Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40–60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae’s potential for tannase and gallic acid production for biotechnological applications.     

Optimisation of extracellular tannase production from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

The tannase production by Paecilomyces variotii was confirmed by high performance thin layer chromatography (HPTLC), and substrate specificity of the tannase was determined by zymogram analysis in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE). A clear band of activity observed after electrophoresis of culture filtrate in non-denaturing gels indicated the production of extracellular tannase by P. varoitii. HPTLC analysis revealed that gallic acid was the enzymatic degradation product of tannic acid during the fermentation process. The optimum condition for tannase production was at 72 h of incubation in shaking condition and addition of 1.5% tannic acid, 1% glucose and 0.2% sodium nitrate at temperature of 35°C and pH of 5–7. The production of extracellular tannase from Paecilomyces variotii was investigated under optimized conditions in solid-state fermentation (SSF), submerged fermentation (SmF) and liquid surface fermentation (LSF) processes. The maximum extracellular tannase production was obtained within 60 h of incubation under SSF followed by SmF and LSF.     

Tannase production by Aspergillus fumigatus MA under solid-state fermentation

A tannase yielding fungal culture identified as Aspergillus fumigatus MA was isolated from the effluent collected from a local small scale tannery. The fungal culture produced high yields of extracellular tannase under solid-state fermentation (SSF) using different agro forest residues such as Amla leaves (Phyllanthus emblica), Ber leaves (Zyzyphus mauritiana), Jamun leaves (Syzygium cumini), Jamoa leaves (Syzygium sp.) and Keekar leaves (Acacia nilotica). Among different substrates used, Jamun leaves yielded maximal extra-cellular production of tannase. Various parameters were studied to optimize the extracellular yield of tannase under SSF. The maximum yield of 174.32 U g⁻¹ was obtained at 25°C after 96 h of incubation at pH 5.0. The tap water was used as a moistening agent. A substrate to tap water ratio of 1:1 was found to best for tannase production. Supplementation of the medium with ammonium sulfate as nitrogen source had enhanced tannase production whereas glucose had decreased the enzyme production. This is the first report on production of tannase by Aspergillus fumigatus MA, giving a much higher yield of enzyme under SSF with Jamun leaves as the substrate.     

Fungal cultures of tar bush and creosote bush for production of two phenolic antioxidants (Pyrocatechol and Gallic acid)

‘Tar bush’ and ‘creosote bush’ were substrates of fungal cultivation for tannase production and gallic acid and pyrocatechol accumulation. Aspergillus niger GH1 grew similarly on both plant materials under solid state culture conditions, reaching maximal levels after 4 d. Fungal strain degraded all tannin content of creosote bush after 4 d of fermentation and >75 % of tar bush after 5 d. Higher level of tannase activity was detected in tar bush fermentation. Biotransformation of tannins to gallic acid was high (93 % in creosote bush and 89 % in tar bush). Pyrocatechol was released poorly. Kinetic parameters of tannin conversion were calculated.     

A new process for simultaneous production of tannase and phytase by <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> in solid-state fermentation of orange pomace

The production of enzymes such as tannases and phytases by solid-state fermentation and their use in animal feed have become a subject of great interest. In the present work, Paecilomyces variotii was used to produce tannase and phytase simultaneously. Solid-state fermentation, a process initially designed for tannase production, was implemented here using orange pomace as substrate. Orange pomace is the waste product of the large orange juice industry in Brazil, and it has also been used as an ingredient in animal feed. In addition to enzymatic production, biotransformation of the phenolic content and antioxidant capacity of the orange pomace were analyzed after fermentation. Fermentation conditions, namely moisture level and tannic acid concentration rate, were studied using CCD methodology. The response surface obtained indicated that the highest tannase activity was 5,000 U/gds after 96 h at 59% (v/w) and 3% (w/w) and that of phytase was 350 U/gds after 72 h at 66% (v/w) and 5.8% (w/w) of moisture level and tannic acid concentration, respectively. The amount of tannase production was similar to the levels achieved in previous studies, but this was accomplished with a 7% (w/w) reduction in the amount of supplemental tannic acid required. These results are the first to show that P. variotii is capable of producing phytase at significant levels. Moreover, the antioxidant capacity of orange pomace when tested against the free radical ABTS was increased by approximately tenfold as a result of the fermentation process.     

