Bacterial Tannase Research Articles

Enterobacter hormaechei Z8b-60: A Tannin Degrading Bacteria Isolated from Slaughterhouse Waste

Tannases or Tannin acyl hydrolases (EC 3.1.1.20) are a family of esterases that catalyze the hydrolysis of ester and depside bonds present in hydrolyzable tannins to release gallic acid. Industrial applications of tannase in food, brewing, chemical, pharmaceutical, and bioremediation, have made it one of the most important enzymes in research. The aim of the present study focused on the degradation of tannic acid by novel bacterial tannase isolated from slaughterhouse waste-enriched soil. Soil containing ruminant microflora is cultured on MSM containing tannic acid. Among four bacterial isolates, a potent strain was cultivated for identification which are then analysed for biochemical characterization and physiological characterization. Bacteria use tannic acid as the sole carbon source for growth and showed maximum growth at 0.6%. The morphology of the isolate shows that it contains gram-negative rods that are nonmotile and capsulated. Based on 16S rRNA and phylogenetic analysis, the isolate was confirmed as Enterobacter hormaechei Z8b-60. Tannase production from bacteria was confirmed by a novel, sensitive plate assay by observing the zone of clearance around colonies and also by dye binding method using methyl gallate as substrate. During the study, TLC and HPLC were performed to analyze the accumulated gallic acid and other metabolites. Intracellular tannase production amplified from 14.98 U/mL to 102.7 U/mL as tannic acid concentration increases from 0.4% to 1.0%, respectively.

Biovalorization of fruit waste to gallic acid synthesis through the application of Bacillus haynesii SSRY4 MN031245 tannase

Present research investigation reports the detection and quantitative estimation of gallic acid synthesis by bacterial tannase catalysed hydrolysis of fruit waste tannins. Partially purified Tannase from Bacillus haynesiiSSRY4 MN031245 was employed for the enzymatic hydrolysis reaction. Tannase could successfully carry out hydrolysis reactions for synthesis of gallic acid from fruit waste. Gallic acid synthesis was detected and confirmed by High-Performance Thin Layer Chromatography (HPTLC) analysis. Partially purified tannase was able to synthesize 20.61 mg/mL gallic acid from the crude tannin extract of black plum seed powder after 24 h of incubation at 40 °C. The results suggest that bacterial tannase can be cost effective for the synthesis of gallic acid from the fruit waste.

Purification, Characterization and Valorization study of <em>Bacillus haynesii </em>SSRY4 MN031245 Tannase for Gallic acid synthesis using fruit waste as substrate

Tannase produced extracellularly by the bacterial strain Bacillus haynesii SSRY4 MN031245 was purified in step-wise manner through ammonium sulphate precipitation, dialysis, followed by anion exchange chromatography. Tannase was purified to 42.0-fold with 36.30% enzyme yield. The enzyme was relatively stable from 30 to 50℃ and pH (4.0–6.0) for up to 4 hours. Partially purified tannase (16.80 U/ml) was able to synthesize 20.304 mg/ml gallic acid from the fruit waste under optimized conditions. The results of application study suggest that bacterial tannase could provide a new source for Gallic acid synthesis from the fruit waste for industrial applications. Our research findings could provide a value chain to fruit waste and help in reducing the waste generation from fruit processing industries.

Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum

Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments. The enzymes were named CbTan1-3, and we show that each one exhibits a unique substrate preference on a range of galloyl ester model substrates; CbTan1 and 3 demonstrated preference toward galloyl esters linked to glucose, while CbTan2 was more promiscuous. All enzymes were also active on oak bark extractives. Furthermore, we solved the crystal structure of CbTan2 and produced homology models for CbTan1 and 3. In each structure, the catalytic triad and gallate-binding regions in the core domain were found in very similar positions in the active site compared with other bacterial tannases, suggesting a similar mechanism of action among these enzymes, though large inserts in each enzyme showcase overall structural diversity. In conclusion, the varied structural features and substrate specificities of the C. butyricum tannases indicate that they have different biological roles and could further be used in development of new valorization strategies for renewable plant biomass.

Biochemical and Structural Characterization of a Novel Bacterial Tannase From Lachnospiraceae bacterium in Ruminant Gastrointestinal Tract.

