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Applied and Environmental Microbiology, January 2003, p. 162-169, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.162-169.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Naoki Tsuruoka,1 Toru Nakayama,1 Masako Ashida,1 Hisashi Hemmi,1 Masahiro Nakao,2 Hiroyuki Minakata,3 Hiroshi Oyama,4 Kohei Oda,4 and Tokuzo Nishino1*

Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579,1 Suntory Research Center,2 Suntory Institute for Bioorganic Research (SUNBOR), Suntory Ltd., Mishima-gun, Osaka 618-8503,3 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan4

Received 27 June 2002/ Accepted 7 October 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (kcat, 5.41 s-1; Km, 32 µM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (kcat, 351 s-1; Km, 214 µM), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Collagen is an insoluble structural protein that accounts for approximately 30% of the total weight of animal proteins and is produced in large quantities as a by-product in livestock industries. It has recently been shown that the enzymatic degradation of collagen as well as that of gelatin, a denatured form of collagen, allows efficient utilization of these structural proteins. This degradation produces peptides, the collagen peptides, which have been shown to have several biological activities of industrial and medical interest (27), leading to the establishment of a wide variety of applications, e.g., an immunotherapeutic agent (7, 9), a moisturizer for cosmetics, a preservative (2), and seasoning and dietary materials (4).

Generally, the enzymatic degradation of collagen is not affected by ordinary digestive proteinases but requires the collagen-specific, Zn2+-dependent metalloproteinases (collagenases). Although microbial collagenases have been found in a wide variety of mesophilic bacterial strains (3), the industrial-scale application of known bacterial collagenases for collagen peptide production has been hampered because of their insufficient stability. In 2000, we searched for a novel collagenolytic proteinase with high catalytic activity and stability under thermoacidophilic conditions (60°C, pH 4.0), which may reduce the possibility of microbial contamination and should be advantageous during industrial production of the peptides. We found that a slightly thermophilic, acidophilic bacterium isolated from the soil of Sendai, Japan, produces the desired collagenolytic activity (17). Recent taxonomic studies of this strain, NTAP-1, revealed that it is a new species of the genus Alicyclobacillus, leading us to name it Alicyclobacillus sendaiensis.

In this study, we describe the purification to homogeneity, characterization, gene cloning, heterologous expression, and primary-structure analysis of the thermoacidophilic collagenolytic enzyme from A. sendaiensis strain NTAP-1 (termed ScpA). Intensive characterization revealed several distinguishing features of ScpA in comparison with known collagenases and other collagenolytic enzymes, and these features warrant the usefulness of ScpA in the efficient production of the collagen peptides. A primary-structure analysis of ScpA revealed that it is a collagenolytic member of the serine-carboxyl proteinase (S53) family (according to the MEROPS database), a class of serine proteinases that has recently been transferred from an aspartic proteinase family (A7) mainly on the basis of the X-ray crystallography results (34). Unexpectedly, ScpA is very similar in primary structure to a member of this family, kumamolysin (15, 26), whose substrate specificity for macromolecular substrates has not been defined thus far.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and bacterial strains.
A. sendaiensis strain NTAP-1 (17) was a stock culture of the Nishino laboratory, Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan. Azocoll (an azo dye-impregnated hide powder), pepstatin, diazoacetyl-DL-norleucine methyl ester (DAN), ß-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane (E-64), and N-ethylmaleimide were purchased from Sigma, St. Louis, Mo. AAFCH2Cl2 (L-alanyl-L-alanyl-L-phenylalanyl-chloromethane) was a product of the Peptide Institute, Osaka, Japan. The substrate peptides, Phe-Gly-Pro-Ala-Gly-Pro-Ile-Gly and Met-Gly-Pro-Arg-Gly-Phe-Pro-Gly-Ser, were synthesized with an automated peptide synthesizer (model 433A; Applied Biosystems Japan, Tokyo, Japan). Collagen (type I, from a bovine Achilles tendon), albumin, casein, and elastin were purchased from Nacalai Tesque, Kyoto, Japan. ScpP (serine-carboxyl proteinase from Pseudomonas sp. strain 101) and kumamolysin, both of which were recombinant enzymes, were obtained as described previously (23, 26). Tyrostatin was purified from a culture filtrate of Kitasatospora sp. strain 55 as described previously (18). Restriction enzymes and other DNA modifying enzymes were purchased from TaKaRa Shuzo, Kyoto, Japan, or Toyobo, Osaka, Japan. A dye terminator cycle sequencing kit was a product of Beckman Coulter (Fullerton, Calif.).

Bacterial cultivation.
A. sendaiensis strain NTAP-1 was grown in a potato dextrose broth (Difco, Kansas City, Mo.) supplemented with 0.2% KH2PO4 (pH 4.8). The cells were grown in 5-liter jar fermentors (Mitsuwa Co., Osaka, Japan), each containing 3 liters of the medium, at 55°C for 24 h. During cultivation, the agitation rate was 300 rpm, and the aeration rate was 1.0 vol/vol/min.

Enzyme assay. (i) Method I.
Collagenolytic activity was routinely assayed at 60°C and pH 4.0 with the chromogenic substrate Azocoll. The standard assay system contained 50 µmol of sodium acetate, pH 4.0, 2 mg of Azocoll, 0.5 mg of Tween 80, and enzyme in a final volume of 0.5 ml. The reaction mixture without the enzyme was previously incubated at 60°C, and the reaction was started by the addition of the enzyme. Incubation was carried out at 60°C with shaking (at 1,000 rpm) for 10 to 60 min (depending on the amount of enzyme to be assayed) with a model E-36 micromixer (TAITEC Co., Saitama, Japan), by which the homogeneous distribution of the Azocoll granules in the reaction mixture was maintained during the incubation. The reaction was terminated by adding 1.0 ml of 1 M potassium phosphate, pH 7.0, and chilling the mixture on ice. Collagenolytic activity was estimated from the increase in absorbance at 540 nm (dA540) of the supernatant of the reaction mixture, which was due to the formation of the azo dye-linked soluble peptides. The enzyme was replaced with water for the blank. It should be noted that the Azocoll substrate remained insoluble in the absence of added enzyme under these assay conditions (at pH 4.0 and 60°C for 1 h). The dA540 of the reaction mixture in which the Azocoll substrate (2 mg) was completely degraded by the addition of an excess amount of collagenase under these assay conditions was also determined and was used for unit calculations. One unit of enzyme was defined as the amount that caused the solubilization of 1 mg of protein per min under these assay conditions. The specific activity was expressed in units per milligram of protein. The protein concentration was determined by using Bradford's method (1) with bovine serum albumin as the standard.