Tannin Degradation by a Novel Tannase Enzyme Present in Some Lactobacillus plantarum Strains

Lactobacillus plantarum is frequently isolated from the fermentation of plant material where tannins are abundant. L. plantarum strains possess tannase activity to degrade plant tannins. An L. plantarum tannase (TanB_Lp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanA_Lp). While all 29 L. plantarum strains analyzed in the study possess the tanB_Lp gene, the gene tanA_Lp was present in only four strains. Upon methyl gallate exposure, the expression of tanB_Lp was induced, whereas tanA_Lp expression was not affected. TanA_Lp showed only 27% sequence identity to TanB_Lp, but the residues involved in tannase activity are conserved. Optimum activity for TanA_Lp was observed at 30°C and pH 6 in the presence of Ca²⁺ ions. TanA_Lp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanA_Lp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanA_Lp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments.     

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.     

Production of tannase from wood-degrading fungus using as substrate plant residues: purification and characterization

In the present study Lenzites elegans, Schizophyllum commune, Ganoderma applanatum and Pycnoporus sanguineus (wood-degrading fungi) were assayed for their tannase producing potential in culture media containing plant residues or/and tannic acid as carbon source. Aspergillus niger was used as positive control for tannase production. We also carried out the isolation, purification and characterization of the enzyme from the fungi selected as the major productor. The highest fungal growth was observed in A. niger and L. elegans in the media containing tannic acid + glucose + plant residues (Fabiana densa). A. niger and L. elegans reached the highest extracellular tannase production in a medium containing tannic acid + F. densa and in a medium supplemented with glucose + tannic acid + F. densa. The produced enzyme by L. elegans was purified by DEAE-Sepharose. Km value was 5.5 mM and relative molecular mass was about 163,000. Tannase was stable at a pH range 3.0–6.0 and its optimum pH was 5.5. The enzyme showed an optimum temperature of 60°C and was stable between 40 and 60°C. This paper is the first communication of tannase production by wood-degrading fungi. Fermentation technology to produce tannase using plant residues and xylophagous fungi could be very important in order to take advantage of plant industrial waste.     

Large-scale production of tannase using the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L⁻¹ when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L⁻¹. The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L⁻¹. Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical.     

Production of tannase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> HA37 growing on tannic acid and Olive Mill Waste Waters

Production of tannase (tannin acyl hydrolase, EC 3.1.1.20) by Aspergillus nigerHA37 on a synthetic culture medium containing tannic acid at different concentrations has been studied. Maximal enzymatic activity increased according to the initial concentration of tannic acid; respectively 0.6, 0.9 and 1.5 enzyme activity units (EU) ml⁻¹ medium in the presence of 0.2%, 0.5% and 1% of tannic acid. Tannase production by A. niger HA37 on fourfold diluted olive mill waste waters (OMWW) as substrate, was between 0.37 and 0.65 EU ml⁻¹. Enzyme production on the diluted OMWW remained globally stable during more than 30 h. Growth of A. niger HA37 on OMWW was correlated with about 70% degradation of phenolic compounds present in the waste. This strain has therefore the capacity to degrade complex wastewaters which cause environmental damage to aquatic streams.     

