1Laboratory of Molecular Enzymology, Graduate School of Bioengineering, Soka University, Hachioji, Tokyo 192-8577 and 2Laboratory of Molecular Enzymology, Graduate School of Agriculture Science, Tohoku University, 11 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
3 To whom correspondence should be addressed. e-mail: ichisima{at}t.soka.ac.jp
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Abstract |
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Keywords: aspzincin/M35 protease/mutagenesis/penicillolysin/thermal stabilization/Zn2+-protease
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Introduction |
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In a previous paper, a new enzymatic method for production of seasoning including 5'-deoxyribonucleotides and amino acids from salmon milt with microbial enzymes was studied (Hayashi et al., 1996). For efficient hydrolysis of salmon milt, we found that a two-step hydrolysis procedure was necessary. Streptomyces griseus protease, actinase AS and nuclease Amano from Penicillum ctrinum were used to obtain the product. The study dealt with the efficient production of seasoning from salmon milt DNA through the combined addition of a microbial protease and a nuclease. The thermal stability of penicillolysin is important because the conversion of milt to peptides and amino acids during the industrial production of seasoning typically takes place at temperatures of 6568°C. The thermal stability of mutant penicillolysin results in a more favorable temperature for enzymatic hydrolysis after the milt protein is decomposed.
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Materials and methods |
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Escherichia coli DH5 was used for DNA manipulation. Aspergillus oryzae niaD 300 (niaD), which is a nitrate reductase gene (niaD)-deficient strain (Minetoki et al., 1996
), and a high-level expression vector plasmid pNAN8142 (9.5 kb) (Minetoki et al., 1995
; Ozeki et al., 1996
), were used for transformation experiments. CzapekDox medium, consisting of 0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·H2O and 2% dextrin, adjusted to pH 5.5, was used for selection of transformants. DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2PO4 and 0.05% MgSO4·7H2O, pH 5.5) was used for recombinant penicillolysin production. Lysing enzyme was purchased from Sigma (St Louis, MO). N-Butoxycarbonyl- L-valyl-L-arginyl-L-arginyl-4-methylcoumaryl-7-amide (Boc-Arg-Val-Arg-Arg-MCA) and other fluorogenic peptide-MCAs were purchased from the Peptide Institute (Mino-shi, Osaka, Japan).
Construction of the expression plasmid
A 1.0 kb SalISphI fragment of the plnC gene was inserted into the SphISalI site of pNAN8142 (9.5 kb) (Minetoki et al., 1995; Ozeki et al., 1996
) and in this way the penicillolysin-producing plasmid pNANplnC (10.5 kb) was constructed.
Fungal transformation
The transformation of A.oryzae was performed basically according to the method of Gomi et al. (1987). In our experiment, the nitrate reductase gene (niaD) and the A.oryzae niaD 300 strain (niaD) were employed as a selectable marker and host, respectively.
Production and purification of penicillolysin and mutant enzymes expressed in A.oryzae
The conidiospores from a selected transformant were inoculated into 50 ml of DPY medium in a 500 ml Sakaguchi flask and cultured at 30°C for 3 days. The filtrate was salted out by adding ammonium sulfate up to a 70%-saturated concentration and centrifuged for 15 min at 10 000 g. The following operations were carried out at below 4°C unless noted otherwise. The precipitate was dissolved in 20 ml of 10 mM sodium acetate buffer, pH 5.0. The solution was dialyzed against 10 mM sodium acetate buffer, pH 5.0 and then applied to a cation-exchange CM Sepharose Fast Flow column (1x 30 cm) (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated in 10 mM sodium acetate buffer, pH 5.0. Elution was effected with a linear gradient of 3075 mM NaCl in 10 mM sodium acetate buffer, pH 5.0. The active enzyme fraction eluted from the CM Sepharose Fast Flow column was dialyzed against Milli-Q water at 4°C. The purified penicillolysin migrated with homogeneity on SDSPAGE, which was performed by the method of Laemmli (1970). Purification procedures of the mutant enzymes were performed using the same method as for wild-type penicillolysin.
