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Tannase production from Bacillus velezensis TA3 was optimized by sequential statistical approach using pomegranate as a solid substrate. Easily available and low-cost substrates such as cow dung, coffee husk, apple peel and pomegranate peel were initially screened. Among these substrates, pomegranate peel was found to enhance tannase activity. Further, pomegranate peel was used as the substrate to optimize the factors by traditional methods. In one-variable-at-a-time approach optimum process conditions were, 48 h incubation (1.92 U/g), 0.3% tannic acid (1.33 U/g), 0.08% minerals (2.3 U/g) with 55% initial moisture content (2.7 U/g). To screen the impact of variables such as fermentation period, moisture content, sodium chloride level, tannic acid concentration and required minerals on tannase production under solid state fermentation, a two-level full factorial design was used. Among these factors, incubation time, moisture and tannic acid concentrations were found to have significant influence on tannase production (p

Made available in DSpace on 2022-04-28T19:47:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-03 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Enzyme immobilization is an important alternative to stabilize enzyme properties favoring the efficiency of derivatives (enzyme + support/matrix) for different purposes. According to this, the current study aimed to immobilize the Aspergillus fumigatus CAS21 tannase and the use of the derivatives in the treatment of the effluent produced by the tannery industry. The tannase was immobilized on sodium alginate, DEAE-Sephadex, amberlite, and glass pearls as supports. Calcium alginate was the most adequate support for tannase immobilization with 100% yield and 94.3% for both efficiency and activity. The best tannase activity for the calcium alginate derivative was obtained at 50°C–60°C and pH 5.0. Thermal and pH stabilities evaluated for 24 h at 30°C–60°C and pH 4–7, respectively, were improved if compared to the stability of the free enzyme. Considering the reuse of the calcium alginate derivative, 78% of the initial activity was preserved after 10 catalytic cycles, and after the 9-month storage at 4°C, the activity was maintained in 70%. This derivative was applied in a packed bed reactor (PBR) for the treatment of tannin-rich effluents from the tannery industry. The reduction of the tannin content was effective reaching degradation of 74–78% after 48 h of PBR operation. The concentration of total phenolic compounds was also reduced, and the color and clarity of the effluent improved. In conclusion, the calcium alginate derivative is an attractive alternative as biocatalyst for large-scale treatment of the effluents from the tannery industry. Instituto de Química de Araraquara—UNESP Departamento de Biologia Faculdade de Filosofia Ciências e Letras de Ribeirão Preto—USP Instituto de Química de Araraquara—UNESP FAPESP: 2016/11311-5

Hutajulu IBE, Kulla PDK, Retnaningrum E. 2021. Diversity of lactic acid bacteria isolated during fermentation of indigenous cassava obtained from Sumba, East Nusa Tenggara, Indonesia. Biodiversitas 22: 2561-2570. Traditional fermented food products hold a great significance in the routine diet of Indonesian inhabitants. The present study investigated the diversity of lactic acid bacteria (LAB) found in fermented indigenous cassava, ubi karet busuk, produced in Sumba, East Nusa Tenggara, Indonesia. The isolated LAB was characterized for their phenotypic, biochemical, and molecular traits, which involved 16S rRNA gene sequencing, determination of lactic acid fermentation pathway, and measurement of b-glucosidase and tannase activity in the LAB strains. During fermentation, 15 LAB strains were obtained from the cassava. Among these, 93.33% strains were found to be homofermentative, whereas 6.73% strains displayed heterofermentative traits. BLASTn and phylogenetic analysis classified 15 LAB strains into six species, namely Enterococcus faecium, Enterococcus faecalis, Enterococcus casseliflavus, Leuconostoc mesenteroides, Lactococcus lactis, and Enterococcus sp. Among these, 10 LAB strains displayed b-glucosidase activity, with highest enzyme activity of 7.13 U/mL recorded in L. lactis F14IS3. Interestingly, two of these strains also showed expression of tannase, with F6IS1 strain displaying highest enzyme activity of 38.23 U/mL. Thus, the study results highlighted the suitability of these b-glucosidase and tannase-producing LAB strains to be used as a starter in the fermentation of cassava and other substrates such as vegetables, fruits, and legumes. These strains can also be utilized in animal feed, food, and beverage industries.