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF. Journal of Industrial Microbiology & Biotechnology (2001) 26, 296–302. Received 07 July 2000/ Accepted in revised form 15 February 2001     

Synthesis of propyl gallate by mycelium-bound tannase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in organic solvent

Aspergillus niger with mycelium-bound tannase activity was employed to investigate the synthesis of propyl gallate from gallic acid and 1-propanol in organic solvents. The effects of various organic solvents (log P: −1.0 to 6.6) on the enzymatic reactions showed that benzene (log P: 2.0) was the most suitable solvent. The water content and protonation state of mycelium-bound enzyme both had significant effects on the activity of tannase. The maximum molar conversion (65%) was achieved with 7.3% (v/v) 1-propanol and 5.56 mM gallic acid at stirring speeds of 200 rev/min, 40 °C in presence of anhydrous sodium sulfate and PEG-10,000. Enzyme specificity for the alcohol portion (C₁–C₈) of the ester showed that the optimum synthesis was observed with alcohols ranging from C₃ to C₅.     

Catechine biotransformation by tannase with sequential addition of substrate

This work studied the effect of a sequential addition of substrate on tannase reaction for the increase of epigallocatechin (EGC) and gallic acid. The addition of 0.5–1% GTE increased the production of gallic acid during 2 h in a single tannase reaction, while the addition of more than 2% in GTE rather showed a decrease in gallic acid level with an increase of EGCG level compared with 1% GTE addition group, suggesting that GTE addition of 2% and over inhibits the reaction of tannase. Examination of sequential addition of 1% GTE on tannase reaction showed that second addition of 1% GTE at 2 h promoted tannase reaction by increasing production of gallic acid, but further addition (2 and 3 h) rather inhibited tannase reaction with lowered gallic acid and enhanced EGCG levels. This result showed that one additional treatment of 1% GTE during tannase reaction is effective in an increase of gallic acid production. Moreover, levels of degallated products including EGC, EC, and GC were increased by 7.3, 4.5, and 3.5-fold, respectively in sequential addition of GTE at 2 h. pH change derived from gallic acid production was not shown to related to tannase activity. Therefore, our study suggests that one sequential addition is a suitable process for desirable production of green tea extracts enriched in active components such as gallic acid and EGC.     

Effects of inoculum concentration,temperature, and carbon sources on tannase production during solid state fermentation of cashew apple bagasse

In this study, the optimization of tannase production by solid state fermentation was investigated using cashew apple bagasse (CAB), an inexpensive residue produced by the cashew apple agroindustry, as a substrate. To accomplish this, CAB was enriched with 2.5% (w/w) tannic acid and 2.5% (w/w) ammonium sulphate and then moistened with water (60 mL/100 g of dry CAB). The influence of inoculum concentration (10⁴ to 10⁷ spores/g), temperature (20, 25, 30, and 35°C) and several additional carbon sources (glucose, starch, sucrose, maltose, analytical grade glycerol, and glycerol produced during biodiesel production) on enzyme production by Aspergillus oryzae was then evaluated. Supplementation with maltose and glycerol inhibited tannase synthesis, which resulted in lower enzyme activity. Starch and sucrose supplementation increased enzyme production, but decreased the enzyme productivity. The maximum tannase activity (4.63 units/g of dry substrate) was obtained at 30°C, using 10⁷ spores/g and 1.0% (w/v) sucrose as an additional carbon source.     

Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity

Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.     

Tannase production by Bacillus licheniformis

Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21 U ml^–1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1 g l^–1) after 18–21 h growth though the organism did not attain maximum growth until 36 h.     

Crystallographic and mutational analyses of tannase from Lactobacillus plantarum

Tannin acylhydrolase (EC 3.1.1.20) referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannins to release gallic acid. Although the enzyme is useful for various industries, the tertiary structure is not yet determined. In this study, we determined the crystal structure of tannase produced by Lactobacillus plantarum. The tannase structure belongs to a member of α/β‐hydrolase superfamily with an additional “lid” domain. A glycerol molecule derived from cryoprotectant solution was accommodated into the tannase active site. The binding manner of glycerol to tannase seems to be similar to that of the galloyl moiety in the substrate. Proteins 2013; 81:2052–2058. © 2013 Wiley Periodicals, Inc.     

