Mol Biotechnol (2015) 57:439–447 DOI 10.1007/s12033-014-9836-z
RESEARCH
Tannase Sequence from a Xerophilic Aspergillus niger Strain and Production of the Enzyme in Pichia pastoris Jose´ Antonio Fuentes-Garibay • Cristo´bal Noe´ Aguilar • Rau´l Rodrı´guez-Herrera • Martha Guerrero-Olazara´n • Jose´ Marı´a Viader-Salvado´
Published online: 9 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Tannin acyl hydrolases, or tannases (EC 3.1.1.20), are enzymes with potential biotechnological applications. In this work, we describe the gene and amino acid sequences of the tannase from Aspergillus niger GH1. In addition, we engineered Pichia pastoris strains to produce and secrete the enzyme, and the produced tannase was characterized biochemically. The nucleotide sequence of mature tannase had a length of 1,686 bp, and encodes a protein of 562 amino acids. A molecular model of mature A. niger GH1 tannase showed the presence of two structural domains, one with an a/b-hydrolase fold and one lid domain that covers the catalytic site, likely being residues Ser-196, Asp-448, and His-494 the putative catalytic triad, which are connected by a disulfide bond between the neighboring cysteines, Cys-195 and Cys-495. A 120-ml shake flask culture with a constructed recombinant P. pastoris strain showed extracellular tannase activity at 48 h induction of 0.57 U/ml. The produced tannase was N-glycosylated, consisted of two subunits, likely linked by a disulfide bond, and had an optimum pH of 5.0 and optimum temperature of 20 °C. These biochemical properties differed from those of native A. niger GH1 tannase. The recombinant tannase could be suitable for food and beverage applications.
J. A. Fuentes-Garibay M. Guerrero-Olazara´n J. M. Viader-Salvado´ (&) Facultad de Ciencias Biolo´gicas, Instituto de Biotecnologı´a, Universidad Auto´noma de Nuevo Leo´n (UANL), Av. Universidad S/N, Col. Ciudad Universitaria, 66455 San Nicola´s De Los Garza, NL, Mexico e-mail: [email protected] C. N. Aguilar R. Rodrı´guez-Herrera DIA-UAdeC/School of Chemistry, Universidad Auto´noma de Coahuila, 25280 Saltillo, Coah, Mexico
Keywords Recombinant and native tannase Tannin acyl hydrolases Aspergillus niger GH1 strain Pichia pastoris Synthetic gene
Introduction Tannin acyl hydrolases, or tannases (EC 3.1.1.20), are enzymes that catalyze the hydrolysis of ester bonds in gallotannins, complex tannins, and gallic acid esters, usually with gallic acid as the main product. Tannases are used in food industries during instant tea manufacture, wine and fruit juice clarification, and for the antinutritional reduction effects of tannins in animal feed [1]. In addition, gallic acid is used for propyl gallate and trimethropim synthesis. The former is used as an antioxidant in fats, oils, and beverages, while trimethropim is an important antibacterial drug [2, 3]. Despite the numerous applications, the practical use of these enzymes is limited due to the lack of an economical process for large-scale production. Recently, Aspergillus niger GH1, a xerophilic fungus, has been reported to be a tannase producer [4–6]. This microorganism was isolated from the Mexican semi-desert and it tolerates extreme conditions that are typical of the region (45 to -15 °C) [5]. This fungus has also been reported to be an invertase producer [7, 8]. The tannase from A. niger GH1 has been shown to be very promising for tannin-rich waste treatment, enhancing the biological activity of tea, and producing important and potent phenolic antioxidants. Compared to other tannases, A. niger GH1 tannase showed high stability to pH, temperature, and other additives that are generally encountered in tannin-rich systems, and has higher specificity constants for several polyphenolic compounds that are also commonly found in such systems. Based on these properties,
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the enzyme may be especially useful in industrial applications [4, 9]. On the other hand, the methylotrophic yeast Pichia pastoris is often used as a host for extracellular recombinant protein production, since it can grow in simple defined media and reach very high cell densities, producing high levels of extracellular recombinant proteins [10]. In this work, we describe the gene and amino acid sequences, and the likely molecular structure of the tannase from A. niger GH1. In addition, we engineered P. pastoris strains with a synthetic gene to produce and secrete the enzyme. Furthermore, the produced recombinant tannase was characterized biochemically, and compared its properties to those of the native tannase from A. niger GH1.
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sequences from five plasmids from different E. coli colonies were determined at the Instituto de Fisiologı´a Celular (UNAM), using T7 and SP6 universal primers. Two internal oligonucleotides were designed (TanC1: 50 -CGTGAAGACC GTCGTAGATG-30 , and TanC2: 50 -CACGTCTTCCCTGCC ACTAT-30 ) and further internal nucleotide sequences were determined for four plasmids. The 18 nucleotide sequences were aligned using the Contig Assembly Program (CAP) module of the BioEdit v7.0.8.0 program [11]. The consensus and deduced amino acid sequences were compared to the database sequences using BLAST tools [12] and sequence identities were calculated using Clustal Omega (http://www. ebi.ac.uk/Tools/msa/clustalo/) with default parameters. Computational Analysis of the A. niger GH1 Tannase Sequence
Materials and Methods Strains, Plasmids, Medium Composition, Chemicals, and Enzymes Pichia pastoris KM71 (his4) and plasmid pPIC9 were purchased from Invitrogen (San Diego, CA). Escherichia coli JM109, GoTaq DNA polymerase, SalI restriction endonuclease, and pGEM-T easy vector were from Promega (Madison, WI). Plasmid pUC57 used for cloning was from GenScript Corp. (Piscataway, NJ). BamHI and AvrII restriction endonucleases and endo Hf glycosidase were from New England Biolabs (Beverly, MA). All oligonucleotides were from Integrated DNA Technologies, Inc. (Coralville, IA). Regeneration dextrose base (RDB), buffered minimal glycerol (BMG), and buffered minimal methanol (BMM supplemented with 0.75 % (v/v) methanol) media were prepared according to the instructions for the Pichia expression kit (Invitrogen). All chemicals were analytical grade and purchased from Sigma-Aldrich Co. (St. Louis, MO) or from Productos Quı´micos Monterrey (Monterrey, Nuevo Leo´n, Mexico). Tannase Gene Sequence from A. niger GH1 Genomic DNA of A. niger GH1 was extracted according to standard protocols. The sequence encoding the mature enzyme was synthesized by polymerase chain reaction (PCR) using a forward primer Tan1 (50 -ACTTCCCTGTCCGATCT C-30 ) and reverse primer Tan2 (50 -AAAAACGGGCATCTT GAA-30 ). The PCR assay was performed with GoTaq DNA polymerase and a 30-cycle amplification program under the following conditions: 94 °C for 1 min, 55 °C for 0.5 min, and 72 °C for 1.75 min, with a first denaturation step at 94 °C for 1 min and a final extension step at 72 °C for 10 min. The amplified product was cloned into the vector pGEM-T according to the manufacturer’s instructions. Nucleotide
