Enzyme and Microbial Technology 63 (2014) 28–33

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Gene encoding a novel invertase from a xerophilic Aspergillus niger strain and production of the enzyme in Pichia pastoris Fabiola Veana a,b , José Antonio Fuentes-Garibay a , Cristóbal Noé Aguilar b , Raúl Rodríguez-Herrera b , Martha Guerrero-Olazarán a , José María Viader-Salvadó a,∗ a Universidad Autónoma de Nuevo León, UANL, Facultad de Ciencias Biológicas, Instituto de Biotecnología, 66450 San Nicolás de los Garza, Nuevo León, Mexico b DIA-UAdeC/School of Chemistry, Universidad Autónoma de Coahuila, 25280 Saltillo, Coahuila, Mexico

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Article history: Received 24 January 2014 Received in revised form 29 April 2014 Accepted 4 May 2014 Available online 10 May 2014 Keywords: Recombinant invertase ␤-Fructofuranosidases Aspergillus niger GH1 strain Pichia pastoris Synthetic gene

a b s t r a c t ␤-Fructofuranosidases or invertases (EC 3.2.1.26) are enzymes that are widely used in the food industry, where fructose is preferred over sucrose, because it is sweeter and does not crystallize easily. Since Aspergillus niger GH1, an xerophilic fungus from the Mexican semi-desert, has been reported to be an invertase producer, and because of the need for new enzymes with biotechnological applications, in this work, we describe the gene and amino acid sequence of the invertase from A. niger GH1, and the use of a synthetic gene to produce the enzyme in the methylotrophic yeast Pichia pastoris. In addition, the produced invertase was characterized biochemically. The sequence of the invertase gene had a length of 1770 bp without introns, encodes a protein of 589 amino acids, and presented an identity of 93% and 97% with invertases from Aspergillus kawachi IFO 4308 and A. niger B60, respectively. A 4.2 L culture with the constructed recombinant P. pastoris strain showed an extracellular and periplasmic invertase production at 72 h induction of 498 and 3776 invertase units (U), respectively, which corresponds to 1018 U/L of culture medium. The invertase produced had an optimum pH of 5.0, optimum temperature of 60 ◦ C, and specific activity of 3389 U/mg protein, and after storage for 96 h at 4 ◦ C showed 93.7% of its activity. This invertase could be suitable for producing inverted sugar used in the food industry. © 2014 Elsevier Inc. All rights reserved.

1. Introduction ␤-Fructofuranosidases or invertases (EC 3.2.1.26) are enzymes that catalyze the hydrolysis of terminal non-reducing ␤-dfructofuranoside residues in ␤-d-fructofuranosides, including the hydrolysis of sucrose into glucose and fructose. Invertases are used for the inversion of sucrose in the preparation of invert sugar and high fructose syrup [1] and as biosensors for sucrose determination in fruit juices [2]. These enzymes are among the most widely used in the food industry, where fructose is preferred over sucrose, especially in the preparation of jams and candies, because it is sweeter and does not crystallize easily [3]. In addition to its higher

∗ Corresponding author at: Universidad Autónoma de Nuevo León, UANL, Facultad de Ciencias Biológicas, Instituto de Biotecnología, Av. Universidad S/N, Col. Ciudad Universitaria, 66451 San Nicolás de los Garza, Nuevo León, Mexico. Tel.: +52 81 8329 4000x6439. E-mail addresses: [email protected], [email protected] (J.M. Viader-Salvadó). http://dx.doi.org/10.1016/j.enzmictec.2014.05.001 0141-0229/© 2014 Elsevier Inc. All rights reserved.

sweetening capacity, fructose is beneficial for diabetics and can potentiate iron absorption in children [4]. Invertases also catalyze the synthesis of fructooligosaccharides (FOS), as 1-kestose, nystose, and fructosyl nystose from sucrose, through a fructotransferase reaction, which occurs mainly at high concentrations of sucrose (>2%, w/v) [5–8]. FOS are promising ingredients for functional foods since they act as prebiotics, and exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, and osteoporosis [9]. Recently, Aspergillus niger GH1, a xerophilic fungus, has been reported to be an invertase producer [10,11]. This microorganism was isolated from the Mexican semi-desert and tolerates extreme conditions typical of this region (45 to −15 ◦ C) [12]. Other enzymes involved in tannin and ellagitannin degradation from this microorganism have also been studied [13,14]. The methylotrophic yeast Pichia pastoris has been developed as a host for the efficient production and secretion of foreign proteins [15]. Usually, a coding sequence under the control of the alcohol oxidase 1 promoter is highly expressed when methanol is used as the carbon source [16]. P. pastoris can grow in simple defined media,