Tannases are a family of esterases that catalyze the hydrolysis of ester and depside bonds present in hydrolyzable tannins to release gallic acid. Here, a novel tannase from Lachnospiraceae bacterium (TanALb) was characterized. The recombinant TanALb exhibited maximal activity at pH 7.0 and 50°C, and it maintained more than 70% relative activity from 30°C to 55°C. The activity of TanALb was enhanced by Mg2+ and Ca2+, and was dramatically reduced by Cu2+ and Mn2+. TanALb is capable of degrading esters of phenolic acids with long-chain alcohols, such as lauryl gallate as well as tannic acid. The Km value and catalytic efficiency (k cat /Km) of TanALb toward five substrates showed that tannic acid (TA) was the favorite substrate. Homology modeling and structural analysis indicated that TanALb contains an insertion loop (residues 341–450). Based on the moleculer docking and molecular dynamics (MD) simulation, this loop was observed as a flap-like lid to interact with bulk substrates such as tannic acid. TanALb is a novel bacterial tannase, and the characteristics of this enzyme make it potentially interesting for industrial use.

Studies on the Effect of Bacterial Tannase Supplementation to Biodegradation of Tannins in Tannery Wastewater

Tannins present in the vegetable tanning process wastewater (VTW) affect the aerobic microbes and reduce the treatment efficiency in the tannery effluent treatment plants. Studies were performed wi...

Bacterial Degradation of Bacteriostatic Polyphenols, Tannins

Tannin degradation by bacteria has not been studied much as tannins are commonly known to be bacteriostatic due to enzyme inhibition, substrate deprivation, and the enzyme activity on the bacterial cell wall. However, about a handful of bacteria have been found to tolerate certain concentrations of tannin. This study focuses on isolating and identifying bacteria from decaying portions of tree bark for tannase production and effective catalysis of ester bond hydrolysis in tannins. Different concentrations of commercial tannic acid were used as the sole carbon source on mineral salt medium (MSM) agar plates, to test the maximum tolerable concentrations (MTCs) by the isolates. Tannin degradation was confirmed by a visual reading method and bacterial tannase activity and the biodegradation percentage were determined. One particular isolate was identified to have 50 g/L MTC of tannin, with a tannase activity of 51.61 U/mL that is optimum after 96 hours of incubation. The 16s rRNA sequencing results showed that the isolate belonged to Bacillus genus and the resulting bacterial strain isolate was found to be a new strain of Bacillus subtilis which was submitted to GenBank under the accession number MH330408.

Diverse methanogens, bacteria and tannase genes in the feces of the endangered volcano rabbit (Romerolagus diazi).

BackgroundThe volcano rabbit is the smallest lagomorph in Mexico, it is monotypic and endemic to the Trans-Mexican Volcanic Belt. It is classified as endangered by Mexican legislation and as critically endangered by the IUCN, in the Red List. Romerolagus diazi consumes large amounts of grasses, seedlings, shrubs, and trees. Pines and oaks contain tannins that can be toxic to the organisms which consume them. The volcano rabbit microbiota may be rich in bacteria capable of degrading fiber and phenolic compounds.MethodsWe obtained the fecal microbiome of three adults and one young rabbit collected in Coajomulco, Morelos, Mexico. Taxonomic assignments and gene annotation revealed the possible roles of different bacteria in the rabbit gut. We searched for sequences encoding tannase enzymes and enzymes associated with digestion of plant fibers such as cellulose and hemicellulose.ResultsThe most representative phyla within the Bacteria domain were: Proteobacteria, Firmicutes and Actinobacteria for the young rabbit sample (S1) and adult rabbit sample (S2), which was the only sample not confirmed by sequencing to correspond to the volcano rabbit. Firmicutes, Actinobacteria and Cyanobacteria were found in adult rabbit samples S3 and S4. The most abundant phylum within the Archaea domain was Euryarchaeota. The most abundant genera of the Bacteria domain were Lachnoclostridium (Firmicutes) and Acinetobacter (Proteobacteria), while Methanosarcina predominated from the Archaea. In addition, the potential functions of metagenomic sequences were identified, which include carbohydrate and amino acid metabolism. We obtained genes encoding enzymes for plant fiber degradation such as endo 1,4 β-xylanases, arabinofuranosidases, endoglucanases and β-glucosidases. We also found 18 bacterial tannase sequences.

Heterologous expression and biophysical characterization of a mesophilic tannase following manganese nanoparticle immobilization