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Functional domain analysis was performed by comparing mature tannase sequence with the Pfam protein families database [13]. A molecular model of A. niger GH1 tannase was constructed by protein threading using the Phyre2 server [14]. Possible N-linked glycosylation sites were predicted with the NetNGlyc 1.0 server (http://www.cbs. dtu.dk/services/NetNGlyc/). The location of disulfide bonds, Kex2 sites, and N-linked glycosylation sites in the molecular model were determined with the Swiss-PdbViewer/DeepView 4.1 program [15]. The system used for residue position identification for the native and recombinant A. niger GH1 tannase corresponded to the methionine of the native signal peptide as the first amino acid residue. Thus, the first residue of the mature native and recombinant A. niger GH1 tannase started at position 20. Construction of P. pastoris Recombinant Strains A synthetic gene (antgs) harboring a nucleotide sequence encoding the Saccharomyces cerevisiae alpha-factor preprosecretion signal, including the BamHI site, fused in frame with a nucleotide sequence encoding the mature A. niger GH1 tannase was designed based on P. pastoris-preferred codons [16]. In addition, AT-rich stretches of more than six nucleotides were removed introducing silent mutations and one AvrII site was introduced at the 30 end. The designed nucleotide sequence, with a full length of 1,961 bp, was synthesized, cloned into vector pUC57, and sequenced by GenScript Corp. (Piscataway, NJ), to generate the plasmid pUC57antgs. The DNA fragment, harboring the antgs sequence, was obtained by digestion of pUC57antgs with BamHI and AvrII restriction enzymes, and then ligated to vector pPIC9 that had previously been digested with the same restriction enzymes, to produce the new expression vector (pPIC9antgs). The correct vector construction was
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confirmed by BamHI-AvrII double-digested restriction analysis and by PCR using 50 and 30 AOX1 primers, directed to the AOX1 promoter and the transcription terminator, as previously described [17]. All DNA manipulations were performed according to standard methods [18]. The P. pastoris host strain, KM71 (his4) was transformed with SalI-digested pPIC9antgs DNA by electroporation, as described elsewhere [19]. Transformants were selected for their ability to grow on histidine-deficient medium (RDB-agar plates) at 30 °C until colonies appeared (His? selection). His? colonies were randomly selected, and the integration of the expression cassette into the genomes of the selected strains was verified by PCR using the 50 and 30 AOX1 primers, as previously described [17]. Production of Recombinant Tannase A His? transformant was reactivated in YPD medium at 30 °C and 250 rpm for 12 h and then used to inoculate 600 ml of BMG medium to an initial optical density at 600 nm (OD600) of 0.3. Further incubation was performed at 30 °C and 250 rpm for 12 h. Cells were harvested by centrifugation (1,7009g, 5 min) and used to inoculate 120 ml of BMM medium to an initial OD600 of 30. Further incubation was carried out at 30 °C for 48 h with continuous shaking at 250 rpm. Methanol was added to a final concentration of 0.75 % every 24 h. Cell-free culture medium was recovered by centrifugation and concentrated 50-fold and diafiltrated by ultrafiltration at 4 °C using 10-kDa Centricon Plus 70 filters (Millipore, MA) and 50 mM sodium citrate buffer (pH 5.0). Aliquots of enzyme concentrates were stored at -20 °C until used for the biochemical tannase characterization. Biochemical Characterization of Recombinant A. niger GH1 Tannase Biochemical characterization of tannase was performed with the enzyme concentrate by ultrafiltration from cellfree culture medium. N-glycosylation was evaluated by assessing the migration shift of endo Hf-treated proteins in a Coomassie blue-stained 12 % SDS-polyacrylamide gel (SDS-PAGE). Reactions were carried out by incubating the tannase concentrate with endo Hf for 1 h at 37 °C, according to the manufacturer’s instructions. The effect of pH on enzymatic activity was determined at 30 °C, using 250 mM glycine–HCl (pH 2.5), 50 mM sodium citrate (pH 4.0, 5.0, and 6.0), 100 mM Tris–HCl (pH 7.0 and 9.0) as buffers. The effect of temperature on enzymatic activity was determined at pH 5 by measuring tannase activity at different temperatures ranging from 10 to 70 °C. All results were compared between statistical
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groups, using analysis of variance (ANOVA) and Tukey’s multiple comparisons, with a significance cutoff P value of \0.05. For tannase-specific activity determination, additional purification was performed with the enzyme concentrate using a Biologic LP chromatography system (Bio-Rad, Hercules, CA) and a HiTrap Q FF anion exchange column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) equilibrated with buffer A (50 mM sodium citrate, pH 5.0). Proteins were eluted with buffer B (50 mM sodium citrate, 1.0 M NaCl, pH 5), using a step-wise gradient elution process (0–10 min, 100 % buffer A; 10–11 min, 0–5 % buffer B; 11–17 min, 5 % buffer B; 17–18 min, 5–35 % buffer B; 18–30 min, 35 % buffer B; 30–31 min, 35–60 % buffer B; 31–36 min, 60 % buffer B; 36–38 min, 60–100 % buffer B; and 38–43 min, 100 % buffer B) at a flow rate of 1 ml/min. Fractions of 1.4 ml were collected, and protein concentration and tannase activity were determined. The highest ratio of volumetric tannase activity to the protein concentration of a fraction was considered as the specific activity of purified tannase. Enzyme kinetics were determined by testing tannase activity at 20 and 30 °C using gallic acid methyl ester, ranging from 0.08 to 10.00 mM, and tannase activity values were fitted to the Michaelis–Menten equation using the DataFit 8.2.79 program (Oakdale Engineering, Oakdale, PA). All protein concentrations were determined by the Bradford protein assay, using bovine serum albumin as the standard. Tannase Activity Assays Tannase activity was determined for cell-free culture medium, protein concentrate obtained by ultrafiltration, biochemical characterization assays, and chromatographic fractions. All samples were diluted in a suitable volume of 50 mM sodium citrate (pH 5.0) and tannase activity was measured by gallic acid–rhodanine chromogen formation using gallic acid methyl ester as substrate [6, 20]. One unit (U) of tannase activity was defined as the amount of enzyme required to release one micromole of gallic acid per minute under the assay conditions (pH 5, 30 °C).
