Tony Marcio da Silva

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t₅₀ of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).

Biotechnological Potential of Agro-Industrial Wastes as a Carbon Source to Thermostable Polygalacturonase Production in Aspergillus niveus

Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. The aim of this work was to study polygalacturonase (PG) production by Aspergillus niveus cultured on liquid or solid media supplemented with agro-industrial wastes. Submerged fermentation (SbmF) was tested using Czapeck media supplemented with 28 different carbon sources. Among these, orange peel was the best PG inducer. On the other hand, for solid state fermentation (SSF), lemon peel was the best inducer. By comparing SbmF with SSF, both supplemented with lemon peel, it was observed that PG levels were 4.4-fold higher under SSF. Maximum PG activity was observed at 55°C and pH 4.0. The enzyme was stable at 60°C for 90 min and at pH 3.0–5.0. The properties of this enzyme, produced on inexpensive fermentation substrates, were interesting and suggested several biotechnological applications.

Biotechnological potential of alternative carbon sources for production of pectinases by Rhizopus microsporus var. rhizopodiformis

Fungi collected from Brazilian soil and decomposing plants were screened for pectinase production. R. microsporus var. rhizopodiformis was the best producer and was selected to evaluate the pectic enzyme production under several nutritional and environmental conditions. The pectinase production was studied at 40oC, under 28 carbon sources-supplemented medium. The inducer effect of several agro-industrial residues such as sugar cane bagasse, wheat flour and corncob on polygalacturonase (PG) activity was 4-, 3- and 2-fold higher than the control (pectin). In glucose-medium, a constitutive pectin lyase (PL) activity was detected. The results demonstrated that R. microsporus produced high levels of PG (57.7 U/mg) and PL (88.6 U/mg) in lemon peel-medium. PG had optimum temperature at 65 oC and was totally stable at 55 oC for 90 min. Half-life at 70 oC was 68 min. These results suggested that the versatility of waste carbon sources utilization by R. microsporus, produce pectic enzymes, which could be useful to reduce production costs and environmental impacts related to the waste disposal.

Increase of the phytase production by Aspergillus japonicus and its biocatalyst potential on chicken feed treatment

Phytase hydrolyzes phytic acid from the plant components of animal feed, releasing inorganic phosphorus. The phytase production by Aspergillus japonicus was optimized using Plackett–Burman designs (PBD), composite central rotational designs (CCRD), and response surface methodology from standard Czapek medium. The enzyme was applied in broiler chicken and laying hen foods. Analysis from PBD showed that KH2PO2, MgSO4 · 7H2O, and yeast extract had significant influences on phytase secretion (p < 0.05). The best results from the CCRD experiments were obtained using (A) 0.040% KH2PO4, (B) 0.050% MgSO4 · 7H2O, and (C) 0.040% yeast extract, enhancing in 49–53 U mg−1 protein. The determination coefficient (R2) was 0.92 and Fcalc was 7.48 times greater than Flisted. Thus, the reduced coded model: Y (U mg−1)=50.29+4.30A−3.35(A)2−4.80(B)2+5.62C−4.26(C)2

Use of cassava peel as carbon source for production of amylolytic enzymes by Aspergillus niveus.

Aspergillus niveus produced high levels of ?-amylase and glucoamylase in submerged fermentation using the agricultural residue cassava peel as a carbon source. In static conditions, the amylase production was substantially greater than in the agitated condition. The optimized culture conditions were initially at pH 5.0, 35°C during 48 hours. Amylolytic activity was still improved (50%) with a mixture of cassava peel and soluble starch in the proportion 1:1 (w/w). The crude extract exhibited temperature and pH optima approximately 70°C and 4.5, respectively. Amylase activity was stable for 1 h at 60°C, and at pH values between 3.0 and 7.0. The enzyme hydrolysed preferentially maltose, starch, penetrose, amylose, isomaltose, maltotriose, glycogen and amylopectin, and not hydrolysed cyclodextrin (? and ß), trehalose and sucrose. In the first hour of reaction on soluble starch, the hydrolysis products were glucose and maltose, but after two hours of hydrolysis, glucose was the unique product formed, confirming the presence in the crude extract of an ?-amylase and a glucoamylase.