Site-directed mutagenesis
Site-directed mutagenesis of the plnC gene was performed by the Kunkel (1985) method. Escherichia coli strain CJ236 was used to generate dU-substituted DNA and single-stranded DNA was isolated using the helper phage M13K07 (Bio-Rad, Hercules, CA). The oligonucleotides used for generation of the mutants were as follows: 5'-TACGGCAGCCgaGTCTGGATCcGCCTCCAAGT-3' for R33E; 5'-AGCACGTCTAcgaGCTGTGGCT-3' for E60R; 5'-GCAGTGACcCCcTaGGATA CTGCGA-3' for T81P; 5'-GATACTGCGAGcCtAACGTGCTaGCTTATACCCT-3' for T87P; 5'-TCTTATCTGCCAcCcTTGGCcGGcACCTGCCATCA-3' for A112P; 5'-CAC GACAACTCTtCgcGAGTTCACCC-3' for H128R; 5'-CAACTCTCCATcAGTTCACCCAT-3' for E129Q; 5'-CCATGAGTTCACtCgaGCACCTGGTG-3' for H132R; 5'-GTGTAT AGCCCcGGgACCGATaACCTGGGATA-3' for D143N; 5'-GTATTGAATGCgaAtTCGTATGCCC-3' for D164N; and 5'-CTGACTCGTATGagCTCTACGCCAACG-3' for A167E. Substituted nucleotides are indicated by lower-case letters. Italic letters show the sites recognized by the restriction endonucleases used. Mutations were verified based on restrictive differences or DNA sequencing before construction of the expression plasmid pNANplnC.
Enzyme assay
The enzyme activity of penicillolysin was routinely assayed with Boc-Arg-Val-Arg-Arg-MCA as a substrate at 30°C in 50 mM TrisHCl buffer, pH 8.0. One katal (kat) of enzyme activity in SI units (Price and Stevens, 1999) is defined as the amount of enzyme that liberates 1 mol of 4-methylcoumaryl-7-amide (MCA) from the fluorogenic peptide substrate per second at 30°C and pH 8.0 (Doi et al., 2003
). The specific activity of the enzyme was expressed as katal/kg protein. The purified enzyme was pre-incubated in 895 µl of 50 mM TrisHCl buffer, pH 8.0, at 30°C for 10 min, to which 5 µl of 10 mM peptide-MCA substrate had been added. The initial rate of increase in the 7-amino-4-methylcoumarin of enzymatic hydrolysates was monitored fluorometrically using a Shimadzu RF-5000 spectrophotometer with excitation at
ex = 360 nm and emission at
em = 440 nm. The kinetic parameters Km and Vmax were determined directly from HanesWoolf plots. Values for kcat were derived from Vmax = kcat[E]0, where [E]0 is the enzyme concentration.
Effect of temperature
The measurements were performed at a low concentrations in 10 mM sodium acetate buffer at pH 5.0. Heating took place in a thermostatically regulated PCR Thermal Cycler 480 thermocycler (Takara, Kyoto, Japan). As the control, unheated samples were kept at 20°C until assayed. The enzyme was incubated at a given temperature for 10 min before being cooled on ice for 10 min and was then assayed for remaining enzymatic activity at pH 8.0 to determine the degree of irreversible thermal denaturation. Thermal stability was quantified as the half-life of activity, T50, being the temperature (°C) of incubation at which 50% of the initial activity of a solution of enzyme is retained in 10 min. The stability of the mutant enzyme is expressed as T50, being the difference in T50 between the wild-type (T50 = 62°C) and mutant enzymes. The error margins in the
T50 values are estimated to be 0.20.4°C.
Protein determination
Protein concentrations were usually measured by the method of Bradford (1976) with bovine serum albumin (BSA) as a standard protein.
CD measurements
Penicillolysin was dialyzed against 10 mM sodium acetate buffer, pH 5.0, then diluted in the same buffer to 0.2 mg protein/ml. Far-UV CD spectra were measured with a Jasco J-720 spectrophotometer at 25°C in a cuvette with a 1 mm pathlength (Yang et al., 1986). Unfolding curves were measured at 222 nm in the temperature scan mode with a gradient of 1°C/min until a temperature of 80°C was reached.