F. Veana et al. / Enzyme and Microbial Technology 63 (2014) 28–33

reach a very high cell density, and produce high concentrations of intra- or extracellular protein [16]. In this work, we describe the gene and amino acid sequence of the invertase from A. niger GH1, and the use of a synthetic gene to produce the enzyme in the methylotrophic yeast P. pastoris. In addition, the produced invertase was characterized biochemically. 2. Materials and methods 2.1. Strains, plasmids, medium composition, chemicals, and enzymes P. pastoris KM71 (his4), plasmid pPIC9, and PTM1 trace salts were purchased from Invitrogen (San Diego, CA). Escherichia coli JM109, Taq 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). XhoI and NotI restriction endonucleases and PNGase F 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), buffered minimal methanol (BMM supplemented with 0.75% (vol/vol) methanol), buffered minimal glycerol yeast extract (BMGY), and fermentation basal salts (FBS) media were prepared as described elsewhere [17,18]. All chemicals were analytical grade and purchased from Sigma–Aldrich Co. (St. Louis, MO) or from Productos Químicos Monterrey (Monterrey, Nuevo Leon, Mexico). 2.2. Invertase gene sequence from A. niger GH1 Genomic DNA of A. niger GH1, extracted by standard protocols, was the source of the invertase gene. The complete sequence encoding the enzyme was synthesized by PCR using a forward primer Inv1 (5 -ATGAAGCTTCAAACGGCTTCCGTA-3 ) and reverse primer Inv2 (5 -TCACCGAACCCAAGTACTCAACG-3 ). The PCR was performed with Taq DNA polymerase and a 35-cycle amplification program under the following conditions: 95 ◦ C for 1 min, 54 ◦ C for 1 min, and 72 ◦ C for 1.5 min, with a first denaturation step at 95 ◦ C for 5 min and a final extension step at 72 ◦ C for 5 min. The amplified product was cloned into the vector pGEM-T according to the manufacturer’s instructions. Nucleotide sequences from seven plasmids from different E. coli JM109 colonies were determined at the Instituto de Fisiología Celular (UNAM), using T7 and SP6 universal primers. Two internal oligonucleotides were designed (FV1: 5 -GGGAGTACCTCGGCCAAT-3 , and FV2: 5 -TAAGAGGAGAAGCCCCAATC-3 ) and further internal nucleotide sequences were determined for four plasmids. The 22 nucleotide sequences were aligned using the Contig Assembly Program (CAP) module of the BioEdit v7.0.8.0 program [19]. The consensus sequence and deduced amino acid sequence were compared with sequences of databases using BLAST tools [20]. The putative signal peptide sequence was predicted with SignalP 3.0 [21] and Signal3L [22] servers. Putative functional domains were determined by comparing the protein sequence with the Pfam database [23]. Potential N-linked glycosylation sites were predicted with the NetNGlyc 1.0 server [24]. The A. niger GH1 invertase molecular model was constructed by homology modeling using SWISS-MODEL 8.05 [25].

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determined by the Bradford method and UV absorption at 280 nm in a NanoPhotometer Pearl (Implen GmbH, München, Germany) using the theoretical molar absorption coefficient (112,300 M−1 cm−1 ) and molecular mass (62.3 kDa) for mature A. niger GH1 invertase, calculated with the ProtParam tool [30], which is available from the ExPASy server. The final cell concentrations were estimated based on 1 OD600 unit, approximately corresponding to 107 cells/mL [31].

