Characterization of a novel metallocarboxypeptidase from Streptomyces cinnamoneus TH-2
© Wan et al. 2016
Received: 11 December 2015
Accepted: 6 April 2016
Published: 18 April 2016
Carboxypeptidases are exopeptidases that catalyze the release of amino acids from the C-terminus of peptides or proteins. The peptides consisting of hydrophobic amino acids taste bitter. Therefore, the hydrolytic capability of carboxypeptidase toward hydrophobic amino acids at the C-terminus of peptides is useful for the degradation of bitter peptides.
Using the genome data of Streptomyces cinnamoneus TH-2, we expressed and characterized a novel metallocarboxypeptidase (TH2-CP) in Streptomyces lividans. TH2-CP had a molecular mass of 37.7 kDa. As TH2-CP possesses a zinc-binding consensus motif (HXXE……H) and N-terminal prosegment residues, we suggest that TH2-CP could be classified into the M14A subfamily. In the presence of Z-Gly-Leu as the substrate, TH2-CP showed optimum activity at pHs 7 and 8 in potassium phosphate and Tris–HCl buffers, respectively. The optimum temperature for activity was 51 °C. Furthermore, 50 % activity was conserved after incubation at 38 °C for 30 min. TH2-CP showed broad substrate specificity, with a preference for hydrophobic amino acids, as demonstrated by casein hydrolysate breakdown.
A novel metallocarboxypeptidase, TH2-CP, from S. cinnamoneus TH-2 was characterized. TH2-CP preferred substrates with hydrophobic amino acids at the C-terminal position for casein peptides. This property indicates that TH2-CP can be used to decrease the bitterness of peptides in food industries.
Streptomycetes produce a wide variety of enzymes, such as peptidases, lipases, and glucohydrolases. Such enzymes have industrial applications. To date, we have discovered several enzymes from Streptomyces cinnamoneus TH-2 (formerly named S. septatus) by screening enzymatic activity. This strain produces extracellular enzymes including phospholipase D, aminopeptidases, and metalloendopeptidases (Arima et al. 2004; Hatanaka et al. 2002, 2005). For these reasons, this strain has the potential to be a resource of useful enzymes.
Carboxypeptidases are exopeptidases that hydrolyze peptide bonds from the C-terminus of peptides or proteins. They function in a variety of physiological processes such as fibrinolysis, inflammation, blood coagulation, and prohormone and neuropeptide processing (Arolas et al. 2007; Vendrell et al. 2000). They can also be used to decrease the bitterness of peptides in food industries (Fu et al. 2011; Ge and Zhang 1996; Komai et al. 2007; Umetsu et al. 1983). According to the presence of particular active site residues, carboxypeptidases are classified as metallocarboxypeptidases, serine carboxypeptidases, or cysteine carboxypeptidases. Among them, metallocarboxypeptidases possess a conserved active site motif, (HXXE……H) or (HEXXH), which requires their binding to divalent metal ions for activity (Lee et al. 1996). To date, there are few reports concerning the characterization of metallocarboxypeptidase from the genus Streptomyces. Using genome data from S. cinnamoneus TH-2, we succeed in expressing and characterizing a novel metallocarboxypeptidase, TH2-CP. TH2-CP shows broad substrate specificity, with a preference for hydrophobic amino acids.
A protein assay kit and gels for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad Laboratories Inc. (USA). All peptides were purchased from Bachem AG (Bubendorf, Switzerland). PD-10 desalting columns were purchased from GE Healthcare (USA). Snake venom l-amino acid oxidase (LAAO) was purchased from Sigma Chemical (USA) and used without further purification. Horseradish peroxidase was purchased from Wako Chemicals (Osaka, Japan). Endoprotease from a Bacillus sp. (Bioprase SP) was a gift from Nagase ChemteX (Fukuchiyama, Japan). EZ:faast™ kits (Phenomenex, USA) for amino acid analysis of protein hydrolysates were purchased from SHIMADZU GLC Ltd. (Tokyo, Japan). All other chemicals were of the highest purity available.