(ii) Method II.
Synthetic peptides were used for the determination of kinetic parameters. The reaction mixture consisted of 100 mM sodium acetate, pH 4.0, various concentrations (20 to 600 µM) of a synthetic peptide, and 0.185 nM enzyme in a final volume of 250 µl. The mixture without the enzyme was preincubated at 60°C, and the reaction was started by the addition of the enzyme. After incubation at 60°C for 10 min, the reaction was stopped by adding 1.0 ml of 1 M potassium phosphate, pH 7.0, and chilling the mixture on ice. The remaining peptide in the reaction mixture was routinely analyzed by a reversed-phase high-performance liquid chromatography (HPLC) system (termed system A) by using a Gilson 305 system equipped with a Shimadzu SPD-10A VP UV-VIS detector: column, YMC-Pack ODS-A A-303 (4.6 by 250 mm); flow rate, 0.7 ml/min; solvent A, 0.1% (vol/vol) trifluoroacetic acid; solvent B, 0.1% (vol/vol) trifluoroacetic acid in 60% (vol/vol) acetonitrile. After injection (50 µl) onto a column that was equilibrated with 36% solvent B, the column was initially developed isocratically for 5 min, followed by linear gradients from 36 to 45% solvent B in 15 min and from 45 to 100% solvent B in 1 min. The column was then washed isocratically with 100% solvent B for 5 min, followed by a linear gradient from 100 to 36% solvent B in 1 min. The chromatograms were obtained with detection at 215 nm, and the amounts of peptides were determined from peak integrals with authentic samples, which were used for calibration. Kinetic parameters were estimated by fitting the initial velocity data to the Michaelis-Menten equation by nonlinear regression analysis.

Purification of ScpA.
Purification of ScpA from the culture filtrate of A. sendaiensis strain NTAP-1 was completed at 0 to 5°C, unless otherwise stated, as follows. Buffer A was a 10 mM potassium phosphate buffer, pH 7.5. The culture broth of the strain NTAP-1 was centrifuged at 8,000 x g and 4°C for 15 min. The pH of the resultant supernatant (5 liters) was adjusted to 7.5 with 1 M NaOH, and the enzyme solution was applied to a column (400 ml) of DEAE-Sepharose Fast Flow (Amersham Biosciences, Piscataway, N.J.) equilibrated with buffer A. The column was washed with the same buffer. The enzyme was eluted with a linear gradient of 0 to 1 M NaCl in buffer A. The active fractions were combined. To the enzyme solution, ammonium sulfate was slowly added to a final concentration of 20% saturation. The enzyme solution was then applied to a column (40 ml) of Phenyl-Sepharose HP (Amersham Biosciences) equilibrated with buffer A containing ammonium sulfate at 20% saturation. The column was washed with the equilibration buffer. The enzyme activity was eluted with a linear gradient of ammonium sulfate (from 20 to 0% saturation) in buffer A (200 ml each). The active fractions were combined, dialyzed thoroughly against buffer A, and then subjected to Fast protein liquid chromatography on Mono Q HR10/10 (Amersham Biosciences) equilibrated with the same buffer. After loading the enzyme solution onto the column, followed by an extensive washing of the column with buffer A, the enzyme was eluted with a linear gradient of 0.15 to 0.5 M NaCl in buffer A in 75 min at a flow rate of 0.5 ml/min. The active fractions were combined and applied to a Gigapite hydoxyapatite column (40 ml; Seikagaku Co., Tokyo, Japan) equilibrated with buffer A. The column was washed with buffer A. The enzyme was eluted with a linear gradient of 10 to 400 mM potassium phosphate, pH 7.5 (80 ml each). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli (10). The proteins on the gels were visualized by silver staining or by staining with Coomassie brilliant blue R-250. Gelatin zymography of SDS-PAGE gels was performed according to the method of Wilson et al. (33), in which the heat treatment (at 97°C for 3 min) of the enzyme samples prior to electrophoresis was omitted.

