Tannase Production Research Articles

Production and Partial Purification of Tannase from Serratia Marcescens Isolated from Different Sources

Tannase has different benefits in food, chemical and pharmaceutical fields. Seventeen Serratia marcescens isolates were collected from septicemia, wound infections and hospital environment(babies incubators).These isolates were identified by biochemical tests and Vitek 2 system that contained Vitek GNI card then conformed by16S rRNA gene products(amplified size 179 bp) for genotypic detection. After that, they screened for higher tannase production and Serratia marcescens b9 was a better producer of tannase with a larger diameter of a dark green zone. The tannase activity was increased to 63U/ml when this isolate was cultivated under the optimal conditions which consisted of using nutrient broth supplemented with ber leaves at pH value 5.5 and a temperature equals to 37°C for 72 hours. In the partial purification of tannase, ammonium sulfate was more efficient than organic solvents, since it was found that 70% saturation of ammonium sulfate led to precipitate of tannase with tannase activity of 80U/ml. In contrast, 30% of ethanol, acetone, and isopropanol led to precipitate of tannase with different levels of activity ranged between 45-47U/ml. Consequently, ber leaves have a potential as an effective and much cheaper (economical) substrate for tannase production in comparison with traditionally used substrates like tannic acid.

Co-production of tannase and gallic acid by a novel Penicillium rolfsii (CCMB 714)

A novel tannase and gallic acid-producing Penicillium rolfsii (CCMB 714) was isolated from cocoa leaves from the South of Bahia. The influence of nutritional sources and the simultaneous effect of parameters involved in the fermentation process were available. Tannase (9.97 U mL−1) and gallic acid (9 mg mL−1) production were obtained in 48 h by submerged fermentation in non-optimized conditions. Among the carbon sources, tested gallic acid and tannic acid showed the highest tannase production (p<.05) when compared with methyl gallate and glucose. After optimization using the temperature and tannic acid concentration as variables with the Central Compound Rotational Design (CCRD), the maximal tannase production (25.6 U mL−1) was obtained at 29.8 °C and 12.7%, respectively, which represents an increase of 2.56 times in relation to the initial activity. The parameters optimized for the maximum production of gallic acid (21.51 mg mL−1) were 30 °C and 10% tannic acid. P. rolfsii CCMB 714 is a new strain with a high tannase and gallic acid production and the gallic acid produced is very important, mainly for its applications in the food and pharmaceutical industry.

Tannases: Production, properties, applications

Abstract Microbial tannases are industrially important enzymes belong to the family of esterases which catalyze the generation of gallic acid and glucose by the degradation of polyphenolic compounds such as tannins. In spite of wastewater treatment and gallic acid production, tannases have wide application in the processing of food, beverage and animal feed. For tannase production, tannins containing low-value agro-industrial wastes are being extensively used in industries. Downstream processing of tannase through conventional methods is cheaper, but have lower purification fold of tannase, whereas advanced methods like ion exchange and size exclusion chromatography provide high purification. Tannases have a vast molecular weight range 31–310 kDa, furthermore, they have much stability to work in broad ranges of pH (3–10) and temperature (30–70 °C). An overview of production, downstream processing, properties, applications and recent advances of tannase is discussed in this review.

Kinetic, thermodynamic parameters and in vitro digestion of tannase from Aspergillus tamarii URM 7115

ABSTRACTTannase is an enzyme used in various industries and produced by a large number of microorganisms. The aim of this study was to evaluate tannase production to determine the biochemical, kinetic, and thermodynamic properties and to simulate tannase in vitro digestion. The tannase-producing fungal strain was isolated from “jamun” leaves and identified as Aspergillus tamarii. Temperature at 26°C for 67 h was the best combination for maximum tannase activity (6.35-fold; initial activity in Plackett–Burman design—15.53 U/mL and average final activity in Doehlert design—98.68 U/mL). The crude extract of tannase was optimally active at 40°C, pH 5.5 and 6.5. Moreover, tannase was stimulated by Na+, Ca2+, Mg2+, and Mn2+. The half-life at 40°C lasted 247.55 min. The free energy of Gibbs, enthalpy, and entropy, at 40°C, was 81.47, 16.85, and −0.21 kJ/mol · K, respectively. After total digestion, 123.95% of the original activity was retained. Results suggested that tannase from A. tamarii URM 7115 is an enzyme of interest for industrial applications, such as gallic acid production, additive for feed industry, and for beverage manufacturing, due to its catalytic and thermodynamic properties.