Analysis of genome sequence of S. cinnamoneus TH-2
Genomic DNA was prepared from the strain TH-2 by the method of Hopwood et al. (1985). Sequencing of the genomic DNA was performed using a Hiseq next-generation sequencer (illumina, USA) and an Ion PGM™ next-generation sequencer (Thermo Fisher Scientific, USA). Sequence reads were assembled into a draft genome sequence (ca. 6.3 Mbp) using CLC Genomics Workbench (CLC Bio, Aarhus, Denmark). The draft sequence was sent to the Microbial Genome Annotation Pipeline (MiGAP, Database Center for Life Science, Japan) for annotation.
Cloning, expression and preparation of TH2-CP for enzymatic assays
TH2-CP was amplified by PCR using the sense primer 5′-CATATGTCCCGAAAACGCCTGGCAGGGC-3′ (the underlined region represents the NdeI site and includes the initiation codon) and the antisense primer 5′-AAGCTTCAGTCGGCCGACCG-3′ (the underlined region represents the HindIII site and is adjacent to the stop codon). PCR was performed using the Tks Gflex DNA Polymerase system (Takara, Shiga, Japan). The PCR product (1.4 kb) was cloned into the pCR-Blunt II-TOPO Vector system (Invitrogen, USA) and correct cloning was confirmed by sequencing. The inserted fragment was subcloned into the NdeI–HindIII gap of pTONA5a (Hatanaka et al. 2008). The constructed pTONA5a-TH2-CP was then transformed in S. lividans 1326 by conjugation, as described by Hatanaka et al. (2008). The resultant S. lividans 1326 transformants were incubated and grown in 50-ml medium (8 g of KH2PO4/l, 20 g of glucose/l, 0.5 g of MgSO4·7H2O/l, 5 g of polypeptone/l, and 5 g of yeast extract/l) in a 500-ml baffled flask at 30 °C for 3 days with rotary shaking at 150 rpm. After centrifugation of the culture, the supernatant was loaded onto PD-10 columns (GE Healthcare) for buffer exchange with 20 mM phosphate buffer (pH 7) or distilled water. The enzyme solution obtained was used for the characterization of TH2-CP2. Simultaneously, the supernatant was concentrated by ammonium sulfate precipitation at 70 % (w/v) saturation at 4 °C. The precipitates were collected by centrifugation at 20,000g for 1 h at 4 °C, and then dissolved in 1 ml of 20 mM Tris–HCl (pH 8) buffer containing 0.15 M NaCl. After centrifugation of the dissolved solution, the resultant supernatant was filtered and subsequently loaded onto a HiLoad™ 16/60 Superdex™ 75 pg column (GE Healthcare). The active fraction eluted with 20 mM Tris–HCl (pH 8) containing 0.25 M NaCl was used for the determination of N-terminal sequences of TH2-CP.
The optimum pH was determined as follows. A mixture of 5 µl of 200 mM NaOAc (pH 4–5.6) or 200 mM potassium phosphate buffer (pH 5.5–8) or 200 mM Tris–HCl (pH 7–8.5) buffer, 2.5 µl of substrate solution (100 mM Z-Gly-Leu), and 37.5 µl of distilled water was added to 5 µl of supernatant enzyme solution from a culture at 30 °C. The enzymatic reaction mixture was incubated at 40 °C for 30 min, and then the reaction was stopped by heat treatment (95 °C, 5 min). The free amino acids released by the hydrolysis of peptides were detected by the 4-aminoantipyrine-phenol method coupled with the reaction of LAAO (Arima et al. 2006). After heat treatment, 20 µl of the reaction liquid was added to 180 µl of a premix solution containing LAAO (0.2 mg/ml). After incubation for 30 min at 37 °C, the absorbance of the solution was determined at 505 nm. This assay was performed in triplicate, and the mean of absorbance at each pH is shown as the percentage of the mean in Tris–HCl buffer (pH 8). Note that the method of evaluating the free amino acids was used for all subsequent procedures.