A total of 117 Bacillus strains were isolated from Miang, a culture relevant fermented tea of northern Thailand. These strains were collected from 16 sampling sites in north Thailand. In this collection 95 isolates were tannin-tolerant Bacillus capable of growth on nutrient agar supplemented with 0.5% (w/v) total tannins from tea leaves extract (TE). The strains were also positive for pectinase, xylanase and amylase activity, while 91 and 86 isolates were positive for cellulase and &beta, mannanase, respectively. Only 21 isolates producing extracellular tannase were selected for further characterization. Identification by 16S rRNA gene sequence analysis revealed that more than 50% (11 of 21 isolates) were Bacillus tequilensis, whereas the remaining were B. siamensis (3), B. megaterium (3), B. aryabhattai (3) and B. toyonensis (1). B. tequilensis K34.2 produced the highest extracellular tannase activity of 0.60 U/mL after cultivation at 37 &deg, C for 48 h. In addition, all 21 isolates were resistant to 0.3% (w/v) bile salt, sensitive to gentamicin, erythromycin, vancomycin and kanamycin and also tolerant to acidic condition. Cell hydrophobicity varied from 9.4 to 80.4% and neutralized culture supernatants of some Bacillus isolates showed bacteriocin producing potentiality against Samonella enterica serovar Typhimurium TISTR 292. All tested probiotic properties indicated that B. tequilensis K19.3, B. tequilensis K34.2 and B. siamensis K19.1 had high probiotic potential. This is the first report describing tannin-tolerant Bacillus and their extracellular tannase producing capability in Miang, a traditional fermented tea of Thailand.

Made available in DSpace on 2020-12-12T01:18:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The Aspergillus fumigatus CAS21 tannase was spray dried with β-cyclodextrin, Capsul® starch, soybean meal, lactose, and maltodextrin as adjuvants. The moisture content and water activity of the products ranged from 5.6 to 11.5% and from 0.249 to 0.448, respectively. The maximal tannase activity was achieved at 40–60 ºC and pH 5.0–6.0 for the powders containing β-cyclodextrin and Capsul® starch, which was stable at 40 ºC and 40–60 ºC for 120 min, respectively. For all the dried products, tannase retained its activity of over 80% for 120 min at pH 5.0 and 6.0. Salts and solvents influenced the activity of the spray-dried tannase. The activity of the spray-dried tannase was maintained when preserved for 1 year at 4 ºC and 28 ºC. Spray-dried tannase reduced the content of tannins and polyphenolic compounds of leather effluent and sorghum flour and catalyzed the transesterification reaction. The spray drying process stabilized the tannase activity, highlighting the potential of dried products for biotechnological applications. Instituto de Química de Araraquara – UNESP, Avenida Professor Mário Degni s/nº, Quitandinha Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo USP, Av. do Café s/n Faculdade de Filosofia Ciências e Letras de Ribeirão Preto – USP, Avenida Bandeirantes 3900 Instituto de Química de Araraquara – UNESP, Avenida Professor Mário Degni s/nº, Quitandinha CNPq: 142389/2017-0 FAPESP: 2016/11311-5

Background High 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. Results Different 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. Conclusions In 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. Electronic supplementary material The online version of this article (10.1186/s13068-018-1093-0) contains supplementary material, which is available to authorized users.

