A new carboxypeptidase from Aspergillus niger with good thermostability, pH stability and broad substrate specificity

Cloning and sequence analysis of the carboxypeptidase gene

The A. niger CBS 513.88 genome (EMBL AM270980-AM270998) has been sequenced¹⁷, and a novel suspected carboxypeptidase (CapA) gene has been identified by analyzing its genome information and BLAST. The cDNA of A. niger F0510 was extracted as a template, and the gene was successfully amplified using CapA-F and CapA-R as primers. The recombinant plasmid pPIC-CapA was constructed. Sequencing confirmed that CapA had an intact open reading frame (ORF), and the nucleotide sequence was identical to the published sequence (Locus tag: ANI_1_238044) of the A. niger CBS 513.88 genome. The serine carboxypeptidase gene contains 1479 bp and encodes 492 amino acids, and analysis of the predicted protein sequence suggested that CapA has a signal peptide of 19 amino acids in the N terminus.

Clustal X2 was used to analyze and compare the amino acid sequences of serine carboxypeptidases from different sources, which are listed and summarized in Fig. 1. There are four conserved domains involved in the substrate binding and catalysis of CapA, among which domain 1 is the conserved substrate binding domain, and domains 2–4 are the conserved catalytic domains, which include the conserved triplet of serine (S), aspartic acid (D) and histidine (H)^3,10. A conserved G-X-S-X sequence (located in domain 1) spatially includes the active serine residues²². There is a conserved glutamate (Glu) in front of the catalytic serine residue (located in domain 2), which is thought to be the reason serine carboxypeptidase performs the best catalysis under acidic conditions²². Similar to other carboxypeptidases, CapA belongs to the S10 family of SC carboxypeptidases²³.

CapA was further compared with the amino acid sequences reported for serine carboxypeptidases using MEGA 5.0, and a phylogenetic tree was constructed, as shown in Fig. 2. The phylogenetic tree reflects the genetic distance between 10 different serine carboxypeptidases, and a short genetic distance and clustering indicate a close genetic relationship. The amino acid sequence similarity between CapA and other serine carboxypeptidases ranges from 15.76% (SpCap) to 93.09% (AlCap), with an average of 30.45%.

Expression and induction of carboxypeptidase CapA

The recombinant plasmid pPIC-CapA was linearized with the restriction enzyme Sac I and electrically transformed into P. pastoris GS115. His⁺ transformants growing on YPD agar plates with G418 at two concentrations (0.5 and 2.0 mg mL⁻¹) after previously growing on MD plates at 30 °C for 3 d were selected. A single colony from the YPD plate with 2.0 mg mL⁻¹ G418 was inoculated into BMGY to an absorbance value of 4.0–6.0 at 600 nm and transferred into BMMY for expression and induction of the recombinant protein. The maximum enzyme activity of the crude enzyme reached 209.3 U mg⁻¹ after 120 h of culture with methanol in a shaking flask. After successive purification of the crude CapA enzyme solution by salting out, dialysis and ion exchange chromatography, the specific activity of purified CapA reached 495.7 U mg⁻¹ (Table 1). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 3) analysis showed that the molecular weight of CapA was approximately 60.0 kDa, which was slightly higher than the theoretical molecular weight of 52.7 kDa; this difference was speculated to be caused by N-glycosylation. N-Glycosylation is one of the most common modifications occurring in the synthesis of proteins in P. pastoris³⁰. This speculation was supported by the prediction of N-glycosylation sites by the NetNGlyc 1.0 Server: Asn29, Asn97, Asn233 and Asn337 were potential N-glycosylation sites in the mature peptide of CapA based on the locations in consensus NxT/S sequences³¹ (Fig. 4).

Enzymatic properties of carboxypeptidase

Optimal temperature and temperature stability

Carboxypeptidase activity was detected in the range of 30–70 °C, and the results are shown in Fig. 5a. The optimal reaction temperature of CapA was 45 °C, and the relative activity could be maintained at more than 70% at temperatures of 30–55 °C. The thermal stability study (Fig. 5b) showed that after incubation at 30–50 °C for 1 h, the enzyme activity of CapA remained greater than 80%, and after incubation at 60 °C and 70 °C for 2 h, the enzyme activity remained greater than 30% and 10%, respectively.

The optimal reaction temperature and thermal stability of CapA were significantly higher than those of A. oryzae carboxypeptidase³, which had an optimal reaction temperature of 30 °C and less than 10% enzyme activity after 30 min incubation at 60 °C. The optimal values of CapA were also higher than the optimal temperature (30 °C) and thermal stability of S. cerevisiae-derived recombinant carboxypeptidase Y (after 60 °C incubation for 1 h, the enzyme activity was almost undetectable)²⁵. CapA has better heat resistance than the other carboxypeptidases, resulting in the advantages of a simplified process, improved the efficiency and reduced the cost in application³².