Novel optimization strategy for tannase production through a modified solid-state fermentation system

BackgroundHigh amounts of insoluble substrates exist in the traditional solid-state fermentation (SSF) system. The presence of these substrates complicates the determination of microbial biomass. Thus, enzyme activity is used as the sole index for the optimization of the traditional SSF system, and the relationship between microbial growth and enzyme synthesis is always ignored. This study was conducted to address this deficiency. All soluble nutrients from tea stalk were extracted using water. The aqueous extract was then mixed with polyurethane sponge to establish a modified SSF system, which was then used to conduct tannase production. With this system, biomass, enzyme activity, and enzyme productivity could be measured rationally and accurately. Thus, the association between biomass and enzyme activity could be easily identified, and the shortcomings of traditional SSF could be addressed.ResultsDifferent carbon and nitrogen sources exerted different effects on microbial growth and enzyme production. Single-factor experiments showed that glucose and yeast extract greatly improved microbial biomass accumulation and that tannin and (NH4)2SO4 efficiently promoted enzyme productivity. Then, these four factors were optimized through response surface methodology. Tannase activity reached 19.22 U/gds when the added amounts of tannin, glucose, (NH4)2SO4, and yeast extract were 7.49, 8.11, 9.26, and 2.25%, respectively. Tannase activity under the optimized process conditions was 6.36 times higher than that under the initial process conditions. The optimized parameters were directly applied to the traditional tea stalk SSF system. Tannase activity reached 245 U/gds, which is 2.9 times higher than our previously reported value.ConclusionsIn this study, a modified SSF system was established to address the shortcomings of the traditional SSF system. Analysis revealed that enzymatic activity and microbial biomass are closely related, and different carbon and nitrogen sources have different effects on microbial growth and enzyme production. The maximal tannase activity was obtained under the optimal combination of nutrient sources that enhances cell growth and tannase accumulation. Moreover, tannase production through the traditional tea stalk SSF was markedly improved when the optimized parameters were applied. This work provides an innovative approach to bioproduction research through SSF.

Optimization of physicochemical parameters of tannase post-purification and its versatile bioactivity.

The present study investigates the optimization of tannase production from Aspergillus nidulans for various physicochemical parameters and harvests tannase for its chemical characterization. The maximum tannase activity was observed on the third day of incubation at 35°C and the stability was observed at pH 5.5-6.0 by holding its 100% activity. The tannase was partially purified from A. nidulans [FT10] by ammonium sulfate precipitation at different concentrations, and it was found that at 80% of ammonium sulfate concentration, the precipitate exhibited the maximum activity for tannase of 96 U/ml. LCMS showed its M/Z value as 162.3 which was reconfirmed by SDS-PAGE. The UV spectrum and FTIR confirmed the presence of two oxy- and three hydroxyl groups in the benzene ring structure. The antibacterial activity of tannase was enhanced with antibiotics such as streptomycin and ceftazidime whereas the biofilm formation was significantly inhibited by the purified tannase. The scavenging activity was greatly increased with purified component and when the concentration of the purified tannase, FT10 was increased. To the best of our knowledge, this is one of the few reports where microbial species was used as the source for producing tannase enzyme and its role in various bioactivities such as antibacterial, anti-biofilm and antioxidant activity was evaluated.

Occurrence of a novel tannase (tan BLP ) in endophytic Streptomyces sp. AL1L from the leaf of Ailanthus excelsa Roxb.

The tannase production ability by endophytic actinobacteria and the genetic identity of responsible tannase gene were determined. The studied strains were isolated from surface-sterilized leaf discs of Ailanthus excelsa Roxb. Four strains were found to hydrolyze tannic acid on solid media containing 0.4% tannic acid. The strain AL1L was found as tanBLP indicating production of tannase with diverse of substrate affinity. The tannase production from the potential strain AL1L was performed in liquid tannic acid broth (0.4%, w/v). The strain was later identified as Streptomyces sp. AL1L on the basis of 16S rDNA homology. Highest enzyme activity was observed at 48h of incubation at the exponential growth phase. The enzyme was purified by ammonium sulfate precipitation followed by dialysis (15 kD cut off). This enzyme, with molecular weight 180 kD shows highest catalytic activity at 35°C, pH 6 with substrate concentration 0.1g%. The purified enzyme possesses 1.4×10-3Km and 11.15 U/ml as Vmax. The above study indicates high industrial prospective of endophytic actinobacteria as source of tannase of potential biotechnological applications.

Exploring the Degradation of Gallotannins Catalyzed by Tannase Produced by Aspergillus niger GH1 for Ellagic Acid Production in Submerged and Solid-State Fermentation.

