Biomphalaria glabrata: lysozyme activities in the hemolymph, digestive gland, and headfoot of the intermediate host of Schistosoma mansoni.

Lysozyme (mucopeptide N-acetylmuramylhydrolase EC 3.2.1.17) activity has been found in the hemolymph, digestive gland, and headfoot extracts of Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. Partial purification of the bacteriolytic enzyme was attained by gel chromatography on Sephacryl S-200 and active lytic fractions were concentrated by Amicon filtration. The properties of the lytic enzymes from the three tissue extracts were identical. Enzyme activity was determined by the rate of lysis of cell wall suspension of Micrococcus lysodeikticus. Lysis of the cell walls was accompanied by a release of reducing sugar groups and N-acetylhexosamines. The enzyme was stable to heating at 100 C for 2 min and had an optimum activity at pH 4.5 to 5.0 in 0.066 M glycylglycine buffer. Low concentrations (5 mM) of NaCl, KCl, and LiCl increased the activity of the enzyme, whereas high concentrations (25 mM) of the same ions caused about 50% inhibition of the enzyme activity. MgCl₂ and CaCl₂ also inhibited the enzyme activity. Addition of 1 mM EDTA or EGTA resulted in about a twofold increase in enzyme activity. Double reciprocal plots of enzyme velocities and substrate concentrations yielded an apparent Michaelis-Menten constant (K_m) of 0.05 ± 0.01 mg/ml of M. lysodeikticus.     

Heat-Moisture Treatment of Cereal Starch Observed by X-ray Diffraction

Chitinases I and II were purified from the culture supernatant of Aeromonas sp. 10S-24 by ammonium sulfate precipitation, SP-Sephadex C-50 chromatography, Sephacryl S-200 gel filtration, and chromatofocusing. Both enzymes were most active at pH 4.0 and the optimum temperature for I and II were 50°C and 60°C. Chitinase I was stable at pHs between 4 and 9 and at temperatures below 50°C and chitinase II was stable at pHs between 5 and 7 and at temperatures below 45°C. The molecular weights were estimated by 8D8 polyacrylamide gel electrophoresis to be 112,000 and 115,000 for I and II respectively, while gel filtration showed the molecular weight to be 114,000 for both types of the enzyme. The pIs for I and II were 7.9 and 8.1, respectively. The activities of both enzymes were inhibited by Ag⁺ and iodoacetic acid.     

Synthesis of (±)-Xanthoxins

The enzymatic properties of P2-2 enzyme were determined by using cells of M. radiodurans. The enzyme was: most active at 60°C incubation temperature, stable at 40°C in neutral buffer, and inactivated by heating at 80°C for 15min. Maximal lytic activity occurred at pH 8.5 in Tris-HCl buffer. The range of enzyme stability was between pH 5.5 and 8. Bivalent metal ions, p-chloromercuribenzoate and monoiodo acetate inhibited lytic activity. The molecular weight was estimated to be 16,000 daltons by gel filtration on Sephadex G-75. The enzymatic digestion of peptidoglycans from the cell walls of M. radiodurans and M. lysodeikticus liberated free amino groups, but neither reducing groups nor N-acetylhexosamine, indicating that the enzyme was an endopeptidase. From analysis of the N-terminal amino acids of the digests, it is suggested that the P2-2 enzyme cleaves the peptide bond at the carboxyl group of D-alanine in peptidoglycan.     

Purification and partial characterisation of tentatively classified acid phosphatase from the earthworm Eisenia veneta

In order to use leakage of lysosomal acid phosphatase (AP) as a biomarker of stress to earthworms, more information about AP’s in earthworms are needed. This paper describes the details about tentatively classified APs in the earthworm Eisenia veneta. Two isoenzymes (enzyme I and II) of acid phosphatase (AP) and one alkaline phosphatase (enzyme III) from the earthworm E. veneta were separated by gel filtration. All three enzymes were further purified and concentrated on a Con A Sepharose 4B column. Enzyme I was inhibited by tartrate, showed an optimal pH range between 4.0 and 5.0 and was assumed to be of lysosomal origin. Enzyme II was the major enzyme showing the highest activity of the three enzymes. It was expected to be a lysosomal AP under physiological conditions. Enzyme II had a molecular mass 113 kDa and was composed of apparently identical polypeptide chains of 36 kDa each. This enzyme was inhibited by tartrate, showed an optimal pH in the range 6.0–7.5 and was slowly degraded at temperatures above 40°C. Enzyme III is not inhibited by tartrate and has a pH-optimum >9. The subcellular location under physiological conditions was assumed to be the cytosol.     