Tannins are polyphenolic compounds produced by plants and they are used in industrial vegetable tanning of leather. Tannins represent one of the low biodegradability substances in tannery wastewaters with high recalcitrant soluble chemical oxygen demand, furthermore high concentration of tannins can inhibit biological treatment. In the present study, four novel rotating submerged packed bed reactors were inoculated with a selected fungal strain to reach a biological degradation of tannins in non-sterile conditions. The selected fungal strain, Aspergillus tubingensis MUT 990, was immobilised in polyurethane foam cubes carriers and inserted inside a submerged rotating cage reactors. The reactors were feed with a solution composed of four tannins: Quebracho (Schinopsis spp.), Wattle (Mimosa spp.), Chestnut (Castanea spp.) and Tara (Caesalpinia spp.). Four reactors with a volume of 4 L each were used, the co-substrate was pure malt extract, the hydraulic retention time was 24 h and the pH setpoint was 5.5. The reactors configuration was chosen to allow the study of the effect of rotation and the co-substrate addition on tannins removal. The experiment lasted two months and it was achieved 80% of chemical oxygen demand and up to 90% dissolved organic carbon removal, furthermore it was detected an important tannase activity.

Made available in DSpace on 2020-12-10T17:01:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Aspergillus ochraceus biofilm, developed on an inert support, can produce tannase in Khanna medium containing 1.5% (w/v) tannic acid as the carbon source, at an initial pH of 5.0, for 72 h at 28 degrees C. Addition of 0.1% (w/v) yeast extract increased enzyme production. The enzyme in the crude filtrate exhibited the highest activity at 30 degrees C and pH 6.0. At 50 degrees C, the half-life was 60 min and 260 min at pH 6.0. In general, addition of detergents and surfactants did not affect tannase activity significantly. Tannase has potential applications in various biotechnological processes such as the production of propyl gallate and in the treatment of tannin-rich effluents. The content of tannins and total phenolic compounds in effluents from leather treatment was reduced by 56-83% and 47-64%, respectively, after 2 h of enzyme treatment. The content of tannins and total phenolic compounds in the sorghum flour treated for 120 h with tannase were reduced by 61% and 17%, respectively. Interestingly, the same A. ochraceus biofilm was able to produce tannase for three sequential fermentative process. In conclusion, fungal biofilm is an interesting alternative to produce high levels of tannase with biotechnological potential to be applied in different industrial sectors. UNESP, Inst Quim Araraquara, Ave Prof Mario Degni S-N, BR-14800900 Araraquara, SP, Brazil Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Ave Bandeirantes 3900, Ribeirao Preto, SP, Brazil UNESP, Inst Quim Araraquara, Ave Prof Mario Degni S-N, BR-14800900 Araraquara, SP, Brazil FAPESP: 2016/11311-5

In this research, optimization of the production medium to enhance tannase production by Bacillus gottheilii M2S2 in laboratory-scale packed bed reactor was studied. Amount of substrate Triphala, moisture content, aeration rate, and fermentation period was chosen for optimization study. During one variable at a time optimization, the highest tannase activity of 0.226 U/gds was shown with Triphala as a substrate at the fermentation period of 32 h. Furthermore, the optimum conditions predicted by response surface methodology (RSM) and genetic algorithm (GA) were found to be 11.532 g of substrate Triphala, 47.071% of the moisture content, and 1.188 L/min of an aeration rate with uppermost tannase activity of 0.262 U/gds. In addition, the single hidden layer feedforward neural network (SLFNN) and the radial basis function neural network (RBFNN) of an artificial neural network (ANN) were adopted to compare the prediction performances of the RSM and GA. It revealed that the ANN models (SLFNN, R(2) = 0.9930; and RBFNN, R(2) = 0.9949) were better predictors than the RSM (R(2) = 0.9864). Finally, the validation experiment exhibited 0.265 U/gds of tannase activity at the optimized conditions, which is an 11-fold increase compared to unoptimized media in shake flask.

