SAF,a New Cell Aggregation Factor Produced by Streptomyces murinus Strain No. A-2805

We searched for a new cell aggregation factor, and found one we called SAF in the mycelia of a strain of Streptomyces murinus. SAF was purified by active carbon and ion exchange column chromatographies and gel filtration of Sepharose 2B from the homogenized mycelia by sonication. SAF was stable from pH 7 to 9 at 37°C and up to 40°C at pH 8.0. The aggregation activity of SAF was maximum around pH 8.0 at 30°C, and the factor required for its activity metallic ions such as calcium and manganese. The aggregation activity of SAF was inhibited by laminarin, but it was not influenced by various other saccharides. SAF aggregated E. coli, S. aureus, M. luteus, sarcoma 180, and HeLa cells as well as S. marcescens, above all, its highest activity was toward B. subtilis, but P. vulgaris, P. aeruginosa, C. albicans, each type of human erythrocytes, and hepatoma 109A cells were quite resistant to SAF. These properties has proved that SAF is completely different from the other aggregation factors so far reported.     

Purification of saliva agglutinin of<Emphasis Type="Italic"> Streptococcus intermedius</Emphasis> and its association with bacterial aggregation and adherence

Streptococcus intermedius strain 1208-1 cells were aggregated in the presence of saliva. The saliva agglutinin was purified by centrifugation, filtration, and gel filtration. SDS-PAGE analyses indicated that the purified agglutinin consisted of two high-molecular-mass proteins. Aggregation was dependent on calcium over pH 5.5, with 1 mM being the most effective concentration. Boiling inactivated purified agglutinin. S. intermedius strain 3 and Streptococcus mutans strain 1 were aggregated in the purified agglutinin. After adsorption with strain 1208-1 cells, the saliva sample did not exhibit any aggregation activity, and the agglutinin bands were no longer visible by SDS-PAGE. Adherence analyses demonstrated that the purified agglutinin immobilized on the surfaces of polystyrene wells, actinomyces cells, and apatite beads accounted for the binding of streptococcus cells. Agglutinin also effectively inhibited adherence to apatite beads coated with native saliva.     

Isolation and Purification of Ascorbate Oxidase from Acremonium sp. HI-25

A screening test was undertaken to isolate microorganisms that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of a fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions, optimum conditions for enzyme production were found; strain HI-25 was aerobically cultured by a jar fermenter at 25°C in a medium containing 5% glycerol, 2% defatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH₂PO₄, 0.02% MgSO₄ ·7H₂O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80,000 by SDS polyacrylamide gel electrophoresis and 76,000 by native gel filtration. This enzyme was most active at pH 4.0, 45°C, and was most stable between pH 6.0–10.0 and at temperatures below 60°C.     

Purification and characterization of tannase from Paecilomyces variotii: hydrolysis of tannic acid using immobilized tannase

An extracellular tannase (tannin acyl hydrolase) was isolated from Paecilomyces variotii and purified from cell-free culture filtrate using ammonium sulfate precipitation followed by ion exchange and gel filtration chromatography. Fractional precipitation of the culture filtrate with ammonium sulfate yielded 78.7% with 13.6-folds purification, and diethylaminoethyl–cellulose column chromatography and gel filtration showed 19.4-folds and 30.5-folds purifications, respectively. Molecular mass of tannase was found 149.8 kDa through native polyacrylamide gel electrophoresis (PAGE) analysis. Sodium dodecyl sulphate–PAGE revealed that the purified tannase was a monomeric enzyme with a molecular mass of 45 kDa. Temperature of 30 to 50°C and pH of 5.0 to 7.0 were optimum for tannase activity and stability. Tannase immobilized on alginate beads could hydrolyze tannic acid even after extensive reuse and retained about 85% of the initial activity. Thin layer chromatography, high performance liquid chromatography, and ¹H-nuclear magnetic resonance spectral analysis confirmed that gallic acid was formed as a byproduct during hydrolysis of tannic acid.     