Effects of Alloxan Diabetes and Hypophysectomy on Growth,and Plasma and Liver Free Amino Acids of Rats Fed Excess Glycine Diets

Three chitinases (EC 3.2.1.14) were purified from yam, Dioscorea opposita THUMB, by fractionation with ammonium sulfate, chromatographies on DEAE-Cellulose and DEAE-Sephadex A-50, chromatofocusing and gel filtration on Bio-Gel P-60. The purified enzymes (E-l, E-2 and E-3) showed single bands on sodium dodecylsulfate polyacrylamide gel electrophoresis, and the molecular weights were estimated to be 33,500. The pIs were 4.05 (E-l), 4.0 (E-2) and 3.8 (E-3). All enzymes were glycoproteins and the neutral sugar contents were 3.6% (E-l), 3.6 (E-2) and 0.9% (E-3). The N-terminal amino acids of E-l and E-3 were the same and determined to be histidine. All enzymes hydrolyzed glycolchitin, but not p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside or Micrococcus lysodeikticus cell walls. E-l and E-3 were stable in the pH range of 5 ~ 11, and below 60°C. These enzymes showed two optimum pHs around 3.5 and 8.0 or 8.5 with glycolchitin as substrate.     

Lytic Enzyme from Streptomyces globisporus 1829 Strain

The maximum yield of lytic enzyme was obtained from shake flask cultures of Streptomyces globisporus 1829 which were grown at 30°DC for 48 hr in a medium containing 2% dextrin, 0.5% soybean meal, 0.2% polypeptone, 0.5% Na₂HP0₄. 12H₂0, 0.1% KH₂P0₄, 0.1% MgS0₄·7H₂0, 1.0% NaCI and 0.02% CaCl₂, pH 7.5. The activity of successively transferred substrains of St. globisporus 1829 gradually decreased. However, a mutant strain obtained by ultra-violet irradiation has been shown not to have lost any lytic activity for 2 years. The enzyme exhibited maximum activity at 60°C in the pH range of 6 to 6.5 and was lytic against the intact cells of Streptococci, Lactobacilli and Bacilli but inert against the intact cells of Staphylococcus aureus and Micrococcus lysodeikticus.     

An Improved Assay Method for Antifouling Substances Using the Blue Mussel,Mytilus edulis

The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37°C and pH 8.5 in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10 mm Tris-HCl, 7 mm 2-mercaptoethanol, 7 mm MnCl₂ and 50 mm NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5′-GACGTC-3′, cuts between T and C and produces a 3′-tetranucleotide extension in the presence of MnCl₂, as it does in the presence of MgCl₂.     

Purification and Properties of Pectinesterase from Acrocylindrium

The crude enzyme fraction of precipitates resulting from the addition of 70% alcohol to the culture filtrate of A. lunatus was separated by CM-Sephadex and Sephadex G-75 chromatography into 13 fractions having lytic activity for M. radiodurans, M. lysodeikticus and P. radiora. Five of the fractions showed similar lytic activity spectra, but the other fractions were separated by the specificities of their lytic activities. This result indicates that the wide lytic spectrum of the crude enzyme against microorganisms is attributable to the action of many lytic enzymes. All fractions, except for P2-2 fraction (designated as the P2-2. enzyme), contained at least two proteins as determined by disc gel electrophoresis. The P2-2 enzyme was purified 34-fold by rechromatography on Sephadex G-75, and appeared to be homogeneous on disc gel electrophoresis. The enzyme was able to lyse intact cells of M. radiodurans and M. lysodeikticus without detergent, and those of P. radiora with detergent, but was not able to digest casein.     