Biocatalytic synthesis of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> ECU0401

The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25°C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(−)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(−)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(−)-mandelic acid g⁻¹ dry cell weight (dcw) cell after 20 cycles of repeated use.     

Selective consumption of acorns by the Japanese wood mouse according to tannin content: a behavioral countermeasure against plant secondary metabolites

We examined the presence of selective consumption against tannins in acorns as a pre-ingestive countermeasure to plant secondary metabolites by using the Japanese wood mouse (Apodemus speciosus) and acorns of Quercus serrata, which contained ca. 6.4% tannins on a dry weight basis. In addition, the presence of selective consumption against proteins was also examined. In the acorn-feeding experiment, 18 wood mice were allocated to two groups: the experienced group (N = 9), which had previous experience in feeding on acorns, and the inexperienced group (N = 9), which had no experience. Mice of both groups were fed only acorns for 3 nights. Selectivity against tannins (an index estimated from the tannin content in control, remaining, and ingested acorns) was significantly positive in the experienced group, indicating the presence of selective consumption against tannins. In contrast, mice in the inexperienced group did not show significant selectivity against tannins. Comparing the selectivity indices directly between two groups, however, they did not differ significantly. Selective consumption against proteins rather than that for proteins also occurred in the experienced group, but it was thought to be a byproduct resulted from the selectivity against tannins. Selective consumption against tannins can mitigate the negative effects of tannins by decreasing tannin intake.     

Isolation of tannase‐producing microbiota from the gastrointestinal tracts of some freshwater fish

Tannins are the most abundant among the plant‐derived antinutrients that bind readily with protein and other macromolecules to form indigestible complexes, thereby reducing the nutritional value of the plant feedstuffs. Presence of tannase‐producing gut microbiota in herbivorous animals has been suggested to overcome the antinutritional effects of tannins. However, this topic has been less investigated in herbivorous/omnivorous fish species. The present study was undertaken to evaluate the presence of tannase‐producing autochthonous microbiota in the gastrointestinal (GI) tracts of some culturable freshwater teleosts and to identify most promising tannase‐producing strains by molecular methods. Isolation and enumeration of tannase‐producing autochthonous microbiota have been carried out in the gut of ten culturable freshwater teleosts, namely catla (Catla catla), silver carp (Hypophthalmichthys molitrix), rohu (Labeo rohita), grass carp (Ctenopharyngodon idella), mrigal (Cirrhinus mrigala), common carp (Cyprinus carpio), bata (Labeo bata), kalbasu (Labeo calbasu), tilapia (Oreochromis mossambicus), and Nile tilapia (Oreochromis niloticus). Culturable heterotrophic and tannase‐producing microbial populations evaluated on tryptone soya agar and selective tannic acid agar media, respectively, revealed the maximum in the hindguts of all fish species studied. Out of 72 tannase‐producing colonies, 18 randomly selected isolates were maintained as pure cultures and evaluated quantitatively for tannase production. Among these, four most promising tannase producers were identified by 16S/26S rDNA sequencing following nucleotide blast and deposited in the National Centre for Biotechnology Information (NCBI) GenBank. The strain LR01 isolated from rohu was a bacterium, Enterobacter asburae (GenBank Accession No. GU939631 ). However, the strains CM02, OM01 and LR03 isolated from mrigal, tilapia and rohu were yeasts and identified as Pichia kudriavzevii (GenBank Accession No. GU939629 ), Candida tropicalis (GenBank Accession No. GU911469 ) and Candida parapsilosis (GenBank Accession No. GU939630 ), respectively. To the authors' knowledge, the present study is the first to report tannase‐producing autochthonous microbiota in the gut of freshwater teleosts. Tannin‐degrading microbiota detected in the present study may endow the fish with some ecological advantages by enabling them to overcome the anti‐nutritional effects of plant tannins.