Production, purification, genetic characterization and application studies of tannase enzyme from marine fungus Aspergillus awamori

Marine fungi remain totally unexplored as a source of industrial enzyme and prospective applications. Further tannase production by a marine organism has so far not been established. The primary objective of this study included the evaluation of the potential of Aspergillus awamori isolated from sea water as part of an earlier study and available in the culture collection of the Microbial technology laboratory for tannase production through different fermentation methods, optimization of bioprocess variables by statistical methods, purification and characterization of the enzyme, genetic study, and assessment of its potential applications.

Propyl Gallate Synthesis Using Acidophilic Tannase and Simultaneous Production of Tannase and Gallic Acid by Marine Aspergillus awamori BTMFW032

Marine Aspergillus awamori BTMFW032, recently reported by us, produce acidophilic tannase as extracellular enzyme. Here, we report the application of this enzyme for synthesis of propyl gallate by direct transesterification of tannic acid and in tea cream solubilisation besides the simultaneous production of gallic acid along with tannase under submerged fermentation by this fungus. This acidophilic tannase enabled synthesis of propyl gallate by direct transesterification of tannic acid using propanol as organic reaction media under low water conditions. The identity of the product was confirmed with thin layer chromatography and Fourier transform infrared spectroscopy. It was noted that 699 U/ml of enzyme could give 60% solubilisation of tea cream within 1 h. Enzyme production medium was optimized adopting Box–Behnken design for simultaneous synthesis of tannase and gallic acid. Process variables including tannic acid, sodium chloride, ferrous sulphate, dipotassium hydrogen phosphate, incubation period and agitation were recognized as the critical factors that influenced tannase and gallic acid production. The model obtained predicted 4,824.61 U/ml of tannase and 136.206 μg/ml gallic acid after 48 h of incubation, whereas optimized medium supported 5,085 U/ml tannase and 372.6 μg/ml of gallic acid production after 36 and 84 h of incubation, respectively, with a 15-fold increase in both enzyme and gallic acid production. Results indicated scope for utilization of this acidophilic tannase for transesterification of tannic acid into propyl gallate, tea cream solubilisation and simultaneous production of gallic acid along with tannase

Solid-state fermentation: a continuous process for fungal tannase production

Truly continuous solid-state fermentations with operating times of 2-3 weeks were conducted in a prototype bioreactor for the production of fungal (Penicillium glabrum) tannase from a tannin-containing model substrate. Substantial quantities of the enzyme were synthesized throughout the operating periods and (imperfect) steady-state conditions seemed to be achieved soon after start-up of the fermentations. This demonstrated for the first time the possibility of conducting solid-state fermentations in the continuous mode and with a constant noninoculated feed. The operating variables and fermentation conditions in the bioreactor were sufficiently well predicted for the basic reinoculation concept to succeed. However, an incomplete understanding of the microbial mechanisms, the experimental system, and their interaction indicated the need for more research in this novel area of solid-state fermentation. (C) 2004 Wiley Periodicals, Inc.

Modelling the uptake and growth kinetics of Penicillium glabrum in a tannic acid-containing solid-state fermentation for tannase production

A mathematical growth model for the batch solid-state fermentation process for fungal tannase production was developed and tested experimentally. The unstructured model describes the uptake and growth kinetics of Penicillium glabrum in an impregnated polyurethane foam substrate system. In general, good agreement between the experimental data and model simulations was obtained. Biomass, tannase and spore production are described by logistic kinetics with a time delay between biomass production and tannase and spore formation. Possible induction mechanisms for the latter are proposed. Hydrolysis of tannic acid, the main carbon source in the substrate system, is reasonably well described with Michaelis-Menten kinetics with time-varying enzyme concentration but a more complex reaction mechanism is suspected. The metabolism of gallic acid, a tannase-hydrolysis product of tannic acid, was shown to be growth limiting during the main growth phase. (c) 2004 Elsevier Ltd. All rights reserved.

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

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

Extracellular tannase from Emericella nidulans showing hypertolerance to temperature and organic solvents

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

Characterization of a tannase from Emericela nidulans immobilized on ionic and covalent supports for propyl gallate synthesis

The extracellular tannase from Emericela nidulans was immobilized on different ionic and covalent supports. The derivatives obtained using DEAE-Sepharose and Q-Sepharose were thermally stable from 60 to 75 °C, with a half life (t50) >24 h at 80 °C at pH 5. 0. The glyoxyl-agarose and amino-glyoxyl derivatives showed a thermal stability which was lower than that observed for ionic supports. However, when the stability to pH was considered, the derivatives obtained from covalent supports were more stable than those obtained from ionic supports. DEAE-Sepharose and Q-Sepharose derivatives as well as the free enzyme were stable in 30 and 50 % (v/v) 1-propanol. The CNBr-agarose derivative catalyzed complete tannic acid hydrolysis, whereas the Q-Sepharose derivative catalyzed the transesterification reaction to produce propyl gallate (88 % recovery), which is an important antioxidant. © 2012 Springer Science+Business Media Dordrecht.

