Red Yeast Cell Lysis by Red Yeast Cell Wall Lytic Enzyme and Protease

The purified red yeast cell wall lytic enzyme of Penicillium lilacinum No. 2093 has a potent saccharifying activity against cell walls, but the living cell lytic activity of it is considerably lower than that of the culture filtrate. Therefore, the living cell lytic factors in the culture filtrate were examined. The alkaline protease of Pen. lilacinum played an important role for living cell lysis. The synergistic effect on living cell lysis was also detected, when acid proteases from various origins were combined with the cell wall lytic enzyme. These results indicated that the protein layers of red yeast cell surface inhibited the action of a glycanase,cell wall lytic enzyme, and the protein molecule contributed to retain the rigid structure of the wall.     

Distribution of Yeast Cell Wall Lytic Activity among Microorganisms

An α-glucosidase has been isolated from the mycelia of Penicillium purpurogenum in electrophoretically homogeneous form, and its properties have been investigated. The enzyme had a molecular weight of 120,000 and an isoelectric point of pH 3.2. The enzyme had a pH optimum at 3.0 to 5.0 with maltose as substrate. The enzyme hydrolyzed not only maltose but also amylose, amylopectin, glycogen, and soluble starch, and glucose was the sole product from these substrates. The Km value for maltose was 6.94×10^?4 m. The enzyme hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside. The enzyme had α-glucosyltransferase activity, the main transfer product from maltose being maltotriose. The enzyme also catalyzed the transfer of α-glucosyl residue from maltose to riboflavin.     

Proteolytic Activity against Model Peptides of the Cell Wall Lytic Enzyme from Chlamydomonas

A cell wall lytic enzyme (gamete wall-autolysin) from Chlamydomonasreinhardtii specifically cleaved several synthetic model peptides,-neo-endorphin, dynorphin (1–13), neurotensin and mastoparan,at the peptide bonds between consecutive hydrophobic amino-acidresidues. The cleavage was not significantly affected by high-saltconditions which are known to inhibit digestion of the cellwall. (Received December 14, 1989; Accepted April 5, 1990)     

Mode of Action of Clostridium Phage HM 7-Induced Lytic Enzyme on Clostridium saccharoperbutylacetonicum Cell Wall Peptidoglycan

The action of Clostridium phage HM 7-induced lytic enzyme on the cell wall peptidoglycan of Clostridium saccharoperbutylacetonicum was investigated. The cell wall peptidoglycan of this strain contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00: 2.08: 0.97; 0.92: 0.68. It was strongly digested when incubated with the lytic enzyme. This digestion was accompanied by the release of NH₂-terminal l-alanine without a concomitant release of COOH-terminal amino acids and reducing groups. Chromatography of the lytic enzyme digest resulted in only two fractions, each of which was chromatographically homogeneous. One was a polysaccharide consisting of glucosamine and muramic acid in molar ratios 1.00: 0.78, and other was a peptide composed of glutamic acid, alanine and diaminopimelic acid in molar ratios of 1.00: 2.09: 1.05. These results indicate that phage HM 7-induced lytic enzyme is N-acetylmuramyl-l-alanine amidase, which cleaves the linkage between N-acetylmuramic acid and l-alanine.

A possible structure for the cell wall peptidoglycan was also proposed.     

Taxonomic Characters and Culture Conditions of a Bacterium which Produces a Lytic Enzyme on Rhizopus Cell Wall

A bacterium R–4 which produces a novel type of lytic enzyme which lyses fungal and yeast cell walls was isolated from the air and was identified to belong to the genus Bacillus.

Production of the enzyme appeared to require a high concentration of nitrogen source in medium. No inducing substance was needed for the enzyme production.

A crude preparation of the enzyme was used to characterize the lytic activity. From the lytic spectrum, the enzyme seemed to have the highest activity toward the cell walls of species in the genus Rhizopus among various fungi and yeasts tested, A proteolytic activity was shown to be parallel with the lytic activity. The lytic activity was also accompanied with the liberation of reducing sugars from Rhizopus cell wall, but no activity on some known carbohydrates tested was detected in the preparation.     