WOS: 000482118200004, In this study, 17 L. brevis strains obtained from fermented products in previous studies were purified and these selected isolates were identified by the DuPont Qualicon RiboPrinter (R) system. Spectrophotometric and visual reading method indicated that isolate L. brevis A6X showed the highest activity. It was observed that pyrogallol product with the decarboxylase activity of gallic acid was composed of tannic acid using the thin layer chromatography. The proximate molecular weights of the enzyme produced by the selected isolate of L. brevis were determined by the SDS-PAGE method, and also the effect of different temperature, pH, substrate concentration, the concentration of tannic acid and various minerals on activity were all investigated. As a result of the test, it was found that the optimum temperature was 37 degrees C, pH 5.0, methyl gallate concentration used as the substrate 7 mM and tannic acid concentration 1.75 mM. While enzyme activity increased in the presence of Ca+, Zn+ and K+ ions, it did not show any changes in the presence of surfactant (Tween 80), inhibitor (DMSO), denaturing agent (urea). In addition, in presence of Mg+, Hg+ and Zn+ ions caused the tannase activity to decrease., Anadolu University of Scientific Research Project Program, This work was financially supported by the Anadolu University of Scientific Research Project Program.

including cellulase, tannase and pectinase. Isolates were morphologically and molecularly identified as Aspergillus niger, Aspergillus tubingensis, Aspergillus japonicus and Aspergillus aculeatus. Isolates were screened for enzyme production ability on solid and in liquid media. Enzymatic activity was determined in the culture filtrate of liquid medium. A total of six isolates were found to produce multiple hydrolytic enzymes. The highest activity of cellulase was produced by A. japonicus ZGM4 and A. aculeatus ZGM6 as 40 and 35 U g-1 dry biomass, respectively. All the isolates exhibited high level of tannase activity in the range of 150-343 U g-1 dry biomass after 24 h of incubation. A. tubingensis ZGM5 and A. aculeatus ZGM6 were found to produce the highest pectinase activity at a level of 130 and 117 U g-1 dry biomass, respectively. In the light of these results, isolates can be used for multiple hydrolytic enzyme production in industry.

Tannase (EC 3.1.1.20) is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable effects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun (Syzygium cumini (L.) Skeels) leaves, and identified as Pestalotiopsis guepinii, in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL). Response surface methodology was employed to evaluate the effect of variables (pH and temperature), and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. Km was found to be 7.18·10-4 mol/L and vmax =250.00 U/mL. The tannase activity was the highest in the presence of Ca2+ at a concentration of 5·10-3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3% of its original activity after 6 h. The study contributes to the identification of microbial species that produce tannase, with potential application in biotechnology.

Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry. The recombinant protein was expressed with 6× His tag and purified using nickel affinity beads. The enzyme was purified up to homogeneity, with approximate molecular weight of 66 kDa. Purified tannase exhibited specific activity of about 716 U/mg. Optimum enzyme activity was found to be 40 °C at pH 7.0. Biochemical characterization revealed; metal ions such as Zn2+, Fe2+, Fe3+ and Mn2+ inhibited tannase activity, and SDS at lower concentration, increased tannase activity. Non polar organic solvents increased the tannase activity and polar solvents inhibited the tannase activity. Tannase immobilization studies show protection of the enzyme under wide range of pH and temperature. Also in this study we report a method for recovery and repeated use of the tannase.

Lactobacillus plantarumMTCC 1407 represents a valuable source of an economically attractive stable long-life tannase with potential for application in various industries. The effect of fermentation parameters such as pH, temperature and agitation speed on the growth of biomass and production of tannase using liquid medium were determined at the end of fermentation period. The optimum values of pH, reaction temperature and agitation speed for tannase activity were 6.0, 30 °C and 125 rpm respectively. The maximum tannase activity was found to be 9.29 U/mL.

