Mycelial glucoamylases produced by the thermophilic fungus Scytalidium thermophilum strains 15.1 and 15.8: purification and biochemical characterization / Glucoamilases miceliais produzidas pelas linhagens 15.1 e 15.8 do fungo termofílico Scytalidium thermophilum: purificação e caracterização bioquímica

Two strains (15.1 and 15.8) of the thermophilic fungus Scytalidium thermophilum produced high levels of intracellular glucoamylases, with potential for industrial applications. The isoform I of the glucoamylase produced by 15.1 strain was sequentially submitted to DEAE-Cellulose and CM-Cellulose chromatography, and purified 141-fold, with 5.45 percent recovery. The glucoamylase of strain 15.8 was purified 71-fold by CM-Cellulose and Concanavalin A-Sepharose chromatography, with 7.38 percent recovery. Temperature and pH optima were in the range of 50-60ºC and 5.0-6.0, respectively, using starch and maltose as substrates. The glucoamylase of S. thermophilum 15.8 was more stable (t50 > 60 min) than that of S. thermophilum 15.1 (t50= 11-15 min), at 60ºC. The glucoamylase activities were enhanced by several ions (e.g. Mn2+ and Ca2+) and inhibited by β-mercaptoethanol. The glucoamylase from 15.1 strain showed a Km of 0.094 mg/ml and 0.029 mg/ml and Vmax of 202 U/mg prot and 109 U/mg prot, for starch and maltose, respectively. The hydrolysis products of starch and maltose, analyzed by TLC, demonstrated glucose as end product and confirming the character of the enzyme as glucoamylase. Differences were observed in relation to the products formed with maltose as substrate between the two strains studied. S. thermophilum 15.8 formed maltotriose in contrast with S. thermophilum 15.1.

Duas linhagens (15.1 e 15.8) do fungo termofílico Scytalidium thermophilum se mostraram produtoras de grandes quantidades de glucoamilases, com potencial aplicação industrial. A isoforma I de glucoamilase produzida pela linhagem 15.1 foi submetida seqüencialmente a cromatografia em colunas de DEAE-celulose e CM-celulose, sendo purificada 141 vezes com porcentagem de recuperação de 5,45 por cento. A glucoamilase da linhagem 15.8 foi purificada 71 vezes através do uso de colunas de cromatografia de CM-celulose e Concanavalina A-sepharose com porcentagem de recuperação de 7,38 por cento. Temperatura e pH ótimo foram de 50-60ºC e 5,0-6,0 respectivamente, utilizando-se amido e maltose como substratos. A glucoamilase de S. thermophilum 15.8 se mostrou mais estável (t50 > 60 min) que a de S. thermophilum 15.1 (t50 =11-15min) a 60ºC. As glucoamilases tiveram suas atividades enzimáticas aumentadas na presença de vários íons (ex: Mn2+, e Ca2+) e inibidas por β-mercaptoetanol. A glucoamilase da linhagem 15.1 apresentou um Km de 0,094 mg/ml e 0,029 mg/ml and Vmax de 202U/mg prot e 109U/mg prot, para amido e maltose respectivamente. A análise do produto da hidrólise de amido e maltose por TLC, demonstrou que o produto final era glucose, confirmando as características da enzima como glucoamilase. Diferenças entre as duas linhagens foram observadas com relação aos produtos formados tendo maltose como susbstrato, a linhagem 15.8 de S. thermophilum produziu maltotriose como produto final em contrate com a linhagem 15.1.