Studies on Bacteriolytic Enzymes

About 100 soil samples were subjected to screening for microorganisms which were capable of producing lytic enzyme toward Staphylococcus aureus. A strain belonging to Streptomyces was isolated and found to produce lytic enzyme(s) noninduciblly, when grown aerobically at 37°C for 25 hr in a medium containing 7.5% soybean cake extract, 2% dextrin, 0.6% K₂HPO₄, 0.02% each of MgSO₄·7H₂O and KCl, pH 7.0. The crude enzyme preparation was active at pH values of 8.5 and 5.8 toward S. aureus, B. subtilis, L. bulgaricus and Str. faecalis but was completely inert against M. lysodeikticus, indicating the enzyme(s) to be distinguished from other bacteriolytic enzymes of Streptomyces so far reported.     

Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.     

Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1

Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn²⁺ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure. Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

Serratia marcescens-Lytic Enzyme Produced by Micromonospora sp. Strain No. 152

A strain of Micromonospora sp. producing a lytic enzyme toward Serratia marcescens was isolated from soil. The lytic enzyme, called 152-enzyme, was purified from the culture filtrate by salting-out with ammonium sulfate, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-75. The molecular weight of 152-enzyme was 17,000 and the isoelectric point was pH 7.3. The 152-enzyme showed lytic activity toward S. marcescens, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Bacillus subtilis, but was completely intert toward Staphylococcus aureus. The enzyme also showed caseinolytic activity. The lytic and caseinolytic activities of 152-enzyme were maximum around pH 11.0 and at 60°C. Both activities were inhibited by DFP and API-2c. Liberation of amino groups from cell walls of P. aeruginosa by incubation with 152-enzyme suggested that the enzyme was a kind of cell wall-lytic peptidase.     

Purification and Some Properties of Two Aminopeptidases from Sardines

Two fish aminopeptidases designated as aminopeptidases I and II were purified by DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and isoelectric focusing. The final preparations of enzymes I and II were judged nearly homogenous by polyacrylamide gel I, electrophoresis. The molecular weights of enzymes I and II were determined by gel filtration to be 370,000 and 320,000, respectively. The isoelectric points were 4.1 (I) and 4.8 (II), Both enzymes were inhibited by EDTA and activated by Co⁺⁺. Bestatin could inhibit enzyme I but not enzyme II. Enzymes I and II rapidly hydrolyzed not only synthetic substrates containing alanine or leucine but also di-, tri-, and tetra-alanine. Judged from all of these properties, sardine aminopeptidases resemble human alanine aminopeptidase. Enzyme I retained more than 70% of its original activity in 15% NaCl, suggesting the enzyme participates in hydrolyzing fish proteins and peptides during fish sauce production.     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Formation of ε-(2-Formyl-5-hydroxy-methyl-pyrrol-1-yl)-l-norleucine in the Maillard Reaction between d-Glucose and l-Lysine

An exo-β-1,3-glucanase was purified from the commercial enzyme preparation “Kitalase” which is a yeast cell wall lytic enzyme preparation. The purification procedures consisted of following steps: ammonium sulfate fraction, SP-Sephadex C-50 and CM-Cellulose C-32 column chromatography, and Sephadex G-100 gel filtration. The optimum pH value was 5.8, and the optimum temperature was 55°C. The enzyme was stable in the pH range of 5.1 to 9.8 and at temperatures below 53°C. The isoelectric point and the molecular weight were estimated to be pH 9.3 and 73000, respectively. The enzyme was shown to bypass β-1,6-linkaged branches to cleave β-1,3-linkages when scleroglucan was used as substrate. The Km values for laminariri, laminari-pentaose, laminaritetraose and laminaritriose were 0.16, 2.01, 2.24 and 1.34 mM, respectively.     

