INTRODUCTION

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Commercial use of proteases having different origins for hydrolysis of food proteins appears to be very promising due to the nonchemical origin of enzymes. However, this technology has been hindered by the production of a bitter hydrolysate (6, 7). The bitterness has been ascribed to various peptides that are produced during enzymatic hydrolysis (8, 13, 14, 15). From the other side, free amino acids and short peptides play positive roles and elicit characteristic tastes of foods. Carboxypeptidases and aminopeptidases having different specificities, therefore, could be expected to be "natural" boosters of flavor during hydrolysis of food proteins. It has been shown that the debittering effects of both aminopeptidases (13) and carboxypeptidases (1, 23, 24) occur due to liberation of hydrophobic amino acids from the bitter peptides.

Serine carboxypeptidases (EC 3.4.16.1) are widely distributed in fungi, as well as in higher plants and animals, and they are particularly abundant in filamentous fungi (5). These enzymes are exopeptidases that specifically release amino acids from the C termini of peptides and proteins by utilizing a catalytic triad consisting of Ser, His, and Asp in their active sites. Their ability to cleave C-terminal amino acids during hydrolysis and their ability to replace C-terminal amino acid residues in transpeptidase reactions without internal cleavage of peptides have also attracted attention in peptide synthesis (3) and C-terminal sequencing (5) studies.

Here we describe a novel carboxypeptidase from Aspergillus oryzae which appears to be a major carboxypeptidase of Flavourzyme.

	MATERIALS AND METHODS

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Reagents. The chemicals used as buffers and reagents were commercial products and were at least reagent grade. Flavourzyme, a commercial fungal extract obtained from A. oryzae fermentation broth, was obtained from Novo Nordisk A/S (Copenhagen, Denmark). Flavourzyme is produced by fermentation of a selected strain of A. oryzae and contains both endoprotease and exopeptidase activities. All peptide substrates were obtained from Sigma Chemical Co. (St. Louis, Mo.). Purification was performed with a Pharmacia fast protein liquid chromatography device with column supports and resins obtained from the same source. The Tricine gels and polyvinylidene difluoride membranes used for peptide separation and sequencing were obtained from Novex (San Diego, Calif.). All assays were performed with a Hitachi model U-2001 spectrophotometer. Quartz cuvettes with a 1-cm path length were used. The model 476A protein sequencer used was obtained from Applied Biosystems.

Purification of A. oryzae carboxypeptidase I. Flavourzyme (20 ml) was diluted twofold in 20 mM sodium phosphate buffer (pH 7.0). Then the preparation was centrifuged at 11,000 × g for 10 min and filtered through a 0.45-µm-pore-size filter (Nalgene). The enzyme solution was diluted so that the conductivity was ~2.4 mS and the pH was 7.0 by using 20 mM sodium phosphate buffer (pH 7.0) and then was loaded onto a Q-Sepharose Big Bead column (XK 26; ~80 ml of resin) which had been preequilibrated with 20 mM sodium phosphate buffer (pH 7.0). The first gradient used for elution was a linear gradient from 100% 20 mM sodium phosphate buffer (pH 7.0) to 100% 20 mM sodium acetate buffer (pH 5.0) (flow rate, 5 ml/min; gradient volume, 300 ml; fraction size, 12.5 ml); and the final gradient was a linear pH gradient from 20 mM sodium acetate buffer (pH 4.3) to 100% 20 mM sodium acetate buffer (pH 3.5) (gradient volume, 400 ml; fraction size, 12.5 ml). The active fractions in the gradient from 20 mM sodium acetate buffer (pH 4.3) to 20 mM sodium acetate buffer (pH 3.5) were pooled, and the pH was adjusted to 7.0 with 20 mM dibasic sodium phosphate. A sample was then loaded onto a 20-ml Mono Q 16/10 column that had been preequilibrated with 20 mM sodium phosphate buffer (pH 7.0). The carboxypeptidase was eluted with a 0 to 0.3 M NaCl gradient at a flow rate of 5 ml/min (gradient volume, 500 ml; fraction size, 12.5 ml).

Physicochemical characterization. Molecular mass and purity were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by using a minicell apparatus and 8 to 16% Tris-glycine gels (Novex). Isoelectric focusing (IEF) was performed by using a Novex pH 3 to 10 IEF gel. The manufacturer's suggested method was used. Bio-Rad and Pharmacia IEF markers were used.