Yeast Cell Wall Bound Proteolytic Enzymes

Nearly all the amino group-producing activity of the autolysate of cells of Saccharomyces sake was recovered in the cell wall fraction obtained from the autolysis residue. The activity of the cell wall fraction was not lost even after repeated use.

The proteolytic activity of the fraction was not solubilized by incubation with detergents, disruption with cell mill or by freezing and thawing method, but was solubilized to some extent by incubation with a commercial yeast cell-lytic enzyme preparation.

The cell wall fraction hydrolysed casein to about 50%. When casein was previously treated with certain proteinases, more than 60% was digested. The activity of the fraction was significantly increased by the addition of Zn²⁺ while it was decreased by several proteolytic enzyme inhibitors. The interesting fact was that in the presence of EDTA the cell wall fraction showed only carboxypeptidase-like activity, and attacked the oxidized insulin B-chain to release two amino acids from the carboxyl terminal in known order.     

The Isolation and Properties of the Lytic Enzyme of the Cell Wall Released by Mating Gametes of Chlamydomonas reinhardtii

The lytic enzyme of the cell wall, which is excreted into theculture medium by mating gametes of Chlamydomonas reinhardtii,was characterized through its purification, and a convenientquantitative test for activity which uses glutaraldehyde-fixedzoosporangia was devised. The enzyme was stabilized at a highsalt concentration (200 mM NaCl) then purified by gel filtrationand sucrose density gradient centrifugation. The results showthat the lytic enzyme is a molecule(s) of about 130,000 daltons. (Received August 28, 1980; Accepted December 18, 1980)     

Cell Wall Glycopolymers as a Diagnostic Trait of Arthrobacter crystallopoietes

Microbiology - The composition and structure of the cell wall glycopolymers from Arthrobacter crystallopoietes VKM Ac-1107T (family Micrococcaceae, phylum Actinobacteria), previously assigned to...     

Method for Fingerprinting Yeast Cell Wall Mannans

Controlled acetolysis of yeast mannans yields mixtures of oligosaccharides with (1-->2) and (1-->3) linkages between the mannose units, whereas the less stable (1-->6) linkages of the polysaccharide backbone are cleaved. The "fingerprints," obtained by gel filtration of the oligosaccharide mixtures, can be used to distinguish between the different yeast mannans. The general method may be useful for determining the taxonomy of yeasts and for making correlations between immunochemical reactivity and mannan structure.     

Investigation of the Structure of Rhizopus Cell Wall with Lytic Enzymes

The components and structure of the cell wall of Rhizopus delemar were investigated using purified lytic enzymes, protease and chitosanase from Bacillus R-4 and chitinase II from Streptomyces orientalis. When these enzymes were used individually they only partially lysed the cell wall, but when allowed to react on the cell wall together, a complete lysis was achieved by cooperative action. These modes of action on the cell wall and the chemical and morphological data suggested that the cell wall structure was different in Rhizopus delemar of Zygomycetes from filamentous fungi of Euascomycetes and that its wall structure might be composed mainly of chitin fibers cemented by chitosan and protein or peptides scattered in a mosaic manner.     

Porosity of the Yeast Cell Wall and Membrane

The limiting sizes of molecules that can permeate the intact cell wall and protoplast membrane of Saccharomyces cerevisiae were determined from the inflection points in a triphasic pattern of passive equilibrium uptake values obtained with a series of inert probing molecules varying in molecular size. In the phase identified with the yeast protoplast, the uptake-exclusion threshold corresponded to a monodisperse ethylene glycol of molecular weight = 110 and Einstein-Stokes hydrodynamic radius (r(ES)) = 0.42 nm. In the cell wall phase, the threshold corresponded to a polydisperse polyethylene glycol of number-average molecular weight ( M(n)) = 620 and average radius (r(ES)) = 0.81 nm. The third phase corresponded to complete exclusion of larger molecules. The assessment of cell wall porosity was confirmed by use of a second method involving analytical gel chromatographic analyses of the molecular weight distribution for a single polydisperse polyglycol before and after uptake by the cells, which indicated a quasi-monodisperse threshold for the cell wall of M(n) = 760 and r(ES) = 0.89 nm. The results were reconciled with two situations in which much larger protein molecules previously have been reported able to penetrate the yeast cell wall.     