Characterization of a glucose- and solvent-tolerant extracellular tannase from Aspergillus phoenicis

Tannases have attracted wider attention because of their biotechnological potential, especially enzymes from filamentous fungi and other microorganisms. However, the biodiversity of these microorganisms has been poorly explored, and few strains were identified for tannase production and characterization. This article describes the production, purification and characterization of a glucose- and solvent-tolerant extracellular tannase from Aspergillus phoenicis. High enzymatic levels were obtained in Khanna medium containing tannic acid up to 72 h at 30 °C under 100 rpm. The purified enzyme with 65% of carbohydrate content had an apparent native molecular mass of 218 kDa with subunits of 120 kDa and 93 kDa and was stable at 50 °C for 1 h. Optima of temperature and pH were 60 °C and 5.0-6.5, respectively. The enzyme was not affected significantly by most ions, detergents and organic solvents. While glucose did not affect the tannase activity, the addition of a high concentration of gallic acid did. The Km values were 1.7 mM (tannic acid), 14.3 mM (methyl-gallate) and 0.6 mM (propyl-gallate). The enzyme was able to catalyze the transesterification reaction to produce propyl-gallate. All biochemical properties suggest the biotechnological potential of the glucose- and solvent-tolerant tannase from A. phoenicis. © 2012 Elsevier B.V. All rights reserved.

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

Background: Tannases are enzymes that may be used in different industrial sectors as, for example, food and pharmaceutical. They are obtained mainly from microorganisms, as filamentous fungi. However, the diversity of fungi stays poorly explored for tannase production. In this article, Aspergillus ochraceus is presented as a new source of tannase with interesting features for biotechnological applications. Results: Extracellular tannase production was induced when the fungus was cultured in Khanna medium with tannic acid as carbon source. The extracellular tannase was purified 9-fold with 2% recovery and a single band corresponding to 85 kDa was observed in SDS-PAGE. The native apparent molecular mass was estimated as 112 kDa. Optima of temperature and pH were 40 degrees C and 5.0, respectively. The enzyme was fully stable from 40 degrees C to 60 degrees C during 1 hr. The activity was enhanced by Mn2+ (33-39%) and NH4+ (15%). The purified tannase hydrolyzed tannic acid and methyl gallate with Km of 0.76 mM and 0.72 mM, respectively, and Vmax of 0.92 U/mg protein and 0.68 U/mg protein, respectively. The analysis of a partial sequence of the tannase encoding gene showed an open read frame of 567 bp and a sequence of 199 amino acids were predicted. TLC analysis revealed the presence of gallic acid as a tannic acid hydrolysis product. Conclusion: The extracellular tannase produced by A. ochraceus showed distinctive characteristics such as monomeric structure and activation by Mn2+, suggesting a new kind of fungal tannases with biotechnological potential. Further, it was the first time that a partial gene sequence for A. ochraceus tannase was described.

Combined bioremediation and enzyme production by Aspergillus sp. in olive mill and winery wastewaters

Olive mill wastewaters (OMW) and vinasses (VS) are effluents produced respectively by olive mills and wineries, both sectors are of great economic importance in Mediterranean countries. These effluents cause a large environmental impact, when not properly processed, due to their high concentration of phenolic compounds, COD and colour. OMW may be treated by biological processes but, in this case, a dilution is necessary, increasing water consumption. The approach here in proposed consists on the bioremediation of OMW and VS by filamentous fungi. In a screening stage, three fungi (Aspergillus ibericus, Aspergillus uvarum, Aspergillus niger) were selected to bioremediate undiluted OMW, two-fold diluted OMW supplemented with nutrients, and a mixture of OMW and VS in the proportion 1:1 (v/v). Higher reductions of phenolic compounds, colour and COD were achieved mixing both residues; with A. uvarum providing the best results. In addition, the production of enzymes was also evaluated during this bioremediation process, detecting in all cases lipolytic, proteolytic and tannase activities. A. ibericus, A. uvarum and A. niger achieved the highest value of lipase (1253.7 ± 161.2 U/L), protease (3700 ± 124.3 U/L) and tannase (284.4 ± 12.1 U/L) activities, respectively. Consequently, this process is an interesting alternative to traditional processes to manage these residues, providing simultaneously high economic products, which can be employed in the same industries.

Purificação e caracterização bioquímica de tanases produzidas pelo fungo filamentoso Aspergillus phoenicis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de tanases por Emericella nivea : purificação e caracterização bioquímica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção e caracterização das Tanases do fungo filamentoso Aspergillus carbonarius

Pós-graduação em Biotecnologia - IQ