Properties of the enzyme. (i) Carboxypeptidase assay. Two methods were used for the carboxypeptidase assay. The first method was based on direct monitoring of decreases in the absorbance at 340 nm (A₃₄₀) as a result of hydrolysis of N-(3-[furyl]acryloyl)-Ala-Lys (1 mM in 50 mM phosphate buffer, pH 6.0). The second method (the o-phthaldialdehyde [OPA] method) was based on detection of a free amino group. At 25°C, 50 µl of enzyme solution was added to 1 ml of a 0.5 mM carbobenzoxy (Z)-Ala-Glu solution in 20 mM sodium acetate buffer (pH 4.5). The release of free glutamic acid was monitored by using OPA and dithiothreitol (18). One unit of activity was defined as the quantity of enzyme which liberated 1 µmol of glutamic acid per min under the conditions described above for the second method.

(ii) Inhibition study. The purified carboxypeptidase was incubated with 1,10-phenanthroline (which is known as an effective Zn chelator) at a concentration of 5 mM and phenylmethylsulfonyl fluoride at a concentration of 1 mM at pH 4.5 (20 mM acetate buffer). The residual activity was measured by using N-(3-[furyl]acryloyl)-Ala-Lys as a substrate at pH 4.5 in 20 mM acetate buffer. The release of free lysine was monitored by measuring the decrease in A₃₄₀.

(iii) pH and temperature optima. The optimal pH for enzymatic activity was determined by using a 50 mM sodium acetate-50 mM sodium phosphate-50 mM sodium citrate universal buffer at pH values ranging from 2.9 to 7.5. Z-Ala-Ile (concentration, 0.5 mM) was used as the substrate. The OPA method was used to quantitate the Ile that was released. The optimal temperature for enzymatic activity was determined by using 0.5 mM Z-Ala-Ile in 50 mM sodium acetate buffer (pH 4.0). One milliliter of substrate was preincubated for 30 min before the enzyme sample was added.

(iv) Thermal stability. Thermal stability was studied by using 50 mM sodium acetate buffer (pH 4.0). A 50-µl enzyme solution was added to 450 µl of preheated buffer. The enzyme sample was incubated for 10 min, and residual activity was measured by using Z-Ala-Ile at pH 4.0. Incubations were performed at temperatures ranging from 35 to 75°C in 5°C increments.

(v) Substrate specificity. The substrate specificity of carboxypeptidase was established by determining the bimolecular constants for a series of Z-Ala-X-OH compounds by using the carboxypeptidase assay method described above. The k_cat/K_m ratio was determined by using the Michaelis-Menten equation.

(vi) Sequential release of C-terminal amino acid residues from natural peptides. Bradykinin and -amyloid were dissolved in 1.4 and 1.0 ml of 50 mM acetate buffer (pH 4.5), respectively, to a final concentration of 0.55 mM. Carboxypeptidase was added to the peptide samples to final concentrations of 0.114 and 0.0878 U/ml for bradykinin and -amyloid, respectively. After incubation at room temperature (21°C), 150-µl aliquots were added to 150-µl portions of 60 mM HCl to terminate the reactions. The resulting samples were then analyzed with a Beckman model 6300 automatic amino acid analyzer.

Cloning and expression. (i) Construction of cDNA library. Total cellular RNA was extracted from A. oryzae mycelia harvested from a Flavourzyme production fermentation (Novo Nordisk A/S). Double-stranded cDNA was synthesized from 5 µg of poly(A)⁺ RNA as described by Kofod et al. (11). A library consisting of 10⁶ independent clones was stored as individual pools (25,000 to 30,000 CFU/pool) in 20% glycerol at 80°C.

(ii) PCR amplification of carboxypeptidase I coding sequences. Based on amino acid sequence data obtained from cyanogen bromide fragments of purified carboxypeptidase I, the following PCR primers were synthesized: forward primer 5'-dTAYGGNGGICAYTAYGGICCNG-3' and reverse primer 5'-dATRAARTTIGGCCAIACRAARTC-3' (R = A or G; Y = C or T; N = G, A, C, or T). PCR mixtures (100 µl) were prepared by using 1 µg of an A. oryzae cDNA library as the template. In addition, each reaction mixture contained the following components: 40 pmol of forward primer, 40 pmol of reverse primer, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 200 µM dTTP, 1× Taq polymerase buffer (Perkin-Elmer Corp., Branchburg, N.J.), and 2.5 U of Taq polymerase (Perkin-Elmer Corp.). The reaction mixtures were incubated in a Perkin-Elmer model 480 thermal cycler programmed as follows: cycle 1 consisting of 95°C for 5 min, 45°C for 2 min, and 67°C for 2 min; and cycles 2 through 30 consisting of 95°C for 2 min, 45°C for 1 min, and 67°C for 2 min. The reaction products were isolated on a 1% agarose gel and cloned into a pCRII vector (Invitrogen, San Diego, Calif.).

