Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus.

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was purified from the culture filtrates of the thermophilic fungus Thermomyces lanuginosus and was established to be homogeneous by a number of criteria. The enzyme was a glycoprotein with an average molecular weight of about 57 000 and a carbohydrate content of 10-12%. The enzyme hydrolysed successive glucose residues from the non-reducing ends of the starch molecule. It did not exhibit any glucosyltransferase activity. The enzyme appeared to hydrolyse maltotriose by the multi-chain mechanism. The enzyme was unable to hydrolyse 1,6-alpha-D-glucosidic linkages of isomaltose and dextran. It was optimally active at 70 degrees C. The enzyme exhibited increase in the Vmax. and decreased in Km values with increasing chain length of the substrate molecule. The enzyme was inhibited by the substrate analogue D-glucono-delta-lactone in a non-competitive manner. The enzyme inhibited remarkable resistance towards chemical and thermal denaturation.

Engineering thermostable fungal cellobiohydrolases

Meeting the world's growing energy demands while protecting our fragile environment is a challenging issue. Second generation biofuels are liquid fuels like long-chain alcohols produced from lignocellulosic biomass. To reduce the cost of biofuel production, we engineered fungal family 6 cellobiohydrolases (Cel6A) for enhanced thermostability using random mutagenesis and recombination of beneficial mutations. During long-time hydrolysis, engineered thermostable cellulases hydrolyze more sugars than wild-type Cel6A as single enzymes and binary mixtures at their respective optimum temperatures. Engineered thermostable cellulases exhibit synergy in binary mixtures similar to wild-type cellulases, demonstrating the utility of engineering individual cellulases to produce novel thermostable mixtures. Crystal structures of the engineered thermostable cellulases indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by proline substitutions. At high temperature, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A and wild-type Cel6A. The mechanism of thermal inactivation in this cellulase family is consistent with disulfide bond degradation and thiol-disulfide exchange. Enhancing the thermostability of Cel6A also increases tolerance to pretreatment chemicals, demonstrated by the strong correlation between thermostability and tolerance to 1-ethyl-3-methylimidazolium acetate. Several semi-rational protein engineering approaches – on the basis of consensus sequence analysis, proline stabilization, FoldX energy calculation, and high B-factors – were evaluated to further enhance the thermostability of Cel6A.

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Localization and partial characterization of thermostable glucoamylase produced by newly isolated Thermomyces lanuginosus TO3 in submerged fermentation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of Different Substrates on the Production of a Mutant Thermostable Glucoamylase in Submerged Fermentation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungus Scytalidium thermophilum

An extracellular (conidial) and an intracellular (mycelial) alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum were purified by DEAE-cellulose and Concanavalin A-Sepharose chromatography. These enzymes showed allosteric behavior either in the presence or absence of MgCl2, BaCl2, CuCl2, and ZnCl2. All of these ions increased the maximal velocity of both enzymes. The molecular masses of the conidial and mycelial enzymes, estimated by gel filtration, were 162 and 132 kDa, respectively. Both proteins migrated on SDS-PAGE as a single polypeptide of 63 and 58.5 kDa, respectively, suggesting that these enzymes were dimers of identical subunits. The best substrate for the conidial and mycelial phosphatases was p-nitrophenylphosphate, but,beta -glycerophosphate and other phosphorylated compounds also served as substrates. The optimum pH for the conidial and mycelial alkaline phosphatases was 10.0 and 9.5 in the presence of AMPOL buffer, and their carbohydrate contents were about 54% and 63%, respectively. The optimum temperature was 70-75 degreesC for both activities. The enzymes were fully stable up to 1 h at 60 degreesC. These and other properties suggested that the alkaline phosphatases of S. thermophilum might be suitable for biotechnological applications.

Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evidence of thermostable amylolytic activity from Rhizopus microsporus var. rhizopodiformis using wheat bran and corncob as alternative carbon source

Rhizopus microsporus var. rhizopodiformis produced high levels of alpha-amylase and glucoamylase under solid state fermentation, with several agricultural residues, such as wheat bran, cassava flour, sugar cane bagasse, rice straw, corncob and crushed corncob as carbon sources. These materials were humidified with distilled water, tap water, or saline solutions-Segato Rizzatti (SR), Khanna or Vogel. The best substrate for amylase production was wheat bran with SR saline solution (1:2 v/v). Amylolytic activity was still improved (14.3%) with a mixture of wheat bran, corncob, starch and SR saline solution (1:1:0.3:4.6 w/w/w/v). The optimized culture conditions were initial pH 5, at 45 degrees C during 6 days and relative humidity around 76%. The crude extract exhibited temperature and pH optima around 65 degrees C and 4-5, respectively. Amylase activity was fully stable for 1 h at temperatures up to 75 degrees C, and at pH values between 2.5 and 7.5.

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)