Synthesis and regulation of extracellular beta (1-3) glucanase and protease by cytophaga sp. In batch and continuous culture

Lytic enzyme systems with the ability to break whole cells of yeast are a mixture of several enzymes and virtually all contain beta(1-3)glucanases and some protease. It appears that the presence of these two enzyme activities is necessary to break the two layers of the rigid cell wall. The enzyme system of Cytophaga NCIB 9497 has a high activity towards the walls of yeast and also of bacteria. This article describes the production of this extracellular lytic enzyme system in batch and continuous culture-it was found to be inducible. The synthesis and regulation of the two main constituent enzymes, beta(1-3)glucanase and protease, have been investigated. The synthesis of beta(1-3)glucanase is regulated by bothinduction (by an unknown inducer) and catabolite repression. Highbeta(1-3)glucanase activities were obtained in continuous culture at low dilution rates over a narrow range (0.05-0.10 h(-1)), and there is evidence of the presence of more than one glucanase enzyme. Proteolytic activity appears subject to catabolite repression and made up of the activities of more than one protease enzyme. Productivity and enzyme concentration were increased several fold in continuous culture when compared to batch culture.     

Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells

Growth of Micromonospora chalcea on a defined medium containing laminarin as the sole carbon source induced the production of an extracellular enzyme system capable of lysing cells of various yeast species. Production of the lytic enzyme system was repressed by glucose. Incubation of sensitive cells with the active component enzymes of the lytic system produced protoplasts in high yield. Analysis of the enzyme composition indicated that beta(1-->3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids. The system also displayed weak chitinase and beta(1-->6) glucanase activities whilst devoid of mannanase activity. Our observations suggest that the glucan supporting the cell wall framework of susceptible yeast cells is not directly accessible to the purified endo-beta(1-->3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells. We propose that protease acts in synergy with beta(1-->3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis.     

Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12.

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.     

Lyticase: Endoglucanase and Protease Activities That Act Together in Yeast Cell Lysis

Yeast lytic activity was purified from the culture supernatant of Oerskovia xanthineolytica grown on minimal medium with insoluble yeast glucan as the carbon source. The lytic activity was found to consist of two synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The first component was a β-1,3-glucanase with a molecular weight of 55,000. The K_m for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of β-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the second lytic component, an alkaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a molecular weight of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity, however. Neither pronase nor trypsin could substitute for this protease in promoting yeast cell lysis. A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination. Lyticase has been applied in spheroplast, membrane, and nucleic acid isolation, and has proved useful in yeast transformation procedures.     

Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12.

Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5.     

Kinetics of enzymatic lysis and disruption of yeast cells: I. Evaluation of two lytic systems with different properties

Many microorganisms produce enzymes which lyse the walls of yeasts, fungi, and bacteria. The proportions of different enzyme activities present in the lytic system, their action patterns, synergism, and dependence on inhibitors, constitute the activity profile of the lytic system. Taken together, the activity profile and process conditions for lysis determine the reaction rate and the distribution of products from lysis of any given type of cells. Kinetics of glucan hydrolysis, proteolysis, and lysis of brewer's yeast were compared for two extracellular yeast-lytic enzyme systems with different properties. The enzyme sources used were filtered culture broths from Cytophaga sp. NCIB 9497 grown in batch culture and from Oerskovia xanthineolytica LL-G109, grown under carbon limitation in continuous culture. Rate and extent of cell hydrolysis, and the accumulation of soluble proteins, peptides, and carbohydrates from the lysed yeast cells, are discussed in terms of the activity profiles and potential applications of the two enzyme systems.     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

Isolation of a yeast-lyzing Arthrobacter species and the production of the lytic enzyme complex in batch and continuous-flow fermentors

