Extracellular Lytic Enzymes of Myxococcus virescens

The aim of the investigation was to obtain large amounts of the bacteriolytic enzymes of Myxococcus virescens and to separate these enzymes from the non-bactcriolytic protemases produced by this organism. The bacteria were grown in Casitone broth. When the bacteriolytic activity had reached its maximal value, the cells were removed from the culture medium by centrifugation. Polyethylene glycol 4000 (20 g/1) and potassium phosphate (about 210 g/1) were added to the cell-free solution. The additions resulted in the formation of two liquid phases. The bottom layer was removed, and polyethylene glycol was added to it at a final concentration of 10 g/1. Again two liquid phases formed. The two top phases thus obtained were pooled and 1.6 volumes of cold acetone were added to the mixture. The precipitate formed was dissolved in water and desalted on a Sephadex G-25 column. The desalted protein solution was applied to a carboxymethyl-cellulose column equilibrated with 0.025 M sodium phosphate buffer of pH 6.0. Most of the proteins and the proteinases but none of the bacteriolytic enzymes passed the column unadsorbed. The column was washed with 0.05 M glycine-NaOH buffer of pH 8.8, whereupon the adsorbed bacteriolytic enzymes together with small amounts of proteinases and other proteins were eluted with 0.2 M ammonium carbonate. The material not adsorbed on the CM-cellulose column contained 22 % of the proteolytic activity of the initial cell-free solution and had a 26-fold higher specific activity. The enzyme solution eluted with carbonate contained 24 and 0.3 %, respectively, of the initial bacteriolytic and proteolytic activities. The specific activity of the bacteriolytic enzyme system was about 5000-fold higher than that of the original solution.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Extracellular Lytic Enzymes of Myxococcus virescens II. Purification of Three Bacteriolytic Enzymes and Determination of their Molecular Weights and Isoelectric Points

The bacteriolytic enzymes produced by Myxococcus virescens and previously concentrated and separated from most of the non-bacteriolytic proteins have been further separated and purified. The bacteriolytic enzyme solution was concentrated by lyo-philization. When applied to a Sephadex G-100 column, three peaks of bacteriolytic activity were eluted. Polyacrylamide gel electrophoresis showed that all the three enzyme fractions were contaminated with at least four non-bacteriolytic proteins. In the first enzyme fraction the bacteriolytic enzymes could be freed from the contaminating proteolytic activity by adsorption on a hydroxylapatite column. The bacteriolytic enzymes could then be adsorbed on a CM-cellulose column. The remaining contaminating proteins passed the column un-adsorbed while the bacteriolytic enzymes could be eluted with a gradient of 0.02–0.10 M ammonium hydrogen carbonate solution. The second enzyme fraction was adsorbed on a CM-cellulose column and then eluted with 0.03–0.15 M NH4 HCO₃. After rechromatography on a new column under the same conditions, all of the contaminating proteins had disappeared. For purification of the third enzyme fraction chro-matography on one single CM-cellulose column was sufficient. The elution of the adsorbed enzymes was performed with a gradient of 0.15–0.30 M NH₄HCO₃. The recovery of activity for each of the ion-exchange chromatography separations was at least 90%. The purity of the enzymes was tested by polyacrylamid gel electrophoresis. Each of the purified enzymes gave only one coloured band which coincided with the enzyme activity assayed in sliced gels. The molecular weights of the enzymes were determined by electrophoresis on acryl-amide gels containing sodiumdodecylsulphate. The molecular weights determined in this way (about 40,000, 30,000 and 20,000, respectively) were about 10,000 daltons higher than those obtained by gel chromatography on Sephadex G-100. This discrepancy seems to depend on interactions between the enzymes and the dextran molecules probably caused by the strongly basic nature of the enzymes or by formation of enzyme-substrate complexes.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

ATP- and polyphosphate-dependent bacterial NAD+ kinases

Measurable levels of activity of NAD⁺ kinases of actinomycetesMicrococcus luteus andCoryne-bacterium ammoniagenes were observed after substituting inorganic tripolyphosphate for ATP, whereas the enzyme from the eubacteriumEscherichia coli was not active with this substrate. Gradient PAGE found two molecular isoforms of NAD⁺ kinase inC. ammoniagenes andE. coli; four forms were found inM. luteus. All isoforms of this enzyme found inC. ammoniagenes andM. luteus displayed NADP-synthesizing activity in the presence of either ATP or tripolyphosphate. Because of its capability of utilizing inorganic tripolyphosphate,M. luteus is the most promising NADP producer organism.     