Co-immobilization of fungal endo-xylanase and α-L-arabinofuranosidase in glyoxyl agarose for improved hydrolysis of arabinoxylan

Plant cell-wall arabinoxylans have a complex structure that requires the action of a pool of debranching (arabinofuranosidases) and depolymerizing enzymes (endo-xylanase). Two Aspergillus nidulans strains over-secreting endo-xylanase and arabinofuranosidase were inoculated in defined 2% maltose-minimum medium resulting in the simultaneously production of these enzymes. To study the synergistic hydrolysis was used arabinoxylan with 41% of arabinose and 59% of xylose residues. Thus, it was adopted different approaches to arabinoxylan hydrolysis using immobilized arabinofuranosidase and endo-xylanase: (i) endo-xylanase immobilized on glyoxyl agarose; (ii) arabinofuranosidase immobilized on glyoxyl agarose; (T1) hydrolysis of arabinoxylan with arabinofuranosidase immobilized on glyoxyl agarose for debranching, followed by a second hydrolysis with endo-xylanase immobilized on glyoxyl agarose; (T2) hydrolysis using (i) and (ii) simultaneously; and (T3) hydrolysis of arabinoxylan with endo-xylanase and arabinofuranosidase co-immobilized on glyoxyl agarose. It was concluded that arabinoxylan hydrolysis using two derivatives simultaneously (T2) showed greater hydrolytic efficiency and consequently a higher products yield. However, the hydrolysis with multi-enzymatic derivative (T3) results in direct release of xylose and arabinose from a complex substrate as arabinoxylan, which is a great advantage as biotechnological application of this derivative, especially regarding the application of biofuels, since these monosaccharides are readily assimilable for fermentation and ethanol production.

THERMOSTABLE SACCHAROGENIC AMYLASE PRODUCED UNDER SUBMERGED FERMENTATION BY FILAMENTOUS FUNGUS Penicillium purpurogenum

The effect of several nutritional and environmental parameters on Penicillium purpurogenum growth and sacharogenic amylase production was analyzed. High enzyme levels (68.2 U mg-1) were obtained with Khanna medium at initial pH 6.0, incubated at 30oC for 144 hours. The optimum pH and temperature activities were 5.0 and 65oC, respectively. The enzyme presented a half-life (t50) of 60 min, at 65oC. Only glucose was detected after 24 hours of reaction using soluble starch as substrate.

Evidence of high production levels of thermostable dextrinizing and saccharogenic amylases by Aspergillus niveus

The aim of this work was to analyze the effect of several nutritional and environmental parameters on amylase production by a novel, isolated from the thermotolerant filamentous fungus Aspergillus niveus. This strain produced high levels of amylolytic activity in Khanna liquid medium supplemented with commercial starch, initial pH 6.5, under static conditions for 72 h. Among the tested carbon sources, milled corn, oatmeal, soluble potato starch and maisena were the best inducers of enzymatic secretion (220, 180, 170 and 150 U/mL), respectively. The main products of hydrolysis analyzed by thin layer chromatography were glucose, maltose and traces of maltooligosaccharides, suggesting the presence of -amylase and glucoamylase activities in the crude extract. The optimal pH were 4.5 and 5.5 and the optimum temperature was 65°C. The enzymes were fully stable up to 1 h at 55°C. It was possible to verify the presence of three bands with amylolytic activity in non-denaturing polyacrylamide gel electrophoresis (PAGE). These aspects and other properties suggested that the amylases produced by A. niveus might be suitable for biotechnological applications.

Gel Electrophoresis for Investigating Enzymes with Biotechnological Application

Maria de Lourdes T. M. Polizeli1*, Simone C. Peixoto-Nogueira1, Tony M. da Silva1, Alexandre Maller2 and Hamilton Cabral3 1Biology Department, Faculty of Philosophy Sciences and Letters of Ribeirao Preto, Sao Paulo University 2Biochemistry and Immunology Department School of Medicine of Ribeirao Preto, Sao Paulo, Sao Paulo University 3Science Pharmaceutical Department School of Pharmaceutical Science of Ribeirao Preto, Sao Paulo University Brazil