Cloning of the scpA gene.
The chromosomal DNA was prepared and purified from strain NTAP-1 cells as described previously (29) and used as a template for PCR amplification of the partial scpA gene. The following degenerate oligonucleotide primers were synthesized: primer S1 (5'-GCITGGCARGARCAYGCIAAYGT-3') and primer A1 (5'-ATRTCRTGRAAIACRTCIGCIGG-3'), where I indicates inosine and R and Y indicate degenerate sites (R, A/G; Y, C/T). The PCR amplifications were completed by using a reaction mixture consisting of 1x TaKaRa PCR buffer (TaKaRa Shuzo), 0.2 mM concentrations of the deoxynucleoside triphosphates, 1 µM (each) primers S1 and A1, and 2.5 U of ExTaq DNA polymerase (TaKaRa Shuzo) with 500 ng of DNA template. The thermal cycling conditions were 95°C for 3 min for denaturation, followed by 5 cycles of 95°C for 30 s for denaturation, 45°C for 30 s for annealing, and 72°C for 1 min for extension and then 25 cycles of 95°C for 30 s for denaturation, 55°C for 30 s for annealing, and 72°C for 1 min for extension. The PCR product, which was 0.26 kbp in length, was digoxigenin (DIG) labeled by using a DNA labeling and detection kit (Roche Diagnostics, Mannheim, Germany). The genomic DNA was partially digested with the restriction enzyme Sau3A1 and was sized by 0.7% agarose gel electrophoresis. DNA fragments (3 to 6 kb) were recovered and were ligated with BamHI-digested and dephosphorylated pUC18. Escherichia coli DH5{alpha} cells were transformed with the ligation mixture, and ampicillin-resistant transformants were selected. The genomic library of ~20,000 clones was screened by colony hybridization with the DIG-labeled PCR fragment (see above) as a probe. The nucleotide sequences of positive clones were determined by using a CEQ2000XL DNA analysis system (Beckman Coulter). The GENETYX program (Software Development, Tokyo, Japan) was used for the analysis of nucleotide and deduced amino acid sequences. Because the positive clones were found to lack a stop codon, we further analyzed the genomic DNA clone containing the 3'-terminal region of the scpA gene by PCR amplification with primers S2 (5'-CGGGTTACGAGGTCGTGATCGAC-3', based on an internal stretch of the scpA gene) and A2 (5'-CGCCAGGGTTTTCCCAGTCACGAC-3', based on the vector sequence). The amplified fragments were further subjected to nested PCR with primers S3 (5'-TACCTGAACCCACGCTGTACCAG-3', based on an internal stretch of the scpA gene) and A3 (5'-CGACGTTGTAAAACGACGGCCAGT-3', based on a vector sequence). The PCR product was extracted from the gel after separation on a 0.8% agarose gel, directly subcloned into the pCR2.1-TOPO vector (Invitrogen, Groningen, Netherlands), and used for sequence analysis.

Expression and purification of recombinant ScpA (rScpA).
The full-length scpA gene was amplified by PCR with the chromosomal DNA of A. sendaiensis NTAP-1 as a template and primers 5'-GAGTGGAAGGTCATGAGCGAGCATGGAAAAACC-3' and 5'-AAAACTCGAGTCACGGCTGGGGTTGTGAGG-3', where the underlining indicates the BspHI and XhoI sites and the double underlining indicates the translation initiation and stop codons. The entire nucleotide sequence of the amplified DNA was confirmed by sequencing in both orientations. The amplified DNA was digested with BspHI and XhoI and inserted into the NcoI-XhoI sites of the pET15b (Novagen, Madison, Wis.) vector to obtain the plasmid pScpA, which carries the full-length scpA gene and was used to transform E. coli BL21(DE3) for the expression of the scpA gene.

Transformant cells harboring the plasmid pScpA were grown at 37°C in Luria-Bertani medium (1 liter) containing 50 µg of ampicillin/ml until the A600 reached 0.6. Expression of the scpA gene was attained by adding isopropyl-ß-D-thio-galactopyranoside to a final concentration of 0.8 mM, followed by further cultivation for 3 h. The cells were harvested by centrifugation (5,000 x g at 4°C for 10 min), suspended in an appropriate volume of a 0.05 M sodium acetate buffer, pH 4.0, and disrupted at 4°C by 10 cycles of ultrasonication (at 10 kHz for 1 min followed by an interval of 1 min). The cell debris was removed by centrifugation (18,000 x g at 4°C for 10 min), and the resultant supernatant (pH 4.0) was incubated at 55°C for 5 h, followed by centrifugation. Almost all of the endogenous E. coli proteins were precipitated by centrifugation, and the resultant supernatant contained the 37-kDa species of rScpA (see Results) with >96% homogeneity. The supernatant was concentrated by ultrafiltration with a YM-10 membrane and an Amicon 8200 unit. The concentrate was subjected to gel filtration on Superdex 200 HR 10/30 (Amersham Biosciences) equilibrated in buffer A containing 0.15 M NaCl. Active fractions contained homogeneous rScpA.

pH activity profiles.
The solubilization of Azocoll was assayed by method I, except that the reaction mixture contained 50 µmol of one of the following buffers: pH 2.5 to 3.5, Gly-HCl; pH 3.5 to 5.5, sodium acetate; pH 6.0 to 7.5, potassium phosphate; pH 8.0 to 9.0, Tris-HCl; and pH 9.0 to 10.0, Gly-NaOH.

Stability studies.
To examine the temperature stability of ScpA, the enzyme (0.1 mg/ml) was incubated in a 0.05 M sodium acetate buffer, pH 4.0, at 60°C. At the appropriate time intervals, an aliquot of the mixture was removed and assayed for residual enzyme activity by assay method I.

To examine the pH stability of ScpA, the enzyme (0.1 mg/ml) was incubated at 60°C in the following buffers (final concentrations, 0.05 M): pH 2.5 to 3.5, Gly-HCl; pH 3.5 to 5.5, sodium acetate; pH 6.0 to 7.5, potassium phosphate; pH 8.0 to 9.0, Tris-HCl; and pH 9.0 to 10.0, Gly-NaOH. After incubation for 30 min, the remaining activity was assayed by assay method I.

Protein chemical analyses.
To determine the internal amino acid sequences of native ScpA, the purified enzyme (200 pmol) was digested with trypsin (5 pmol) as described previously (30), and the resultant peptides were separated by the following reversed-phase HPLC system (termed system B): column, YMC-Pack ODS-A A-303 (4.6 by 250 mm); flow rate, 0.7 ml/min; solvent A, 0.1% (vol/vol) trifluoroacetic acid; solvent B, 0.1% (vol/vol) trifluoroacetic acid in 80% (vol/vol) acetonitrile. After injection of peptides onto a column that was equilibrated with 0% solvent B, the column was initially developed isocratically for 3 min, followed by a linear gradient from 0 to 100% solvent B in 45 min. The column was then washed isocratically with 100% solvent B for 5 min, followed by a linear gradient from 100 to 0% solvent B in 1 min. The amino acid sequences of the purified peptides were determined by automated Edman degradation with a gas-phase Shimadzu PPSQ-10 sequencer equipped with an online PTH amino acid analyzer LC10A.

