LYTIC ENZYMES OF SORANGIUM SP.: SOME ASPECTS OF ENZYME PRODUCTION IN SUBMERGED CULTURE

1965 ◽  
Vol 43 (12) ◽  
pp. 1927-1933 ◽  
Author(s):  
D. R. Whitaker ◽  
F. D. Cook ◽  
D. C. Gillespie
Keyword(s):  
Submerged Culture ◽  
Aeration Rate ◽  
Lytic Activity ◽  
Lytic Enzyme ◽  
Arthrobacter Globiformis ◽  
Growth Substrate ◽  
Critical Factors ◽  
Lytic Enzymes ◽  
Mineral Salts ◽  
Casamino Acids

Data are reported on growth, substrate utilization, and production of lytic activity by a species of Sorangium grown in a fermentor containing 130 liters of a glucose – Casamino acids – mineral salts medium. The results indicate that pH and aeration rate are critical factors in the production of lytic enzyme. Under the conditions tested, increases in lytic activity in the medium were accompanied by roughly parallel increases in hydrolase activity towards hydroxyethylchitin and casein. A method for assay of lytic activity toward Arthrobacter globiformis cells is described.

GROWTH INTERACTIONS OF ARTHROBACTER GLOBIFORMIS AND PSEUDOMONAS SP. IN RELATION TO THE RHIZOSPHERE EFFECT

1961 ◽  
Vol 7 (5) ◽  
pp. 759-767 ◽  
Author(s):  
E. C. S. Chan ◽  
H. Katznelson
Keyword(s):  
Soil Extract ◽  
Soil Microflora ◽  
Biologically Active ◽  
Arthrobacter Globiformis ◽  
Pseudomonas Sp ◽  
Mineral Salts ◽  
Root Extracts ◽  
Cultural Conditions ◽  
Alkaline Conditions ◽  
Casamino Acids

Studies on the influence of plant roots on the soil microflora have shown that the number of Gram-negative rods in the rhizosphere is relatively higher than the number of Gram-positive rods, coccoid rods, and sporeforming types. This ecological phenomenon was duplicated and studied in a model system using a Pseudomonas sp. as representative of the rhizosphere and Arthrobacter globiformis as representative of the soil flora. Growth of A. globiformis was strongly suppressed in the presence of the pseudomonad in root extracts of mature plants and in a medium containing casamino acids, yeast extract, glucose, and mineral salts (CAYG medium); suppression was less marked in soil extract. The pseudomonad was unaffected in the association. A. globiformis was inhibited in the first 48-hour incubation in CAYG medium by acid elaborated by the pseudomonad; the pH dropped to 5.3 in 16 hours but rose steadily to alkaline conditions after 48 hours, resulting in a delayed increase in the number of A. globiformis to approximately that of a pure culture, in 5 days. Under cultural conditions favoring pigment production by the pseudomonad, growth of A. globiformis was completely inhibited throughout this period. Another toxic principle was also produced by the pseudomonad. This substance was biologically active in mixed culture against the Arthrobacter but was present in low concentration.

A Computational Method for the Identification of Endolysins and Autolysins

2020 ◽  
Vol 27 (4) ◽  
pp. 329-336 ◽  
Author(s):  
Lei Xu ◽  
Guangmin Liang ◽  
Baowen Chen ◽  
Xu Tan ◽  
Huaikun Xiang ◽  
...  
Keyword(s):  
Support Vector Machine ◽  
Cell Wall ◽  
Experimental Results ◽  
Computational Method ◽  
Lytic Enzyme ◽  
Support Vector ◽  
Lytic Enzymes ◽  
Data Set ◽  
Optimal Feature ◽  
Better Than