Searching for the role of protein phosphatases in eukaryotic microorganisms

Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism

Serine/threonine protein phosphatases and a protein phosphatase 1 inhibitor from Neurospora crassa

The major spontaneously active serine/threonine (Ser/Thr) protein phosphatase activities in N. crassa wild type (FGSC 424) were type 1 (PP1), type-2A (PP2A) and type-2C (PP2C). PP1 and PP2C predominantly dephosphorylated phosphorylase a and casein, respectively. PP2A acted on both substrates, but was two-fold more active against casein. PPI activity was inhibited by protamine, heparin, okadaic acid (IC50 50 nM) and mammalian inhibitor- 1 (lC50 2 nM). On the other hand, PP2A activity was inhibited by much lower concentrations of okadaic acid (IC50 0.2 nM) and also by protamine, but not by heparin or inhibitor-l. About 80 per cent of total PP1 activity was associated with the particulate fraction and could be partially extracted with 0.5 M NaCl. Seventy and ninety percent of PP2A and PP2C activities, respectively, were found in the soluble fraction. In addition we have partially purified an acid and thermostable PP1 inhibitor which effectively inhibits both N. crassa and mammalian PP1.

Cell wall deficiency in slime strains of Neurospora crassa: osmotic inhibition of cell wall synthesis and Beta-D-glucan synthase activity

1. The RCP-3 S/H mutant of Neurospora crassa was obtained by vegetative selection in medium of high osmolarity of a mycelial form an fz, sg, os-1 ("smile"-like) segregant. The mutant exhibits spheroplast-hyphal dimorphism conditioned by the osmolarity of the culture medium (pietro et al. (1990). Journal of General Microbiology, 136: 121-129). The carbohydrate composition of the cell wall of the mutant was different from that of the wild type in the absence of an alkalisoluble galactosaminoglycan polymer. Furthermore the mutant cell wall had a somewhat lower content of ß-glucan relative to that of chitin. 2. Increasing concentrations of sorbitol in the culture medium of the mutant inhibited by 10-fold the formation of cell wall relative to toal biomass. The cell wall of the mutant cultured in the presence of sorbitol lacked mannose-and galactose-containing polymers, and also showed progressively lower amounts of ß-glucan relative to chitin. 3. The activity of membrane-bound (1-3)-ß-D-glucan synthase from the mutant grown in the absence of sorbitol shared several properties with the wil type enzyme (i.e., Km app, Vmax, stability at 30ºC, activation by GTPyS, and dissociability by treatment with NaCl and Tergitol NP-40 into a membrane-bound catalytic center and GTP-binding activating protein). On the other hand, the enzyme from the mutant but not that from the wild type was inactivated by about 15 per cent by treatment with NaCl and detergent. 4. At high concentrations of sorbitol (1.0M) the RCP-3 S/H mutant exclusively produced spheroplasts devoid of (1-3)-ß-D-glucan synthase activity. The defect was at the level of the membrane-bound catalytic center. The activity of the GTP-binding activating factor was apparently normal in these cells. 5. These results suggest that the definitive loss of cell wall in the N. crassa "slime" RCP-3 S/H mutant was due to a defect in (1-3)-ß-D-glucan synthase activity which wass exaggerated in the presence of high osmolyte concentrations

Mating type response in Neurospora crassa: early and transient changes in the patters of protein synthesis in sexually stimulated mycelia

1. Pulse labeling with [35S]-methionine, one-dimensional SDS-polyacrylamide gel electrophoresis and florography were used to study the pattern of protein synthesis in Neurospora crassa mycelia undergoing sexual development. 2. Contact of sexually-competent mycelium with cells of the opposite mating type elicited a rapid and transient increase in the synthesis of two predominant proteins of 58 KDa and yoKDa and 40 KDa localized in the cytosol fraction. 3. Marked changes in the pattern of protein synthesis were also observed in the 12,000 g particulate fraction, predominantly mitochondrial, where the synthesis of a 34 KDa polypeptide was most prominent among others. 4. Poly(A) + RNA extracted from mycelia 2 h after sexual stimulation supported the in vitro synthesis of the 58 KDa and yoKDa major polypeptides synthesized in vivo. 5. No differences were observed in the pattern of protein synthesis of treated cultures and controls 24 h after the sexual stimulus