To determine the N-terminal amino acid sequence of the 38- and 37-kDa species of rScpA (see Results), the enzyme samples were subjected to SDS-PAGE without prior boiling of the samples. Protein bands in the gel were transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting, and the membrane was stained with Coomassie brilliant blue R-250. Stained portions of the membrane corresponding to each rScpA species were excised with dissecting scissors and subjected to automated Edman degradation.

Separation and sequencing of collagen peptides were performed as follows. The reaction mixture (final volume, 1 ml) consisted of 5 mg of collagen and 0.5 µg of ScpA in a 0.1 M sodium acetate buffer, pH 4.0. The reaction was started by the addition of enzyme, and the mixture was allowed to stand at pH 4.0 at 60°C for 1 h. The resultant peptides (collagen peptides) were separated and purified until homogeneous by reversed-phase HPLC (system B) followed by sequencing by automated Edman degradation.

MALDI-TOF (MS) analysis of ScpA.
Protein samples of 2 to 3 pmol were mixed with a matrix solution (0.1% trifluoroacetic acid-acetonitrile [2:1], saturated with 3,5-dimethoxy-4-hydroxycinnamic acid). The mixture was dried on a target table and analyzed by matrix-assisted laser desorption ionization-time of flight (mass spectrometry) MALDI-TOF (MS) (REFLEX III; Bruker).

Nucleotide sequence accession number.
The nucleotide sequence of scpA reported in this paper has been submitted to the DNA Data Bank of Japan (DDBJ) with the accession number AB085855.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification, molecular mass, and stability of ScpA.
ScpA was constitutively and extracellularly produced by A. sendaiensis strain NTAP-1. ScpA could be purified in high yield (52%) from the culture filtrate of strain NTAP-1 by four chromatographic steps consisting of anion exchange, hydrophobic interaction, and hydroxyapatite chromatographies (Table 1). SDS-PAGE of the purified enzyme yielded a single protein band. Gelatin zymography showed that this band was positive to gelatinolytic activity (Fig. 1). MALDI-TOF (MS) analysis of the same enzyme preparation yielded an m/z of 37,100 ± 450 (see also legend to Fig. 1). The native molecular mass of the purified ScpA was estimated to be 40 kDa by gel permeation chromatography on a Superdex 200HR column. These results show that the enzyme was monomeric. ScpA was stable at pH 3.5 to 5.0 (at 60°C for 2 h) and retained more than 80% of its activity after incubation at 60°C and pH 4.0 for 4 h. However, it was completely inactivated after incubation at 60°C and pH 7.0 for 30 min.


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  		TABLE 1. Purification of ScpA

 


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  		FIG. 1. SDS-PAGE and gelatin zymography of purified ScpA from the culture filtrate of A. sendaiensis strain NTAP-1. The purified ScpA (lane 1) and marker proteins (lane M) were simultaneously electrophoresed by using the conditions of Laemmli (10) and stained by silver staining. The purified enzyme was also analyzed for gelatinolytic activity by gelatin zymography (lane 2) by using the conditions of Wilson et al. (33). Molecular sizes of marker proteins are indicated by arrows. It should be noted that the apparent molecular mass estimated based on the electrophoretic mobility in SDS-PAGE (46 kDa) was significantly larger than the m/z values obtained from the MALDI-TOF (MS) analysis of the gel-purified enzymes (e.g., m/z 37,100 ± 450) and the value calculated from the deduced amino acid sequence of mature rScpA (36,702 Da). This arises from anomalously low electrophoretic mobility of ScpA in SDS-PAGE, which is probably due to the relatively high contents of acidic amino acid residues in the enzyme, as suggested by Zirwes et al. (35). Similar observations are also reported for scytalidopepsin B (31) and kumamolysin (26).

 
Catalytic properties and substrate specificity.
The optimum pH and temperature for degradation of Azocoll by purified ScpA were pH 3.9 and 60°C, respectively, under the conditions described in Materials and Methods. The specific activities of the purified enzyme for the degradation of hide powder (Azocoll), collagen, albumin, and casein at pH 4.0 and 60°C are summarized in Table 2. Among these macromolecular substrates, the highest specific activities were found with Azocoll and collagen. The enzyme showed none of the keratinolytic and elastin-degrading activities. Thus, ScpA was a thermoacidophilic proteinase highly specific for collagen. A variety of commercially available peptides were also examined for their ability to serve as the substrate for ScpA (Table 3). However, none of them, including Suc-Gly-Pro-Leu-Gly-Pro-4-methyl-coumaryl-7-amide (MCA), which was a general substrate for bacterial collagenases (8), acted as a substrate for ScpA. To obtain specific peptide substrates suitable for the kinetic characterization of ScpA, collagen was digested with purified ScpA, and the resultant peptides were separated by reversed-phase HPLC. More than 50 peptide peaks were generated upon digestion. Several of the resultant peptides were analyzed for their amino acid sequences, which were subsequently compared with the known primary structure of the substrate. Although we did not determine all of the possible cleavage sites in collagen, we did identify the following: Glu108*Gly109, Arg183*Gly184, Arg403*Gly404, Ala489*Gly490 (for the {alpha}1 chain), and Arg40*Gly41 (for the {alpha}2 chain), where asterisks denote the cleavage sites. On the basis of these results, we designed the following peptides, each of which has a partial amino acid sequence around a cleavage site in the collagen {alpha}1 chain: peptide I, Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly; and peptide II, Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser. We then analyzed the ability of these peptides to serve as the substrate for ScpA by HPLC. The results showed that these were good substrates for ScpA, and automated Edman degradation analysis of the product peptides revealed that cleavage took place at the expected site (indicated by an asterisk). The kinetic parameters of peptides I and II were determined by assay method II and are summarized in Table 3.