A gram-negative bacterium strongly lytic toward living cells of the food yeast Saccharomyces fragilis was isolated by continuous-flow enrichment from compost. The organism was identified as a species of Arthrobacter. The extracellular lytic enzyme complex produced by this bacterium contained β-1,3-glucanase, mannan mannohydrolase, and proteolytic activities. The polysaccharases were inducible by whole yeast cells. In chemostat cultures on chemically defined media, synthesis of the polysaccharases was very slight and only detectable at dilution rates below 0.02 hr^?1. Enzyme production in defined media was not solely dependent on growth rate but also was influenced by the growth limiting substrate and the culture history. The production of individual depolymerases and of the lytic activity was studied in batch and chemostat cultures containing yeast as the limiting substrate. The maximum specific growth rate of the Arthrobacter under these conditions was 0.22 hr^?1. β-1,3-Glucanase and proteolytic activities were synthesized by exponentially growing bacteria but maximum lytic titers did not develop until the specific growth rate was declining, at which time mannan mannohydrolase syntheses was induced. In yeast limited chemostats polysaccharase syntheses were greatest at the lowest dilution rates examined, namely 0.02 hr^?1. Further optimization of enzyme production was achieved by feeding the Arthrobacter culture to a second-stage chemostat. A comparison of lytic enzyme productivities in batch and chemostat cultures has been made.     

Enzymes of the yeast lytic system produced by Arthrobacter GJM-1 bacterium and their role in the lysis of yeast cell walls.

Yeast lytic system produced by Arthrobacter GJM-1 bacterium during growth on baker's yeast cell walls contains a complete set of enzymes which can hydrolyze all structural components of cell walls of Saccharomyces cerevisiae. Chromatographic fractionation of the lytic system showed the presence of two types of endo-beta-1,3-glucanase. Rapid lysis of isolated cell walls of yeast was induced only by endo-beta-1,3-glucanase exhibiting high affinity to insoluble beta-1,3-glucans and releasing laminaripentaose as the main product of hydrolysis of beta-1,3-glucans. This enzyme was able to lyse intact cells of S. cerevisiae only in the presence of an additional factor present in the Arthrobacter GJM-1 lytic system, which was identified as an alkaline protease. This enzyme possesses the lowest molecular weight among other identified enzyme components present in the lytic system. Its role in the solubilization of yeast cell walls from the outer surface by endo-beta-1,3-glucanase could be substituted by preincubation of cells with Pronase or by allowing the glucanase to act on cells in the presence of thiol reagents. The mechanism of lysis of intact cells and isolated cell walls by the enzymes of Arthrobacter GJM-1 is discussed in the light of the present conception of yeast cell wall structure.     

Cytochemical study of the yeast and mycelial cell walls ofParacoccidioides brasiliensis

Cell wall structure and macromolecular organization of the various growth forms ofParacoccidioides brasiliensis were investigated using chemical, enzymatic, and cytochemical methods. The wall of yeast-like cells was not sensitive to periodic acid and β(1 → 3) glucanase treatments, but bound calcofluor suggesting that it is composed of an α(1 → 3) glucan and of chitin. The fibrillar outer layer of bud cell initials and of abscision areas, were characterized by the presence of a strongly periodic acid- and concanavalin A-reactive substance that was sensitive to the lytic action of protease, presumably composed of mannan and proteins. The outermost layer of the mycelium was sensitive to protease, and periodic acid and peanut agglutinin positive indicating that it was composed of galactomannan and protein. The inner layer is mainly composed of a β(1 → 3), (1 → 6) glucan (sensitive to snail enzyme but not to an exo β(1 → 3) glucanase), and chitin. Septa were brightly fluorescent with calcofluor. Our results are compared with models previously proposed by other authors.     

Purification and properties of a β-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp.

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.     

Optimization of enzymatic lysis of yeast

In enzymatic lysis of yeast for the recovery of intracellular proteins, the rupture of whole cells is caused by the action of a lytic system consisting primarily of protease and glucanase. A first-principles mechanism for the lytic reaction based on a two-layer model of the wall structure and a burst model for the disruption of cells is pre sented. The fed-batch model results in a dynamic optimization problem, with the enzymes, activities being the control variables. Orthogonal collocation is applied to discretize the state equations, and the resulting non linear program is solved using successive quadratic pro gramming to determine the enzyme and protease inhibitor add-in rates and pH control profiles that maximize the recovery of intracellular protein. Applying the proposed approach, optimal profiles were determined such that a significant increase of the production of proteins in a fed-batch reactor is realized. Also, the optimal control policies in a series of continuous-flow stirred tank reactors (CFSTRs) are determined.     

