Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T)

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.     

Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.     

Purification and characterization of an endo-polygalacturonase. From Aspergillus awamori

An extracellular endo-polygalacturonase (PGase) produced by Aspergillus awamori IFO 4033 was isolated from the culture filtrate. The enzyme was purified to a homogeneous preparation with cation-exchange and size-exclusion chromatographies. Its properties were investigated, comparing them with that of recombinant pgx2 gene product, a PGase having protopectinase activity. This enzyme was a monomeric protein of 41 kDa, with an isoelectric point of pH 6.1. The characteristics of this PGase substantially coincide, with that of recombinant pgx2 gene product, and the PGase is assumed to be native pgx2 gene product. The production of PGase-X2 was confirmed to be regulated by ambient pHs.     

Purification and characterization of extracellular tannin acyl hydrolase from <Emphasis Type="Italic">Aspergillus heteromorphus</Emphasis> MTCC 8818

A tannase (E.C. 3.1.1.20) producing fungal strain was isolated from soil and identified as Aspergillus heteromorphus MTCC 8818. Maximum tannase production was achieved on Czapek Dox minimal medium containing 1% tannic acid at a pH of 4.5 and 30°C after 48 h incubation. The crude enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography. Diethylaminoethyl-cellulose column chromatography led to an overall purification of 39.74-fold with a yield of 19.29%. Optimum temperature and pH for tannase activity were 50°C and 5.5 respectively. Metal ions such as Ca²⁺, Fe²⁺, Cu¹⁺, and Cu²⁺ increased tannase activity, whereas Hg²⁺, Na¹⁺, K¹⁺, Zn²⁺, Ag¹⁺, Mg²⁺, and Cd²⁺ acted as enzyme inhibitors. Various organic solvents such as isopropanol, isoamyl alcohol, benzene, methanol, ethanol, toluene, and glycerol also inhibited enzyme activity. Among the surfactants and chelators studied, Tween 20, Tween 80, Triton X-100, EDTA, and 1, 10-o-phenanthrolein inhibited tannase activity, whereas sodium lauryl sulfate enhanced tannase activity at 1% (w/v).     

Purification and characterization of tannase from Paecilomyces variotii: hydrolysis of tannic acid using immobilized tannase

An extracellular tannase (tannin acyl hydrolase) was isolated from Paecilomyces variotii and purified from cell-free culture filtrate using ammonium sulfate precipitation followed by ion exchange and gel filtration chromatography. Fractional precipitation of the culture filtrate with ammonium sulfate yielded 78.7% with 13.6-folds purification, and diethylaminoethyl–cellulose column chromatography and gel filtration showed 19.4-folds and 30.5-folds purifications, respectively. Molecular mass of tannase was found 149.8 kDa through native polyacrylamide gel electrophoresis (PAGE) analysis. Sodium dodecyl sulphate–PAGE revealed that the purified tannase was a monomeric enzyme with a molecular mass of 45 kDa. Temperature of 30 to 50°C and pH of 5.0 to 7.0 were optimum for tannase activity and stability. Tannase immobilized on alginate beads could hydrolyze tannic acid even after extensive reuse and retained about 85% of the initial activity. Thin layer chromatography, high performance liquid chromatography, and ¹H-nuclear magnetic resonance spectral analysis confirmed that gallic acid was formed as a byproduct during hydrolysis of tannic acid.     

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.     

Aspergillus niger contains the cryptic phylogenetic species A. awamori

Aspergillus section Nigri is an important group of species for food and medical mycology, and biotechnology. The Aspergillus niger 'aggregate' represents its most complicated taxonomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergillus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins β-tubulin (benA), calmodulin (CaM), and the translation elongation factor-1 alpha (TEF-1α) of a population of A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population, A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between A. niger and the cryptic A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B?, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-γ-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies.     

从黑茶中分离和筛选产单宁酶菌株

茶叶中富含单宁化合物。从分离自黑茶的真菌菌株中,筛选高产单宁酶的菌株;进而分离纯化单宁酶,分析单宁酶对茶汤的转溶效果。从不同产地的3个黑茶样品中,共分离获得44个真菌分离物;经初步鉴定,这些真菌分离物以曲霉属（Aspergillus）、青霉属（Penicillium）和散囊菌属（Eurotium）的真菌居多。以单宁酸为底物的鉴别培养基初筛表明,其中26个真菌分离物在鉴别平板上产生透明圈,显示单宁水解酶活性;通过固体发酵复筛,筛选到1株产单宁酶活性较高的菌株,初步鉴定为青霉属（Penicillium）菌株,命名为青霉MP-24菌株。青霉MP-24可以以茶叶、茶梗和麸皮等农副产品作为原料固体发酵产生单宁酶。以麸皮为原料的发酵产物经过硫酸铵分级沉淀、DEAE阴离子交换层析和葡聚糖G-150凝胶层析等分离纯化步骤,得到分子量为70 kDa的单一蛋白质条带,单宁酶活力达到603.68 U/mg。纯化获得的单宁酶对茶汤有良好的转溶效果。研究结果表明,在黑茶相关微生物中含有丰富的产单宁酶菌株,是工业酶制剂的重要资源。     

Purification, characterization, and partial primary sequence of a major-maltotriose-producing alpha-amylase, ScAmy43, from Sclerotinia sclerotiorum

A novel alpha-amylase (alpha-1,4-alpha-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal alpha- amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and 55oC with an apparent Km value of 1.66 mg/ml and Vmax of 0.1 micromol glucose x min-1 x ml-1. ScAmy43 activity was strongly inhibited by Cu2+, Mn2+, and Ba2+, moderately by Fe2+, and was only weakly affected by Ca2+ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and beta-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.     