The optimum temperature was determined as follows. A mixture of 5 µl of 200 mM potassium phosphate buffer (pH 7), 2.5 µl of substrate solution (100 mM Z-Gly-Leu), and 37.5 µl of distilled water was added to 5 µl of enzyme solution (20 mM potassium phosphate buffer, pH 7). Then, the enzymatic reaction mixture was incubated at 40–60 °C for 30 min. Finally, the free amino acids released were detected. This assay was performed in triplicate, and the mean absorbance at each temperature is shown as the percentage of the mean at 51 °C.
Thermostability was determined as follows. A mixture of 5 µl of 200 mM potassium phosphate buffer (pH 7), 5 µl of enzyme solution (20 mM potassium phosphate buffer, pH 7), and 37.5 µl of distilled water was prepared and incubated at 5–45 °C for 30 min. Then, 2.5 µl of substrate solution (100 mM Z-Gly-Leu) was added to the mixture to start the enzymatic reaction at 40 °C for 30 min. Finally, the free amino acids released were detected. This assay was performed in triplicate, and the mean absorbance at each temperature is shown as the percentage of the mean at 5 °C.
pH stability was determined as follows. An enzyme mixture of 0.56 µl of 200 mM potassium phosphate buffer (pH 5.5–8), and 5 µl of enzyme solution (distilled water), was prepared and incubated at 5 °C overnight. Then, a substrate mixture of 2.5 µl of substrate solution (100 mM Z-Gly-Leu), 5 µl of 200 mM potassium phosphate buffer (pH 7), and 36.94 µl distilled water was prepared and added to the enzyme mixture to start the enzymatic reaction at 40 °C for 30 min. Finally, the free amino acids released were detected. This assay was performed in triplicate, and the mean absorbance at each pH is shown as the percentage of the mean in potassium phosphate buffer (pH 7).
Except for two tripeptides, Phe-Phe-Phe and Tyr-Tyr-Tyr, the specific activity and kinetics of other substrates were evaluated as follows. A mixture of 5 µl of substrate solution (specific activity evaluation, 50 mM; kinetics evaluation, 3–50 mM), 5 µl of 200 mM sodium phosphate buffer (pH 7), and 35 µl distilled water was added to 5 µl of enzyme solution (20 mM sodium phosphate buffer, pH 7). Then, the enzymatic reaction mixture was incubated at 40 °C for 30 min. For Phe-Phe-Phe and Tyr-Tyr-Tyr, the enzymatic reaction mixture consisted of 40 µl of substrate solution (1 mM), 5 µl of 200 mM sodium phosphate buffer (pH 7), and 5 µl of 160-fold diluted enzyme solution (20 mM sodium phosphate buffer, pH 7). Finally, the free amino acids released were detected. This assay was performed in triplicate and the free amino acids were quantified by a standard curve of 0, 0.5, and 1 mM free l-leucine. K m and V max were calculated from the Hanes–Woolf plots obtained using the Michaelis–Menten equation.
Determination of N-terminal amino acid sequence
The purified TH2-CP was blotted onto a PVDF membrane after 12 % SDS-PAGE under denaturing conditions. The protein blotted on the PVDF membrane was used for determining the N-terminal amino acid sequence using a protein sequencer (Shimadzu PPSQ-31A).
Assay of TH2-CP activity inhibition
The assay of TH2-CP activity inhibition was performed using phenylmethylsulfonyl fluoride, iodoacetic acid, 2-iodoacetamide, N-ethylmaleimide, and 1,10-phenanthroline, respectively. An enzyme mixture of 0.56 µl of 10 mM inhibitor or distilled water, and 5 µl of enzyme solution (20 mM potassium phosphate buffer, pH 7), was prepared and incubated overnight at 5 °C. Then, a substrate mixture of 2.5 µl of substrate solution (100 mM Z-Gly-Leu), 5 µl of 200 mM potassium phosphate buffer (pH 7), and 36.94 µl distilled water was prepared and added to the enzyme mixture to start the enzymatic reaction at 40 °C for 30 min. Finally, the free amino acids released were detected. This assay was performed in triplicate, and the mean absorbance for each inhibitor is shown as the percentage of the mean in distilled water.
