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.

Plant extract assisted extracellular tannase production by Aspergillus sp MIK 23

An extracellular tannase (E.C. 3.1.1.20) producing fungal strain was isolated from soil and identified as Aspergillus sp MIK23. Out of various plant extracts, Terminalia chebula powder (TCP) in the optimized medium enhanced enzyme production. Maximum yield of tannase (3 IU ml-1) was obtained with glucose (10 g/L), urea (2 g/L), and yeast extract (2.5 g/L) when inoculated with 10% inoculum in 48 h. An initial medium at pH 6.0 and a cultivation temperature of 37 0C was found to be optimum for enzyme production. Metal ions Mg2+, Zn2+, Ca2+, Cu2+ and Cd2+ did not improve enzyme activity, whereas, Ca2+, Fe2+ and Hg2+ repressed enzyme activity. The enzyme was purified using ammonium sulfate precipitation followed by Q-sepharose ion-exchange chromatography. The enzyme was purified to 42-fold with an overall recovery of 20. The pH and temperature optima of the purified tannase were found to be 7.0 and 37°C, respectively.

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.

真菌单宁酶发酵五倍子和有机溶剂中酶法合成没食子酸丙酯的研究

本文报告了丝状真菌单宁酶发酵五倍子及有机溶剂中酶法合成没食子酸丙酯的研究。利用单宁和/或五倍子诱导丝状真菌产生单宁 酶的原理，借助二级发酵程序，对从天然源得到的75株菌进行了生物转化实验研究。选择出既能水解单宁或五倍子成没食子酸，又 能把没食子酸和丙醇合成没食子酸丙酯，而且生物催化活性都较高的1株菌，这株菌经初步鉴定为黑曲霉(Aspergillus niger No.17)。随后对它开展了产酶条件和参数优化实验，得出了最佳培养条件。立足于参数优化实验方案的基础上，经由液体培养发酵 制备单宁酶制剂，并把该酶通过化学手段共价结合到一种新型载体—聚乙烯醇和戊二醛反应生成的缩醛上，制备得到固定化单宁酶 。这种固定化生物催化剂在两种有机介质体系中都具有逆向催化合成没食子酸丙酯的能力。最后建立起来一条有效可行的微生物酶 法制备没食子酸的技术途径，没食子酸产率达到70%。对这种物质进行元素 分析：含C,49.45%;含H,3.63%。它的熔点为237℃～243 ℃，三种溶剂系统的TLC均只给出一个斑点。这些数据都与标准品一致。有机溶剂中酶法合成没食子酸丙酯的技术途径已经建立。 水溶性单宁酶在潜溶剂体系中也能催化上述酯化反应，反应混合物中的PG浓度为16.4mmol/L，制备薄层被用于分离反应混合物所含 的PG，这种产物被红外、质谱及三种溶剂系统的TLC等方法鉴定，确证为目标产物。在这一学位论文的实验研究过程中，还包括一 些生化分析方法的建立和应用，这些方法用于鉴定底物和产物及测定它们的浓度，其内容主要包括TLC定性/半定量分析、元素分析 、质谱、红外等手段的综合运用。本工作为开发我国特有的天然产物资源—五倍子的生物化工加工技术及非水相生物催化技术的开 发，提供了有用的基础数据资料，具有应用基础研究工作的重要性。In this thesis, the studies on the fermentation of Chinese gallotannin by filamentous fungi with tannase activity and enzymatic synthesis of propyl gallate(PG) in organic solvents were described through these biocatalysts. Based on the principles of induction enzyme, the tannase produced from filamentous fungi by adding tannic acid(TA) and/or Chinese gallotannin into media was investigated, and the screening experiments of bioconversion were done with 75 strains by means of a two-stage fermentation procedure. These strains were isolated with the enrichment culture technique from natural sources. Hence we selected one strain (Aspergillus niger No.17) that can not only catalyze the hydrolyses of TA and/or Chinese gallotannin into gallic acid(GA) in the liquid cultures, but also be used to synthesize PG from propanol and GA in the non-aqueous media. At the same time both of its biocatalytical activities were higher. This strain was calssified to be Aspergillus niger by the primary identification. Then optimum conditions for production of the tannase and its parameters were examined. In this way, one set of optimum culture conditions was selected. Making use of the optimum proposal, the tanase was prepared through a liquid fermentation procedure. The enzyme was convalently coupled to a new type of carrier which was made chemically from polyvinyl alcohol(PVA)and glutaraldehyde. The immobilized enzymes were able to synthesize PG reversely in two organic media. Finally, an effective enzymatic technique for production of GA was developed. The yield of GA products was up to 70%。Element analysis for this substance: calce: C, 49.42%; H, 3.56%; found: C, 49.45%, H, 3.63%. Its melting point was 237℃～ 243℃ and TLCs on three solvent systems gave only one spot respectively. These data were identical with theauthentic GA. The enzymatic synthesis of PG in organic solvents was extablished with reverse route of tannase catalytical hydrolysis. Aqueous enzyme perparation also catalyzed above esterification in a buffer system. The PG concentration in the reaction mixture was 16.4mmol/L. The reparative-scale TLC was used to isolate PG from the reaction mixture. This product separated was identified by IR, MS and TLC on three solvent systems. In this study of thesis, some biochemical analytical mehtods were developed and used to identify substrates and products, and to determinate their concentration. These methods, including TLC qualitative/half quantitative analysis, element analysis, MS, IR and so on, were useful, available and performable. This work provided basic data and information for developing the biochemical engineering and bio-processing of Chinese gallotannin-a special natural resource in China and the non-aqueous phase biocatalysis. Thus, this study possesses importance in the applied and basic research work.

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