Due to great interest on producing bioactive compounds for functional foods and biopharmaceuticals, it is important to explore the microbial degradation of potential sources of target biomolecules. Gallotannins are polyphenols present in nature, an example of them is tannic acid which is susceptible to enzymatic hydrolysis. This hydrolysis is performed by tannase or tannin acyl hydrolase, releasing in this way, biomolecules with high-added value. In the present study, chemical profiles obtained after fungal degradation of tannic acid under two bioprocesses (submerged fermentation (SmF) and solid state fermentation (SSF)) were determined. In both fermentation systems (SmF and SSF), Aspergillus niger GH1 strain and tannic acid as a sole carbon source and inducer were used (the presence of tannic acid promotes production of enzyme tannase). In case of SSF, polyurethane foam (PUF) was used like as support of fermentation; culture medium only was used in case of submerged fermentation. Fermentation processes were monitored during 72h; samples were taken kinetically every 8h; and all extracts obtained were partially purified to obtain polyphenolic fraction and then were analyzed by liquid chromatography-mass spectrometry (LC-MS). Molecules like gallic acid and n-galloyl glucose were identified as intermediates in degradation of tannic acid; during SSF was identified ellagic acid production. The results obtained in this study will contribute to biotechnological production of ellagic acid.

English

Tannase (tannin acyl hydrolse, E.C.3.1.1.20) is an enzyme that catalyzes the hydrolysis of ester and depside bonds of hydrolysable tannins. Among the filamentous fungi the genus Penicillium is recognized as the second best producer of tannase. This enzyme presents applications in several industrial segments, such as in the production of judges and teas. The industrial production of tannase can be performed through solid state fermentation (SSF) by the use of tannin rich fruit residues, in order to minimize production costs. In the present study 31 species of Penicillium isolated from Caatinga and Atlantic Forest were tested quantitatively for the production of tannase. The species were kept in the Collection of Cultures Micoteca - URM (WDCM604) under mineral oil. The enzyme production was carried out under SSF using “caja” (Spondias lutea L.) and Manga (Mangifera indica L.) residues that are rich in tannins. Mango residue proved to be the best inducer of the production of tannase by Penicillium species, most of which presented high tannase activity, ranging from 14.48 to 89.48 U mL-1. The three best producers were Penicillium rolfssi URM6216 with 89.49 U mL-1, Penicillium janthinellum URM5993 with 85.93 U mL-1 and Penicillium glabrum URM6092 with 59.26 U mL-1. Therefore, P. rolfssi URM6216 is being indicated for optimization of tannase production by SSF, using mango residue as a substrate. This is the first study on the potential of hog-plum (caja) and mango wastes as substrates for tannase production by filamentous fungi. Key words: Penicillium rolfssi URM6216, tannin acyl hydrolase, waste, mango, caja.

Diversity of lactic acid bacteria from Miang, a traditional fermented tea leaf in northern Thailand and their tannin-tolerant ability in tea extract.

The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041T. Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.

Evaluation of model parameters for growth, tannic acid utilization and tannase production in Bacillus gottheilii M2S2 using polyurethane foam blocks as support.

Production of tannase from B. gottheilii M2S2 was studied under solid-state fermentation with an optimized medium consisting of polyurethane foam matrix of dimension 40×40×5mm, impregnated with a liquid medium comprising (w/v): 4% tannic acid; 2% NH4NO3; 0.1% KH2PO4; 0.2% MgSO4; 0.1% NaCl and 0.05% CaCl2·2H2O in distilled water, having a pH of 4.7. Maximum tannase production of 56.87 U/L was obtained after 32h of fermentation at 32°C in static condition. This study deals with the evaluation of unstructured kinetic models to understand the behavior of biomass, tannase production and tannic acid degradation, with the fermentation time. The growth rate of B. gottheilii M2S2 was 0.0703h-1 at 32h of fermentation. Product (Yx/s) and biomass yield (Yp/s) coefficients were estimated as 1.77 U/g of tannic acid and 0.276g of biomass/g of tannic acid. All the kinetic constants µ, α, β, m and n were evaluated using MATLAB 2015Rb program. The experimental and model-generated data showed a good correlation, which indicated that these models will describe tannase production and fermentation process.