Purification, immobilization and characterization of tannase from Penicillium variable

Tannase from Penicillium variable IARI 2031 was purified by a two-step purification strategy comprising of ultra-filtration using 100 kDa molecular weight cutoff and gel-filtration using Sephadex G-200. A purification fold of 135 with 91% yield of tannase was obtained. The enzyme has temperature and pH optima of 50 degrees C and 5 degrees C, respectively. However, the functional temperature range is from 25 to 80 degrees C and functional pH range is from 3.0 to 8.0. This tannase could successfully be immobilized on Amberlite IR where it retains about 85% of the initial catalytic activity even after ninth cycle of its use. Based on the Michaelis-Menten constant (Km) of tannase, tannic acid is the best substrate with Km of 32 mM and Vmax of 1.11 micromol ml(-1)min(-1). Tannase is inhibited by phenyl methyl sulphonyl fluoride (PMSF) and N-ethylmaleimide retaining only 28.1% and 19% residual activity indicating that this enzyme belongs to the class of serine hydrolases. Tannase in both crude and crude lyophilized forms is stable for one year retaining more than 60% residual activity.     

Purification and characterization of acid-stable protopectinase produced by Aspergillus awamori in solid-state fermentation

Aspergillus awamori IFO 4033 produced an acid-stable protopectinase in solid-state fermentation using wheat bran as the medium. The enzyme was purified to a homogeneous preparation with anion-exchange, hydrophobic, and size-exclusion chromatography. The enzyme was a monomeric protein of 52 kDa, by SDS-PAGE analysis, with an isoelectric point of pH 3.7. The optimum pH for enzyme activity was 2.0, and it was most active at 50 degrees C (at pH 2.0) and was stable up to 50 degrees C (at pH 2.0). The enzyme showed pectin-releasing activity toward protopectins from various origins, especially on lemon protopectin. An outstanding characteristic of the enzyme was its extreme stability in acidic conditions: the enzyme activity was not lost after incubating at pH 2.0 and 37 degrees C for 24 h.     

Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal-0.77% (w/v); (NH(4))(2)SO(4)-0.1m; KH(2)PO(4)-0.05 m; rice bran oil-2% (v/v); CaCl(2)-0.05 m; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35°C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6-fold increase in lipase production was achieved. Partial purification by (NH(4))(2)SO(4) precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40°C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.     

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.     

Production, partial purification and characterization of feruloyl esterase by Aspergillus awamori in submerged fermentation

Microbial feruloyl esterases acting on plant cell wall polymers represent key tools for the degradation of plant cell wall. In this paper, we describe in detail the microbial production, partial purification and characterization of feruloyl esterase from a culture medium of Aspergillus awamori strain IFO4033 obtained from a crude hemicellulose preparation of wheat straw, corncobs and wheat germ. Feruloyl esterase was extracted using centrifugation and dialysis, and then purified by ion exchange chromatography and microfiltration to homogeneity, which was checked by SDSPAGE and isoelectric focusing-PAGE. Protein content and activity of the enzyme were measured in each step of extraction and purification. Biomass was determined by the dry weight method. pH and temperature optima of feruloyl esterase enzyme were also determined. The effects of culturing time, and carbon and nitrogen sources on enzyme production were systematically investigated. Finally, enzyme activities under different storage conditions were examined.     

Expression of Aspergillus niger N5-5 in E. coli and purification and identification of products

Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields. However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a large-scale. Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper Aspergillus niger N5-5 was expressed using E. coli system. Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification. Then, tannase gene tan was connected to expression vector by NdeI/HindIII enzyme cutting. In this way, recombinant expression vector tan-pET43.1a was formed. Then, the expression vector pET43.1a by NdeI/HindIII enzyme cutting was transformed into E. coli BL21 (DE3) to induce expression of Aspergillus niger N5-5. When the induced fungi were disrupted by the ultrasonic wave, the crude enzyme was extracted and purified by using the IMAC, and then the activity of the crude enzyme and pure enzyme was determined. According to the results of determination of the tannase activity, the tannase activity of the crude enzyme was greatly improved after the crude enzyme was purified, and the specific activity of the pure enzyme was about 8 times of that of the crude enzyme. The results of SDS-PAGE of the pure enzyme showed that the molecular mass of the pure enzyme was about 65 kDa/64–65 kDa, which was consistent with the expected result (64.2 kDa), It can be concluded that the crude enzyme solution was purified successfully. The results of pure enzyme’s protein identification by Western Blotting showed that clear protein bands pro-3 were observed. Molecular mass of clear protein bands pro-3 was about 65 kDa, which was in line with the expected results (64.2 kDa). It can be seen that the aforementioned expression protein could be specifically combined with His tag. It proved expression protein to be a recombinant fusion protein with 6 His tag.     