Gallic acid production in a batch bioreactor was evaluated using as catalytic material the mouldy polyurethane solids (MPS) obtained from a solid-state fermentation (SSF) bioprocess carried out for tannase production by Aspergillus niger GH1 on polyurethane foam powder (PUF) with 5 % (v/w) of tannic acid as inducer. Fungal biomass, tannic acid consumption and tannase production were kinetically monitored. SSF was stopped when tannase activity reached its maximum level. Effects of washing with distilled water and drying on the tannase activity of MPS were determined. Better results were obtained with dried and washed MPS retaining 84 % of the tannase activity. Maximum tannase activity produced through SSF after 24 h of incubation was equivalent to 130 U/gS with a specific activity of 36 U/mg. The methylgallate was hydrolysed (45 %) in an easy, cheap and fast bioprocess (30 min). Kinetic parameters of tannase self-immobilized on polyurethane particles were calculated to be 5 mM and 04.1×102 mM/min for K M and V max, respectively. Results demonstrated that the MPS, with tannase activity, can be successfully used for the production of the antioxidant gallic acid from methyl-gallate substrate. Direct use of PMS to produce gallic acid can be advantageous as no previous extraction of enzyme is required, thus reducing production costs., Authors thank the National Council for Science and Technology (CONACYT-Mexico) for the financial support. The present work was performed as part of a cooperative agreement between DIA-Universidad Autonoma de Coahuila (Mexico) and IBB-Universidade do Minho (Portugal) within a specific training stay undertaken at the DEB-UM. Part of the research was funded by a project SEP-CONACYT-CB-2011.

Bacteria with tannase activity were isolated from the feces of the Japanese large wood mouse, Apodemus speciosus. They were largely classified into two groups: Gram-positive cocci and Gram-positive bacilli. Genotypic analysis using a species-specific PCR assay as well as biochemical tests identified all cocci as Streptococcus gallolyticus. A PCR assay targeting a genus-specific sequence in the 16S/23S rDNA spacer region and additional 16S rDNA sequencing indicated that the bacilli belong to the genus Lactobacillus, with L. animalis and L. murinus being closely related taxa. Subsequent estimation of guanine-plus-cytosine content, amplified ribosomal DNA restriction analysis, and DNA/ DNA hybridization assay confirmed that the bacilli are homologous to each other but different from L. animalis or L. murinus. Consequently, a novel species of the genus Lactobacillus may be proposed. To date, this study is the first to report on the isolation of tannase-positive bacteria from the feces of a rodent species. These bacteria may play an essential role for the host organism in digesting tannin-rich acorns available in their natural habitats, thereby endowing them with a greater ecological advantage.

A novel colorimetric method to quantify tannase activity of viable tannase-producing bacterial strains was developed through application of a visual reading method that was to detect the activity qualitatively. The novel method was sensitive enough to quantify the marginal tannase activity of strains that could not be otherwise measured by conventional spectrophotometric or colorimetric methods.

[Background] Herbivores have developed mechanisms to overcome adverse effects of dietary tannins through the presence of tannin-resistant bacteria. Tannin degradation is an unusual characteristic among bacteria. Streptococcus gallolyticus is a common tannin-degrader inhabitant of the gut of herbivores where plant tannins are abundant. The biochemical pathway for tannin degradation followed by S. gallolyticus implies the action of tannase and gallate decarboxylase enzymes to produce pyrogallol, as final product. From these proteins, only a tannase (TanBSg) has been characterized so far, remaining still unknown relevant proteins involved in the degradation of tannins., [Results] In addition to TanBSg, genome analysis of S. gallolyticus subsp. gallolyticus strains revealed the presence of an additional protein similar to tannases, TanASg (GALLO_0933). Interestingly, this analysis also indicated that only S. gallolyticus strains belonging to the subspecies “gallolyticus” possessed tannase copies. This observation was confirmed by PCR on representative strains from different subspecies. In S. gallolyticus subsp. gallolyticus the genes encoding gallate decarboxylase are clustered together and close to TanBSg, however, TanASg is not located in the vicinity of other genes involved in tannin metabolism. The expression of the genes enconding gallate decarboxylase and the two tannases was induced upon methyl gallate exposure. As TanBSg has been previously characterized, in this work the tannase activity of TanASg was demonstrated in presence of phenolic acid esters. TanASg showed optimum activity at pH 6.0 and 37°C. As compared to the tannin-degrader Lactobacillus plantarum strains, S. gallolyticus presented several advantages for tannin degradation. Most of the L. plantarum strains possessed only one tannase enzyme (TanBLp), whereas all the S. gallolytcius subsp. gallolyticus strains analyzed possesses both TanASg and TanBSg proteins. More interestingly, upon methyl gallate induction, only the tanB Lp gene was induced from the L. plantarum tannases; in contrast, both tannase genes were highly induced in S. gallolyticus. Finally, both S. gallolyticus tannase proteins presented higher activity than their L. plantarum counterparts., [Conclusions] The specific features showed by S. gallolyticus subsp. gallolyticus in relation to tannin degradation indicated that strains from this subspecies could be considered so far the best bacterial cellular factories for tannin degradation., This work was financially supported by grants AGL2011-22745, S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