Background: Cell lytic enzyme is a kind of highly evolved protein, which can destroy the cell structure and kill the bacteria. Compared with antibiotics, cell lytic enzyme will not cause serious problem of drug resistance of pathogenic bacteria. Thus, the study of cell wall lytic enzymes aims at finding an efficient way for curing bacteria infectious. Compared with using antibiotics, the problem of drug resistance becomes more serious. Therefore, it is a good choice for curing bacterial infections by using cell lytic enzymes. Cell lytic enzyme includes endolysin and autolysin and the difference between them is the purpose of the break of cell wall. The identification of the type of cell lytic enzymes is meaningful for the study of cell wall enzymes. Objective: In this article, our motivation is to predict the type of cell lytic enzyme. Cell lytic enzyme is helpful for killing bacteria, so it is meaningful for study the type of cell lytic enzyme. However, it is time consuming to detect the type of cell lytic enzyme by experimental methods. Thus, an efficient computational method for the type of cell lytic enzyme prediction is proposed in our work. Method: We propose a computational method for the prediction of endolysin and autolysin. First, a data set containing 27 endolysins and 41 autolysins is built. Then the protein is represented by tripeptides composition. The features are selected with larger confidence degree. At last, the classifier is trained by the labeled vectors based on support vector machine. The learned classifier is used to predict the type of cell lytic enzyme. Results: Following the proposed method, the experimental results show that the overall accuracy can attain 97.06%, when 44 features are selected. Compared with Ding's method, our method improves the overall accuracy by nearly 4.5% ((97.06-92.9)/92.9%). The performance of our proposed method is stable, when the selected feature number is from 40 to 70. The overall accuracy of tripeptides optimal feature set is 94.12%, and the overall accuracy of Chou's amphiphilic PseAAC method is 76.2%. The experimental results also demonstrate that the overall accuracy is improved by nearly 18% when using the tripeptides optimal feature set. Conclusion: The paper proposed an efficient method for identifying endolysin and autolysin. In this paper, support vector machine is used to predict the type of cell lytic enzyme. The experimental results show that the overall accuracy of the proposed method is 94.12%, which is better than some existing methods. In conclusion, the selected 44 features can improve the overall accuracy for identification of the type of cell lytic enzyme. Support vector machine performs better than other classifiers when using the selected feature set on the benchmark data set.

scholarly journals Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other IdentifiedEnterococcus faecalisPhage Endolysins

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Hongming Zhang ◽  
Bettina A. Buttaro ◽  
Derrick E. Fouts ◽  
Salar Sanjari ◽  
Bradley S. Evans ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Amino Acid Identity ◽  
Lytic Infection ◽  
Mass Spectrometry Analysis ◽  
Lytic Enzyme ◽  
Lytic Enzymes ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Wide Range

ABSTRACTϕEf11 is a temperateSiphoviridaebacteriophage that infects strains ofEnterococcus faecalis. The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for anN-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathioneS-transferase tag. It produced rapid, profound lysis inE. faecalispopulations and was active against 73 of 103 (71%)E. faecalisstrains tested. In addition, it caused substantial destruction ofE. faecalisbiofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+. Liquid chromatography-mass spectrometry analysis ofE. faecaliscell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as anN-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than anN-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterizedE. faecalisbacteriophages.IMPORTANCEThe emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains ofEnterococcus faecalis. The lysin is broadly active against most of the testedE. faecalisstrains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced byE. faecalisbacteriophages.

scholarly journals Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

2008 ◽  
Vol 74 (24) ◽  
pp. 7490-7496 ◽  
Author(s):  
Yu Pei Tan ◽  
Philip M. Giffard ◽  
Daniel G. Barry ◽  
Wilhelmina M. Huston ◽  
Mark S. Turner
Keyword(s):  
Cell Wall ◽  
Lactococcus Lactis ◽  
Transmembrane Protein ◽  
Random Mutagenesis ◽  
Lytic Enzyme ◽  
Control Strain ◽  
Lytic Enzymes ◽  
Positive Bacterium ◽  
Peptidoglycan Biosynthesis ◽  
Cell Wall Lytic Enzymes

ABSTRACT Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.