Results Aspergillus niger GH1 Tannase Sequence The nucleotide sequence of mature tannase had a length of 1,686 bp, without introns, and encodes a protein of 562 amino acids (GenBank accession no. KP273835). The amino acid sequence had identities of 78.6–99.4 % with the six more similar fungal tannases (Aspergillus kawachii
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IFO 4308 tannase [GenBank accession no. GAA91900.1] 99.4 %, A. niger CBS 513.88 tannase [GenBank accession no. XP_001402486.1] 98.0 %, A. niger ATCC 1015 tannase [GenBank accession no. EHA22262.1] 97.1 %, Aspergillus terreus NIH2624 tannase [GenBank accession no. XP_001216558.1] 80.0 %, A. niger tannase [GenBank accession no. ABX89592.1] 79.2 %, Aspergillus ruber CBS 135680 tannase and feruloyl esterase [GenBank accession no. EYE96818.1] 78.6 %), with nucleotide sequence identities ranging from 73.3 to 98.6 % to the same fungal tannases. Compared to the most related tannase (A. kawachii IFO 4308), the A. niger GH1 tannase has three differences, all of which are conservative (T20S, S62A, and N82D). According to the Pfam protein families database, A. niger GH1 tannase has a functional domain from residue 53–545 of the Tannase PF07519 family where other tannases, feruloyl esterases, and several bacterial proteins of unknown function are grouped. This family belongs to the clan AB_hydrolase (CL0028) which its catalytic domain being found in a wide range of enzymes, since this clan contains 67 members. Currently, 757 amino acid sequences distributed in 345 species are reported in the PF07519 family: 375 (49.5 %) sequences from bacteria, 271 (78.6 %) species, and 382 (50.5 %) sequences from eukaryotes, 74 species (21.4 %). Among eukaryotes highlight fungi with 365 sequences (48.2 % of all sequences) distributed in 69 species (20.0 %). In the PF07519 family, 15 architectures of domain organization have been described, having A. niger GH1 tannase the simplest architecture of a single tannase domain. Only one three-dimensional structure has been experimentally determined by X-ray crystallography for the PF07519 family (PDB code: 3WMT), which corresponds to a feruloyl esterase from Aspergillus oryzae [21]. The Phyre2 server constructed a full molecular model of the mature A. niger GH1 tannase; 512 residues (91 %) were modeled with the 3WMT structure because Phyre2 considered the 3WMT protein as a true homologue of A. niger GH1 tannase with 100 % confidence. Also, 50 residues (9 %) were modeled using ab initio techniques. The A. niger GH1 tannase molecular model (Fig. 1a) showed the presence of 16 alpha helices and 16 beta sheets covering 29 and 13 % of the protein, respectively, forming two structural domains. One domain has an a/b-hydrolase fold, constituting the catalytic domain, and one lid domain that covers the catalytic site. From the molecular model and by comparisons with the A. oryzae feruloyl esterase 3WMT, residues Ser-196, Asp-448, and His-494 likely constitute the putative catalytic triad. In addition, these serine and histidine residues are likely directly connected by a disulfide bond between neighboring cysteines, Cys-
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195 and Cys-495 (Fig. 1b), forming the recently described structural motif (CS-D-HC) [21]. Construction of a Tannase P. pastoris Overproducer Strain DNA sequencing of the pUC57 plasmid confirmed the correct nucleotide sequences of the antgs gene. The tannase synthetic gene with the nucleotide sequence encoding the S. cerevisiae alpha-factor prepro-secretion signal had a full length of 1,961 bp. The DNA sequence was engineered to produce the A. niger GH1 tannase as a mature polypeptide of 562 amino acids. PCR analysis of the recombinant plasmid, using AOX1 primers, showed a 2,158 bp product that confirmed the pPIC9antgs construct. Transformation of P. pastoris KM71 with the SalIdigested pPIC9antgs plasmid gave about 15 His? transformants. PCR analysis of the genomic DNAs isolated from P. pastoris KM71ANT (His? transformants) showed a 2,158-bp band that corresponds to the alpha-factor prepro-secretion signal (255 bp) and synthetic tannase (1,689 bp) coding sequences, and fragments of the multiple cloning site from the pPIC9 vector (21 bp), AOX1 promoter (94 bp), and transcription terminator (99 bp). Production and Biochemical Characterization of Recombinant A. niger GH1 Tannase Cell-free culture medium from a 48 h methanol-induced recombinant strain culture showed volumetric tannase activity of 0.57 U/ml, and 46.71 mg/l of total protein. With the ultrafiltration process applied to the cell-free culture medium, a tannase concentrate (25.7 U/ml) was obtained to carry out the biochemical characterization. Figure 2 shows the migration shift results in SDS-PAGE for the recombinant tannase preparation after treatment with endo Hf glycosidase. Recombinant tannase without endo Hf treatment showed two broad bands of apparent molecular masses of 45.0–54.8 and 41.8–45.0 kDa, with a higher intensity at molecular masses of 48.7 and 42.4 kDa, on the SDS-PAGE (Fig. 2, lane 2). After N-deglycosylation by endo Hf, the two bands shifted to apparent molecular masses of 34.6 and 30.3 kDa, respectively, (Fig. 2, lane 3). These results clearly indicate that recombinant tannase is highly N-glycosylated. The analysis with the NetNGlyc 1.0 server showed that the mature A. niger GH1 tannase amino acid sequence has eleven potential N-glycosylation sites (19NGTL, 54NVTV, 130NGSI, 237NATI, 265NLTS, 280NYTS, 304NGSV, 383NVTY, 451NTTY, 508NATV, and 535NSSF). The recombinant tannase had its optimum activity at pH 5.0 (Fig. 3a) and showed more than 80 % of its maximum activity in the pH range between 4.0 and 5.0. Tannase
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Fig. 1 Molecular model (a) of the A. niger GH1 tannase constructed using the Phyre2 server showing the presence of two structural domains. Putative catalytic triad (b), Ser-196, Asp448, and His-494, directly connected by a disulfide bond between neighboring cysteines, Cys-195 and Cys-495. Kex2 recognition site (Lys-309-Arg310) at the protein surface in a flexible loop of the lid domain (c)
Fig. 2 SDS-polyacrylamide gel of recombinant tannase concentrated by ultrafiltration and treated with or without endo Hf glycosidase. Lane M Molecular mass marker. Lane 1 endo Hf. Lanes 2–3 recombinant tannase without and with endo Hf treatment, both samples were treated with 2-mercaptoethanol in the loading buffer
activity showed a profile with a broad temperature range (10–50 °C) with more than 40 % of its maximum activity, and an optimum temperature of 20 °C (Fig. 3b).
Residual activity in the stability assays followed zeroorder kinetics with a reaction rate of 0.01 and 0.48 %/h at 4 and 30 °C, respectively, decreasing to 98.9 and 44.0 % after 120 h (Fig. 3c). The anion-exchange chromatogram for tannase preparation showed two predominant peaks by UV detection at 280 nm. One of the peaks, eluted at 0.35 M NaCl, showed tannase activity (data not shown). The specific activity (20 °C, pH 5.0) at the maximum of this peak was 50.0 U/ mg protein. The recombinant tannase followed typical Michaelis– Menten kinetics at both 20 and 30 °C (R2 = 0.947 and 0.978, respectively). The Michaelis constants (Km) and the maximum rates of reaction (Vmax) were estimated to be 1.98 ± 0.50 mM and 2.01 ± 0.14 lmol/min, and 0.18 ± 0.04 mM and 0.48 ± 0.02 lmol/min at 20 and 30 °C, respectively.
Discussion Recently, the xerophilic A. niger GH1 has been described as a tannase producer in both submerged and solid-state cultures [5, 6, 22]. The biochemical properties of native A.