Extracellular Lytic Enzymes of Myxococcus virescens

An easy and rapid method for the purification of a bacteriolytic endopeptidase produced by Myxococcus virescens is described. The bacteria were grown in casitone media and the cells were sedimented by centrifugation. About 1.2 g of montmorillonite were added per liter of cell-free culture solution. The clay was sedimented by centrifugation and the enzyme was then eluted by 0.05 M Na-phosphate buffer pH 6.0, containing 0.4 M NaCl. The enzyme was diluted with water and chromatographed on carboxymethyl-cellulose columns. The purified enzyme liberated free amino groups but no reducing sugars or N-acetylhexosamines when acting on purified N-acetylated cell walls of Micrococcus lysodeikticus. Analysis of N- and C-terminal amino acids in the digestion products showed that the enzyme had liberated about 110 nmoles of lysine ε-amino groups and 60 nmoles of alanine carboxyl groups per mg of cell wall. When it acted on a bisdisaccharide pentapeptide dimer isolated from M. lysodeikticus cell walls, it cleaved about 30% of the alanyl-lysine linkages. Consequently the enzyme was an alanyl-lysine endopeptidase. It had no muramyl-alanine amidase activity.     

Immunochemical subfractionation of a microsomal fraction of rat liver with antibody-coated Staphylococcus aureus cells

The procedure for immunochemical adsorption of vesicles with specific antigen on their outer surfaces was improved. When microsomal vesicles were mixed with Staphylococcus aureus cells coated with the antibody against NADPH-cytochrome c reductase, more than 90% of the enzyme activity was adsorbed on the cell, whereas, only about 10% of the activity was adsorbed on cells coated with the same amount of anti-ovalbumin antibody. NADH-cytochrome c reductase and aldehyde dehydrogenase activities were adsorbed on the cell to the same extent as was NADPH-cytochrome c reductase activity. Under this condition, there was no adsorption of the activities of the marker enzymes of lysosomes and Golgi apparatus, whereas large amounts of the activities of the plasma membrane enzymes were adsorbed. The specific activity of NADPH-cytochrome c reductase in the adsorbed vesicles from the microsomal fractions increased considerably. In contrast, marker enzymes of the Golgi or of the plasma membranes could be enriched in unadsorbed vesicles from the Golgi fractions.     

Bacteriolytic activity of human interleukin-2

In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme — chicken egg white lysozyme — by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcus luteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases.     

Variants of Myxococcus virescens Exhibiting Dispersed Growth. Growth and Production of Extracellular Enzymes and Slime

The growth of two strains of Myxococcus virescens exhibiting dispersed growth was followed in casamino acids (N III-C) media and casitone media. The changes in optical density, pH, pigmentation as well as the secretion of bacteriolytic and proteolytic enzymes, DNA and polysaccharides during growth were recorded. In both media the bacteria grew exponentially with a generation time of 4 (casitone) and 20 hours (N III-C) respectively. The maximal cell mass was about 4 times higher in casitone than in casamino acids media. The amounts of bacteriolytic enzymes produced by the two strains in N III-C medium were different but in casitone medium they were about equal and considerably higher. The maximal values of proteolytic enzymes were about the same in both media and always occurred later than the bacteriolytic maxima. Both activity peaks appeared before the phase of decline. The polysaccharide production reached a maximum during the stationary growth phase in both media. A higher value was reached during growth in casitone medium than in N III-C medium. During the phase of decline a second increase of polysaccharide in the medium appeared. No DNA could be detected in the cell-free solutions until the beginning of the phase of decline.     

Production and characterization of the milk-clotting protease of<Emphasis Type="Italic"> Myxococcus xanthus</Emphasis> strain 422

The cheese industry is seeking novel sources of enzymes for cheese production. Microbial rennets have several advantages over animal rennets. (1) They are easy to generate and purify and do not rely on the availability of animal material. (2) The production of microbial clotting enzymes may be improved by biotechnological techniques. In this work, the biochemical characterization of a novel milk-clotting extracellular enzyme from Myxococcus xanthus strain 422 and a preliminary evaluation of its cheese-producing ability are reported. Strain 422 was selected from four M. xanthus strains as the best producer of extracellular milk-clotting activity, based on both its enzyme yield and specific milk-clotting activity, which also afforded lower titration values than enzymes from the three other M. xanthus strains. The active milk-clotting enzyme from M. xanthus strain 422 is a true milk-clotting enzyme with a molecular mass of 40 kDa and a pI of 5.0. Highest milk-clotting activity was at pH 6 and 37 °C. The enzyme was completely inactivated by heating for 12 min at 65 °C. The crude enzyme preparation was resolved by anion-exchange chromatography into two active fractions that were tested in cheese production assays of compositional (dry matter, fat content, fat content/dry-matter ratio, and moisture-non-fat content) and physicochemical properties (firmness, tensile strength, pH and Aw) of the milk curds obtained. Purified protein fraction II exhibited a significantly higher milk-clotting ability than either protein fraction I or a total protein extract, underlining the potential usefulness of M. xanthus strain 422 as a source of rennet for cheese production.     