LYTIC ENZYMES OF SORANGIUM SP.: ISOLATION AND ENZYMATIC PROPERTIES OF THE α- AND β-LYTIC PROTEASES

1965 ◽  
Vol 43 (12) ◽  
pp. 1935-1954 ◽  
Author(s):  
D. R. Whitaker
Keyword(s):  
Substantial Part ◽  
Arthrobacter Globiformis ◽  
Lytic Enzymes ◽  
Trace Impurities ◽  
Citrate Buffer ◽  
Egg White Lysozyme ◽  
First Order Kinetics ◽  
Zero Order Kinetics ◽  
Low Concentrations ◽  
Hydrolyze Casein

Procedures are described for the isolation of two lytic enzymes from culture filtrates of a species of Sorangium. The enzymes, designated α-lytic protease and β-lytic protease, are responsible for most of the filtrate's lytic activity towards Arthrobacter globiformis cells. The enzymes were adsorbed from the filtrate by Amberlite CG50, separated by displacement from the resin with citrate buffer containing a gradient of sodium citrate concentration, and refractionated on columns of the same resin. Trace impurities in the β-enzyme were removed by precipitation of the enzyme with ammonium sulfate. The β-enzyme has been crystallized.On electrophoresis in Tris buffer of pH 8.0, the α-enzyme migrates slightly faster than egg-white lysozyme, the β-enzyme slightly slower. The absorptivity of the α-enzyme at 280 mμ was estimated to be 0.89; that of the β-enzyme 2.05.Low concentrations of the β-enzyme lyse suspensions of Arthrobacter globiformis cells completely, and moderately higher concentrations lyse suspensions of Micrococcus lysodeikticus cells completely; corresponding concentrations of the α-enzyme lyse the suspensions incompletely. The concomitant changes in A660 of the suspensions are consistent with zero-order kinetics for the β-enzyme and first-order kinetics for the α-enzyme. Untreated and partially lysed suspensions of Arthrobacter cells show little difference in the dependence of their absorbances on wavelength; corresponding suspensions of Micrococcus cells show marked differences in this respect, and the nature of the change suggests that the breakup of clumps of cells is responsible for a substantial part of the change in absorbance measured during lysis of suspensions of Micrococcus cells. Phase-contrast photomicrographs of cells undergoing lysis show that individual cells vary greatly in their rates of lysis; swelling and decreases in the refractive index precede fragmentation of the cell and may contribute appreciably to the change in absorbance of the suspension.Both enzymes hydrolyze casein. The α-enzyme has the greater activity towards this substrate.

scholarly journals The N-Terminal Region of the Oenococcus oeniBacteriophage fOg44 Lysin Behaves as a Bona Fide Signal Peptide inEscherichia coli and as a cis-Inhibitory Element, Preventing Lytic Activity on Oenococcal Cells

2000 ◽  
Vol 182 (20) ◽  
pp. 5823-5831 ◽  
Author(s):  
Carlos São-José ◽  
Ricardo Parreira ◽  
Graça Vieira ◽  
Mário A. Santos
Keyword(s):  
Signal Peptide ◽  
Lethal Effect ◽  
Oenococcus Oeni ◽  
Terminal Region ◽  
Lytic Activity ◽  
Signal Peptidase ◽  
Evolutionary Significance ◽  
Wall Structure ◽  
Dependent Manner ◽  
Lytic Enzymes

ABSTRACT The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed inE. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeniand Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.

scholarly journals Partial Characterization of an Enzyme Fraction with Protease Activity Which Converts the Spore Peptidoglycan Hydrolase (SleC) Precursor to an Active Enzyme during Germination of Clostridium perfringens S40 Spores and Analysis of a Gene Cluster Involved in the Activity

2001 ◽  
Vol 183 (12) ◽  
pp. 3742-3751 ◽  
Author(s):  
Seiko Shimamoto ◽  
Ryuichi Moriyama ◽  
Kazuhiro Sugimoto ◽  
Shigeru Miyata ◽  
Shio Makino
Keyword(s):  
Clostridium Perfringens ◽  
Catalytic Domain ◽  
Early Stage ◽  
Amino Acid Sequences ◽  
Active Enzyme ◽  
Enzyme Inactivation ◽  
Lytic Enzyme ◽  
Lytic Enzymes ◽  
Enzyme Fraction ◽  
Protein Components

ABSTRACT A spore cortex-lytic enzyme of Clostridium perfringensS40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55°C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl2 indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5′ end ofsleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.