Effects of divalent metal ions on TH2-CP activity
The effects of divalent metal ions on TH2-CP activity was investigated using cobalt chloride (CoCl2), nickel chloride (NiCl2), zinc chloride (ZnCl2), magnesium chloride (MgCl2), manganese chloride (MnCl2), and calcium chloride (CaCl2), respectively. An enzyme mixture of 0.56 µl of 10 mM divalent metal ion solution or distilled water, and 5 µl of enzyme solution (20 mM potassium phosphate buffer, pH 7), was prepared and incubated overnight at 5 °C. A substrate mixture of 2.5 µl of substrate solution (100 mM Z-Gly-Leu), 5 µl of 200 mM potassium phosphate buffer (pH 7), and 36.94 µl distilled water was prepared and added to the enzyme mixture to start the enzymatic reaction at 40 °C for 30 min. Finally, the free amino acids released were detected. This assay was performed in triplicate, and the mean absorbance for each metal ion is shown as the percentage of the mean in distilled water.
Assay of TH2-CP-catalyzed casein hydrolysate
A mixture of 33 µl of Bioprase SP (100 proteolytic units/mg, 10 mg/ml) and 10 ml of 0.6 % (w/v) aqueous dispersion of casein (40 mM NaH2PO4, pH 7.5) was prepared. It was incubated at 40 °C overnight and then at 80 °C for 30 min to inactivate Bioprase SP. After centrifugation, 250 µl of the resultant supernatant was added to 100 µl of enzyme solution (20 mM NaH2PO4, pH 7), or 100 µl of 20 mM NaH2PO4 (pH 7) buffer. The reaction solution was incubated at 40 °C for 6 h with rotary shaking at 1400 rpm, and then at 80 °C for 30 min to inactivate TH2–CP2. Both of these reactions were performed in triplicate. For the amino acid analysis of casein hydrolysate, a 100-µl aliquot of each of the triplicate reaction solutions was obtained to be acidulated with 0.2 M HCl, and then analyzed by gas chromatography (GC-2025, SHIMADZU GLC Ltd.) using an EZ:faast™ kit. The released free amino acids were quantified using a 250 µM standard mixture of free amino acids. To calculate the rate of casein hydrolysis by TH2-CP, the hydrolysis of casein hydrolysate by 6 M HCl was also assayed. In this assay, 2 ml of aqueous dispersion of casein (10 mg/ml, 6 M HCl) was heated at 110 °C for 15 h, and then 100 µl of the solution was taken for analysis using an EZ:faast™ kit. This assay was also performed in triplicate.
Results and discussion
Expression of carboxypeptidase (CP) genes
A good number of candidate genes encoding various enzymes were predicted by MiGAP analysis. Among them, there are six candidate genes encoding CP enzymes. Sequence analysis using the Signal P 4.1 Server (http://www.chs.dtu.dk/services/SignalP/) indicated that three of the six were predicted to possess signal peptides. We cloned these three genes into the pTONA5a vector and expressed them in S. lividans 1326. Only the culture supernatant from TH2-CP expression proved to possess carboxypeptidase activity (data not shown).
N-terminal amino acid sequence of TH2-CP
The N-terminal amino acid sequence of TH2-CP of S. cinnamoneus TH-2 was determined to be VTDDA (Fig. 1). Excluding the signal peptide and pro-segment, the theoretical molecular weight of TH2-CP2 was 37.7 kDa. This value corresponds to the observed result of SDS-PAGE (Fig. 2).