Atomic absorption spectrometric (AAS) analysis
Zinc analysis of the recombinant penicillolysin was carried out byAAS using a Shimadzu Model AA-660 (P/N206-1000-02) instrument.
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Results and discussion |
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A recombinant penicillolysin was expressed in A.oryzae. The system is capable of making as much as 15 mg of enzyme per liter of culture. The recombinant penicillolysin was secreted as an active form in culture broth and purified to a homogeneous state by one-step column chromatography. The molecular mass was determined as 23.7 kDa by SDSPAGE. The N-terminal amino acid sequence of the enzyme was found to be TKET. These results suggest that the recombinant enzyme was accurately processed and the number of amino acid residues was assumed to be 177 from the predicted amino acid sequence described previously (Matsumoto et al., 1994). The recombinant penicillolysin contained 1 mol of zinc per mol of enzyme with a molecular mass of 18 525 Da.
Penicillolysin does not hydrolyze small synthetic peptides (Yamaguchi et al., 1993). To obtain a sensitive small substrate of penicillolysin, 45 kinds of fluorogenic peptide-MCAs were tested. The recombinant penicillolysin displayed hydrolytic activity towards Boc-Arg-Val-Arg-Arg-MCA (Boc-RVRR-MCA), with optimal activity at pH 8.0. These findings indicate that penicillolysin exhibits a similar specificity to that of deuterolysin (Doi et al., 2003
). The properties of the recombinant penicillolysin expressed in A.oryzae are summarized in Table I.
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Protein engineering is a promising tool for obtaining stable proteins. Comparisons between homologous penicillolysin and deuterolysin can reveal the features responsible for the enhanced stability of penicillolysin. Thermally unstable penicillolysin has 68% sequence identity with the thermally stable deuterolysin from A.oryzae; however, in the N-terminal region from amino acid residues 1 to 60, only 60% identity was observed (Figure 1B). This shows that the region was a target for mutation. Thermally unstable penicillolysin had Arg33 and Glu60, whereas thermally stable deuterolysin had Glu33 and Arg60. From the crystal structure of deuterolysin from A.oryzae (Figure 1A), the N-terminal region of the enzyme is composed of three helices, A,
B and
C, and a single ß-structure, ß1, as shown in Figure 1B (McAuley et al., 2001
). Glu33 in deuterolysin is located in the
A long helix region and Arg60 in the
C long helix. In this study, our choice of mutation was R33E/E60R in the N-terminal structure of penicillolysin. For thermal stabilization of penicillolysin, we expected that on the polar surface of the helix, electrostatic interactions between acidic and basic residues at Glu33 and Arg60 would copy the N-terminal sequence of deuterolysin. We generated the mutant, R33E/E60R.
Generally, proline residues have less conformational freedom in unfolded structures than any other residues since the proline side chain is fixed by a covalent bond to the main chain. From the structure of deuterolysin, it is evident that three proline residues, Pro81, Pro87 and Pro112, are present in the molecule, but not in penicillolysin (Figure 1B). Pro81 and Pro87 are located in the loop region between ß3 and ß4 and Pro112 is located in the loop region between D and
E. We decided on the position and species of amino acids to be replaced to give enhanced thermal stability. We generated the mutants T81P, T87P and A112P.
In /ß proteins,
-helices and ß-sheets alternate along the peptide chain. For example, the greater the fraction of the non-polar surface buried, the lower is the stability of the protein, suggesting that the alternative polar surface buried, in hydrogen-bonded form, contributes more to stability (Darby and Creighton, 1993
).
In GfMEP from G.frondosa, the active-site helix, helix D, contains two zinc ligands, His117 and His121, and the catalytic residue Glu118 as components of the HExxH motif (Hori et al., 2001
). The loop
D
E includes the GTxDxxYG segment, which is strictly conserved among aspzincins and contains Asp130 as the third zinc ligand. Asp130 makes a unique bifurcated interaction with the zinc ion. Such a bifurcated interaction has not observed in other metalloendopeptidases whose third zinc ligand is Asp or Glu. Thr125 in penicillolysin, Thr125 in the
E helix of deuterolysin and Thr114 in the
D helix of GfMEP from G.frondosa were conserved in the common TxxHExxH motif at the C-terminal end of aspzincin family enzymes and the
E of deuterolysin and
D of GfMEP from G.frondosa are called the active-site helix.