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  		TABLE 2. Substrate specificity

 

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  		TABLE 3. Substrate specificity

 
Inhibitors.
The enzyme was partially inhibited by 100 µM AAFCH2Cl (residual activity, 16%), 100 µM leupeptin (21%), 1 mM Fe2+ (26%), and 1 mM Hg2+ (30%). The enzyme showed more than 50% residual activities with 1 mM metal ions (Ca2+, Cd2+, Co2+, Cu2+, Mg2+, Mn2+, Ni2+, Sn2+, and Zn2+), inhibitors (EDTA, pepstatin, DAN, ß-mercaptoethanol, PMSF, E-64, N-ethylmaleimide [1 mM each], and tyrostatin [1 µM]), and 1% detergent (Tween 80, Tween 20, and Triton X-100).

Cloning and expression of the scpA gene.
Partial amino acid sequences of the purified ScpA were determined. We designed the PCR primers (S1 and A1) (see Materials and Methods) on the basis of these sequences and executed PCR by using the chromosomal DNA of A. sendaiensis as a template and these primers. The 266-bp fragment encoding the partial amino acid sequences of ScpA was amplified and then DIG labeled. Using this fragment as a probe, the genomic library was screened for the scpA gene. A positive clone with the longest insert was found to contain a partial scpA sequence of 1,343 bp lacking the 3'-terminal region. To reveal the 3'-terminal region of the scpA gene, we carried out a PCR with the genomic library as a template. The primers used for this PCR (S2 and A2) (see Materials and Methods) were designed on the basis of the nucleotide sequences of the partial scpA gene and the pUC18 vector, respectively. Subsequent nested PCR with an alternative set of primers (S3 and A3) yielded a single PCR product of 1.5 kbp, which was found to contain the lacking portion of the scpA gene as analyzed by DNA sequencing. The open reading frame of ScpA consisted of 1,659 bp and encoded a protein of 553 amino acid residues with a calculated molecular mass of 57,167 Da.

The scpA gene was expressed under the control of the T7 lacI promoter in E. coli BL21(DE3) cells as a soluble protein at a level of 10 mg of protein/liter. The recombinant enzyme, rScpA, could be efficiently purified from the crude extracts of transformant cells by a two-step purification procedure: an acid treatment of the extracts at 55°C followed by gel filtration chromatography (for details, see Materials and Methods). MALDI-TOF (MS) analysis of the enzyme preparation thus obtained yielded an m/z of 36,960 ± 450, and automated Edman degradation of the enzyme revealed an N-terminal amino acid sequence starting from Ala190, suggesting that the precursor rScpA of 57 kDa should undergo processing into a mature form of 37 kDa. When the rScpA was purified with an acid treatment at 4°C (instead of 55°C) prior to gel filtration, the resultant preparation contained two distinct rScpA species of different sizes (Fig. 2); one yielded an m/z of 38,200 ± 450 and an N-terminal amino acid sequence starting from Phe173 (termed the 38-kDa species), while another was the mature 37-kDa species mentioned above. When the acid treatment was performed in the presence of 100 µM AAFCH2Cl, which was an ScpA inhibitor (see above), rScpA remained a mixture of the 38- and 37-kDa species (Fig. 2); thus, the 37-kDa species should be produced from the 38-kDa species in an autocatalytic manner. Thus, during the maturation of ScpA, the first cleavage took place at the His172*Phe173 site of the 57-kDa precursor, probably by the action of an endogenous proteinase of E. coli, to give rise to the 38-kDa species, which then underwent autocatalytic cleavage at Ala189*Ala190 to produce the mature 37-kDa form of the enzyme (Fig. 2). Because one of the partial amino acid sequences determined with the native ScpA (Leu540 to Pro553) (Fig. 3) was found to contain the C terminus of the deduced amino acid sequence, the ScpA precursor contains no C-terminal pre (or pro) sequence. The molecular mass, substrate specificity, catalytic properties, and thermostability of the 37-kDa species of rScpA were indistinguishable from those of the native ScpA.



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  		FIG. 2. Detection of a 38-kDa precursor of rScpA by SDS-PAGE. The recombinant enzyme, rScpA, was purified from the crude extracts of transformant cells by acid treatment (at pH 4.0 for 5 h) under different conditions (lanes 2 to 4, see below) followed by gel filtration chromatography. Purified rScpA species were subsequently analyzed by SDS-PAGE as described in Materials and Methods. Native ScpA purified from the culture filtrate of strain NTAP-1 (lane 1) was also electrophoresed simultaneously. Acid treatment conditions were as follows: lane 2, at 4°C; lane 3, at 55°C; and lane 4, at 55°C in the presence of 100 µM AAFCH2Cl. Open and closed circles indicate the 37- and 38-kDa species of rScpA as identified by MALDI-TOF (MS) analysis, respectively, and the 37-kDa species was the mature form of rScpA.

 


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  		FIG. 3. Alignment of the amino acid sequences of the mature forms of ScpA and other S53 family enzymes: kumamolysin (26) (DDBJ/EMBL/GenBank accession number AB070740), ScpP (23) (accession no. D37970), ScpX (19) (accession no. D83740), and CLN2 (32) (accession no. O14773). These sequences are labeled with amino acid numbering, where the N termini of the mature ScpA, kumamolysin, ScpP, ScpX, and CLN2 are Ala190, Ala189, Ala216, Ala238, and Leu194, respectively. Identical amino acid residues among all of the proteinases are shown in white type on a black background, and those identical among 3 or 4 proteinase sequences are shadowed. Putative active-site residues are indicated by asterisks above the ScpA sequence. The Gly-Xaa-Ser motif unique to serine hydrolases is boxed.