Effects of pH and temperature on TH2-CP activity
Effects of inhibitors and divalent mental ions on TH2-CP activity
Effects of various inhibitors on TH2-CP activity
Inhibitor final 1 mM
Residual activity (%)
Effects of divalent metal ions on TH2-CP activity
Divalent metal ion final 1 mM
Residual activity (%)
Substrate specificity and kinetic analysis of TH2-CP
Kinetic parameters for hydrolysis of Z derivatives by TH2-CP
K m mM
V max mmol min−1 mg−1
V max/K m
1.49 ± 0.13
1.19 ± 0.06
1.10 ± 0.14
1.40 ± 0.06
4.97 ± 0.34
4.82 ± 0.39
Profiles of casein hydrolysate hydrolyzed by TH2-CP
The comparison of the substrate specificities against the synthetic peptides tested showed that TH2-CP could hydrolyze the C-terminal residues (Pro, Asp, Glu, and Lys) of peptides from casein hydrolysate hydrolyzed by Bioprase SP. This result is most likely due to the different lengths and penultimate residues between the synthetic peptides and the casein hydrolysate hydrolyzed by Bioprase SP. Figure 5b shown that the rates of hydrolysis of Met, Leu, Tyr, Phe, Val, and Ile were 74, 48, 47, 36, 30, and 10 %, respectively. These six amino acids are all hydrophobic amino acids. Protein hydrolysates obtained using proteolytic enzymes are frequently accompanied by bitter compounds formed during hydrolysis (Bumberger and Belitz 1993; Clegg et al. 1974; Matoba et al. 1970). Bitter compounds in dairy products have been well studied in cheese manufacturing. Many bitter peptides in cheese are mainly produced by the proteolytic digestion of casein. The bitterness is generally due to peptides with high contents of hydrophobic amino acids (Matoba and Hata 1972). Additionally, the higher the contents of hydrophobic amino acids at the C-terminus, the higher the level of bitterness (Ishibashi et al. 1987a, b; Kanehisa et al. 1984; Otagiri et al. 1984; Shinoda et al. 1986). The tripeptides Phe-Phe-Phe and Tyr-Tyr-Tyr are bitter, and both of them are fivefold bitterer than caffeine (Ishibashi et al. 1987b). In contrast, their corresponding dipeptides Phe-Phe and Tyr-Tyr have 83 and 43 % the bitterness of caffeine, respectively (Ishibashi et al. 1987b). Among the synthetic peptides tested, Phe-Phe-Phe and Tyr-Tyr-Tyr were the optimum two substrates of TH2-CP (Fig. 4). This finding indicates that TH2-CP can decrease the bitterness of peptides. It was confirmed that carboxypeptidases from wheat, pancreatin, and Actinomucor elegans (Fu et al. 2011; Ge and Zhang 1996; Komai et al. 2007; Umetsu et al. 1983),which can effectively hydrolyze hydrophobic amino acids at the C-terminus, can decrease the bitterness of peptides, including the peptides produced by proteolytic digestion of casein. Thus, we consider that the metallocarboxypeptidase TH2-CP from S. cinnamoneus TH-2 could also be used to decrease the bitterness of peptides in food industry, since it has a good hydrolytic activity against the C-terminal hydrophobic amino acids of peptides.
A metallocarboxypeptidase from S. cinnamoneus TH-2 (TH2-CP) has the pH optima of 7.0 and 8.0 in potassium phosphate buffer and Tris–HCl buffer, respectively. However, its stability in potassium phosphate buffer was much higher than that in Tris–HCl buffer. Moreover, the optimum temperature for activity was 51 °C, and 50 % of activity was conserved after incubation at 38 °C for 30 min. Using Z-Gly-Gly-Leu as a substrate, the K m and V max values were 4.97 ± 0.34 mM and 4.82 ± 0.39 µmol min−1mg−1, respectively. TH2-CP can hydrolyze 18 amino acids from the C-terminal residues of casein hydrolysate using the endoprotease from Bacillus sp. TH2-CP prefers hydrophobic amino acids at the C-terminal position of casein peptides as substrates. This property indicates that TH2-CP could be used for decreasing the bitterness of peptides in food industries.
TH designed the experiments and modified the manuscript. KW carried out all experiment except the determination of the N-terminal amino acid sequence for TH2-CP, which was carried out by JA. KW drafted the manuscript. MU gave advice to KW regarding his experiment and also modified the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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