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Fig. 3 Effects of pH (a) at 30 °C and temperature (b) at pH 5.0 on recombinant tannase activity. Tannase activities are expressed as relative values. Residual activity for the tannase stability assays (c) at
4 °C (filled circle) and 30 °C (filled square). Points represent the means for at least three independent enzyme assays (coefficient of variation \5 %)
niger GH1 tannase have also been described [4] and these properties were compared for A. niger GH1 tannase produced by solid-state and submerged fermentations [9]. In this paper, we report the gene and amino acid sequences for the mature A. niger GH1 tannase. In addition, we used the P. pastoris expression system to produce the enzyme, using a synthetic gene, and characterized the produced recombinant tannase. The A. niger GH1 tannase gene and amino acid sequences appeared to be similar but not identical to the A. kawachii IFO 4308 tannase. Because no three-dimensional structure with a height sequence identity with the A. niger GH1 tannase is available in the Protein Data Bank, constructing a molecular model by homology modeling is not suitable [23]. Nevertheless, fold recognition by protein threading methods can accurately model protein sequences with less than 20 % sequence identity with a known protein. The Phyre2 server uses protein threading methods for protein molecular modeling and was placed it among the best servers in the Critical Assessment of Protein Structure Prediction (CASP) [14]. Therefore, we used the Phyre2 server to construct a molecular model of the A. niger GH1 tannase and found that the model
has a fold that is similar to the structure of the A. oryzae feruloyl esterase 3WMT (two structural domains: a/bhydrolase and a lid domain), which has a 24 % amino acid sequence identity with the tannase from A. niger GH1. In addition, both structures have a catalytic triad forming the structural motif CS-D-HC. To the best of our knowledge, this is the first report of a putative molecular structure from a tannase of the Tannase PF07519 family with a fold very similar to a feruloyl esterase, harboring two structural domains and the structural motif CS-D-HC. Nevertheless, the substrate specificity of the two enzymes is different, likely because the substrate-binding pockets are formed by different residues in the two enzymes: Phe-225, Gln-228, Gln-229, Glu-365, Ser-279, Gly-381, and Ile-451 for A. niger GH1 tannase, and Phe-232, Leu-235, Thr-236, Tyr-348, Phe-354, Tyr-356, and Ile-419 for A. oryzae feruloyl esterase [21]. We engineered P. pastoris strains to produce the A. niger GH1 tannase as an extracellular mature polypeptide of 562 amino acids, since we had removed the putative native signal sequence (19 residues) predicted by SignalP 4.1 [24] for the A. niger CBS513.88 tannase from the recombinant construction. In addition, the mature
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polypeptide was fused in frame with the S. cerevisiae alpha-factor prepro-secretion signal, which is known to be a good secretion signal for P. pastoris [10]. The production of seven tannases in microorganisms have been reported, including Lactobacillus plantarum, Klebsiella pneumoniae, Bacillus licheniformis, Aspergillus awamori, A. oryzae, and A. niger [25–31]. Among these tannases, six were native tannases and only one from A. oryzae was produced as a recombinant [30, 31]. Among the native tannases, five have been produced under conventional submerged culture and only the A. niger GH1 tannase was extracellularly produced in solid-state culture being the highest production level (2,291 U/l in 20 h) compared to the other native tannases. However, a decline in tannase activity was observed after 20 h of incubation associated with a concomitant increase in protease activity [6]. Aspergillus oryzae tannase has already been produced with the P. pastoris expression system, both extracellularly [31] and intracellularly [30], resulting 7 U/ml after 96 h of induction for a fed-batch culture in a 3-L bioreactor in the first case, and 0.96 U/ml after 72 h of induction for a shake-flask culture in the second case, both with a Muts strain. However, in any case synthetic genes were used based on P. pastoris-preferred codons and optimized AT content for the tannase gene and/or the sequence encoding the S. cerevisiae alpha-factor prepro-secretion signal, neither the biochemical properties of the recombinant tannases were not described, except for the formation of the doublestranded structure. Because high cell densities of P. pastoris can be achieved in a simple bioreactor in a controlled form, a 10- to 100-fold increase in production of the recombinant A. niger GH1 tannase produced in sake-flask can be expected, as described for other proteins produced in P. pastoris [32]. The NetNGlyC 1.0 server predicted eleven potential N-glycosylation sites for A. niger GH1 tannase. Among them, three potential N-glycosylation sites (130NGSI, 265NLTS, and 451NTTY) are more likely to be N-glycosylated since the molecular model of A. niger GH1 tannase showed that these sites are at the protein surface. In any case, experimental data is needed to verify whether or not a recombinant protein is N-glycosylated by the host, since the presence of N-glycosylation sites is not sufficient to conclude that an asparagine residue would be N-glycosylated. Our results from endo Hf treatment clearly demonstrate that recombinant A. niger GH1 tannase was N-glycosylated, since a band was not observed on the SDSPAGE corresponding to the theoretical molecular mass of mature A. niger GH1 tannase (61.4 kDa), judging from the amino acid sequence deduced from the tannase gene. Nevertheless, two broad bands were mainly observed (Fig. 2, lane 2) at lower molecular masses (48.7, and
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42.4 kDa) than the expected theoretical molecular mass for mature A. niger GH1 tannase, that shifted to two defined bands at molecular masses of 34.6 and 30.3 kDa (Fig. 2, lane 3) after N-deglycosylation by endo Hf glycosidase. The N-glycosylation pattern of recombinant A. niger GH1 tannase should be similar to that described for other recombinant proteins produced in P. pastoris [33–35]. These findings also indicate that the recombinant tannase preparation had a high degree of purity. Furthermore, the recombinant tannase consisted of two subunits, likely linked by a disulfide bond, generated by a Kex2 protease cleavage of the tannase gene product, since the mature A. niger GH1 tannase sequence has two pairs of basic amino acids (Lys-309-Arg-310, and Lys-347-Arg-348) that are a typical Kex2 recognition site. This type of structure has already been described for a native A. oryzae tannase [36], and a recombinant A. oryzae tannase produced either extracellularly [31] or intracellularly [30] in P. pastoris. Of the two Kex2 recognition sites, the Lys-309-Arg-310 site is more likely to be recognized by the Kex2 protease, since the molecular model showed that this site is at the protein surface in a flexible loop of the lid domain (Fig. 1c). The Lys-347-Arg-348 site, in contrast, is located at an alpha helix of the down side of the lid domain and is not so exposed to the protein surface than the Lys-309-Arg-310 site. A cleavage by Kex2 beside Arg-310 would render two peptides of 31.2 and 30.2 kDa. The disulfide bond between Cys-195 and Cys-495 would link both peptides. All of the biochemical properties for the recombinant A. niger GH1 tannase were determined using the enzyme concentrate prepared by ultrafiltration from cell-free culture medium. A buffer exchange was performed during the ultrafiltration process, to remove low molecular weight peptides and other compounds that might affect the biochemical properties. In addition, the SDS-PAGE analysis of the tannase concentrate prepared by ultrafiltration showed that the preparation had a high degree of protein purity (Fig. 2). All of the biochemical properties described in this paper are relative values, to the maximum value or to the initial value (Fig. 3). Therefore, if some residual impurities could affect the biochemical properties of the tannase concentrate preparation, they would not affect the relative values of the biochemical properties. Native and recombinant A. niger GH1 tannase have the same amino acid sequence. Therefore, any differences in their biochemical properties (pH and temperature profiles, and stability) must be due to their differing degrees of N-glycosylation and/or their different structure since native A. niger GH1 tannase is mainly a single glycopolypeptide strand of 102–105 kDa [9]. N-glycosylation affects biochemical properties such as molecular mass and pH optimum [37]. While native A. niger GH1 tannase displayed optimal pH of 6.0–7.0 and optimal temperature of 60 °C [9,
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38], recombinant A. niger GH1 tannase showed optimal pH of 5.0 and optimal temperature of 20 °C. The produced recombinant tannase is the first tannase described with an optimum temperature of 20 °C. In addition, recombinant A. niger GH1 tannase had a higher relative activity compared to native A. niger GH1 tannase over a range of 10–40 °C, which would be advantageous for many food and beverage applications [39]. Native and recombinant tannases showed high residual activity at 4 °C for at least 120 h. Notably, biochemical properties of a double-stranded Aspergillus tannase were not described until very recently [40]. Nevertheless, in that publication a very similar biochemical properties for native and recombinant tannase were described, likely because both had a double-stranded structure, this is not our case. Based on its pH and temperature profiles, the tannase produced in this study could be used in food and beverage processing at low temperatures. Acknowledgments We thank Fabiola Veana for her technical support and Luis V. Dura´n-Rodrı´guez for his technical suggestions. We also thank Glen D. Wheeler for his stylistic suggestions in the preparation of the manuscript. J.A.F-G. thanks CONACYT for fellowship.
