Use of Cassava Peel as Carbon Source for Production of Amylolytic Enzymes by Aspergillus niveus

Aspergillus niveus produced high levels of alpha-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 degrees 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 degrees C and 4.5, respectively. Amylase activity was stable for 1 h at 60 degrees 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 (alpha and beta), 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 alpha-amylase and a glucoamylase.

Effects on starch and amylolytic enzymes during Lepidium meyenii Walpers root storage

The high water content in maca (Lepidium meyenii W.) roots combined with the damage produced during or after harvest makes them vulnerable to attack by enzymes and microorganisms. Although starch degradation has been extensively studied, in maca roots there is a paucity of research regarding the starch reserves. In this paper, parameters of starch degradation are shown to be related to the action of amylolytic enzymes during storage at room temperature. Over the course of three weeks, the starch and protein content, soluble sugar, total amylolytic activity, and alpha- and beta-amylase activity were measured. In addition, the integrity of starch granules was observed by scanning electron microscopy. Despite the evidence of dehydration, there were no significant differences (p <= 0.5) in the total starch content or in the activities of alpha- and beta-amylase. After the third week the roots remained suitable for consumption. The results indicate a postharvest latency that can lead to sprout or to senescence, depending on the environmental conditions. (C) 2012 Elsevier Ltd. All rights reserved.

Sequential production of amylolytic and lipolytic enzymes by bacterium strain isolated from petroleum contaminated soil

Amylases and lipases are highly demanded industrial enzymes in various sectors such as food, pharmaceuticals, textiles, and detergents. Amylases are of ubiquitous occurrence and hold the maximum market share of enzyme sales. Lipases are the most versatile biocatalyst and bring about a range of bioconversion reactions such as hydrolysis, inter-esterification, esterification, alcoholysis, acidolysis, and aminolysis. The objective of this work was to study the feasibility for amylolitic and lipolytic production using a bacterium strain isolated from petroleum contaminated soil in the same submerged fermentation. This was a sequential process based on starch and vegetable oils feedstocks. Run were performed in batchwise using 2% starch supplemented with suitable nutrients and different vegetable oils as a lipase inducers. Fermentation conditions were pH 5.0; 30 degrees C, and stirred speed (200 rpm). Maxima activities for amyloglucosidase and lipase were, respectively, 0.18 and 1,150 U/ml. These results showed a promising methodology to obtain both enzymes using industrial waste resources containing vegetable oils.

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t(50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).

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

An alpha-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The alpha-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pl value of 4.5. Temperature and pH optima were 60 degrees C and 4.0, respectively. The enzyme was stable for 1 h at 55 degrees C, showing a t(50) of 53 min at 60 degrees C. Starch protected the enzyme against thermal inactivation. The a-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 alpha-amylase on Reagen (R) and Sigma (R) 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 alpha-amylases from Bacillus sp. These results confirmed that the studied enzyme was an a-amylase ((1 -> 4)-alpha-glucan glucanohydrolase). (C) 2010 Elsevier Ltd. All rights reserved.

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 107 CFU ml−1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 105 to 108 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD50 to BALB/c mice was found to be 109 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections

Partitioning of the glucoamylase activity at the cell surfaces in cultures of Saccharomyces

Glucoamylases have been used with alpha-amylases for the industrial conversion of starch into glucose. However, little is known about the properties of this glycosylated protein retained in the cell wall of Saccharomyces as well as its role in the saccharification and fermentation of amylaceous substrates, notably in high cell density processes. In most of the strains assayed, decreases in biomass formation were followed by increases in glucoamylase secretion (expressed as U/mg(biomass) in 1 ml of culture) when glucose was exchanged for starch as carbon source or the growth temperature was raised from 30 to 35 degrees C. Despite the losses in viability, significant increases in the activity of the wall fraction occurred when cultures of thermotolerant yeasts propagated at 30 degrees C or washed cells resuspended in buffer solution were heated to 60 degrees C for 60-80 min prior to amylolytic assays. Thus, intact cells of thermotolerant yeasts can be used as colloidal biocatalysts in starch degradation processes. (C) 2005 Published by Elsevier Ltd.

Produção de etanol a partir de sorgo sacarino com tratamento enzimático

Pós-graduação em Microbiologia Agropecuária - FCAV

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.