The characterization of a pectin-degrading enzyme from Aspergillus oryzae grown on passion fruit peel as the carbon source and the evaluation of its potential for industrial applications

An extracellular pectinase (PEC-I) was isolated from the crude extract of Aspergillus oryzae when grown on passion fruit peel (PFP) as the carbon source and partially purified by ultra filtration, gel filtration and ion-exchange chromatography procedures. Pectinase activity was predominantly found in the retentate. The pectinase from retentate (PEC-Ret) was most active at 50?°C and pH 7.0 and stable at 50?°C with a half-life of approximately 8?h. PEC-I showed higher activity at pH 4.5 and 55?°C, 70?°C and 75?°C and was inhibited by cations (Ag⁺, Fe²⁺, Fe³⁺, Co²⁺, Ca²⁺ and Hg²⁺), EDTA, tannic acid and vanillin. On the other hand, PEC-I was activated by Cu²⁺, ferulic acid, cinnamic acid and 4-hydroxybenzoic acid. The gel under denaturing conditions of PEC-Ret and PEC-I samples showed a protein band of ～45?kDa coincident with that found by staining for pectinase activity. In the bioscouring of cotton fabric the PEC-Ret pectinase preparation led to a better wettability and removed more pectin from the cotton fibers than the commercial enzyme preparation Viscozyme L, but was less effective than a commercial alkaline pectate lyase preparation and alkaline scouring. The incubation of PEC-Ret with guava juice resulted in a 4.15% decrease in juice viscosity.     

Purification of Acetyl Coenzyme A: Deacetylacephalosporin C O-Acetyltransferase from Acremonium chrysogenum

Acetyl CoA: deacetylcephalosporin C O-acetyltransferase, which catalyzes the final step of the biosythetic pathway to cephalosporin C, was stabilized by a buffer solution containing 7-aminocephalosporanic acid and purified over 1300-fold from Acremonium chrysogenum. The purified enzyme has a molecular weight of 55,000 as measured by gel filtration. SDS-polyacrylamide gel electrophoresis showed two subunit bands corresponding to molecular weights of 27,000 and 14,000. The enzyme has an isoelectoric point at pH 4.0 and optimum activity at pH 7.5.     

Specific aggregation of poly(α-L-glutamic acid) and hysteresis effects in aqueous solutions. I. Influence of temperature-dependent aggregation on the optical rotation of PGA

The instability of aqueous solutions of poly(α-L -glutamic acid) (PGA) at low pH is due to two distinguishable phenomena: precipitation, favored above 40°C., and aggregation, favored below 20°C. The aggregated form of PGA can be isolated by gel permeation chromatography. Both aggregation and precipitation increase with decreasing pH, i.e., with decreasing ionization of the side chain carboxyl groups. Temperature-induced aggregation and disaggregation give rise to a reproducible hysteresis loop which can be followed by optical rotation, light scattering, sedimentation, viscosity, and chromatography. Hysteresis has been observed with different PGA samples, and in several aqueous buffered or unbuffered solvents and organic-aqueous solvent mixtures and in the pH range 4.1–4.5. Aggregation manifests itself as an increase in negative optical rotation in the visible and ultraviolet spectral range. The specific relation at 233 mμ is sensitive to aggregation and also reflects the hysteresis. Measurements of optical rotatory dispersion indicate that a₀ reflects the hysteresis but b₀ does not, the latter revealing only reversible changes with aggregation and disaggregation. The helix-coil equilibrium is apparently unperturbed by aggregation, as is the thermal stability of the helix structure. For fully aggregated PGA it is estimated that a₀ increases by about 300 degrees, which suggests that a₀ may be a sensitive parameter to measure aggregation in other systems. The rate of aggregation increases with decreasing temperature. The disaggregation, upon heating, is more rapid. However, kinetics measurements have not yet been done. The temperature M at which all aggregates are disrupted increases with decreasing pH, but is independent of total PGA concentration, at constant pH. No molecular weight dependence of M was detected in the range 20–100 × 10³. The shape and size of the hysteresis loop depends upon pH and molecular weight, which is interpreted as a dependence on the extent of aggregation. One branch of the loop, representing the helix–coil transition of isolated molecules, is reversible, while the others, representing the formation and disruption of the aggregates, are not. The system exhibits both ascending and descending scanning curves, which are typical of a true hysteresis.     