Synthesis and characterization of nanoparticles conjugated tannase and using it for enhancement of antibacterial activity of tannase produced by Serratia marcescens

Fourteen isolates of Serratia marcescens were collected from patients suffering from septicemia. All theseisolates revealed different levels in tannase production. Tannase was partially purified from Serratia marcescens b9 by precipitation method at 70% saturation of ammonium sulfate. Au, Pt, SnO2 and SiO2 nanoparticles were prepared by laser ablation and examined by transmission electron microscopy (TEM), X-ray diffraction pattern and UV-Visible absorption spectroscopy. Conjugation of SiO2 nanoparticles to tannase by feeding and pulses methods were prepared and characterized by TEM, X-ray diffraction pattern and UV-Visible spectrum. SiO2 nanoparticles conjugated partially purified tannase by feeding showed the higher effectiveness and higher significant level against all tested UTI causing in comparison with ciprofloxacin antibiotic, SiO2 nanoparticles alone, partially purified tannase alone and partially purified tannase by pulses. So that we can conclude that feeding method was the best method for enhancement partially purified tannase activity to maximum level thus SiO2 nanoparticles conjugated partially purified tannase may be a useful antibacterial agent for the treatment of urinary tract infection.

Isolation and screening of fungal isolates from bambara (&lt;i&gt;Vigna subterranea&lt;/i&gt;) nuts for tannase production

Tannase (Tannin acyl hydrolase, EC 3.1.1.20) is an enzyme produced in the presence of tannic acid by various filamentous fungi. They are produced principally by fungi of the genus Aspergillus and Penicillium. The enzyme is used in the food and beverage industry as a clarifying agent for wines, beers and fruit juices. In Africa, billions of dollars are expended yearly on the importation of commercial enzymes for the food and pharmaceutical industries and this increases the cost of production and the finished goods. This study was carried out to isolate tannase producing fungal species using Bambara nuts as a substrate in a bid to finding alternatives to the importation of tannase. Fresh Bambara nuts were collected from different locations in Nigeria. They were cleaned, sorted and intermittently moistened with water to encourage fungal growth for fourteen days. The different fungi obtained after fourteen days were inoculated onto Potato Dextrose Agar plates and incubated at 25°C for five days. Subculturing of fungal isolates was carried out to obtain pure cultures of isolates. Tannilytic activity (hydrolysis of tannin) of isolates was assessed by inoculating them in media containing tannin. The plates were incubated at 25°C for 2-5 days after which the plates were observed and zones of hydrolysis measured. A total of eighteen isolates were obtained. They were all members of the Aspergillus genus. 56% (10) of the isolates were able to degrade tannin acid with mean zone of hydrolysis of 39mm ±23.7 mm (Range 10-70mm). This study established members of the Aspergillus genus isolated from Bambara nuts as viable fungi for application in the production of tannase. This study adds to existing reports on fungal production of tannase.

Screening, Selection and Optimization of the Culture Conditions for Tannase Production by Endophytic Fungi Isolated from Caatinga

Screening, Selection and Optimization of the Culture Conditions for Tannase Production by Endophytic Fungi Isolated from Caatinga

English

Tannase is an inducible extracellular enzyme produced by filamentous fungi, yeasts, and bacteria by solid-state fermentation (SSF) or submerged fermentation (SmF). Among the filamentous fungi, Aspergillus and Penicillium are recognized as the most efficient producers of tannase. The aims of this study were to evaluate the production of tannase under SSF by isolation from Aspergillus and Penicillium preserved in the Collection of Cultures Micoteca - URM (WDCM604); select the best enzyme producer, and use the crude extract to clarify mangaba and tamarind juices. The optimal conditions were determined by using the Placket-Burman Planning (PB) and response surface methodology (RSM). All tested crops produced activity between 2088.19 and 238.93 U/gds, and Aspergillus carneus URM5577 was the best producer. Through MSR, the best parameters for producing tannase were found to be 70 h of cultivation at pH 6.0, 7% tannic acid at 28°C and, as the response variable, 5449.31 activity U/gds. The optimum purification conditions were the molecular weight of PEG 8000 (g/mol), concentration of PEG 15% (w/w), 25% citrate (w/w), and pH 8.0. Its application in mangaba juice reduced the tannin content by 49.66% after 90 min and in tamarind by 51.82% at 120 min incubation at 37°C. Key words: Tannase, agroforestry waste, Aspergillus, Penicillium.