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RESEARCH
Tannase Sequence from a Xerophilic Aspergillus niger Strain and Production of the Enzyme in Pichia pastoris Jose´ Antonio Fuentes-Garibay • Cristo´bal Noe´ Aguilar • Rau´l Rodrı´guez-Herrera • Martha Guerrero-Olazara´n • Jose´ Marı´a Viader-Salvado´
Published online: 9 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Tannin acyl hydrolases, or tannases (EC 3.1.1.20), are enzymes with potential biotechnological applications. In this work, we describe the gene and amino acid sequences of the tannase from Aspergillus niger GH1. In addition, we engineered Pichia pastoris strains to produce and secrete the enzyme, and the produced tannase was characterized biochemically. The nucleotide sequence of mature tannase had a length of 1,686 bp, and encodes a protein of 562 amino acids. A molecular model of mature A. niger GH1 tannase showed the presence of two structural domains, one with an a/b-hydrolase fold and one lid domain that covers the catalytic site, likely being residues Ser-196, Asp-448, and His-494 the putative catalytic triad, which are connected by a disulfide bond between the neighboring cysteines, Cys-195 and Cys-495. A 120-ml shake flask culture with a constructed recombinant P. pastoris strain showed extracellular tannase activity at 48 h induction of 0.57 U/ml. The produced tannase was N-glycosylated, consisted of two subunits, likely linked by a disulfide bond, and had an optimum pH of 5.0 and optimum temperature of 20 °C. These biochemical properties differed from those of native A. niger GH1 tannase. The recombinant tannase could be suitable for food and beverage applications.
J. A. Fuentes-Garibay M. Guerrero-Olazara´n J. M. Viader-Salvado´ (&) Facultad de Ciencias Biolo´gicas, Instituto de Biotecnologı´a, Universidad Auto´noma de Nuevo Leo´n (UANL), Av. Universidad S/N, Col. Ciudad Universitaria, 66455 San Nicola´s De Los Garza, NL, Mexico e-mail: [email protected] C. N. Aguilar R. Rodrı´guez-Herrera DIA-UAdeC/School of Chemistry, Universidad Auto´noma de Coahuila, 25280 Saltillo, Coah, Mexico
Keywords Recombinant and native tannase Tannin acyl hydrolases Aspergillus niger GH1 strain Pichia pastoris Synthetic gene
Introduction Tannin acyl hydrolases, or tannases (EC 3.1.1.20), are enzymes that catalyze the hydrolysis of ester bonds in gallotannins, complex tannins, and gallic acid esters, usually with gallic acid as the main product. Tannases are used in food industries during instant tea manufacture, wine and fruit juice clarification, and for the antinutritional reduction effects of tannins in animal feed [1]. In addition, gallic acid is used for propyl gallate and trimethropim synthesis. The former is used as an antioxidant in fats, oils, and beverages, while trimethropim is an important antibacterial drug [2, 3]. Despite the numerous applications, the practical use of these enzymes is limited due to the lack of an economical process for large-scale production. Recently, Aspergillus niger GH1, a xerophilic fungus, has been reported to be a tannase producer [4–6]. This microorganism was isolated from the Mexican semi-desert and it tolerates extreme conditions that are typical of the region (45 to -15 °C) [5]. This fungus has also been reported to be an invertase producer [7, 8]. The tannase from A. niger GH1 has been shown to be very promising for tannin-rich waste treatment, enhancing the biological activity of tea, and producing important and potent phenolic antioxidants. Compared to other tannases, A. niger GH1 tannase showed high stability to pH, temperature, and other additives that are generally encountered in tannin-rich systems, and has higher specificity constants for several polyphenolic compounds that are also commonly found in such systems. Based on these properties,
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the enzyme may be especially useful in industrial applications [4, 9]. On the other hand, the methylotrophic yeast Pichia pastoris is often used as a host for extracellular recombinant protein production, since it can grow in simple defined media and reach very high cell densities, producing high levels of extracellular recombinant proteins [10]. In this work, we describe the gene and amino acid sequences, and the likely molecular structure of the tannase from A. niger GH1. In addition, we engineered P. pastoris strains with a synthetic gene to produce and secrete the enzyme. Furthermore, the produced recombinant tannase was characterized biochemically, and compared its properties to those of the native tannase from A. niger GH1.
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sequences from five plasmids from different E. coli colonies were determined at the Instituto de Fisiologı´a Celular (UNAM), using T7 and SP6 universal primers. Two internal oligonucleotides were designed (TanC1: 50 -CGTGAAGACC GTCGTAGATG-30 , and TanC2: 50 -CACGTCTTCCCTGCC ACTAT-30 ) and further internal nucleotide sequences were determined for four plasmids. The 18 nucleotide sequences were aligned using the Contig Assembly Program (CAP) module of the BioEdit v7.0.8.0 program [11]. The consensus and deduced amino acid sequences were compared to the database sequences using BLAST tools [12] and sequence identities were calculated using Clustal Omega (http://www. ebi.ac.uk/Tools/msa/clustalo/) with default parameters. Computational Analysis of the A. niger GH1 Tannase Sequence
Materials and Methods Strains, Plasmids, Medium Composition, Chemicals, and Enzymes Pichia pastoris KM71 (his4) and plasmid pPIC9 were purchased from Invitrogen (San Diego, CA). Escherichia coli JM109, GoTaq DNA polymerase, SalI restriction endonuclease, and pGEM-T easy vector were from Promega (Madison, WI). Plasmid pUC57 used for cloning was from GenScript Corp. (Piscataway, NJ). BamHI and AvrII restriction endonucleases and endo Hf glycosidase were from New England Biolabs (Beverly, MA). All oligonucleotides were from Integrated DNA Technologies, Inc. (Coralville, IA). Regeneration dextrose base (RDB), buffered minimal glycerol (BMG), and buffered minimal methanol (BMM supplemented with 0.75 % (v/v) methanol) media were prepared according to the instructions for the Pichia expression kit (Invitrogen). All chemicals were analytical grade and purchased from Sigma-Aldrich Co. (St. Louis, MO) or from Productos Quı´micos Monterrey (Monterrey, Nuevo Leo´n, Mexico). Tannase Gene Sequence from A. niger GH1 Genomic DNA of A. niger GH1 was extracted according to standard protocols. The sequence encoding the mature enzyme was synthesized by polymerase chain reaction (PCR) using a forward primer Tan1 (50 -ACTTCCCTGTCCGATCT C-30 ) and reverse primer Tan2 (50 -AAAAACGGGCATCTT GAA-30 ). The PCR assay was performed with GoTaq DNA polymerase and a 30-cycle amplification program under the following conditions: 94 °C for 1 min, 55 °C for 0.5 min, and 72 °C for 1.75 min, with a first denaturation step at 94 °C for 1 min and a final extension step at 72 °C for 10 min. The amplified product was cloned into the vector pGEM-T according to the manufacturer’s instructions. Nucleotide