In the current study, we analyzed the efficacy of manganese oxide nanoparticle (MnNP)-water dispersion as an immobilization matrix for bacterial tannase. The tannase-secreting Bacillus subtilis strain NJKL.tan.2 obtained from tannery effluent soil was subsequently purified and cloned in pET20b vector. The activity of MnNP-tan (tannase activated by manganese nanoparticles) was 1.51- and 3.5-fold higher at 20 °C and 80 °C, respectively, compared with the free enzyme. MnNP-tan decreased Km by 41.66 % and 3-fold, whereas free tannase showed two-fold and six-fold improvement in Kcat at 37 °C and 80 °C, respectively. MnNP-tan showed an increase in (half-life)t1/2and Ed by 13-fold and 50.05 units, respectively, at 80 °C, in contrast to the native enzyme. MnNP-tan retained its residual activity by 78.2 % at 37 °C and 34.24 % at 80 °C after 180 min of incubation when compared with untreated set. MnNP-tan retained 51 % of its activity after 120 days with the native enzyme losing ∼50 % functionality following 40 days of incubation. The MnNP-mediated tannase immobilization technique is being reported for the first time. The technique has numerous advantages due to the use of MnNP as a potential matrix for biomolecule immobilization, which can be further extended to immobilize other biocatalysts used in agro-industrial and lab-based applications.

Contributions of protein microenvironment in tannase industrial applicability: An in-silico comparative study of pathogenic and non-pathogenic bacterial tannase

Tannase is an inducible industrially important enzyme, produced by several microorganisms. A large number of bacteria have reported as tannase producers; however, some of them are pathogenic in nature. Therefore, it is quite uncertain whether the application of these tannase enzymes from such pathogenic bacteria is suitable for industries and human welfare. Till date, there is no clear evidence regarding which group of bacteria (non-pathogenic or pathogenic) is better suited for their application in the edge of industries with particular reference to the food industry. The present study is following the findings of the above queries. In this study, a large number of tannase protein sequences have been retrieved from the databases, including both non-pathogenic and pathogenic bacterial species. Physiochemical and evolutionary properties of those sequences have been evaluated. Results have shown that non-pathogenic bacterial tannase possesses a high number of acidic and basic amino acid residues as compared to their pathogenic counterparts. The acidic and basic amino acid residues of tannase provide unique microenvironment to it. In the other hand, the numbers of disorder forming residues are higher in tannase sequences of pathogenic bacteria. The study of tannase microenvironment leads in the formation of salt bridges, which finally favoring the stability and proper functioning of tannase. This is the first report of such observation on tannase enzyme using in silico approach. Study of the microenvironment concept will be helpful in protein engineering.

Effect of Mixed Inoculums Volume and pH on Anti Nutritional Level in Cabbage Fermentation using Saccharomyces cerevisiae and Lactobacillus plantarum

Cabbage (Brassica oleracea var. capitata) that is one of the widely consumed vegetables, contains significant amounts of anti-nutritional components, such as tannin. This component can be reduced by fermentation, where bacterial tannase activity is able to degrade tannin into glucose and gallic acid. A simultaneously mixed cultures fermentation using Saccharomyces cerevisiae and Lactobacillus plantarum has been developed because they possess some mutualism interaction that can be beneficial in the fermentation process. Therefore, this research was focused on the effect of mixed inoculums volume of S. cerevisiae and L. plantarum and initial culture pH on the tannin and glucose level during cabbage fermentation. The sliced cabbage was inoculated with simultaneously mixed cultures (volume ratio of yeast and bacteria used was 1:1) at a different volume range 5%-25% (v/v) and also various pH range from 4 to 7, and then incubated at room temperature in anaerobic condition for 4 days. Tannin and glucose level in fermented cabbage (both biomass and filtrate) were determined using Folin-Denis and phenol-sulphuric method, respectively. The using of 5% mixed inoculums volume and initial pH around 7 were found to be the optimum condition for tannin degradation in cabbage. The remained tannin level in these fermented cabbage decreased up to 18.58% (54.061 mg/100 g FW). This experiment reveals that the amounts of inoculums and initial culture pH affected tannin degradation during cabbage fermentation using mixed cultures of yeast and lactic acid bacteria.

Bacterial tannases: classification and biochemical properties.

Tannin acyl hydrolases, also known as tannases, are a group of enzymes critical for the transformation of tannins. The study of these enzymes, which initially evolved in different organisms to detoxify and/or use these plant metabolites, has nowadays become relevant in microbial enzymology research due to their relevant role in food tannin transformation. Microorganisms, particularly bacteria, are major sources of tannase. Cloning and heterologous expression of bacterial tannase genes and structural studies have been performed in the last few years. However, a systematic compilation of the information related to all recombinant tannases, their classification, and characteristics is missing. In this review, we explore the diversity of heterologously produced bacterial tannases, describing their substrate specificity and biochemical characterization. Moreover, a new classification based on sequence similarity analysis is proposed. Finally, putative tannases have been identified in silico for each group of tannases taking advantage of the use of the "tannase" distinctive features previously proposed.