Arylsulphatases in human brain: separation, purification, and certain properties of the two soluble arylsulphatases

Abstract— Arylsulphatases (aryl-sulphate sulphohydrolases; E.C. 3.1.6.1) in the soluble subcellular fraction (105000g, 2 h) of human brain were partially purified by ammonium sulphate fractionation, ion exchange chromatography, and Sephadex gel filtration. Potassium-4-methylumbelliferone-sulphatase (MUS-sulphatase) adsorbed on DEAE-cellulose was purified approximately 700-fold over activity in the soluble fraction and the unadsorbed MUS-sulphatase was similarly purified approximately 600-fold. The arylsulphatase adsorbed to DEAE-cellulose exhibited a K_m value for MUS of 12.5 mM and a pH optimum of 5.7, whereas the unadsorbed arylsulphatase exhibited a K_m value for MUS of 8.3 mM and a pH optimum of 5.4. The molecular weights of the two enzymes were approximately 109,600 and 51,300, respectively. Sulphate (0.5 mM) showed pronounced mixed inhibition only of the unadsorbed arylsulphatase. Ag⁺ ions (0.25 mM) showed 96 per cent inhibition of the adsorbed arylsulphatase, whereas an activation of the unadsorbed arylsulphatase was observed.     

Effect of Fatty Acids on the Activity of Bacteriolytic Enzymes

The ability of fatty acids to sensitize gram-negative and gram-positive bacterial cells to the action of bacteriolytic enzymes was studied. By synergetic effects between bacteriolytic enzymes and fatty acids isolated from Myxococcus such bacteria, which were otherwise resistant to the enzymes, could be lysed. Isobranched and unbranched acids with 11–15 carbon atoms were active and could sensitize Bacillus megaterium and Aerobacter aerogenes to the action of bacteriolytic enzymes from myxobacteria and to lysozyme. The sensitizing activity of tetradecanoic acid was enhanced with increasing concentration even after the solution was saturated. Neither ethylene diaminetetraacetate (0.1 and 1 mM) nor Triton X-100 (1 0/00) could sensitize resistant bacteria to the action of bacteriolytic enzymes. However, they were active in combination and they could also increase the effect of tetradecanoic acid.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

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 of several bacteriolytic enzymes by affinity chromatography on lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with sepharose.

Using lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with Sepharose, several bacteriolytic enzymes were purified from crude preparations of animal and microbial origin. Quail egg-white, human milk and salivary lysozymes [EC 3.2.1.17] were adsorbed onto the adsorbent at pH 5-7 and eluted with 2M NaCl at pH 10. By means of these treatments, lysozymes were purified 20-250 fold with activity recoveries of 60-80%, and the quail lysozyme thus purified was shown to be discelectrophoretically homogeneous. Some bacteriolytic enzymes of microbial origin were also highly purified by using this affinity adsorbent. A bacterial lysozyme from Bacillus sp. ML-208 showed high affinity for the ligand and was not eluted under the conditions mentioned above, but was recovered by elution with 2M guanidine-HCl at pH 5.8, resulting in a 500-fold increase in the specific activity. A Pseudomonas-lytic enzyme from Streptomyces sp. P-51 was easily released from the adsorbent by elution with 0.5M NaCl at pH 5.0. A staphylolytic F2 enzyme from S. griseus S-35 and a chitinase [EC 3.2.1.14] from yam, both of which were completely inert toward M. lysodeikticus cell wall, passed through the adsorbent column. A modified ligand, in which muramic acid and glucosamine residues were N,O-acetylated, failed to adsorb any of these animal and bacterial lysozymes. Some of the enzymatic properties and bacteriolytic action spectra of these purified enzymes are also described in this paper in comparison with those of hen egg-white lysozyme.     

Extracellular Lytic Enzymes of Myxococcus virescens

Two bacteriolytic hexosaminidases isolated from Myxococcus virescens were characterized. When acting on purified cell walls of Micrococcus lysodeikticus they liberated reducing groups and N-acetylhexosamines. By chromatography on Sephadex G-50 and G-25 columns disaccharides were isolated from degraded cell walls. After reduction of the disaccharides with sodium borohydride the acid hydrolysis products were identified by thin layer chromatography. Only pale spots of glucosamine appeared after this treatment but the spots of muramic acid remained unchanged. The enzymes were found to be devoid of exo-β-N-acetylglucosaminidase activity. These results are compatible with the action of endo-β-N-acetylglucosaminidases.     