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Functional domain analysis was performed by comparing mature tannase sequence with the Pfam protein families database [13]. A molecular model of A. niger GH1 tannase was constructed by protein threading using the Phyre2 server [14]. Possible N-linked glycosylation sites were predicted with the NetNGlyc 1.0 server (http://www.cbs. dtu.dk/services/NetNGlyc/). The location of disulfide bonds, Kex2 sites, and N-linked glycosylation sites in the molecular model were determined with the Swiss-PdbViewer/DeepView 4.1 program [15]. The system used for residue position identification for the native and recombinant A. niger GH1 tannase corresponded to the methionine of the native signal peptide as the first amino acid residue. Thus, the first residue of the mature native and recombinant A. niger GH1 tannase started at position 20. Construction of P. pastoris Recombinant Strains A synthetic gene (antgs) harboring a nucleotide sequence encoding the Saccharomyces cerevisiae alpha-factor preprosecretion signal, including the BamHI site, fused in frame with a nucleotide sequence encoding the mature A. niger GH1 tannase was designed based on P. pastoris-preferred codons [16]. In addition, AT-rich stretches of more than six nucleotides were removed introducing silent mutations and one AvrII site was introduced at the 30 end. The designed nucleotide sequence, with a full length of 1,961 bp, was synthesized, cloned into vector pUC57, and sequenced by GenScript Corp. (Piscataway, NJ), to generate the plasmid pUC57antgs. The DNA fragment, harboring the antgs sequence, was obtained by digestion of pUC57antgs with BamHI and AvrII restriction enzymes, and then ligated to vector pPIC9 that had previously been digested with the same restriction enzymes, to produce the new expression vector (pPIC9antgs). The correct vector construction was
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confirmed by BamHI-AvrII double-digested restriction analysis and by PCR using 50 and 30 AOX1 primers, directed to the AOX1 promoter and the transcription terminator, as previously described [17]. All DNA manipulations were performed according to standard methods [18]. The P. pastoris host strain, KM71 (his4) was transformed with SalI-digested pPIC9antgs DNA by electroporation, as described elsewhere [19]. Transformants were selected for their ability to grow on histidine-deficient medium (RDB-agar plates) at 30 °C until colonies appeared (His? selection). His? colonies were randomly selected, and the integration of the expression cassette into the genomes of the selected strains was verified by PCR using the 50 and 30 AOX1 primers, as previously described [17]. Production of Recombinant Tannase A His? transformant was reactivated in YPD medium at 30 °C and 250 rpm for 12 h and then used to inoculate 600 ml of BMG medium to an initial optical density at 600 nm (OD600) of 0.3. Further incubation was performed at 30 °C and 250 rpm for 12 h. Cells were harvested by centrifugation (1,7009g, 5 min) and used to inoculate 120 ml of BMM medium to an initial OD600 of 30. Further incubation was carried out at 30 °C for 48 h with continuous shaking at 250 rpm. Methanol was added to a final concentration of 0.75 % every 24 h. Cell-free culture medium was recovered by centrifugation and concentrated 50-fold and diafiltrated by ultrafiltration at 4 °C using 10-kDa Centricon Plus 70 filters (Millipore, MA) and 50 mM sodium citrate buffer (pH 5.0). Aliquots of enzyme concentrates were stored at -20 °C until used for the biochemical tannase characterization. Biochemical Characterization of Recombinant A. niger GH1 Tannase Biochemical characterization of tannase was performed with the enzyme concentrate by ultrafiltration from cellfree culture medium. N-glycosylation was evaluated by assessing the migration shift of endo Hf-treated proteins in a Coomassie blue-stained 12 % SDS-polyacrylamide gel (SDS-PAGE). Reactions were carried out by incubating the tannase concentrate with endo Hf for 1 h at 37 °C, according to the manufacturer’s instructions. The effect of pH on enzymatic activity was determined at 30 °C, using 250 mM glycine–HCl (pH 2.5), 50 mM sodium citrate (pH 4.0, 5.0, and 6.0), 100 mM Tris–HCl (pH 7.0 and 9.0) as buffers. The effect of temperature on enzymatic activity was determined at pH 5 by measuring tannase activity at different temperatures ranging from 10 to 70 °C. All results were compared between statistical
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groups, using analysis of variance (ANOVA) and Tukey’s multiple comparisons, with a significance cutoff P value of \0.05. For tannase-specific activity determination, additional purification was performed with the enzyme concentrate using a Biologic LP chromatography system (Bio-Rad, Hercules, CA) and a HiTrap Q FF anion exchange column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) equilibrated with buffer A (50 mM sodium citrate, pH 5.0). Proteins were eluted with buffer B (50 mM sodium citrate, 1.0 M NaCl, pH 5), using a step-wise gradient elution process (0–10 min, 100 % buffer A; 10–11 min, 0–5 % buffer B; 11–17 min, 5 % buffer B; 17–18 min, 5–35 % buffer B; 18–30 min, 35 % buffer B; 30–31 min, 35–60 % buffer B; 31–36 min, 60 % buffer B; 36–38 min, 60–100 % buffer B; and 38–43 min, 100 % buffer B) at a flow rate of 1 ml/min. Fractions of 1.4 ml were collected, and protein concentration and tannase activity were determined. The highest ratio of volumetric tannase activity to the protein concentration of a fraction was considered as the specific activity of purified tannase. Enzyme kinetics were determined by testing tannase activity at 20 and 30 °C using gallic acid methyl ester, ranging from 0.08 to 10.00 mM, and tannase activity values were fitted to the Michaelis–Menten equation using the DataFit 8.2.79 program (Oakdale Engineering, Oakdale, PA). All protein concentrations were determined by the Bradford protein assay, using bovine serum albumin as the standard. Tannase Activity Assays Tannase activity was determined for cell-free culture medium, protein concentrate obtained by ultrafiltration, biochemical characterization assays, and chromatographic fractions. All samples were diluted in a suitable volume of 50 mM sodium citrate (pH 5.0) and tannase activity was measured by gallic acid–rhodanine chromogen formation using gallic acid methyl ester as substrate [6, 20]. One unit (U) of tannase activity was defined as the amount of enzyme required to release one micromole of gallic acid per minute under the assay conditions (pH 5, 30 °C).