 
Sequence similarity.
In the extensive search for amino acid sequence similarity, ScpA was found to show high similarity to members of the S53 family, which contains the serine-carboxyl proteinases of Pseudomonas sp. strain 101 (ScpP, 31% identity) (23), Xanthomonas sp. strain T22 (ScpX, 30%) (19), and humans (CLN2, 31%) (32) (Fig. 3). Unexpectedly, the highest similarity (85% identity) was found with kumamolysin, produced by Bacillus sp., species nova, strain MN-32 (15), whose primary structure has been published very recently (26) (Fig. 3). Although the substrate specificity of kumamolysin has been extensively studied by using synthetic peptides of 8 amino acids (21), the substrate specificity of kumamolysin toward macromolecular substrates, including collagen, has not been defined (15), and the physiological substrate of kumamolysin has not been established. These circumstances prompted us to examine the collagenolytic activity of the homogeneous preparation of the recombinant kumamolysin. It was revealed that kumamolysin was also a collagenolytic enzyme (Table 2). For comparison, we also determined the collagenolytic activity of the homogeneous preparation of ScpP, which has been shown to be specific for globular proteins (data not shown). ScpP showed only low specific activity (6.3 U/mg, Azocoll assay) at pH 4.0 and 37°C, which was 2% of that of ScpA.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A specificity analysis of homogeneous ScpA with several macromolecular substrates revealed that it could effectively degrade hide powder and was highly specific for collagen. Because strain NTAP-1 could not grow on peptone but could utilize hide powder, collagen, or gelatin as a sole proteinaceous nitrogen source (N. Tsuruoka, T. Nakayama, and T. Nishino, unpublished data), the observed specificity of ScpA should be of physiological significance for the growth of this bacterium. The primary-structure analysis of peptides derived from collagen upon digestion with ScpA suggested that the enzyme should preferably act on the Pro-Xaa*Gly-Yaa-Zaa sequence of the {alpha}1 and {alpha}2 chains of collagen, and one of the preferred amino acids at the P1 position (Xaa) in collagen was suggested to be Arg; this was corroborated by the observation that the kcat/Km value of peptide II containing this amino acid at its P1 position greatly exceeded the kcat/Km value of peptide I, which has Ala at the same position (Table 3). Xaa, Yaa, and Zaa denote arbitrary amino acids. The amino acid residues around the cleavage site of the substrate are designated P4-P3-P2-P1*P1'-P2', where the residues on the acyl side of the scissile peptide bond are numbered P1, P2, and P3, etc., and those towards the C terminus are labeled P1' and P2', etc., with the scissile bond (indicated by the asterisk) located between P1 and P1'.

Intensive characterization of purified ScpA established several distinguishing features of this collagenolytic enzyme in comparison with known bacterial collagenases and other collagenolytic enzymes. Namely, ScpA was essentially a carboxyl proteinase with an optimum pH for catalytic activity at 3.9, in striking contrast with collagenases and collagenolytic serine proteinases, whose activities show the maximum at pH 8 to 9. However, ScpA was insensitive to specific inhibitors of aspartic proteinases, such as pepstatin and DAN. It was also insensitive to specific inhibitors of metalloproteinases (EDTA), cysteine proteinases (N-ethylmaleimide), and serine proteinases (PMSF)(it has been shown that some serine-carboxyl proteinases are also insensitive to PMSF [15, 20, 22]). For comparison, known collagenases are strongly inhibited by metal chelators, and the collagenolytic serine proteinases are inhibited by PMSF. In addition, ScpA was one of the most thermostable enzymes among the collagen-specific proteinases reported to date; it retained more than 80% of the original activity after incubation at pH 4.0 and 60°C for 1 h. A recent report shows that a novel collagenolytic proteinase of Bacillus sp. strain MO-1, a serine proteinase, was thermostable as well, being active after incubation at pH 7.5 and 60°C for 30 min (24). In contrast, however, the collagenases of Streptomyces parvulus subsp. citrinus (Nacalai Tesque) and Clostridium histolyticum were completely inactivated after a 5-min incubation at 60°C and pH 7.5 (17).

The observed catalytic properties and stability of ScpA revealed several potential practical advantages of ScpA in the industrial production of collagen peptides. Namely, the observed high thermostability of ScpA should allow the efficient enzymatic production of collagen peptides on an industrial scale, which has been hampered by the insufficient stability of known collagenases. Moreover, ScpA was most active under thermoacidophilic conditions (at 60°C and pH 4), which can effectively eliminate the possibility of microbial contamination of the reaction system; this is of particular importance for the processing of collagen and gelatin, which are nutritionally rich for microbial growth. These results also suggest that ScpA may be applicable to the continuous production of peptides from gelatin by an immobilized system under such thermoacidophilic conditions. ScpA could efficiently degrade hide powder, suggesting that the use of ScpA should also allow the utilization of such relatively low-grade collagenous materials, instead of purified collagen and gelatin, as starting materials for the production of collagen peptides. At neutral pHs, the enzyme exhibited virtually no enzyme activity and was highly unstable at 60°C, indicating that the enzymatic reaction can be easily stopped by shifting the pH of the reaction system to 7.0; this would also be a practical advantage in an industrial application of this enzyme.

In order to clarify to which proteinase family this unique collagenolytic enzyme belongs, we cloned the gene coding for ScpA and sequenced it. It was revealed that the scpA gene encoded a protein of 553 amino acid residues with a calculated molecular mass of 57,167 Da, which was significantly larger than the value (molecular mass, 37 kDa) determined with purified preparations of the native and recombinant enzymes. The analysis suggested that, in the E. coli transformant cells, the 57-kDa precursor of rScpA should undergo the two-step removal of the N-terminal portions of the precursor to produce the mature 37-kDa species. Primary-structure analysis of ScpA revealed that ScpA showed sequence similarities to the members of the S53 family of proteinases, i.e., kumamolysin (15), ScpP (22, 23), ScpX (19, 20), and CLN2 (32). Until recently, these S53 family members had been classified into the A7 family of aspartic peptidase, mainly on the basis of their optimum pH for activity (pH 3.0 to 4.5) and the results of chemical modification (25) as well as site-directed mutagenesis studies (5). In 2001, however, the crystal structure of ScpP in complex with its specific inhibitor, tyrostatin, was solved, and the overall main chain conformation of ScpP was found to be very similar to that of subtilisin (34), which is a serine proteinase. Moreover, it was revealed that the Ser502 of ScpP covalently bound the inhibitor and appears to constitute the putative catalytic Asp299-Glu295-Ser502 triad, which could be stereoscopically superimposed with the catalytic triad of Asp32-His64-Ser221 identified in the three-dimensional structure of subtilisin. These observations led to the proposal that, despite their optimum pH for activity being at the acidic region, ScpP and its homologs should be essentially serine proteinases and should more appropriately be categorized as a subfamily of serine proteinases, called the serine-carboxyl proteinase family (the S53 family) (34). In the ScpA sequence, we could identify the conservations of the putative catalytic residues (Asp271, Glu267, and Ser467) along with that of the Gly-Xaa-Ser motif, which is ubiquitously found among serine proteinases, including S53 family enzymes (28) (Fig. 3). The optimum pH for activity (pH 3.9) and the inhibitor specificity (insensitivity to pepstatin and DAN) of ScpA are also consistent with the specific properties of this family of enzymes. Taking these lines of evidence together with the fact that the ability of ScpA to degrade collagen was crucial for bacterial growth, as mentioned earlier, we conclude that ScpA is a collagenolytic member of the S53 family.