A hemolysin from the mushroom Pleurotus eryngii

A monomeric 17-kDa hemolysin designated as eryngeolysin was isolated from fresh fruiting bodies of the mushroom Pleurotus eryngii, using a protocol that involved gel filtration on Superdex 75, ion exchange chromatography on Mono Q and gel filtration on Superdex 75. Its N-terminal sequence demonstrated striking homology to that of its counterparts ostreolysin from the oyster mushroom Pleurotus ostreatus and aegerolysin from the mushroom Agrocybe cylindracea. Its hemolytic activity was unaffected over the pH range 4.0–12.0, but no activity was observed at pH 13 and at and below pH 2. The hemolysin was stable between 0 and 30 °C. At 40 °C, only residual activity was detectable. At and above 50 °C, activity was indiscernible. Eryngeolysin exhibited cytotoxicity toward leukemia (L1210) cells but not toward fungi. The hemolysin was inactivated by treatment with trypsin. It exhibited antibacterial activity against Bacillus sp. but not against other species. It inhibited basal as well as ConA-stimulated mitogenic response of murine splenocytes. N-Glycolyneuraminic acid was the only sugar capable of inhibiting the hemolytic activity. Eryngeolysin-induced hemolysis was osmotically protected by polyethylene glycol (PEG) 10000 with a mean hydrated diameter dose to 9.3 nm. However, no protection was offered by PEG 10000 to the anti-mitogenic and antiproliferative activities of eryngeolysin. The susceptibility of erythrocytes from different classes of vertebrates to eryngeolysin was mammalian > avian > reptilian > piscine.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

Biochemical characterization of a lectin from Delonix regia seeds

A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60°C. The lectin required Mn²⁺ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.     

Purification and Some Properties of Chlorogenic Acid Oxidase from Apple (Malus pumila)

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6–8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (?)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The K_m value for the enzyme was found to be 122μM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.     

A Novel Phospholipase C Inhibitor,S-PLI Produced by Streptomyces Sp. Strain No. A-6288

Abstract

S-PLI, an inhibitor of phospholipase C (PLC) produced by Strepromyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37°C and up to 40° at pH 6.0. The inhibitory activity showed pH-and temperature-dependence with a maximum around pH 7.0 at 50°C. S-PLI inhibited phospholipase C in a competitive manner (K_i value; 9.5 × 10-⁶ mM), but did not inhibit S-Hemolysin, phospholipase A₂, phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.     

A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus

A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100. The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each. The SOD was found to be inhibited by NaN₃, but not by KCN or H₂O₂, suggesting that the SOD in T. lanuginosus was of the manganese superoxide dismutase type. The SOD exhibited maximal activity at pH 7.5. The optimum temperature for the activity was 55°C. It was thermostable at 50 and 60°C and retained 55% activity after 60 min at 70°C. The half-life of the SOD at 80°C was approximately 28 min and even retained 20% activity after 20 min at 90°C.     

Purification and characterization of a novel broad-spectrum bacteriocin from Bacillus licheniformis MKU3

A bacterial strain Bacillus licheniformis MKU3, isolated from slaughterhouse sediments showed a strong antimicrobial activity. The antimicrobial substance produced by this strain was found to be a protein that inhibited a broad range of bacterial strains, such as Bacillus sp., Staphylococcus sp., Streptococcus sp., and Listeria monocytogenes. The antimicrobial peptide was purified to homogeneity by cut off membrane filtration followed by gel filtration chromatography. The purified protein with low molecular mass (< 8 kDa) was resolved as single band on Tricine SDS-PAGE. This protein was stable at 100°C for 10 min, but lost its activity at 121°C in 15 min. It was resistant to the proteolytic action of trypsin, proteinase K, and pronase E and stable within a wide range of pH (3.0∼11.0). This protein exhibited lytic activity on selected indicator strain Kurthia gibsonii GCS6.     