Optimal pH and pH stability

Carboxypeptidase activity was detected in the pH range of 4.0–8.0, and the results are shown in Fig. 6a. The optimal reaction pH of CapA was 6.0, and the relative activity could be maintained at more than 60% in the range of pH 5.0–6.5. When the pH was less than 5.0 or greater than 6.0, enzyme activity decreased rapidly. Studies on the pH stability showed (Fig. 6b) that CapA was relatively stable at pH 4.0–8.0, and the enzyme activity remained above 60% after 1 h of incubation. CapA was similar to the carboxypeptidases from other sources, which all exhibited the best hydrolysis in acidic conditions. However, unlike the optimal reaction pH of the carboxypeptidases from most filamentous fungi^3,34 and yeasts³³, which is near 4.0, the optimal reaction pH and stable pH range of CapA are more neutral. Similar to the reported carboxypeptidase Y from S. cerevisiae²⁵, CapA plays a role in reaction systems that proceed at a neutral pH, which is more convenient for subsequent product processing after enzyme catalysis has occurred.

Influence of metal ions and chemical reagents on enzyme activity

The effects of metal ions or chemical reagents on CapA are shown in Table 2. Mg²⁺ significantly increased the CapA activity, Cu²⁺, Fe²⁺ and CO²⁺ significantly inhibited the CapA activity, and Ca²⁺, Zn²⁺ and low concentrations (1–5 mmol L⁻¹) of Mn²⁺ had little effect on the CapA activity, while 10 mmol L⁻¹ Mn²⁺ markedly decreased the activity. CapA is a serine protease, and its active center does not need the assistance of metal ions³⁴, which is also demonstrated by the fact that the enzyme activity is not affected after ion chelation by EDTA. Therefore, metal ions should form coordination bonds with some of the key amino acids of the enzyme and change its conformation, thus affecting the activity of the enzyme³⁵.

The nature and specificity of CapA

PMSF can inhibit the activity of recombinant enzymes by more than 80%. As a specific inhibitor of serine proteases, PMSF inhibits serine carboxypeptidase (OcpC) and Monascus carboxypeptidase³. This result clearly demonstrated that CapA is a carboxypeptidase.

CBZ-Pro-Gly, CBZ-Ala-Lys, CBZ-Gly-Ala, CBZ-Ala-Glu and CBZ-Phe-Leu were used as substrates to determine the enzyme kinetic parameters of CapA, and the results are summarized in Table 3. Among the substrates used, the highest affinity (K_m 0.063 mM) and the highest catalytic efficiency (k_cat/K_m 186.35 mM⁻¹ s⁻¹) were both obtained for CBZ-Phe-Leu. CapA has higher specific activity or higher catalytic efficiency than other reported carboxypeptidases^13,36,37. Therefore, CapA will be more adaptable and less expensive in industrial applications.

Using 6 CBZ-AA-AA as substrates, the hydrolysis specificity of CapA was measured and is summarized in Fig. 7. CBZ-Phe-Leu is the optimal substrate for CapA. The ability of CapA to hydrolyze the 6 substrates is as follows: CBZ-Phe-Leu > CBZ-Gly-Ala > CBZ-Ala-Lys > CBZ-Pro-Gly > CBZ-Ala-Glu > CBZ-Ala-Arg. CapA has a wide range of substrate specificities and prefers the carboxy-terminal hydrophobic amino acids Leu and Lys, which cause oligopeptides to be bitter; thus, it has good potential for application in protein C-terminal sequencing and debittering oligopeptides^8,12. The bitterness of certain peptides is always caused by hydrophobic amino acids. Two carboxypeptidases have been previously used in enzymatic debittering because of their preferred hydrolysis of carboxy-terminal hydrophobic amino acids: a carboxypeptidase from B. subtilis completely removed the bitterness of fermented soybean meal and improved the flavor of soybean meal at the same time³⁸, and a carboxypeptidase from Pseudozyma hubeiensis eliminates the bitterness of bitter peptides generated by proteases³⁹. CapA has a similar substrate preference to the abovementioned carboxypeptidases and the advantage of higher specific activity^13,36,37. Therefore, CapA has the potential to debitter peptides.

There are also reports of using serine carboxypeptidases to accelerate cheese ripening (patent US 20140205717)⁴⁰ and improve the taste of cheese⁴¹. Recently, a carboxypeptidase extracted from A. oryzae was utilized in industry for debittering and improving the flavor of coffee, thereby adding value⁴². The application of CapA in these fields also deserves further investigation.