The entire amino acid sequence of ScpA was very similar to that of kumamolysin (identity, 85%), whose gene cloning, sequencing, and expression have been published very recently (26). However, the preferred macromolecular substrates of kumamolysin are not yet known in detail (15, 21). In addition, the kumamolysin-producing bacterium, Bacillus, species nova, strain MN32, has been shown to have a requirement for a nitrogen source distinct from that of strain NTAP-1; strain MN32 could grow on peptone used as a sole nitrogen source (15), whereas strain NTAP-1 could not grow on peptone but could only grow on gelatin and collagen (see above). Therefore, we examined the ability of kumamolysin to degrade collagen and other macromolecular substrates. The results showed that kumamolysin also showed the highest reactivity toward collagen among the macromolecular substrates compared. Thus, we propose that kumamolysin is also a collagenolytic member of the S53 family. The subsite preference of kumamolysin has been systematically analyzed by using synthetic peptide substrates, and it was revealed that kumamolysin shows a strong preference at the P2 position for small amino acid residues, such as Pro and Ala, whereas bulky amino acid residues are acceptable at the P3, P1', and P2' positions (21). This subsite preference consistently explains the observed preference of kumamolysin for collagen, which contains Gly, Pro, and Ala in abundance in its primary structure. These results strongly suggest that the preference of ScpA for collagen may also be explained in terms of a subsite specificity, which is very similar to that of kumamolysin. Detailed analyses of the subsite specificity of ScpA with the peptide II derivatives, in conjunction with three-dimensional structural studies of ScpA and collagen, are currently under way in this laboratory to further understand the collagenolytic action of ScpA.


   ACKNOWLEDGMENTS
 
This work was supported in part by a grant from the CREST Noike project entitled "Energy recovery and recycling of resources from municipal wastes by advanced technologies for environmental protection."

