytase has an optimum pH of 4.6, an optimum temperature of 40 degrees C and a pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, p-chroloromercuribenzoate (pCMB) and phenylmethylsulfonyl fluoride (PMSF). The phytase displayed a broad substrate specificity and the K(m) for phytate was 0.03 mM. Phytate was sequentially hydrolyzed by the phytase. Furthermore, 1D and 2D NMR analyses and bioassay of myoinositol indicated that the end hydrolysis product of phytate was myoinositol 2-monophosphate.     

木薯块根中β-葡萄糖苷酶的分离纯化及酶学性质

研究了木薯块根中β-葡萄糖苷酶的分离纯化及酶学性质。以缓冲液从木薯块根中获得粗提酶液,粗酶酶活力为9.37 U/g木薯干重;再分别通过丙酮沉淀、离子交换层析和凝胶过滤层析进行纯化,β-葡萄糖苷酶酶活力为1.14 U/g木薯干重,经纯化β-葡萄糖苷酶纯度提高了14.62倍,总活力回收率为12.14%,电泳测得其分子量约70 kDa。该酶米氏常数K_m为3.60 mmol/L,V_max为12.36μmol/(min·mg protein);其最适pH为7.0,pH在6.0～8.0之间有较好的稳定性;在40℃以内有良好稳定性,在4℃存放30 d酶活力剩余81.78%。Mn²⁺和K⁺对酶有一定的促进作用,Al³⁺、Cu²⁺、Mg²⁺、Zn²⁺、Ca²⁺、Ba²⁺、Na⁺、尿素和SDS对酶没有显著影响(P>0.05),而Fe³⁺、F...     

Immobilization of β-galactosidase by bioaffinity adsorption on concanavalin A layered calcium alginate–starch hybrid beads for the hydrolysis of lactose from whey/milk

Aspergillus oryzae β-galactosidase was immobilized on the surface of a novel bioaffinity support: concanavalin A layered calcium alginate–starch beads. The maximum activity of the immobilized β-galactosidase was obtained at 60 °C, approximately 10 degrees higher than that of the free enzyme. The immobilized β-galactosidase exhibited significantly higher stability to heat, urea, MgCl₂, and CaCl₂ than the free enzyme. An enhancement of the activity of immobilized β-galactosidase by up to 5.0% MgCl₂ was seen, whereas the activity of the free enzyme decreased above 3.0% MgCl₂. Immobilized β-galactosidase retained 61%, 50% and 43% activity in the presence of 5% CaCl₂, 5% galactose and 4 m urea, respectively, when incubated for 1 h at 37 °C. The immobilized β-galactosidase had a much higher K_iapp value than the free enzyme, which indicated less susceptibility to product inhibition by galactose. The immobilized β-galactosidase preparation was superior to the free enzyme in hydrolysing lactose in whey or milk in a batch process: it hydrolyzed 89% of the lactose in whey in 3 h and 79% of the lactose in milk in 4 h. The immobilized β-galactosidase retained 61% of its original activity after 2 months storage at 4 °C, while the soluble enzyme showed only 37% of the initial activity under identical conditions.     

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

An extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43,000 and contained 2 g atom Ca/mol. It was active over a pH range 4.8-9.5 and had optimum activity on casein at pH 7.0 with Km = 0.17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 degrees C; above 50 degrees C activity declined rapidly, but significant activity persisted at 4 degrees C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.     

Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF α-GALACTOSIDASE FROM ASPERGILLUS ORYZAE

A crude extract of α-galactosidase obtained by fermenting Aspergillus oryzae on wheat bran was purified 35 fold by ethanol precipitation, gel filtration and ion-exchange chromatography. The final preparation was free of protease activity but contained invertase activity. The molecular weight of the enzyme was estimated as 64,000 daltons. The pH and temperature optima were 4.0 and 60°C, respectively. The enzyme was stable over the pH range 3–7.5 and at temperatures up to 55°C (pH 4.0). The K_m values for p-nitrophenyl α-Dgalactopyranoside (PNPG) and raffinose were 4.0 × 10⁻⁴M and 1 × 10⁻²M, respectively. Divalent cations were not required for activity. More than 80% of the oligosaccharides in soy milk were hydrolyzed after 3 h at 50°C using 0.113 PNPG units/mL milk.     

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.     

Cold-active lipolytic activity of psychrotrophic Acinetobacter sp. strain no. 6

A lipolytic bacterium, strain no. 6, was isolated from Siberian tundra soil. It was a gram-negative coccoid rod capable of growing at 4 degrees C but not at 37 degrees C and was identified as a psychrotrophic strain of the genus Acinetobacter. Strain no. 6 extracellularly produced a lipolytic enzyme that efficiently hydrolyzed triglycerides such as soybean oil during bacterial growth even at 4 degrees C; it degraded 60% of added soybean oil (initial concentration, 1% w/v) after cultivation in LB medium at 4 degrees C for 7 d. Thus, the bacterium is potentially applicable to in-situ bioremediation or bioaugumentation of fat-contaminated cold environments. We partially purified the lipolytic enzyme from the culture filtrate by acetone fractionation and characterized it. The enzyme preparation contained a single species of cold-active lipase with significant activity at 4 degrees C, which was 57% of the activity at the optimum temperature (20 degrees C). The enzyme showed a broad specificity toward the acyl group (C8-C16) of substrate ethyl esters.     

Identification of cellulolytic bacteria associated with tunic softness syndrome in the sea squirt, Halocynthia roretzi

The edible ascidian, sea squirt Halocynthia roretzi (Drasche) is marine invertebrate that is a valuable source of foods and bioactive compounds. A severe disease of the sea squirt characterized by degeneration of tunic fibers formed of bundled cellulose microfibrils has occurred. We hypothesized that bacteria lyse the cellulose fibril structure, cellulase activity may be a causative agent of the disease. Among the bacteria isolated from diseased sea squirt, Pseudoalteromonas sp. NO3 had cellulase activity based on a Congo red overlay assay and starch-reducing activity. Sea squirts exhibited 40–100% cumulative mortality after injection with Pseudoalteromonas sp. NO3 using doses of 2×10⁶?2×10⁸ colony forming unit (CFU)/individual. Dead sea squirts possess thinner and ruptured tunics, which were similar to natural outbreaks. These results suggest that Pseudoalteromonas sp. NO3 possessing cellulase activity is one of the causes of tunic softness syndrome in sea squirt.