The production of tannases by Aspergillus tamarii was evaluated in submerged cultures using tannic acid and gallic acid as substrates. Two tannases, designated as TAH I and TAH II were produced in gallic acid submerged cultures. TAH I, responsible for 70% of the total tannase activity was purified to apparent electrophoretic homogeneity with 18.35% yield. The enzyme is a homodimeric protein with molecular mass of 180 kDa and 40.5% of its weight corresponds to carbohydrates. TAH I exhibited optimal activity at 30°C and pH 5.5 and was stable over a large pH range (3.0 to 9.0) and at temperatures up to 40°C. With methyl gallate as substrate, the enzyme presented a K M of 0.77 mM and a V max of 682.8 U/mg proteins. The enzyme was inhibited by metal ions but showed relative resistance to organic solvents and surfactants. Since the enzyme is active over a wide range of pH and temperature, it is potentially useful in food and pharmaceutical industries. Key words : Aspergillus tamarii , enzyme purification, submerged culture, tannase.

WOS: 000293693100012, A new high performance liquid chromatography (HPLC) method was developed based on an indirect determination of tannase activity through gallic acid measurement using paracetamol as an internal standard. The separation was performed on a C(18) column using a mobile phase consisting of aqueous formic acid solution (1%) and methanol (85:15; v/v) pumped at 1 ml min(-1). Samples (10 mu l) were injected and signals were detected at 254 nm. Repeatability was 0.81 and 1.28 for peak normalization values of gallic acid (4.16 x 10(-5) M) and internal standard (2.02 x 10(-5) M). A good linearity was shown in the range of 1.04 x 10(-5) to 8.32 x 10(-5) M. Limit of detection (LOD) and limit of quantification (LOQ) values were calculated to be 2.2 x 10(-6) M and 6.6 x 10(-6) M, respectively. The applicability of the method was tested using Penicillium spinulosum which produces tannase. The method was highly sensitive, accurate, reliable, and repeatable and at the same time it is applicable to both pure and crude enzyme., Anadolu University [011024], The authors gratefully acknowledge for their financial support of the research project (No: 011024) by the Anadolu University Council of Research Project Fund.

Tannase is an enzyme with important biotechnological applications in the food industry. Previous studies have identified the tannase encoding gene in Lactobacillus plantarum and also have reported the description of the purification of recombinant L. plantarum tannase through a protocol involving several chromatographic steps. Here, we describe the high-yield production of pure recombinant tannase (17 mg/L) by a one-step affinity procedure. The purified recombinant tannase exhibits optimal activity at pH 7 and 40 C. Addition of Ca2þ to the reaction mixture greatly increased tannase activity. The enzymatic activity of tannase was assayed against 18 simple phenolic acid esters. Only esters derived from gallic acid and protocatechuic acid were hydrolyzed. In addition, tannase activity was also assayed against the tannins tannic acid, gallocatechin gallate, and epigallocatechin gallate. Despite L. plantarum tannase representing a novel family of tannases, which shows no significant similarity to tannases from fungal sources, both families of enzymes shared similar substrate specificity range. The physicochemical characteristics exhibited by L. plantarum recombinant tannase make it an adequate alternative to the currently used fungal tannases, This work was supported by grants AGL2005-00470, AGL2008-01052, BFU2007-67404/BMC, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD, and Consolider INGENIO 2007 Factoria de Crystalización (CICYT), RM2008-00002 (INIA), and S-0505/AGR/000153 (CAM). H. Rodríguez and J. A. Curiel were recipients of predoctoral fellowships from the I3P-CSIC Program and FPI-MEC, respectively.