Nitrogen fixation associated with the rice plant grown in water culture

Two bacteriolytic enzymes were produced when Hartmanella glebae was grown in the presence of both Enterobacter aerogenes and Alcaligenes faecalis. The identification of enzyme I as N-acetylmuramidase was reported earlier. Enzyme II was purified by gel filtration on a Bio-Gel A column. A recovery of 68.76% with 72.3-fold purification was obtained. It was found that 5 and 10 mM MgCl2 significantly increased the bacteriolytic activity. It is a basic protein. The cell walls of Micrococcus lysodeikticus were lysed by the enzyme, and the products of digestion were purified by Amberlite CG-120 and Sephadex G-15 chromatography to facilitate the detection of amino sugars. After reduction of the oligosaccharides with sodium borohydride and acid hydrolysis, the amino sugars were identified by paper chromatography. It was found that enzyme II cleaved the glycosidic bond between N-acetylmuramic and and N-acetylglucosamine of the peptidoglycan moiety of the cell walls. Thus, the enzyme was identified as endo-beta-N-acetylmuramidase.     

Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli

Summary Several hundred bacterial isolates were screened for bacteriolytic activity by growing them on agar medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells as the substrate. A Bacillus sp. producing the largest lytic zone was selected. A genomic bank of this selected bacterium was constructed in the multi-functional vector pTZ18R, with partial SauIIIA DNA fragments inserted at the SalI restriction site. Screening of 800 colonies of this bank for cell lysis gave 5 recombinants exhibiting lytic activity, as detected by analysis of extracts of sonicated Escherichia coli cells on denaturing polyacrylamide gels containing autoclaved, lyophilized M. lysodeikticus cells as the substrate. One clone (pBH2500), expressed inE. coli strain NM522, was found to code for a lytic enzyme corresponding, in molecular weight, to the 27 kDa Bacillus sp hydrolase. This clone with an insertion of 2.5 kb was then subcloned as a 929 bp EcoRI-SauIIIA fragment in pTZ18R (pBH929) and showed higher cell lytic activity. A unique open reading frame for a protein of 251 amino acids, followed by a putative terminator sequence, was found after a consensus ribosome binding site. A putative leader sequence was identified in the first 37 amino acids. One truncated subclone (pBH703), corresponding to 196 out of 251 residues from the protein N-terminal end, still possessed lytic activity.     

Purification and Properties of a Dehyrodicaffeic Acid Dilactone-forming Enzyme from a Mushroom,Inonotus sp. K-1410

A dehydrodicaffeic acid dilactone-forming enzyme was purified from the mycelia of a mushroom, Inonotus sp. K-1410 by calcium acetate treatment, ammonium sulfate precipitation and column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and caffeic acid-bound AH-Sepharose 4B. The enzyme was purified about 1200-fold from a crude extract and shown to be almost completely homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 39,000. The optimal pH for the enzymic conversion of caffeic acid to dehydrodicaffeic acid dilactone is around 6.0. The enzyme is stable up to 60°C and preincubation of the enzyme at 40°C for 10 min gives 1.5-fold activation compared with preincubation at 0°C. The optimal temperature for the enzyme reaction is 40°C.     

Purification and Properties of an Aminopeptidase from a Protamine-degrading Marine Bacterium

A protamine-degrading marine bacterium was isolated from marine soil and identified as Aevomonas salmonicida subsp. based on its taxonomical characteristics. An alanine-specific aminopeptidase, called aminopeptidase K, from an extract of the strain was purified and characterized. The aminopeptidase K was purified about 80-fold by fractionation with ammonium sulfate and column chromatography on QA-52 cellulose, Phenyl Superose and Superose 12. The purified enzyme is composed of 6 subunits of 86 kDa with a molecular mass of 520 kDa according to gel filtration and SDS–PAGE. The N-terminal sequence of the enzyme was H · Gly-Gln-GIn-Pro-Gln-Ile-Lys-Try-Tyr-His-Asp-Tyr-Asp-Ala-Pro-Asp-Tyr-Tyr-Ile-Thr-. It is inhibited by monoiodoacetate, N-ethylmaleimide, and puromycin. The Michaelis constant (K_m) and the maximal rate of hydrolysis (V_max) were, respectively, 0.28 mm and 49.4 μmol/min/mg for the l-Ala-β-naphthylamide substrate. The optimum pH and optimum temperature were 6.5 and 45°C, respectively. The purified enzyme was highly specific to l-Ala-β-naphthylamide.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.