Identification of a highly active tannase enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum

BackgroundTannases are tannin-degrading enzymes that have been described in fungi and bacteria as an adaptative mechanism to overcome the stress conditions associated with the presence of these phenolic compounds.ResultsWe have identified and expressed in E. coli a tannase from the oral microbiota member Fusobacterium nucleatum subs. polymorphum (TanBFnp). TanBFnp is the first tannase identified in an oral pathogen. Sequence analyses revealed that it is closely related to other bacterial tannases. The enzyme exhibits biochemical properties that make it an interesting target for industrial use. TanBFnp has one of the highest specific activities of all bacterial tannases described to date and shows optimal biochemical properties such as a high thermal stability: the enzyme keeps 100% of its activity after prolonged incubations at different temperatures up to 45 °C. TanBFnp also shows a wide temperature range of activity, maintaining above 80% of its maximum activity between 22 and 55 °C. The use of a panel of 27 esters of phenolic acids demonstrated activity of TanBFnp only against esters of gallic and protocatechuic acid, including tannic acid, gallocatechin gallate and epigallocatechin gallate. Overall, TanBFnp possesses biochemical properties that make the enzyme potentially useful in biotechnological applications.ConclusionsWe have identified and characterized a metabolic enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum. The biochemical properties of TanBFnp suggest that it has a major role in the breakdown of complex food tannins during oral processing. Our results also provide some clues regarding its possible participation on bacterial survival in the oral cavity. Furthermore, the characteristics of this enzyme make it of potential interest for industrial use.

Gallic acid production under anaerobic submerged fermentation by two bacilli strains.

BackgroundTannase is an enzyme that catalyses the breakdown of ester bonds in gallotannins such as tannic acid. In recent years, the interest on bacterial tannases has increased because of its wide applications. The lactic acid bacteria (LAB) plays an important role in food tannin biotransformation, it has the ability of hydrolyse tannins in ruminants intestine. The finding of tannin hydrolysis by LAB has sparked their use as tannase producer.ResultsThe bacterial strains used in the present work were identified as Bacillus subtilis AM1 and Lactobacillus plantarum CIR1. The maximal tannase production levels were 1400 and 1239 U/L after 32 and 36 h of fermentation respectively, for B. subtilis AM1 and L. plantarum CIR1. Maximum gallic acid release was 24.16 g/L for B. subtilis AM1 and 23.73 g/L for L. plantarum CIR1. HPLC analysis showed the formation of another peaks in the retention time range of 9–14 min, which could be attributed to the formation of di or tri-galloyl glucose.ConclusionsAccording to database, the strains were identified as Bacillus subtilis AM1 and Lactobacillus plantarum CIR1. In conclusion, both strains had the capability to produce good titres of extracellular tannase and release gallic acid.

Purification and characterization of a thermophilic tannase from Klebsiella pneumoniae KP715242

Abstract Tannase (tannin acyl hydrolase EC 3.1.1.20) is an inducible enzyme that catalyzes the hydrolysis of ester and depside bonds present in hydrozable tannins. In the present study, tannase from a novel Klebsiella pneumoniae KP715242 was purified and characterized for the desirable properties. The enzyme was purified with an overall purification of 8.55-fold leading an yield of 9.5%. The molecular mass of 38.20 kDa suggested this enzyme to be one of the smallest tannase of the bacterial source. Optimum temperature and pH for tannase activity were found to be 50 °C and 5.5 respectively. The optimum temperature of 50 °C puts this enzyme in thermostable category of bacterial tannase reported till date. Km of the enzyme for tannic acid was found to be 0.00375 M with Vmax 3.64 mM/S. Effects of various conventional inhibitors, metal salts, organic solvents, surfactants and chelators on tannase activity were assessed. Methyl gallate, sodium thioglycolate and xylene inhibited the enzyme maximally. Among the metal ions Hg2+ had maximum inhibitory effect on the tannase activity whereas Zn2+, Mg2+, Mn2+ were found to be enzyme activators. Surfactants and chelators inhibited the enzyme, however, no significant effect of EDTA was observed on tannase activity.

Tannase Immobilization by Chitin-Alginate Based Adsorption-Entrapment Technique and Its Exploitation in Fruit Juice Clarification