Results Aspergillus niger GH1 Tannase Sequence The nucleotide sequence of mature tannase had a length of 1,686 bp, without introns, and encodes a protein of 562 amino acids (GenBank accession no. KP273835). The amino acid sequence had identities of 78.6–99.4 % with the six more similar fungal tannases (Aspergillus kawachii
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IFO 4308 tannase [GenBank accession no. GAA91900.1] 99.4 %, A. niger CBS 513.88 tannase [GenBank accession no. XP_001402486.1] 98.0 %, A. niger ATCC 1015 tannase [GenBank accession no. EHA22262.1] 97.1 %, Aspergillus terreus NIH2624 tannase [GenBank accession no. XP_001216558.1] 80.0 %, A. niger tannase [GenBank accession no. ABX89592.1] 79.2 %, Aspergillus ruber CBS 135680 tannase and feruloyl esterase [GenBank accession no. EYE96818.1] 78.6 %), with nucleotide sequence identities ranging from 73.3 to 98.6 % to the same fungal tannases. Compared to the most related tannase (A. kawachii IFO 4308), the A. niger GH1 tannase has three differences, all of which are conservative (T20S, S62A, and N82D). According to the Pfam protein families database, A. niger GH1 tannase has a functional domain from residue 53–545 of the Tannase PF07519 family where other tannases, feruloyl esterases, and several bacterial proteins of unknown function are grouped. This family belongs to the clan AB_hydrolase (CL0028) which its catalytic domain being found in a wide range of enzymes, since this clan contains 67 members. Currently, 757 amino acid sequences distributed in 345 species are reported in the PF07519 family: 375 (49.5 %) sequences from bacteria, 271 (78.6 %) species, and 382 (50.5 %) sequences from eukaryotes, 74 species (21.4 %). Among eukaryotes highlight fungi with 365 sequences (48.2 % of all sequences) distributed in 69 species (20.0 %). In the PF07519 family, 15 architectures of domain organization have been described, having A. niger GH1 tannase the simplest architecture of a single tannase domain. Only one three-dimensional structure has been experimentally determined by X-ray crystallography for the PF07519 family (PDB code: 3WMT), which corresponds to a feruloyl esterase from Aspergillus oryzae [21]. The Phyre2 server constructed a full molecular model of the mature A. niger GH1 tannase; 512 residues (91 %) were modeled with the 3WMT structure because Phyre2 considered the 3WMT protein as a true homologue of A. niger GH1 tannase with 100 % confidence. Also, 50 residues (9 %) were modeled using ab initio techniques. The A. niger GH1 tannase molecular model (Fig. 1a) showed the presence of 16 alpha helices and 16 beta sheets covering 29 and 13 % of the protein, respectively, forming two structural domains. One domain has an a/b-hydrolase fold, constituting the catalytic domain, and one lid domain that covers the catalytic site. From the molecular model and by comparisons with the A. oryzae feruloyl esterase 3WMT, residues Ser-196, Asp-448, and His-494 likely constitute the putative catalytic triad. In addition, these serine and histidine residues are likely directly connected by a disulfide bond between neighboring cysteines, Cys-
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195 and Cys-495 (Fig. 1b), forming the recently described structural motif (CS-D-HC) [21]. Construction of a Tannase P. pastoris Overproducer Strain DNA sequencing of the pUC57 plasmid confirmed the correct nucleotide sequences of the antgs gene. The tannase synthetic gene with the nucleotide sequence encoding the S. cerevisiae alpha-factor prepro-secretion signal had a full length of 1,961 bp. The DNA sequence was engineered to produce the A. niger GH1 tannase as a mature polypeptide of 562 amino acids. PCR analysis of the recombinant plasmid, using AOX1 primers, showed a 2,158 bp product that confirmed the pPIC9antgs construct. Transformation of P. pastoris KM71 with the SalIdigested pPIC9antgs plasmid gave about 15 His? transformants. PCR analysis of the genomic DNAs isolated from P. pastoris KM71ANT (His? transformants) showed a 2,158-bp band that corresponds to the alpha-factor prepro-secretion signal (255 bp) and synthetic tannase (1,689 bp) coding sequences, and fragments of the multiple cloning site from the pPIC9 vector (21 bp), AOX1 promoter (94 bp), and transcription terminator (99 bp). Production and Biochemical Characterization of Recombinant A. niger GH1 Tannase Cell-free culture medium from a 48 h methanol-induced recombinant strain culture showed volumetric tannase activity of 0.57 U/ml, and 46.71 mg/l of total protein. With the ultrafiltration process applied to the cell-free culture medium, a tannase concentrate (25.7 U/ml) was obtained to carry out the biochemical characterization. Figure 2 shows the migration shift results in SDS-PAGE for the recombinant tannase preparation after treatment with endo Hf glycosidase. Recombinant tannase without endo Hf treatment showed two broad bands of apparent molecular masses of 45.0–54.8 and 41.8–45.0 kDa, with a higher intensity at molecular masses of 48.7 and 42.4 kDa, on the SDS-PAGE (Fig. 2, lane 2). After N-deglycosylation by endo Hf, the two bands shifted to apparent molecular masses of 34.6 and 30.3 kDa, respectively, (Fig. 2, lane 3). These results clearly indicate that recombinant tannase is highly N-glycosylated. The analysis with the NetNGlyc 1.0 server showed that the mature A. niger GH1 tannase amino acid sequence has eleven potential N-glycosylation sites (19NGTL, 54NVTV, 130NGSI, 237NATI, 265NLTS, 280NYTS, 304NGSV, 383NVTY, 451NTTY, 508NATV, and 535NSSF). The recombinant tannase had its optimum activity at pH 5.0 (Fig. 3a) and showed more than 80 % of its maximum activity in the pH range between 4.0 and 5.0. Tannase
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Fig. 1 Molecular model (a) of the A. niger GH1 tannase constructed using the Phyre2 server showing the presence of two structural domains. Putative catalytic triad (b), Ser-196, Asp448, and His-494, directly connected by a disulfide bond between neighboring cysteines, Cys-195 and Cys-495. Kex2 recognition site (Lys-309-Arg310) at the protein surface in a flexible loop of the lid domain (c)
Fig. 2 SDS-polyacrylamide gel of recombinant tannase concentrated by ultrafiltration and treated with or without endo Hf glycosidase. Lane M Molecular mass marker. Lane 1 endo Hf. Lanes 2–3 recombinant tannase without and with endo Hf treatment, both samples were treated with 2-mercaptoethanol in the loading buffer
activity showed a profile with a broad temperature range (10–50 °C) with more than 40 % of its maximum activity, and an optimum temperature of 20 °C (Fig. 3b).
Residual activity in the stability assays followed zeroorder kinetics with a reaction rate of 0.01 and 0.48 %/h at 4 and 30 °C, respectively, decreasing to 98.9 and 44.0 % after 120 h (Fig. 3c). The anion-exchange chromatogram for tannase preparation showed two predominant peaks by UV detection at 280 nm. One of the peaks, eluted at 0.35 M NaCl, showed tannase activity (data not shown). The specific activity (20 °C, pH 5.0) at the maximum of this peak was 50.0 U/ mg protein. The recombinant tannase followed typical Michaelis– Menten kinetics at both 20 and 30 °C (R2 = 0.947 and 0.978, respectively). The Michaelis constants (Km) and the maximum rates of reaction (Vmax) were estimated to be 1.98 ± 0.50 mM and 2.01 ± 0.14 lmol/min, and 0.18 ± 0.04 mM and 0.48 ± 0.02 lmol/min at 20 and 30 °C, respectively.
Discussion Recently, the xerophilic A. niger GH1 has been described as a tannase producer in both submerged and solid-state cultures [5, 6, 22]. The biochemical properties of native A.