Purification and Properties of Hydroxycinnamic Acid Ester Hydrolase from Aspergillus japonicus

Hydroxycinnamic acid ester hydrolase from the wheat bran culture medium of Aspergillus japonicus was purified 255-fold by ammonium sulfate fractionation, DEAE-Sephadex treatment and column chromatographies on DEAE-Sephadex, CM-Sephadex and various other Sephadexes. The purified enzyme was free from tannase and found to be homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was estimated to be 150,000 by gel filtration and 142,000 by SDS-gel electrophoresis. The isoelectric point of the enzyme was pH 4.80. As to its amino acid composition, aspartic acid and glycine were abundant. The optimum pH and temperature for the enzyme reaction were, respectively, 6.5 and 55°C when chlorogenic acid was used as a substrate. The enzyme was stable between pH 3.0 to 7.5 and inactivated completely by heat treatment at 70°C for 10 min.

All metal ions examined did not activate the enzyme, while Hg⁺⁺ reduced its activity. The enzyme was markedly inhibited by diisopropylfluorophosphate and an oxidizing reagent, iodine, although it was not affected so much by metal chelating or reducing reagents. The purified enzyme hydrolyzed not only esters of hydroxycinnamic acids such as chlorogenic acid, caffeoyl tartaric acid and p-coumaroyl tartaric acid, but also ethyl and benzyl esters of cinnamic acid. However, the enzyme did not act on ethyl esters of crotonic acid and acrylic acid or esters of hydroxybenzoic acids.     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

Purification and Properties of Extracellular Acid Phosphatase from Zizania latifolia-pavasite,stilago esculenta

An extracellular acid phosphatase from Ustilago esculenta was purified to homogeneity on the basis of polyacrylamide gel electrophoresis. It was a glycoprotein with an isoelectric point of 4.7. The molecular weight of the enzyme was estimated to be about 343,000 by gel filtration on Sephadex G-200, whereas on SDS-polyacrylamide gel electrophoresis, the enzyme gave a single protein band with a molecular weight of 116,000. This result suggests that the enzyme consists of three identical subunits. The enzyme showed an optimum activity at pH 4.5, retained 90% of its activity for 10 min at 55°C and had a Km value of 0.25 mm for p-nitrophenylphosphate. No definite substrate specificity of the enzyme was observed.     

Purification and some properties of endopolygalacturonase from Rhizopus sp. LKN

An endopolygalacturonase of Rhizopus sp. strain LKN, one of several isolates from tempe starter (ragi), was purified 235-fold by CM-Sephadex C-50, DEAE-Sephadex A-50 ion exchange chromatographies and Sephadex G-75 gel filtration. The purified enzyme was homogeneous by SDS-PAGE with a M _r of 38.5 kDa. Its K _m value for pectic acid was 2 mg/ml. It was stable at pH 4.5 to 11 and up to 50°C, with optimum activity at pH 4.5 to 4.75 and 55 to 60°C. Some ionic compounds enhanced the enzyme activity, whereas tannic acid at 0.5 mm caused about 90% inhibition.The authors are with the Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan.     

Purification and Some Properties of Carboxylesterase from Arthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.     

Characterization of Endo β-1,3-Glucanase IV from Flavobacterium dormitator var. glucanolyticae FA-5

Endo β-1,3-glucanase IV (E.C. 3.2.1.6, endo-1,3(4)-β-d-glucanase) from Flav. dormitator var. glucanolyticae FA-5 was shown to be a glycoprotein by gel filtration and sodium dodecyl sulfate gel electrophoresis. The carbohydrate moiety was composed of 17 hexose units. The enzyme had an apparent molecular weight of 3.3 x 10⁴, determined by gel filtration, sodium dodecyl sulfate gel electrophoresis and ultracentrifugation. The enzyme showed maximum reactivity at pH 6.0 and 6.5 for living yeast cells and laminaran, respectively. The enzyme predominantly released laminaripen-taose from a variety of linear β-1,3-glucans and showed transglucanosylation activity. The amino acid composition of the enzyme and some of its physicochemical and enzymatic properties are described.