The molecular mechanisms involved in tannic-acid (TA)-mediated cell growth retardation and viability prolongation of Lactobacillus plantarum VP08 strain were evaluated by a proteomic analysis of starved cells grown in the presence of TA or glucose as carbon source. The tannase activity and the cell growth retardation as well as viability prolongation were confirmed by enzymatic assay and growing tests, respectively. In order to gain information about the effect triggered at the molecular level by TA, total proteins (extracted from starved cells grown in 250 mg/L TA, or 2 g/L glucose) were analyzed by a 2D-PAGE/MS approach to detect differentially expressed proteins. A total of 15 spots were found to be down-regulated and 21 up-regulated in TA-grown cells. The results indicate an overall impact of TA on proteins involved in some cellular and metabolic pathways: glycolysis, amino acid metabolism, translation and protein folding. The modulation of specific proteins correlates with the positive effect of TA on the survival of tannase-positive L. plantarum.

The aim of this work was to select strains of Aspergillus niger for tannase production. Growth of colonies in plates with tannic acid-containing medium indicated their ability to synthesize tannase. Tannase activity was also measured in solid-state fermentation. A. niger 11T25A5 was the best tannase producer (67.5 U.g-1/72 hours of fermentation). O objetivo deste trabalho foi selecionar linhagens de Aspergillus niger para síntese de tanase. O crescimento das colônias em meio contendo ácido tânico indicou capacidade de produção de tanase. A atividade enzimática foi determinada em fermentação semi-sólida. A. niger 11T25A5 foi o melhor produtor (67.5 U.g-1/72 horas de fermentação).

Lactobacilli with tannase activity were isolated from human feces and fermented foods. A PCR-based taxonomic assay revealed that the isolates belong to Lactobacillus plantarum , L. paraplantarum , and L. pentosus . Additional studies on a range of Lactobacillus species from established culture collections confirmed that this enzymatic activity is a phenotypic property common to these three species.

Six methods to determine the activity of tannase produced by Aspergillus niger Aa-20 on polyurethane foam by solid state fermentation, which included two titrimetric techniques, three spectrophotometric methods and one HPLC assay were tested and compared. All methods assayed enabled the measurement of extracellular tannase activity. However, only five were useful to evaluate intracellular tannase activity. Studies on the effect of pH on tannase extraction demonstrated that tannase activity was considerably under-estimated when its extraction was carried out at pH values below 5.5 and above 6.0. Results showed that the HPLC technique and the modified Bajpai and Patil methods presented several advantages in comparison to the other methods tested.Seis métodos para determinar a atividade de tannase produzida por Aspergillus niger O Aa-20 em espuma de polyuretano por fermentação em estado sólido foram estudados. Duas técnicas titulométricas , três métodos spectrofotométricos e um método por HPLC foram testados e comparados. Todos os métodos testados permitiram determinar a atividade da tannase produzida extracelularmente. Entretanto, somente cinco se mostraram úteis para avaliar a atividade da tannase produzida intracelularmente. Os estudos do efeito do pH na extração de tannase demonstraram que a atividade de tannase era consideravelmente subestimada quando sua extração foi executada em valores de pH inferiores a 5.5 e superior a pH 6.0. Os resultados demostraram que a técnica de HPLC o método Bajpai and Patil modificado apresentam várias vantagens em comparação aos outros métodos testados.

Made available in DSpace on 2018-11-26T15:28:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results: The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tapwater at a 1:1 ratio as the moisture agent for 72 h at 30 degrees C. Two tannase activity peakswere obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60 degrees C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20-60 degrees C for 120 min, and the half-life (T1/2) at 75 degrees C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion: A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application. (C) 2015 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved. Univ Estadual Paulista, Inst Quim Araraquara, BR-14800900 Sao Paulo, Brazil Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, Dept Biol, BR-14040901 Sao Paulo, Brazil Univ Estadual Paulista, Inst Quim Araraquara, BR-14800900 Sao Paulo, Brazil FAPESP: 2011/50880-1