2.4. Production of recombinant invertase Invertase production from the selected His+ transformant strain was carried out in a 7 L Applikon bioreactor interfaced with the ez-Control and BioXpert Lite program (Applikon Biotechnology B.V., Delft, Netherlands). The inoculum was prepared in 2 L BMGY medium, as described elsewhere [32,33]. The culture was performed in three steps (glycerol batch, glycerol-fed batch, and methanol-fed batch) in FBS medium and in a similar manner to that described previously, with minor modifications [34]. In the methanol-fed batch step, a secondorder polynomial feeding strategy with 100% methanol (12 mL/L 0.02% biotin and PTM1) was used, beginning at 0.11 mL/min and reaching 0.56 mL/min at 72 h induction. The pH of the culture medium was kept constant at pH 5 with 28% NH4 OH, the temperature was maintained at 25 ◦ C, the air flow rate was set to 5 L/min, and the dissolved oxygen (DO) was maintained above 20% saturation increasing the agitation up to 1200 rpm and using an automatic supplementation of oxygen–air mixtures as needed. At each sampling point, the cell growth in cell density (g/L dry cell weight, DCW), total DCW, and specific growth rate () for the methanol-fed batch step were determined as described previously [34]. The total volume of methanol added in the induction step was considered as the methanol consumption volume. The specific substrate uptake rates (qs ) were determined from the first derivative of the polynomial smoothing time profile of the ratio between the methanol consumption (in mmol) and total DCW (in g). Total extracellular invertase activities (in U) were calculated from the volumetric invertase activity (in U/L) of the cell-free culture medium times the cell-free culture medium volume at each induction time, calculated with the cell volume per dry cell (3.6 × 10−3 L/g dry cells), as described elsewhere [32]. Total periplasmic invertase activities (in U) were calculated from the invertase activity per g of cell-wall-lysed dry cells times total DCW. The periplasmic proteins were extracted from 12.5 mg dry cells, which had been previously washed in three steps (20 mL water, 20 mL freshly prepared SED [1 M sorbitol, 25 mM EDTA, 50 mM DTT, pH 8.0], and 20 mL 1 M sorbitol), suspended in 5 mL SCE (1 M sorbitol, 1 mM EDTA, 10 mM sodium citrate, pH 5.8), and cell wall lysed with 2.4 ␮L litycase (5 U/␮L). Invertase activity was determined directly from the cell-wall lysis mixture. Total invertase production was estimated by adding the total extracellular and periplasmic invertase activity. Except for DCW, all of the analytical determinations were carried out at least three times (coefficient of variation less than 5%). The cell-free culture medium was clarified, concentrated 22-fold, and diafiltrated at 4 ◦ C by micro and ultrafiltration, using a QuixStand Benchtop System (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), two different hollow fiber cartridges (0.2 ␮m and 10,000 nominal molecular weight cutoff), and 100 mM sodium acetate (pH 5). Aliquots of the enzyme concentrate were stored at −20 ◦ C until used for the biochemical characterization of the invertase.

2.3. Construction of P. pastoris recombinant strains 2.5. Biochemical characterization of recombinant A. niger GH1 invertase A synthetic gene encoding the A. niger GH1 invertase (invGS) was designed based on P. pastoris-preferred codons [26]. In addition, AT-rich stretches of more than six nucleotides were removed introducing silent mutations. Twelve nucleotides from the 3 terminus of the alpha-factor prepro-secretion signal sequence from Saccharomyces cerevisiae, including the XhoI site, and a NotI site were introduced at the 5 and 3 ends, respectively. The designed nucleotide sequence, with a full length of 1746 bp, was synthesized, cloned into vector pUC57, and sequenced by GenScript Corp. (Piscataway, NJ), to generate the plasmid pUC57invGS. The DNA fragment, harboring the invGS sequence, was obtained by digestion of pUC57invGS with the XhoI and NotI restriction enzymes, and then ligated to vector pPIC9 that had previously been digested with the same restriction enzymes, to produce the new expression vector (pPIC9invGS). This vector harbors the invGS sequence in frame with the S. cerevisiae alpha-factor prepro-secretion signal and between the promoter and transcriptional terminator of the AOX1 gene. The correct construction of the vector was confirmed by XhoI–NotI double-digested restriction analysis and by PCR using 5 and 3 AOX1 primers, directed to the AOX1 promoter and the transcription terminator, as previously described [27]. All DNA manipulations were performed according to standard methods [28]. The P. pastoris host strain, KM71 (his4), was transformed with SalI-digested pPIC9invGS DNA by electroporation, as described elsewhere [29]. The transformants were selected for the ability to grow on histidine-deficient medium (RDB-agar plates) at 30 ◦ C until colonies appeared (His+ selection). His+ colonies from each transformation were randomly selected, and the integration of the expression cassette into the genomes of the selected strains was verified by PCR using the 5 and 3 AOX1 primers, as previously described [27]. P. pastoris recombinant strains from twenty-four His+ colonies were tested in BMG and BMM media to select an overproducer strain for invertase. Cell-free culture medium was recovered by centrifugation, and the protein concentration was

The biochemical characterization of invertase was performed with the diafiltrated enzyme concentrate by ultrafiltration from the cell-free culture medium. N-glycosylation was evaluated by assessing the migration shift of PNGase F-treated proteins in a Coomassie blue-stained 12% SDS-polyacrylamide gel. Reactions were carried out by incubating the invertase concentrate with PNGase F for 1 h at 37 ◦ C, according to the manufacturer’s instructions. The effect of pH on enzymatic activity was determined at 60 ◦ C, using 250 mM glycine–HCl (pH 2.5), 50 mM sodium citrate (pH 4.0), 100 mM sodium acetate (pH 5.0), 100 mM potassium phosphate (pH 7.0), and 100 mM Tris–HCl (pH 9.0) as buffers. The effect of temperature on enzymatic activity was determined at pH 5 by measuring the invertase activity at various temperatures ranging from 30 to 80 ◦ C. All results were compared between statistical groups, using analysis of variance (ANOVA) and Tukey’s multiple comparisons, with a significance cutoff P value of