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Fig. 3 Effects of pH (a) at 30 °C and temperature (b) at pH 5.0 on recombinant tannase activity. Tannase activities are expressed as relative values. Residual activity for the tannase stability assays (c) at
4 °C (filled circle) and 30 °C (filled square). Points represent the means for at least three independent enzyme assays (coefficient of variation \5 %)
niger GH1 tannase have also been described [4] and these properties were compared for A. niger GH1 tannase produced by solid-state and submerged fermentations [9]. In this paper, we report the gene and amino acid sequences for the mature A. niger GH1 tannase. In addition, we used the P. pastoris expression system to produce the enzyme, using a synthetic gene, and characterized the produced recombinant tannase. The A. niger GH1 tannase gene and amino acid sequences appeared to be similar but not identical to the A. kawachii IFO 4308 tannase. Because no three-dimensional structure with a height sequence identity with the A. niger GH1 tannase is available in the Protein Data Bank, constructing a molecular model by homology modeling is not suitable [23]. Nevertheless, fold recognition by protein threading methods can accurately model protein sequences with less than 20 % sequence identity with a known protein. The Phyre2 server uses protein threading methods for protein molecular modeling and was placed it among the best servers in the Critical Assessment of Protein Structure Prediction (CASP) [14]. Therefore, we used the Phyre2 server to construct a molecular model of the A. niger GH1 tannase and found that the model
has a fold that is similar to the structure of the A. oryzae feruloyl esterase 3WMT (two structural domains: a/bhydrolase and a lid domain), which has a 24 % amino acid sequence identity with the tannase from A. niger GH1. In addition, both structures have a catalytic triad forming the structural motif CS-D-HC. To the best of our knowledge, this is the first report of a putative molecular structure from a tannase of the Tannase PF07519 family with a fold very similar to a feruloyl esterase, harboring two structural domains and the structural motif CS-D-HC. Nevertheless, the substrate specificity of the two enzymes is different, likely because the substrate-binding pockets are formed by different residues in the two enzymes: Phe-225, Gln-228, Gln-229, Glu-365, Ser-279, Gly-381, and Ile-451 for A. niger GH1 tannase, and Phe-232, Leu-235, Thr-236, Tyr-348, Phe-354, Tyr-356, and Ile-419 for A. oryzae feruloyl esterase [21]. We engineered P. pastoris strains to produce the A. niger GH1 tannase as an extracellular mature polypeptide of 562 amino acids, since we had removed the putative native signal sequence (19 residues) predicted by SignalP 4.1 [24] for the A. niger CBS513.88 tannase from the recombinant construction. In addition, the mature
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polypeptide was fused in frame with the S. cerevisiae alpha-factor prepro-secretion signal, which is known to be a good secretion signal for P. pastoris [10]. The production of seven tannases in microorganisms have been reported, including Lactobacillus plantarum, Klebsiella pneumoniae, Bacillus licheniformis, Aspergillus awamori, A. oryzae, and A. niger [25–31]. Among these tannases, six were native tannases and only one from A. oryzae was produced as a recombinant [30, 31]. Among the native tannases, five have been produced under conventional submerged culture and only the A. niger GH1 tannase was extracellularly produced in solid-state culture being the highest production level (2,291 U/l in 20 h) compared to the other native tannases. However, a decline in tannase activity was observed after 20 h of incubation associated with a concomitant increase in protease activity [6]. Aspergillus oryzae tannase has already been produced with the P. pastoris expression system, both extracellularly [31] and intracellularly [30], resulting 7 U/ml after 96 h of induction for a fed-batch culture in a 3-L bioreactor in the first case, and 0.96 U/ml after 72 h of induction for a shake-flask culture in the second case, both with a Muts strain. However, in any case synthetic genes were used based on P. pastoris-preferred codons and optimized AT content for the tannase gene and/or the sequence encoding the S. cerevisiae alpha-factor prepro-secretion signal, neither the biochemical properties of the recombinant tannases were not described, except for the formation of the doublestranded structure. Because high cell densities of P. pastoris can be achieved in a simple bioreactor in a controlled form, a 10- to 100-fold increase in production of the recombinant A. niger GH1 tannase produced in sake-flask can be expected, as described for other proteins produced in P. pastoris [32]. The NetNGlyC 1.0 server predicted eleven potential N-glycosylation sites for A. niger GH1 tannase. Among them, three potential N-glycosylation sites (130NGSI, 265NLTS, and 451NTTY) are more likely to be N-glycosylated since the molecular model of A. niger GH1 tannase showed that these sites are at the protein surface. In any case, experimental data is needed to verify whether or not a recombinant protein is N-glycosylated by the host, since the presence of N-glycosylation sites is not sufficient to conclude that an asparagine residue would be N-glycosylated. Our results from endo Hf treatment clearly demonstrate that recombinant A. niger GH1 tannase was N-glycosylated, since a band was not observed on the SDSPAGE corresponding to the theoretical molecular mass of mature A. niger GH1 tannase (61.4 kDa), judging from the amino acid sequence deduced from the tannase gene. Nevertheless, two broad bands were mainly observed (Fig. 2, lane 2) at lower molecular masses (48.7, and
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42.4 kDa) than the expected theoretical molecular mass for mature A. niger GH1 tannase, that shifted to two defined bands at molecular masses of 34.6 and 30.3 kDa (Fig. 2, lane 3) after N-deglycosylation by endo Hf glycosidase. The N-glycosylation pattern of recombinant A. niger GH1 tannase should be similar to that described for other recombinant proteins produced in P. pastoris [33–35]. These findings also indicate that the recombinant tannase preparation had a high degree of purity. Furthermore, the recombinant tannase consisted of two subunits, likely linked by a disulfide bond, generated by a Kex2 protease cleavage of the tannase gene product, since the mature A. niger GH1 tannase sequence has two pairs of basic amino acids (Lys-309-Arg-310, and Lys-347-Arg-348) that are a typical Kex2 recognition site. This type of structure has already been described for a native A. oryzae tannase [36], and a recombinant A. oryzae tannase produced either extracellularly [31] or intracellularly [30] in P. pastoris. Of the two Kex2 recognition sites, the Lys-309-Arg-310 site is more likely to be recognized by the Kex2 protease, since the molecular model showed that this site is at the protein surface in a flexible loop of the lid domain (Fig. 1c). The Lys-347-Arg-348 site, in contrast, is located at an alpha helix of the down side of the lid domain and is not so exposed to the protein surface than the Lys-309-Arg-310 site. A cleavage by Kex2 beside Arg-310 would render two peptides of 31.2 and 30.2 kDa. The disulfide bond between Cys-195 and Cys-495 would link both peptides. All of the biochemical properties for the recombinant A. niger GH1 tannase were determined using the enzyme concentrate prepared by ultrafiltration from cell-free culture medium. A buffer exchange was performed during the ultrafiltration process, to remove low molecular weight peptides and other compounds that might affect the biochemical properties. In addition, the SDS-PAGE analysis of the tannase concentrate prepared by ultrafiltration showed that the preparation had a high degree of protein purity (Fig. 2). All of the biochemical properties described in this paper are relative values, to the maximum value or to the initial value (Fig. 3). Therefore, if some residual impurities could affect the biochemical properties of the tannase concentrate preparation, they would not affect the relative values of the biochemical properties. Native and recombinant A. niger GH1 tannase have the same amino acid sequence. Therefore, any differences in their biochemical properties (pH and temperature profiles, and stability) must be due to their differing degrees of N-glycosylation and/or their different structure since native A. niger GH1 tannase is mainly a single glycopolypeptide strand of 102–105 kDa [9]. N-glycosylation affects biochemical properties such as molecular mass and pH optimum [37]. While native A. niger GH1 tannase displayed optimal pH of 6.0–7.0 and optimal temperature of 60 °C [9,
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38], recombinant A. niger GH1 tannase showed optimal pH of 5.0 and optimal temperature of 20 °C. The produced recombinant tannase is the first tannase described with an optimum temperature of 20 °C. In addition, recombinant A. niger GH1 tannase had a higher relative activity compared to native A. niger GH1 tannase over a range of 10–40 °C, which would be advantageous for many food and beverage applications [39]. Native and recombinant tannases showed high residual activity at 4 °C for at least 120 h. Notably, biochemical properties of a double-stranded Aspergillus tannase were not described until very recently [40]. Nevertheless, in that publication a very similar biochemical properties for native and recombinant tannase were described, likely because both had a double-stranded structure, this is not our case. Based on its pH and temperature profiles, the tannase produced in this study could be used in food and beverage processing at low temperatures. Acknowledgments We thank Fabiola Veana for her technical support and Luis V. Dura´n-Rodrı´guez for his technical suggestions. We also thank Glen D. Wheeler for his stylistic suggestions in the preparation of the manuscript. J.A.F-G. thanks CONACYT for fellowship.
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