Purification and Properties of Lytic Enzymes from Streptomyces globisporus 1829

Two lytic enzymes capable of lysing Streptococcus mutans have been purified to give a single band on disc-gel electrophoresis, respectively. The M–1 and M–2 enzymes were both proved to be N-acetylmuramidases. However, these enzymes were entirely different on their enzymatic properties. The molecular weights were about 20,000 and 11,000 for M–1 and M–2 enzymes, respectively, The maximal lytic activity of M–1 enzyme was obtained at ionic strength 0.05, while lytic activity of M–2 enzyme did not change within the ionic strength range of 0 to 0.05. The M–1 enzyme constituted the majority of the total lytic activity against the cell walls of Streptococcus mutans BHT of cultured filtrate. The M–2 enzyme showed less specific lytic activity on the cell walls of Streptococcus mutans BHT than M–1 enzyme.     

Mannose-specific isolectins with different hemagglutinating potencies isolated from Chinese daffodil (Narcissus tazetta var. chinensis) leaves

Three mannose-specific lectins exhibiting considerable similarities in NH₂-terminal amino acid sequence were isolated from leaves of the Chinese daffodil Narcissus tazetta (Family Amaryllidaceae). The purification protocol involved extraction with an aqueous buffer, anion exchange chromatography on DEAE-cellulose using stepwise elution with increasing salt concentrations, affinity chromatography on mannose-agarose, and FPLC-gel filtration on Superose 12. From the peak unadsorbed on DEAE-cellulose, and two peaks adsorbed on the ion exchanger and eluted respectively with 0.2 M Tris-HCl buffer and 0.5 M NaCl, were prepared fractions which yielded isolectins 1, 2, and 3 after adsorption on mannose-agarose and FPLC-gel filtraton. All three isolectins were homodimers with a molecular weight of 26 kDa. The lectin unadsorbed on DEAE-cellulose had the lowest, while the most strongly adsorbed lectin had the highest hemagglutinating activity.     

Protein purification and nucleotide sequence of a lysozyme from the bacteria-induced larvae of the fall webworm,Hyphantria cunea

A protein with lytic activity against Micrococcus luteus was purified from the hemolymph of the fall webworm, Hyphantria cunea, larvae challenged with live E. coli. A bacteriolytic protein of about 14,000 daltons in mass was purified by cation exchange chromatography and reverse-phased HPLC. The optimum pH and optimum temperature range for activity were around pH 6.2 and 50°C, respectively, in a 100 mM phosphate buffer. The aminoterminal amino acid sequence of this protein was determined and the corresponding cDNA was isolated and analyzed. The deduced protein of 142 amino acid residues was composed of a putative leader sequence of 20 residues and the mature enzyme of 122 residues. The cloned lysozyme gene was strongly induced in response to bacterial injection, implying that the enzyme is a part of the immune response of H. cunea. Comparison with other known lysozyme sequences shows that our lysozyme belongs to the chicken lysozyme. Arch. Insect Biochem. Physiol. 35:335–345, 1997.© 1997 Wiley-Liss, Inc.     

Crystallization and characterization of monoacylglycerol and diacylglycerol lipase from Penicillium camembertii.

A new lipase from Penicillium camembertii U-150, which is specific for monoacylglycerols and diacylglycerols, but not triacylglycerols, was purified as four active components using concanavalin-A-Sepharose column chromatography, crystallized in the form of needles, and its properties investigated. No significant difference was observed in substrate specificity, but molecular mass and other enzymatic properties, such as pH, heat stability and optimum pH and temperature, were clearly different between the unadsorbed and the three adsorbed components on concanavalin-A-Sepharose; the three adsorbed components were similar to each other and more stable than the unadsorbed component. On the other hand, after enzymatic removal of carbohydrates from the three adsorbed components, their enzymatic properties became similar to those of the unadsorbed component. The carbohydrates of this lipase contribute to the stability of the enzyme, but not to its enzyme activity. The amino acid compositions of the four components did not differ from each other, and tryptic mapping of the deglycosylated components and amino acid composition of the tryptic fragments were identical. The carbohydrate compositions of four intact components were, however, different from each other. All four components have the same polypeptide backbone and multiple forms of this lipase are due to the differences in composition of the carbohydrates bound in this lipase.