Biotransformation of hydrolysable tannin to ellagic acid by tannase from Aspergillus awamori

Madhuca indica, locally known as mahua in India is a multipurpose tree species. Mahua, particularly bark contains a significant amount of hydrolysable tannin (17.31%) which can be utilized for ellagic acid production through biotransformation. In the present study, mahua bark utilized not only as a raw material for tannase production but also for ellagic acid a well-known therapeutic compound. After prior confirmation of hydrolysable tannin content in bark, it has been supplemented, as a substrate for tannase production through solid state fermentation of Aspergillus awamori. Tannase production, as well as biodegradation of the hydrolysable tannin reached a maximum at 72 h of incubation time. The optimum conditions for tannase production are solid to liquid ratio of 1:2, 35 °C, pH 5.5 and 72h incubation time which resulted 0.256 mg/mL of an extract of ellagic acid. Maximum tannase activity of 56.16 IU/gds at 35 °C and 72h of incubation time is recorded. It seems that tannase production and biotransformation of hydrolysable tannins using bark powder of mahua can be considered as an appropriate alternative to the existing procedures of ellagic acid production.

Kinetic, logistic, box-behnken model and statistical procedure for media optimization of tannase producing bacteria from tannery effluent and its application in Glue and gelatine production for pharma applications

A key enzyme tannase enables the degradation of gallotannins, a type of hydrolysable tannins. A wide variety of microorganisms such as yeasts, fungi and bacteria leads to the production of tannase. Among these the tannase producing bacteria were commonly reported in dissipated water from paper, leather and forestry industries. In this study we will discuss about diverse bacteria that were isolated from the tannery effluent of several industries and its screening, characterization and the production of extracellular tannase. The production of extracellular tannase is highly influenced by Media Optimization and hence wide range of pH and temperature is utilized for the production of tannase and the optimal activity was found to be at 45°C and at a pH 6.5. The placket-Burman Logistic, Kinectic and Box-Behnken models were employed to optimize the media for tannase producing bacteria. In submerged culture (SMC) and solid-state (SSC) the induction and repression pattern of tannase production can be observed and the results presented that SSC has higher potential to minimize catabolite repression. Assessment of the effect of various additives on tannase activity was also done and thus among the various metal salts tested, the enzyme was found to be strongly inhibited byHgCl2 followed by ZnCl2 and MnCl2. Again it was also found that EDTA and ?- mercaptoethanol had inhibitory effect where as the detergents (Tween-20, -60 and -80 as well as SDS) did not affect the activity of the enzyme1. The recovery of glue and gelatin from tannery effluent was also studied and for that a three step hydrolysis process is best suited for maximum recovery of collagen hydrolysate followed by the glue and gelatine production from it. It was also reported in accordance with the antibiotic sensitivity test (Abst) that the bacteria were very sensitive for streptomycin.

Production of Tannase and Gallic Acid by Utilizing AgroIndustrial Wastes as Economical Raw Material and Purification of Tannase

Production of Tannase and Gallic Acid by Utilizing AgroIndustrial Wastes as Economical Raw Material and Purification of Tannase

Tannase from Aspergillus melleus improves the antioxidant activity of green tea: purification and biochemical characterisation

SummaryTannase is an inducible enzyme used extensively in food, feed, pharmaceutical and chemical industries. In this study, tannase production and its biochemical properties were evaluated. From 42 Aspergillus strains analysed for potential tannase selection, Aspergillus melleus yielded the best results. Production was analysed using a complete factorial planning of 2³. Maximum activity (452.55 U mL−1) was obtained in the optimal conditions of substrate (5.0 g), initial moisture (60%), tannic acid (2%) and 48 h of fermentation. The molecular weight of the purified enzyme was estimated as 69.52 kDa; its optimum temperature and pH were 40 °C and 5.5, respectively. Regarding the chemical effectors used, tannase was inhibited by ZnCl2, ZnSO4, Triton X‐100 and SDS. The addition of tannase to green tea improved its antioxidant potential by approximately 85% when compared to the control. The present results suggest that tannase may be used as an adjuvant to increase the antioxidant potential of green tea.

Optimized Production of Tannase from Cashew Testa using Aspergillus niger MTCC 5898

ABSTRACTThe production of enzymes by bioprocessing is a good alternative to add value to agro-industry residues. Production cost of enzymes, the major constraint in bioprocessing, may be brought down by multifaceted approaches which include the use of cheap agro-residues for the microbial-based production of the enzyme, and the use of cost-efficient aerobic cell growth strategies and anaerobic-combined strategies like solid state fermentation (SSF). The current study investigated the production of tannase by Aspergillus niger CEPC 11 (MTCC 5898) on an abundantly available waste byproduct cashew testa under solid state A. niger growth. The optimum values of parameters obtained through response surface methodology (RSM) were cashew testa (23%), K2HPO4 (3.40 mM), sodium chloride (0.47 mM) and temperature (32–35°C). Optimization of the amount of cashew testa, content of the medium and incubation temperature resulted in a 3.02-fold increase from 97.32–301.70 U/g DS of tannase.