Specific Cell Wall Proteins Confer Resistance to Nisin upon Yeast Cells

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104–3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both CWP1 and CWP2 was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin, and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.     

Lysis of Yeast Cell Wall by Enzymes from Streptomycetes

This paper deals with yeast cell-wall lytic enzymes formed by Streptomyces with regard to the connection with the cell-wall structure.

In the first place, 29 organisms of β-glucanase-producing Streptomycetes were selected among 777 strains belonging to genus Streptomyces by means of a cylinder-plate method employing the yeast glucan as a substrate. As for these organisms, the depolymerizing activity against the yeast glucan was considered to be mainly due to β-1,3-glucanase activity. Against the heat-treated cell of bakers’ yeast, the crude enzymes merely showed poor lytic activities, however, in the combined employment with some protease preparations, especially with an alkaline protease from St. satsumaensis nov. sp., a remarkable increase of the lytic activities was demonstrated. On the other hand, the intact cell wall of bakers’ yeast, or both the heat-treated and the intact cells of Sacch. cerevisiae 18.29 strain were dissolved very easily by a sole action of β-glucanase or of protease, respectively. In consequence, it seemed that the lysis occurred with different mechanisms in response to differences of substrates. On this subject, the results of investigations and discussions were described in special measure. In addition, the possibility, that some other enzymes than β-glucanase or protease might concern to the lysis of the cell wall, was also investigated and discussed.     

Endo-N-acetyl-glucosaminidase from Clostridium perfringens, Lytic for Cell Wall Murein of Gram-Negative Bacteria

An endo-N-acetyl-glucosaminidase which degrades the murein (peptidoglycan) sacculi of the cell walls of Escherichia coli and Spirillum serpens, but not those of Micrococcus lysodeikticus and Sarcina lutea, is present as a contaminant in a "phospholipase C" from Clostridium perfringens. The specificity of enzyme action was elucidated by reduction of liberated glycosidic groups with NaBH(4) and identification of glucosaminol as the reduction product. This finding contradicts previous reports associating cell wall breakdown with specific phospholipase action.     

真菌细胞溶壁酶的产酶培养和应用效果

从土样中分离的７１株木霉（Ｔｒｉｃｈｏｄｅｒｍａ ｓｐ．）筛选出一株分解几丁质和葡聚糖能力较强的菌株。该菌株在麸皮、稻草粉、硫酸铵和诱导物为主成份的固体培养基上,经过２５℃、８４ｈ的培养,产生较高的真菌细胞溶壁酶,能溶解酵母、毛霉、黑曲霉及食用菌等丝状菌丝体的细胞壁,形成原生质体,经选用酵母、毛霉等进行原生质体再生,效果较优。     

Sed1p Is a Major Cell Wall Protein of Saccharomyces cerevisiae in the Stationary Phase and Is Involved in Lytic Enzyme Resistance

A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Amino acid sequence analysis revealed that this protein is the product of the SED1 gene. SED1 was formerly identified as a multicopy suppressor of erd2, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway. Sed1p is very rich in threonine and serine and, like other structural cell wall proteins, contains a putative signal sequence for the addition of a glycosylphosphatidylinositol anchor. However, the fact that Sed1p, unlike other cell wall proteins, has six cysteines and seven putative N-glycosylation sites suggests that Sed1p belongs to a new family of cell wall proteins. Epitope-tagged Sed1p was detected in a β-1,3-glucanase extract of cell walls by immunoblot analysis, suggesting that Sed1p is a glucanase-extractable cell wall protein. The expression of Sed1p mRNA increased in the stationary phase and was accompanied by an increase in the Sed1p content of cell walls. Disruption of SED1 had no effect on exponentially growing cells but made stationary-phase cells sensitive to Zymolyase. These results indicate that Sed1p is a major structural cell wall protein in stationary-phase cells and is required for lytic enzyme resistance.