The Ala167 residue of the C-terminal of penicillolysin was located in the corresponding region of H in deuterolysin. On the other hand, the Glu157 residue in the
F helix in the C-terminal region of aspzincin from G.frondosa (GfMEP), a thermally stable fungal metalloendopeptidase, corresponded to Ala167 in penicillolysin. We expected penicillolysin to have a hydrogen bond between the acidic residue Glu at position 167 and Thr at position 125. We therefore generated the mutant A167E.
The polypeptide backbone of L-amino acid residues is chiral and gives an intense CD signal in the far-UV region (180 240 nm) that is dependent on the conformation of the backbone. In particular, -helices and ß-structures each give characteristically different spectra in the wavelength range. The approximate average content of such structures in a protein can be estimated from the CD spectrum, by comparison to a database of spectra of proteins of known structure (Figure 2). Although the CD spectra of the wild-type and mutants have slight differences (Figure 2), the enzyme activity of the mutants was not largely affected. Kinetic parameters of the mutant R33E/E60R are similar to those of the wild-type enzyme (Table III). Similar kcat/Km parameters were obtained for the two mutants R33E/E60R and T81P. The kcat/Km value of the A167E mutant was 1.6-fold higher than that of the wild-type enzyme.
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When the rate of degradation of enzyme activity is measured, it is more common to express the result in terms of the half-life of enzyme activity, T50, rather than in terms of the rate constant, kd. For a first-order reaction the relationship is t1/2 = ln2/kd = 0.69/kd (Price and Stevens, 1999). In this study, thermal stability was quantified as T50, being the temperature at which 50% of the initial activity was retained for 10 min. The thermal stability of the mutants also depended on temperature, as shown in Figure 4A and B. The double mutant R33E/E60R was found to be fairly thermally stable, as shown in Figure 4A and B. The T50 value, 66°C, of R33E/E60R was higher than that of the wild-type penicillolysin (Table IV). It is interesting that two mutations probably due to favorable interactions between the two
-helices (A and C helices) were generated and these interactions are important in the thermal stability of the mutant enzyme R33E/E60R. The A167E mutant showed the same thermal stability as R33E/E60R. The A167E mutation was also sufficient to increase the T50 value from 62 to 65°C (Table IV). The single A167E mutation was useful for the thermal stabilization of penicillolysin, indicating that the connection between the
E and
H helices may be important. Among the three proline mutants, the T81P mutant significantly increased the T50 value from 62 to 68°C, whereas T87P and A112P had no clear stabilizing effects. For the 50% residual activity, the thermal stability at 65°C was 4, 12, 12 and 21 min for the wild-type, R33E/E60R, A167E and T81P, respectively (Figure 4B). When the T81P mutation was introduced, the enzyme was found to be more thermally stable than the two mutants, R33E/E60R and A167E. It is interesting that T81P similarly enhanced the thermal stability of penicillolysin; however, the other two mutants, T87P and A112P, showed decreased thermal stability. The T81P mutant of penicillolysin may have a structure essentially identical to that of the wild-type of deuterolysin. There are indications that the proline residues contribute to enzyme thermal stability (Matthews et al., 1987
; Suzuki, 1989
). The contribution can be explained in terms of entropic effects (or chain flexibility) since proline is more restricted conformationally in the unfolded state than any other amino acid (Matthews et al., 1987
). Since proline residues are restricted with respect to their
angles, their introduction can easily cause conformational strain in the backbone. Suzuki et al. (1987
) demonstrated that an increase in the frequency of proline in ß-turns and in the total number of hydrophobic residues can enhance protein thermal stability.
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The thermal stability of an enzyme is influenced by many factors, including amino acid sequence, three-dimensional structure, cofactors and pH. However, at present we cannot explain the results obtained. The present findings show that the thermal stability of penicillolysin is increased dramatically by just a few mutations in one particular region of the protein (Table IV).
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Received December 18, 2003; revised March 26, 2004; accepted April 1, 2004 Edited by Taiji Imoto
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