(iii) Identification of carboxypeptidase I clones. The A. oryzae cDNA library was screened by colony hybridization (4) by using PCR-generated probes.

(iv) DNA sequence analysis of A. oryzae ATCC 20386 carboxypeptidase I gene. DNA sequencing of one carboxypeptidase I clone (designated pEJG12) was performed with a model 373A automated DNA sequencer (Applied Biosystems, Inc., Foster City, Calif.); both strands were examined by using the primer walking technique with dye terminator chemistry (9). Oligonucleotide sequencing primers for complementary sequences in the carboxypeptidase I gene were designed and were synthesized with an Applied Biosystems model 394 DNA-RNA synthesizer used according to the manufacturer's instructions.

(v) Construction of a carboxypeptidase I expression vector. We designed the following two synthetic oligonucleotide primers to amplify the A. oryzae carboxypeptidase coding region from pEJG12 for subcloning into Fusarium expression vector pDM181: forward primer 5'-ATTTAAATATGCGTGGCTACGAATT-3' and reverse primer 5'-GGGTTAATTAACTATGCCATACCAA-3' (boldface type indicates the coding sequence).

(vi) Transformation of Fusarium strain and analysis of transformants. The recombinant DNA molecule (pEJG15) was introduced into the host organism by incubating the plasmid with protoplasts of Fusarium venenatum CC1-3. Transformants were selected by growing organisms on a medium containing phosphinothricin, the active ingredient in the herbicide BASTA (Hoechst). BASTA-resistant transformants were screened for carboxypeptidase activity. The vectors containing the F. oxysporum trypsinlike protease promoter and terminator, as well as the protocols used for transformation of F. venenatum, have been described previously (19).

Nucleotide sequence accession number. The gene sequence which we determined has been deposited in the GENESEQN database under accession no. V28620.

	RESULTS

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Biochemical properties of native A. oryzae carboxypeptidase I. The secreted carboxypeptidase obtained from A. oryzae was homogeneous after purification on two anion-exchange columns. When SDS-PAGE was used, a single component with a molecular weight of ca. 67,000 was detected. The protein sample also yielded a single band with a pI near 4.6 during IEF.

View this table: [in this window] [in a new window]   TABLE 2.   Release of C-terminal amino acids from bradykinin (5 nmol) and -amyloid (5.25 nmol) by native A. oryzae carboxypeptidase Ia

View larger version (9K): [in this window] [in a new window]   FIG. 1.   pH dependence of hydrolysis of Z-Ala-Ile catalyzed by the carboxypeptidase (25°C, 50 mM sodium acetate-50 mM sodium phosphate-50 mM sodium citrate universal buffer). A typical error in Km and kcat determinations did not exceed 15%.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Temperature dependence of hydrolysis of Z-Ala-Ile catalyzed by the carboxypeptidase (50 mM acetate buffer, pH 4.0; [Z-Ala-Ile], 1.5 mM; [E], 0.15 mg/ml). The means shown were based on three measurements. A typical error in concentration determinations did not exceed 6%.

Sequence analysis of cDNA encoding A. oryzae carboxypeptidase I. A carboxypeptidase I gene segment consisting of approximately 186 codons (550 bp) was amplified from A. oryzae cDNA with the carboxypeptidase-specific PCR primers. Screening of the A. oryzae cDNA library resulted in six colonies that exhibited strong hybridization signals with the probe. The carboxypeptidase-encoding plasmids were confirmed by DNA sequencing.

View larger version (67K): [in this window] [in a new window]   FIG. 3.   Nucleotide sequence and deduced amino acid sequence of A. oryzae carboxypeptidase. The amino acid sequences of the peptide fragments obtained from cyanogen bromide cleavage of the wild-type carboxypeptidase are underlined. The arrows indicate PCR primers. The putative signal peptide is enclosed in a box. Boldface type indicates the amino acid residues that form the catalytic triad.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Plasmid used for expression of the carboxypeptidase in F. venenatum. The F. oxysporum trypsinlike protease promoter and terminator regions were used for expression of the recombinant carboxypeptidase. The Aspergillus nidulans amdS promoter was used to express the bar gene. The bar gene codes for a Streptomyces hydroscopicus phosphinothricin acetyltransferase. The polyadenylation and transcription termination region was derived from the Aspergillus niger glaA (glucoamylase) gene.

Properties of recombinant carboxypeptidase I. Purified F. venenatum carboxypeptidase I produced two bands at molecular weights near 110,000 and 90,000 on SDS-PAGE gels. This finding was attributed to the presence of two different glycoforms of recombinant carboxypeptidase I. Enzymatic deglycosylation of a preparation containing the two components of F. venenatum carboxypeptidase I with endoglycosidase F/N-glycosidase F resulted in a homogeneous protein with a molecular weight near 60,000 (as determined by SDS-PAGE).