We thank Masayuki Kobayashi, Graduate School of Engineering, Tohoku University, for his kind guidance with the MALDI-TOF (MS) measurements.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 07 Aoba-yama, Sendai 980-8579, Japan. Phone and fax: 81-22-217-7270. E-mail: nishino{at}mail.cc.tohuku.ac.jp. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bradford. M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
  2. Gaffney, P. J., T. A. Edgell, P. A. Dawson, A. W. Ford, and E. Stocker. 1996. A pig collagen peptide fraction. A unique material for maintaining biological activity during lyophilization and during storage in the liquid state. J. Pharm. Pharmacol. 48:896-898.[Medline]
  3. Harrington, D. J. 1996. Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect. Immun. 64:1885-1891.[Medline]
  4. Honda, S. 1998. Dietary use of collagen and collagen peptides for cosmetics. Food Style 21:54-60. (In Japanese.)
  5. Ito, M., S. Narutaki, K. Uchida, and K. Oda. 1999. Identification of carboxyl residues in pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 that participate in catalysis and substrate binding. J. Biochem. 125:210-216.[Abstract/Free Full Text]
  6. Kawabata, S., T. Miura, T. Morita, H. Kato, K. Fujikawa, S. Iwanaga, K. Takada, T. Kimura, and S. Sakakibara. 1988. Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin. Eur. J. Biochem. 172:17-25.[Medline]
  7. Khare, S. D., C. J. Krco, M. M. Griffiths, H. S. Luthra, and C. S. David. 1995. Oral administration of an immunodominant human collagen peptide modulates collagen-induced arthritis. J. Immunol. 155:3653-3659.[Abstract]
  8. Kojima, K., H. Kinoshita, T. Kato, T. Nagatsu, K. Takada, and S. Sakakibara. 1979. A new and highly sensitive fluorescence assay for collagenase-like peptidase activity. Anal. Biochem. 100:43-50.[CrossRef][Medline]
  9. Ku, G., M. Kronenberg, D. J. Peacock, P. Tempst, M. L. Banquerigo, B. S. Braun, J. R. J. Reeve, and E. Brahn. 1993. Prevention of experimental autoimmune arthritis with a peptide fragment of type II collagen. Eur. J. Immunol. 23:591-599.[Medline]
  10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
  11. Mizuno, K., T. Nakamura, S. Takada, S. Sakakibara, and H. Matsuo. 1987. A membrane-bound, calcium-dependent protease in yeast alpha-cell cleaving on the carboxyl side of paired basic residues. Biochem. Biophys. Res. Commun. 144:807-814.[CrossRef][Medline]
  12. Morita, T., H. Kato, S. Iwanaga, K. Takada, and T. Kimura. 1977. New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase. J. Biochem. 82:1495-1498.[Abstract/Free Full Text]
  13. Mumford, R. A., A. W. Strauss, J. C. Powers, P. A. Pierzchala, N. Nishino, and M. Zimmerman. 1980. A zinc metalloendopeptidase associated with dog pancreatic membranes. J. Biol. Chem. 255:2227-2230.[Abstract/Free Full Text]
  14. Murakami, K., T. Ohsawa, S. Hirose, K. Takada, and S. Sakakibara. 1981. New fluorogenic substrates for renin. Anal. Biochem. 110:232-239.[CrossRef][Medline]
  15. Murao, S., K. Ohkuni, M. Nagao, K. Hirayama, K. Fukuhara, K. Oda, H. Oyama, and T. Shin. 1993. Purification and characterization of kumamolysin, a novel thermostable pepstatin-insensitive carboxyl proteinase from Bacillus novo sp. MN-32. J. Biol. Chem. 268:349-355.[Abstract/Free Full Text]
  16. Nagase, H., C. G. Fields, and G. B. Fields. 1994. Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3). J. Biol. Chem. 269:20952-20957.[Abstract/Free Full Text]
  17. Nakayama, T., N. Tsuruoka, M. Akai, and T. Nishino. 2000. Thermostable collagenolytic activity of a novel thermophilic isolate, Bacillus sp. strain NTAP-1. J. Biosci. Bioeng. 89:612-614.
  18. Oda, K., Y. Fukuda, S. Murao, K. Uchida, and M. Kainosho. 1989. A novel proteinase inhibitor, tyrostatin, inhibiting some pepstatin-insensitive carboxyl proteinase. Agric. Biol. Chem. 53:405-415.
  19. Oda, K., M. Ito, K. Uchida, Y. Shibano, K. Fukuhara, and S. Takahashi. 1996. Cloning and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Xanthomonas sp. T-22. J. Biochem. 120:564-572.[Abstract/Free Full Text]
  20. Oda, K., T. Nakazima, T. Terashita, K. Suzuki, and S. Murao. 1987. Purification and properties of an S-PI (pepstatin Ac)-insensitive carboxyl proteinase from a Xanthomonas sp. bacterium. Agric. Biol. Chem. 51:3073-3080.
  21. Oda, K., S. Ogasawara, H. Oyama, and B. M. Dunn. 2000. Subsite preferences of pepstatin-insensitive carboxyl proteinases from prokaryotes: kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase. J. Biochem. 128:499-507.[Abstract/Free Full Text]
  22. Oda, K., M. Sugitani, K. Fukuhara, and S. Murao. 1987. Purification and properties of a pepstatin-insensitive carboxyl proteinase from a gram-negative bacterium. Biochim. Biophys. Acta 923:463-469.[Medline]
  23. Oda, K., T. Takahashi, Y. Tokuda, Y. Shibano, and S. Takahashi. 1994. Cloning, nucleotide sequence, and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Pseudomonas sp. 101. J. Biol. Chem. 269:26518-26524.[Abstract/Free Full Text]
  24. Okamoto, M., Y. Yonejima, Y. Tsujimoto, Y. Suzuki, and K. Watanabe. 2001. A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1. Appl. Microbiol. Biotechnol. 57:103-108.[CrossRef][Medline]
  25. Oyama, H., S. Abe, S. Ushiyama, S. Takahashi, and K. Oda. 1999. Identification of catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryotes by site-directed mutagenesis. J. Biol. Chem. 274:27815-27822.[Abstract/Free Full Text]
  26. Oyama, H., T. Hamada, S. Ogasawara, K. Uchida, H. Matsuo, B. B. Beyer, B. M. Dunn, and K. Oda. 2002. A CLN2-related and thermostable serine-carboxyl proteinase, kumamolysin: cloning, expression, and identification of catalytic residue. J. Biochem. 131:757-765.[Abstract/Free Full Text]
  27. Ravanti, L., and V. M. Kähäri. 2000. Matrix metalloproteinases in wound repair. Int. J. Mol. Med. 6:391-407.[Medline]
  28. Rawlings, N. D., and A. J. Barrett. 1999. Tripeptidyl-peptidase I is apparently the CLN2 protein absent in classical late-infantile neuronal ceroid lipofuscinosis. Biochim. Biophys. Acta 1429:496-500.[CrossRef][Medline]
  29. Saito, H., and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72:619-629.[Medline]
  30. Sawai-Hatanaka, H., H. Ashikari, Y. Tanaka, Y. Asada, T. Nakayama, H. Minakata, N. Kunishima, K. Fukuyama, H. Yamada, Y. Shibano, and T. Amachi. 1995. Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase. Biosci. Biotechnol. Biochem. 59:1221-1228.[Medline]
  31. Shimuta, K., N. Oda-Ueda, M. Washio, H. Oyama, K. Oda, and D. Tsuru. 2000. Expression and secretion of scytalidopepsin B, an acid protease from Scytalidium lignicolum, in yeast. Biosci. Biotechnol. Biochem. 64:1542-1546.[CrossRef][Medline]
  32. Sleat, D. E., R. J. Donnelly, H. Lackland, C. Liu, I. Sohar, R. K. Pullarkat, and P. Lobel. 1997. Association of mutation in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis. Science 277:1802-1805.[Abstract/Free Full Text]
  33. Wilson, M. J., M. Strasser, M. M. Vogel, and A. A. Sinha. 1991. Calcium-dependent and calcium-independent gelatinolytic proteinase activities of the rat ventral prostate and its secretion: characterization and effect of castration and testosterone treatment. Biol. Reprod. 44:776-785.[Abstract]
  34. Wlodawer, A., M. Li, Z. Dauter, A. Gustchina, K. Uchida, H. Oyama, B. M. Dunn, and K. Oda. 2001. Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes. Nat. Struct. Biol. 8:442-446.[CrossRef][Medline]
  35. Zirwes, R. F., M. S. Schmidt-Zachamann, and W. W. Franke. 1997. Identification of a small very acidic constitutive nucleolar protein (NO29) as a member of the nucleoplasmin family. Proc. Natl. Acad. Sci. USA 94:11387-11392.[Abstract/Free Full Text]

Applied and Environmental Microbiology, January 2003, p. 162-169, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.162-169.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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