Biomass production and secretion of hydrolytic enzymes are influenced by the structural complexity of the nitrogen source in Fusarium oxysporum and Aspergillus nidulans

The structural complexity of the nitrogen sources strongly affects biomass production and secretion of hydrolytic enzymes in filamentous fungi. Fusarium oxysporum and Aspergillus nidulans were grown in media containing glucose or starch, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids), peptides (peptone) and protein (gelatin). In glucose, when the initial pH was adjusted to 5.0, for both microorganisms, higher biomass production occurred upon supplementation with a nitrogen source in the peptide form (peptone and gelatin). With a close to neutrality pH, biomass accumulation was lower only in the presence of the ammonium salt. When grown in starch, biomass accumulation and secretion of hydrolytic enzymes (amylolytic and proteolytic) by Fusarium also depended on the nature of the nitrogen supplement and the pH. When the initial pH was adjusted to 5.0, higher growth and higher amylolytic activities were detected in the media supplemented with peptone, gelatin and casamino acids. However, at pH 7.0, higher biomass accumulation and higher amylolytic activities were observed upon supplementation with peptone or gelatin. Ammonium sulfate and casamino acids induced a lower production of biomass, and a different level of amylolytic enzyme secretion: high in ammonium sulfate and low in casamino acids. Secretion of proteolytic activity was always higher in the media supplemented with peptone and gelatin. Aspergillus, when grown in starch, was not as dependent as Fusarium on the nature of nitrogen source or the pH. The results described in this work indicate that the metabolism of fungi is regulated not only by pH, but also by the level of structural complexity of the nitrogen source in correlation to the carbon source.

Digestive Peptidase Evolution In Holometabolous Insects Led To A Divergent Group Of Enzymes In Lepidoptera.

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

Production of Cellulolytic Enzymes by Anaerobic Fungi Cultivated in Different Conditions

The present paper studies the influence of different nutrients for the production of two cellulolytic enzymes: endo beta-1.4 glucanase and exo beta-1.4 glucanase by anaerobic fungi taken from cow rumen, that were fed a diet of corn silage and Brachiaria decumbens grass hay. During the enzymatic degradation assays, it was observed that the addition of some essential nutrients in the formulation of the culture medium contributed positively in the cellulolytic enzyme production, with exception of riboflavin. Such results contributed in the establishment of an effective method for the evaluation of enzymatic activities in anaerobic fibrolytic fungi. In this work, nutrients added to enrich the culture medium have successfully proven that they can be used as inoculating agents (inductors) in diets rich in ensilage with law nutritive value.

Evaluation of hexose and pentose in pre-cultivation of Candida guilliermondii on the key enzymes for xylitol production in sugarcane hemicellulosic hydrolysate

The evaluation of hexose and pentose in pre-cultivation of Candida guilliermondii FTI 20037 yeast on xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes activities was performed during fermentation in sugarcane bagasse hemicellulosic hydrolysate. The xylitol production was evaluated by using cells previously growth in 30.0 gl(-1) xylose, 30.0 gl(-1) glucose and in both sugars mixture (30.0 gl(-1) xylose and 2.0 gl(-1) glucose). The vacuum evaporated hydrolysate (80 gl(-1)) was detoxificated by ion exchange resin (A-860S; A500PS and C-150-Purolite(A (R))). The total phenolic compounds and acetic acid were 93.0 and 64.9%, respectively, removed by the resin hydrolysate treatment. All experiments were carried out in Erlenmeyer flasks at 200 rpm, 30A degrees C. The maximum XR (0.618 Umg (Prot) (-1) ) and XDH (0.783 Umg (Prot) (-1) ) enzymes activities was obtained using inoculum previously growth in both sugars mixture. The highest cell concentration (10.6 gl(-1)) was obtained with inoculum pre-cultivated in the glucose. However, the xylitol yield and xylitol volumetric productivity were favored using the xylose as carbon source. In this case, it was observed maximum xylose (81%) and acetic acid (100%) consumption. It is very important to point out that maximum enzymatic activities were obtained when the mixture of sugars was used as carbon source of inoculum, while the highest fermentative parameters were obtained when xylose was used.