	DISCUSSION

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Our inhibition study and sequence homology results unequivocally show that carboxypeptidase I is a serine carboxypeptidase. Based on preferences for hydrophobic (Phe) and basic (Lys) amino acids at the P1' position, two subclasses (the carboxypeptidase C and carboxypeptidase D subclasses, respectively) were identified (17). The preferences are determined by examining the ratio of the bimolecular constant for Z-Ala-Lys to the bimolecular constant for Z-Ala-Phe. Carboxypeptidase Y from yeast has a low ratio (0.11) and therefore is considered a member of the carboxypeptidase C subclass. Carboxypeptidase AII, a member of the carboxypeptidase D subclass, has by far the highest ratio, 1,300. We found (Table 1) that the ratio of the bimolecular constants for Z-Ala-Lys and Z-Ala-Phe is ~3.75. This finding does not definitively identify carboxypeptidase I as a member of the carboxypeptidase D subclass. P. janthinellum produces a carboxypeptidase (2) that has a ratio of 1.8 (Table 1). This enzyme also cannot be placed into either class definitively. It is reasonable to place these enzymes in a subclass of serine carboxypeptidases which do not exhibit a dramatic preference for basic amino acids over hydrophobic amino acids at the P1' position or vice versa.

Carboxypeptidase I possesses unique properties. It is very favorable for protein hydrolysis that this enzyme does not exhibit a preference for any class of amino acids. Indeed, the K_m values for the nonpolar amino acid Ile, the polar but not ionized amino acid Asn, and the oppositely charged ionogenic amino acids Glu and Arg during catalytic hydrolysis of Z-Ala-X dipeptides are comparable, as are the k_cat values (Table 1). Apparently, Gly and Pro are less preferable amino acids for carboxypeptidase I due to the small size of Gly and the special structure of Pro. In spite of this, carboxypeptidase I effectively goes through both Gly and Pro in bradykinin and -amyloid and even hydrolyzes a Pro-Gly peptide bond. A comparative study of the release of amino acids during hydrolysis of these natural substrates led to the conclusion that both Gly and Pro hinder hydrolysis of the C-terminal amino acid when they are present at the penultimate position.

A study of the pH dependence of catalytic hydrolysis of Z-Ala-Ile (Fig. 1) and Z-Ala-Glu (data not shown) revealed a peak of carboxypeptidase activity around pH 4.0 to 4.5. This pH is not optimal for hydrolysis of, for example, soy protein, which normally starts at neutral pH. However, Fig. 1 shows that the value of the bimolecular constant for hydrolysis of Z-Ala-Ile at pH 4.0 to 4.5 is less than sixfold greater than the value at pH 6.0.

Several other carboxypeptidases with molecular weights near 60,000 have been found in A. oryzae; these enzymes include carboxypeptidase O-1, carboxypeptidase O-2 (22), and carboxypeptidase III (15). Although the previously described data are not sufficient for detailed comparisons of these three enzymes and carboxypeptidase I to be made, the parameter values for hydrolysis of Z-Glu-Tyr by carboxypeptidases O-1 and O-2 obviously show that these two enzymes have much lower (approximately 25-fold lower) specific activities and narrower pH stabilities (pH 5.0 to 6.0) than carboxypeptidase I. In contrast to carboxypeptidase I, carboxypeptidase III (15) has a greater preference for Z-Glu-Tyr than for Z-Ala-Glu and a temperature optimum of 40°C, while carboxypeptidase I is most active at temperatures between 55 and 60°C.

F. venenatum A3/5 has been shown to be a promising host for production of fungal enzymes. Previous studies have demonstrated that this organism is able to produce a variety of recombinant enzymes, such as cellulase, protease, and lipase (19). In addition, its low background levels of proteases and other secreted proteins are advantageous for production of relatively pure enzyme products (19). In the present study, we employed F. venenatum as a host for expression of A. oryzae carboxypeptidase I. Expression of recombinant carboxypeptidase I was confirmed based on activity and protein sequence data. Carboxypeptidase I from F. venenatum and A. oryzae carboxypeptidase were found to be very similar in all parameters except molecular weight. The enzyme from F. venenatum is heavily glycosylated. Both carboxypeptidases exhibited broad substrate specificity, which is uncommon for serine carboxypeptidases (17).

The ability of carboxypeptidase I expressed in the edible fungus F. venenatum A3/5 to release all C-terminal amino acids, including Gly and Pro, and the relatively high specific activity and high thermostability of this enzyme make it a promising candidate for use in production of protein hydrolysates of different origins and in flavor-improving processes in the food industry.