Large-scale purification of fungal glucoamylases using anion-exchange resin chromatography

Anion-exchange chromatography on polystyrene resin is shown to be more effective than DEAE-cellulose for purification of glucoamylase from crude enzyme extracts of Aspergillus awamori or from commercial preparations. The glucoamylase from A. awamori culture medium was purified to electrophoretic homogeneity with yields approaching 80%.     

Production and Partial Purification of Tannase from Aspergillus ficuum Gim 3.6

A novel fungal strain, Aspergillus ficuum Gim 3.6, was evaluated for its tannase-producing capability in a wheat bran-based solid-state fermentation. Thin-layer chromatography (TLC) analysis revealed that the strain was able to degrade tannic acid to gallic acid and pyrogallol during the fermentation process. Quantitation of enzyme activity demonstrated that this strain was capable of producing a relatively high yield of extracellular tannase. Single-factor optimization of process parameters resulted in high yield of tannase after 60 hr of incubation at a pH of 5.0 at 30°C, 1 mL of inoculum size, and 1:1 solid–liquid ratio in the presence of 2.0% (w/v) tannic acid as inducer. The potential of aqueous two-phase extraction (ATPE) for the purification of tannase was investigated. Influence of various parameters such as phase-forming salt, molecular weight of polyethylene glycol (PEG), pH, and stability ratio on tannase partition and purification was studied. In all the systems, the target enzyme was observed to preferentially partition to the PEG-rich top phase, and the best result of purification (2.74-fold) with an enzyme activity recovery of 77.17% was obtained in the system containing 17% (w/w) sodium citrate and 18.18% (w/w) PEG1000, at pH 7.0.     

Isolation and characterization of juvenile hormone esterase from hemolymph of Lymantria dispar by affinity- and by anion-exchange chromatography

Juvenile hormone esterase (JHE), which catalyzes the hydrolysis of juvenile hormone, was isolated from the hemolymph of 5(th) instars of Lymantria dispar by two different procedures. One procedure was based on affinity chromatography and the other on anion-exchange chromatography. The material from both purifications showed bands of approximately 50 kDa when analyzed by SDS-PAGE. Isoelectric focusing (IEF) gels in combination with enzyme activity assays indicated two isoelectric forms with the same pI values (pH 5.1. and 5.3) from affinity purification and from anion-exchange chromatography. Amino acid sequencing of several internal peptides from the 50 kDa band following affinity purification and alignment of these sequences with JHEs from previously purified lepidopteran species (Heliothis virescens, Manduca sexta) showed high homology of these enzymes.The isolated JHE, at least in the stage of insect used, was different from the enzyme reported earlier [Valaitis, A.P., 1991. Characterization of hemolymph juvenile hormone esterase from Lymantria dispar. Insect Biochemistry 21, 583-595] to hydrolyze JH in the hemolymph of gypsy moth, based on molecular weight and amino acid sequence.     

Isolation of the antioxidant pyranonigrin-A from rice mold starters used in the manufacturing process of fermented foods

Rice mold starters prepared from Aspergillus species are commonly used for the manufacture of koji in the production of oriental fermented foods. Methanol extracts of rice mold starters fermented by the Aspergillus species, A. awamori, A. kawachii, A. oryzae, A. saitoi, and A. sojae, were examined for their antioxidative activity by using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging system. The extracts of A. awamori, A. kawachii, and A. saitoi exhibited higher activity than those of A. oryzae and A. sojae. An antioxidant was isolated from the extract of A. saitoi and identified as pyranonigrin-A by 1H-NMR, 13C-NMR, and FAB-MS analyses. The antioxidative activity of pyranonigrin-A was approximately equivalent to that of ferulic acid, an antioxidant in cereal grain. It was present in rice mold starters prepared by A. awamori, A. kawachii, and A. saitoi, although there was no pyranonigrin-A in the A. oryzae and A. sojae starters. The results suggest that the content of pyranonigrin-A in rice mold starters has a correlation with the antioxidative activity, and that it is induced in rice mold starters at the sporulation stage.     

Improvement in tannase recovery using enzymatic disruption of mycelium in combination with reverse micellar enzyme extraction

Summary A high activity tannase (tannin acyl hydrolase EC 3.1.1.20) is synthetized in high yield by Aspergillus niger LCF 8. At the production optimum, the tannase is strongly bound to the mycelium and detachment of the enzyme by classical physical and chemical means, largely failed. Enzymatic hydrolysis of cell walls using a chitinase from Streptomyces griseus followed by reverse micellar tannase extraction resulted in a recovery of 43% active enzyme, i.e. an improvement in yield of 2.5 from a previous process. Best conditions were enzymatic hydrolysis of mycelium with chitinase at pH 6.0 and 25°C for 2.5 h followed by tannase extraction at pH 7.5 with isooctane containing 80 mM cetyl trimethyl ammonium bromide and stripping at pH 4.0 in the presence of 0.35 M NaCl.