1,3- -Glucan synthase in cell-free extracts from mycelium and protoplasts of Sclerotium glucanicum

Kottutz, Elke; Rapp, Peter R.

doi:10.1099/00221287-136-8-1517

Cited by 16 publications

(10 citation statements)

References 22 publications

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“…These could be assigned by their vicinal couplings in an HH-COSY spectrum. [29][30][31][32]. The Lorentzian tails associated with the rather high linewidth of the 1H-NMR resonances (approximately 7 Hz) combined with the low dispersion of the chemical shift precluded an accurate estimate of impurities from the proton and HH-COSY NMR spectra alone.…”

Section: Product Characterizationmentioning

confidence: 99%

Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

Antelo¹,

Cosio

Hertkorn

et al. 1998

FEBS Letters

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A (1 ~ 3)-~-glucan synthase activity was identified in cell membrane preparations from the oomycete Phytophthora sojae, a soybean pathogen. The activity could be solubilized using the zwitterionic detergent CHAPS at relatively low concentrations (3 mg/ml). High salt concentrations were not effective in removing the activity from the membranes. Detergent solubilization of the enzyme resulted in a six-fold increase of calculated Vma× values (2.5 vs. 0.4 nkat/mg protein) but only minor alteration of the Km (10.6 vs. 10.7 raM). Analysis of the reaction product of the solubilized enzyme by enzymatic degradation and by 2D NMR spectroscopy confirmed its identity as a linear high molecular weight (1 ~ 3)-[~-glucan. Glucan synthase activity in both membrane and solubilized preparations was not activated by GTP or divalent cations as reported for other fungal or plant glucan synthases, The activity was inhibited, as expected, in a competitive manner by UDP with a Ki of 2.9 raM. Partial purification of the enzyme was achieved by anion exchange chromatography followed by product entrapment. This procedure resulted in the selective enrichment of a protein band with apparent Mr 108 000 in SDS-PAGE which was not visible in any of the steps preceding product entrapment. The glucan pellets from product entrapment contained up to 3% of the initial enzyme activity present in the fraction used for the procedure.

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Section: Product Characterizationmentioning

confidence: 99%

Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

Antelo¹,

Cosio

Hertkorn

et al. 1998

FEBS Letters

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“…Samples were removed after 1 h and hydrolysis was stopped by heating the samples in a boiling water bath for 10 min. The products were detected by thin-layer chromatography (TLC) (Kottutz and Rapp 1990).…”

Section: Analysis Of Hydrolysis Productsmentioning

confidence: 99%

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

Prokop¹,

Rapp²,

Wagner³

1994

Can. J. Microbiol.

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1994. Production, purification, and characterization of an extracellular endo-a-l,3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-P-1,3-glucanase formation. Can. J. Microbiol. 40: 18-23. Production of extracellular P-1,3-glucanase activity by a monokaryotic Schizophyllum commrtne strain was monitored and results indicated that the P-glucanase activity consisted of an endo-P-l,3-glucanase activity, besides a negligible amount of P-1,6-glucanase and P-glucosidase activity. Unlike the P-I ,3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the P-1.3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo-P-I ,3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing P-1,3-linkages, including lichenan, a P-1 ,3-1,4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular P-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo-P-l,3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass fi-1,3-/P-1,6-glucan. The K, of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50°C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50°C. The enzyme was completely inhibited by 1 mM H~~+ .Growth of the monokaryotic S. commune strain was not affected by its constitutive endo-P-l,3-glucanase formation. . 1994. Production, purification, and characterization of an extracellular endo-P-l,3-glucanase from a monokaryon of Schizophyllutn commune ATCC 38548 defective in exo-P-1,3-glucanase formation. Can. J. Microbiol. 40 : 18-23. Les auteurs ont CtudiC la production d'une activitC P-1,3-glucanase extracellulaire par une souche monocaryote de Schizophyllum cotnmune; les rCsultats ont indiquC que l'activitt P-glucanase est composCe d'une endo-P-l,3-glucanase, en plus d'une quantitC negligeable d'activitC P-1,6-glucanase et P-glucosidase. Contrairement i la production de P-1,3-glucanase de la souche parentale dicaryote de S. commutle ATCC 38548, la synthbse de P-1,3-glucanase du monocaryon n'a pas Ct C dCterminCe par la repression d'un catabolite. L'endo-P-1,3-glucanase du monocaryon a Ct C purifiCe du filtrat de culture par lyophilisation, chromatographie Cchangeuse d'anion sur Mono Q et filtration sur gel de Sephacryl S-100. L'Clectrophorbse en gel dCnaturant de polyacrylamide (SDS-PAGE) a rCvClt une protCine homogbne de masse molCculaire de 3 5 3 kDa et le point isoClectrique a CtC trouvC i 3,95. L'enzyme fut active seulement envers les glucanes contenant des liaisons P-1,3, incluant la lichenane, une p -1 3 1 ...

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“…1974) or some influence derived from the buffer viscosity have been speculated (Kavanagh and Whittaker 1991). It is important to point out that the selection of the optimal stabilizer would be closely related to the lytic enzyme in use (Kottutz and Rapp 1990). In this case, 0·6 mol l −1 MgSO 4 was found optimal when used in conjunction with T. harzianum enzymes.…”

Section: Discussionmentioning

confidence: 99%

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126

2004

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Aims: Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. Methods and Results: Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0AE6 mol l )1 MgSO 4 as osmotic stabilizer was designed to produce approx. 2 · 10 9 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0AE6 mol l )1 sucrose agar media. Conclusions: The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. Significance and Impact of the Study: The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.

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1,3- -Glucan synthase in cell-free extracts from mycelium and protoplasts of Sclerotium glucanicum

Cited by 16 publications

References 22 publications

Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126

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