Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger

Abstract

The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.

FIG. 1.

Alignment of the amino acid sequences of the CGA hydrolases from A. niger CIRM BRFM 131 (ChlE) and CBS 513.88 (An07g04470) with the CLUSTAL W sequence alignment algorithm (43). Perfect matches are represented by asterisks, high amino acid similarities are represented by double dots, and low amino acid similarities are represented by single dots. A six-histidine tag was added to the sequence of recombinant ChlE to facilitate purification of the protein.

FIG. 2.

Southern hybridization analysis of A. niger DNA. Approximately 10 μg of genomic DNA from A. niger CIRM BRFM131 was restricted and separated by electrophoresis on a 0.6% agarose gel. Following transfer to a nylon membrane, the blot was hybridized with a digoxigenin-labeled probe consisting of the chlE gene. Genomic DNA was digested with EcoRV (lane 1), PstI (lane 2), EcoRI (lane 3), NotI (lane 4), XbaI (lane 5), XhoI (lane 6), and BamHI (lane 7).

FIG. 3.

Production, purification, and characterization of CGA hydrolase from A. niger D15-ChlE16. (A1) Extracellular production of the protein in strain D15-ChlE16 over time. CGA activity (▴) and mycelial dry weight (•) are shown. (A2) SDS-PAGE analysis of the culture medium (lane 1), the purified protein (lane 2), and the deglycosylated protein (lane 3) with approximately 20 μg of loaded proteins. Proteins were stained with Coomassie blue. (A3) Purification of CGA hydrolase. Effects of temperature (B1) and pH (B2) on the activity of the purified protein. Various temperatures (37°C to 75°C) and pH values (3.85 to 8.55) were tested under standard conditions. The pH tests used a 100 mM citrate-phosphate buffer for activity assay. Effects of temperature (B3) and pH (B4) on the stability of the purified protein. The selected temperatures were 37°C (⧫), 45°C (▪), 50°C (▴), 60°C (+), and 65°C (•), and the selected pH values were 3.80 (⧫), 4.75 (+), 6.85 (▪), 7.85 (•), and 8.50 (▴). All assays were performed with CGA as the substrate. IMAC, immobilized metal affinity chromatography.

FIG. 4.

Model substrates tested for esterase activity. The backbone of cinnamic acid is replaced with R1, R2, and R3. FA is O-[5-O-(trans-feruloyl)-α-l-Araf], and FAX is O-[5-O-(trans-feruloyl)-α-l-Araf]-(1→3)d-Xylp.

FIG. 5.

Sequence alignment of ChlE with the fungal esterase EstA and mouse esterase AChE. Asterisks denote residues of the catalytic triad, solid circles indicate residues of the acyl pocket, and solid triangles indicate the potentially glycosylated amino acids. The secondary structural elements of EstA (PDB accession no. 1UKC) are indicated above the sequences.

FIG. 6.

Overall view of the predicted structure of ChlE. The image was generated with PyMOL (DeLano Scientific, San Carlos, CA). The ribbon diagram shows the 11 central stranded β sheets in blue and the surrounding α helix in brown. The hypothetical Asn-linked glycan moieties are displayed in purple. The catalytic triad residues are displayed in red at the center of the ChlE molecule. term, terminus.