Present study aimed to immobilize the bacterial tannase on suitable carrier and its subsequent applicability in fruit juice clarification. Tannase from Bacillus subtilis PAB2 was immobilized on chitin-alginate bead by adsorption-entrapment technique. Maximum immobilization (82 %) with an activity yield of 67 % was achieved in presence of 1.5 % (w/v) sodium alginate, 1 M CaCl2 and 1 % (w/v) flake chitin, incubation at 4 °C temperature, and adsorption period of 90 min. Thermal and pH stabilities of immobilized tannase increased in respect to free tannase. The storage stability and reusability of the immobilized tannase were also improved significantly, retaining 83 % activity after storing 90 days at 4 °C and it showed 79 % residual activity after 10 times repeated use. Then, immobilized tannase was applied for clarification of jamun and cashew apple juice to investigate its debittering property. Enzymatic treatment removed 60 and 51 % tannin from jamun and cashew apple juice, respectively, after incubation at 40 °C for 120 min. Other qualitative indicator viz., total titratable acidity, color, and viscosity also reduced, whereas, flavonoid (460.49 μg/ml; 994.44 μg/ml), glucose (6.95 mg/ml; 9.19 mg/ml), and antioxidant activity (69.28 %; 78.44 %) were significantly increased in clarified jamun and cashew apple juice, respectively.

Characterization of a tannin acyl hydrolase from Streptomyces sviceus with substrate preference for digalloyl ester bonds.

The search for new tannases with novel enzymatic properties suitable for industrial applications has been a continuous effort since the first discovery of the enzyme more than a century ago. A tannase gene (Ss-Tan) from the Gram-positive bacterium Streptomyces sviceus was identified, chemically synthesized, and cloned into a C-terminal His-tagged vector for expression in Escherichia coli. The tannase possesses the active site motif of GXSXG that is conserved for serine hydrolases. The residues that constitute the catalytic triad and galloyl binding site in bacterial tannases are found conserved in Ss-Tan, which include Ser209, Asp452, His484 and Lys370, Glu384, Asp454, respectively. Ss-Tan was overexpressed in E. coli BL21-AI cells with high productivity. Enzymatic assay revealed that the enzyme displays tannase activities to hydrolyze both the ester bonds and depside bonds in hydrolyzable tannins. Kinetic analysis indicated that the enzyme preferentially acts on depside bonds with considerably higher substrate affinity and catalytic efficiency. The enzyme showed maximum activity around pH 8.0 and at 50 °C with the highest melting temperature close to 70 °C. The high depsidase activity and thermostablility of Ss-Tan may make the enzyme suitable for potential industrial applications to achieve complete digestion of hydrolyzable tannins.

Enhancement of tannase production by Lactobacillus plantarum CIR1: validation in gas-lift bioreactor

The optimization of tannase production by Lactobacillus plantarum CIR1 was carried out following the Taguchi methodology. The orthogonal array employed was L18 (2(1)×3(5)) considering six important factors (pH and temperature, also phosphate, nitrogen, magnesium, and carbon sources) for tannase biosynthesis. The experimental results obtained from 18 trials were processed using the software Statistical version 7.1 using the character higher the better. Optimal culture conditions were pH, 6; temperature, 40°C; tannic acid, 15.0 g/L; KH2PO4, 1.5 g/L; NH4Cl, 7.0 g/L; and MgSO4, 1.5 g/L which were obtained and further validated resulting in an enhance tannase yield of 2.52-fold compared with unoptimized conditions. Tannase production was further carried out in a 1-L gas-lift bioreactor where two nitrogen flows (0.5 and 1.0vvm) were used to provide anaerobic conditions. Taguchi methodology allowed obtaining the optimal culture conditions for the production of tannase by L. plantarum CIR1. At the gas-lift bioreactor the tannase productivity yields increase 5.17 and 8.08-fold for the flow rates of 0.5 and 1.0vvm, respectively. Lactobacillus plantarum CIR1 has the capability to produce tannase at laboratory-scale. This is the first report for bacterial tannase production using a gas-lift bioreactor.

Comparison of three tannases cloned from closely related lactobacillus species: L. Plantarum, L. Paraplantarum, and L. Pentosus

BackgroundTannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum.ResultsThe genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917T, respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The Km values of the enzymes on each galloyl ester were comparable; however, the kcat/Km values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity.ConclusionTwo tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases.

Contribution of a tannase from Atopobium parvulum DSM 20469T in the oral processing of food tannins

During oral passage, food tannins interact with the microbiota present in the oral cavity. Atopobium parvulum strains are inhabitants of the human oral cavity. A gene encoding a protein similar to bacterial tannases is present in A. parvulum strains. The tanAAp (apar_1020) gene was cloned and expressed in Escherichia coli BL21 (DE3). The overproduced TanAAp protein was purified to homogeneity. It exhibited optimal activity at pH6.0 and broad temperature range, being these properties compatible with its action during food oral processing. However, purified TanAAp protein presented the lowest specific activity among bacterial tannases (3.5U/mg) and was unable to hydrolyze complex tannin, such as tannic acid. These biochemical properties discard a main role of TanAAp in the breakdown of complex food tannins during oral processing.