Germination of macroconidia and growth of Sporidesmium sclerotivorum in vitro

1981 ◽  
Vol 27 (7) ◽  
pp. 664-669 ◽  
Author(s):  
W. A. Ayers ◽  
E. A. Barnett ◽  
P. B. Adams
Keyword(s):  
Organic Nitrogen ◽  
Nitrogen Sources ◽  
Soluble Starch ◽  
Mineral Salts ◽  
Ethanolic Extracts ◽  
Carbon Substrates ◽  
Agar Media ◽  
Casamino Acids ◽  
Inorganic And Organic Nitrogen

Macroconidia of Sporidesmium sclerotivorum, a mycoparasite of Sclerotinia spp., were induced to germinte by aqueous and ethanolic extracts of sclerotia of Sclerotinia minor. Paper chromatography of sclerotial extracts indicated the presence of several amino acids and carbohydrates, chiefly glucose. Glucose was identified as the principal germination stimulant in ethanolic extracts. Glucose, fructose, mannose, cellobiose, sucrose, maltose, trehalose, soluble starch, and glycerol at 0.1% (w/v) stimulated macroconidia to germinate in 3–6 days at 25 °C. Crude sclerotial extracts, and glucose combined with inorganic and organic nitrogen sources, supported germination of greater numbers of macroconidia than glucose alone. Yeast extract, Casaminc acids, peptone, and several carbon substrates alone did not support germination. Macroconidia germinated well (> 30%) over the range of pH 3–7; maximum germination (> 80%) occurred at pH 5.0–5.5. Mycelial growth in a glucose – Casamino acids - mineral salts medium was also greatest in the range of pH 5.0–5.5, but growth fell off sharply below pH 4.5 and above pH 6.0. The fungus grew slowly on several complex agar media adjusted to pH 5.5.

scholarly journals Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells

2021 ◽  
Vol 22 (17) ◽  
pp. 9536
Author(s):  
Agnieszka Morzywolek ◽  
Magdalena Plotka ◽  
Anna-Karina Kaczorowska ◽  
Monika Szadkowska ◽  
Lukasz P. Kozlowski ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Bioinformatics Analysis ◽  
Analytical Ultracentrifugation ◽  
Lipoteichoic Acid ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Lytic Enzyme ◽  
Gram Positive ◽  
Genes Encoding

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

scholarly journals Thermophile Lytic Enzyme Fusion Proteins that Target Clostridium perfringens

Antibiotics ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 214 ◽  
Author(s):  
Steven M. Swift ◽  
Kevin P. Reid ◽  
David M. Donovan ◽  
Timothy G. Ramsay
Keyword(s):  
Cell Wall ◽  
Clostridium Perfringens ◽  
Animal Feed ◽  
Lytic Enzyme ◽  
Necrotic Enteritis ◽  
Lytic Enzymes ◽  
Thermostable Enzymes ◽  
Catalytic Domains ◽  
Binding Domains ◽  
Cell Wall Binding

Clostridium perfringens is a bacterial pathogen that causes necrotic enteritis in poultry and livestock, and is a source of food poisoning and gas gangrene in humans. As the agriculture industry eliminates the use of antibiotics in animal feed, alternatives to antibiotics will be needed. Bacteriophage endolysins are enzymes used by the virus to burst their bacterial host, releasing bacteriophage particles. This type of enzyme represents a potential replacement for antibiotics controlling C. perfringens. As animal feed is often heat-treated during production of feed pellets, thermostable enzymes would be preferred for use in feed. To create thermostable endolysins that target C. perfringens, thermophile endolysin catalytic domains were fused to cell wall binding domains from different C. perfringens prophage endolysins. Three thermostable catalytic domains were used, two from prophage endolysins from two Geobacillus strains, and a third endolysin from the deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2). These domains harbor predicted L-alanine-amidase, glucosaminidase, and L-alanine-amidase activities, respectively and degrade the peptidoglycan of the bacterial cell wall. The cell wall binding domains were from C. perfringens prophage endolysins (Phage LYtic enzymes; Ply): PlyCP18, PlyCP10, PlyCP33, PlyCP41, and PlyCP26F. The resulting fifteen chimeric proteins were more thermostable than the native C. perfringens endolysins, and killed swine and poultry disease-associated strains of C. perfringens.

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