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.     

Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast

In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell. Some of the chitin is free and the remainder attached to beta(1-3)glucan or beta(1-6)glucan. We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to beta(1-3)glucan at that site. Here, we devised a novel strategy for the analysis of chitin cross-links in [14C]glucosamine-labeled cell walls, involving solubilization in water of alkali-treated walls by carboxymethylation. Intact cell walls or their digestion products with beta(1-3)glucanase or beta(1-6)glucanase were carboxymethylated and fractionated on size columns, and the percentage of chitin bound to different polysaccharides was calculated. Chitin dispersed in the wall was labeled in maturing unbudded cells and that of the ring in early budding cells. The former was mostly attached to beta(1-6)glucan and the latter to beta(1-3)glucan. This confirmed our hypothesis and indicated that the cell has mechanisms to attach chitin, a water-insoluble substance, synthesized here through chitin synthase III, to different acceptors, depending on location. In contrast, most of the chitin synthase II-dependent chitin of the primary septum was free, with the remainder linked to beta(1-3)glucan.     

Lytic β-1,3 Glucanase from Arthrobacter: Pattern of Action

The action pattern of lytic β-1,3 glucanase (glucanase I) from Arthrobacter which liberates predominantly laminaripentaose from various β-glucans has been studied. The enzyme was not active on short linear laminaridextrins, but was active on an enzymatically synthesized, linear β-1,3 oligoglucan preparation. Any intactness of the glucose residues of the chain ends of a substrate did not seem to be necessary for the action of the enzyme. The results of determination of laminaripentaose during a relatively early phase of the reaction suggested that about half of the reducing power liberated in the medium might be explained by the formation of the sugar. It seems that the formation of laminaripentaose relates to the initial attack of glucanase 1 on β-1,3 glucan chains.     

Synthesis and release of proteases byBacteroides fragilis

Protease production byBacteroides fragilis ATCC 25285 was determined in batch and continuous cultures. During exponential growth in batch culture, the majority of proteolysis was cell associated. However, as the bacteria reached stationary phase, most of the intracellular proteases were released into the culture medium. Measurements of alkaline phosphatase and -galactosidase, which are respectively periplasmic and cytoplasmic marker enzymes inB. fragilis, showed that secretion of proteases in the stationary phase was a discrete event and was not associated with a general release of cytoplasmic contents. When the bacterium was grown in continuous culture, cell-associated protease activity increased concomitantly with dilution rate (D=0.03–0.23/h). The ratio of intracellular to whole cell protease activity also increased with growth rate (11 at D=0.03/h; 11.7 at D=0.23/h). Extracellular protease activity was detected only in trace amounts in continuous cultures at the lowest dilution rate. Determinations of the distribution of extracellular protease activity in batch culture after 48 h incubation showed that the majority of proteolysis (ca. 90%) was soluble. Nevertheless, a proportion was associated with particulate fractions, which had high specific activities.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.     

Purification and Properties of Lytic β-Glucanase from an Arthrobacter Bacterium

A glucanase was isolated from a culture fluid of an Arthrobacter bacterium. The purified enzyme preparations consisted of the glucanase components having the same enzymatic activity. The enzyme was stable in a broad pH range, but lost its activity rapidly at above 60°C. Optimum pH values were found to be 5.5~6.5.

The glucanase attacked the following glucan preparations and liberated a relatively small amount of reducing power: Saccharomyces cerevisiae glucan, Candida albicans glucan, Saccharomyces fragilis glucan, pachyman, curdlan and laminaran. The most prominent sugar spot on the chromatogram of the digest from yeast glucan was identified with laminan-pentaose, and the other faint spots with a series of laminaridextrins. The β-1,6 glucosidic bonds in yeast glucan were not hydrolyzed and concentrated in a soluble fraction which was found near the origin of the chromatogram.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.