Substrate specificity of carboxyl proteinase fromPycnoporus coccineus,a wood-deteriorating fungus

A carboxyl proteinase was purified from submerged-culture filtrate of a wood-deteriorating basidiomycete,Pycnoporus coccineus. The purified enzyme was found to be essentially homogeneous in disc gel electrophoresis tests at pH 9.4 and 2.3. The specificity and mode of action ofP. coccineus carboxyl proteinase I_a were investigated with the oxidized B-chain of insulinP. coccineus carboxyl proteinase I_a hydrolyzed primarily three peptide bonds, Ala¹⁴-Leu¹⁵, Tyr¹⁶-Leu¹⁷, and Phe²⁴-Phe²⁵ bonds, in the oxidized B-chain of insulin.     

Protective Effect of Some Anions on the Heat-Denaturation of Ovotransferrin

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 °C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H₂O₂, 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P₁ position, in descending order. The k _cat, K _m and k _cat/K _m values for Z-Val-Lys-Met-MCA were 16.8±0.14 min^?1, 5.1±0.28 μM, and 3.3±0.28 μM^?1 min^?1 respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.     

Purification and Properties of Extracellular Carboxyl Proteases of Acid-tolerant Bacteria,Isolated from Compost

Four strains of acid-tolerant and protein-using bacteria were isolated from compost. Two of them, Gram-negative strains MB8 and MB11, were identified as a new genus close to Stenotrophomonas species MB8 and Burkholderia species MB11, respectively. Both bacteria produced extracellular carboxyl proteases (CP) in acid-casein-starch medium. The enzymes, termed CP MB8 and CP MB11, purified through ion exchange and gel filtration chromatographies had molecular weights of 61,000 (CP MB8) and 36,000 (CP MB11) as estimated by SDS-PAGE, and showed optimum activities with hemoglobin as a substrate at pH 3.5 (CP MB8) and pH 3.7 (CP MB11) at 55°C. Both of the enzymes were not inhibited by pepstatin, DAN, or EPNP. These results suggest that both enzymes are members of the pepstatin-insensitive carboxyl proteinase family (EC 3.4.23.33), except for a larger molecular weight of the CP MB8 enzyme.     

Streptomyces Pepsin Inhibitor-Insensitive Carboxyl Proteinase from Lentinus edodes

A Streptomyces-pepsin inhibitor (S-PI or Pepstatin Ac), and DAN-insensitive carboxyl proteinase was found in a still culture filtrate of Lentinus edodes. The new carboxyl proteinase was purified, and about 9 mg purified enzyme was obtained from 19 liters of culture filtrate, with 11% recovery. The enzyme showed a single band on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were 40,000 and pH 4.2, respectively. The enzyme did not contain histidine and was composed of 387 amino acid residues. The enzyme was most active between pH 2.7 ~ 2.9, and stable over a pH of 3.2 ~ 5.2 for 3 hr at 37°C. The enzyme was not inhibited by S-PI or synthetic pepsin inhibitors such as DAN and EPNP. The physicochemical and enzymological properties were very similar to those of Scytalidium lignicolum carboxyl proteinase A, which was reported to be an S-PI-, and DAN-insensitive carboxyl proteinase.     

1 '-(6-Hydroxyoctanoyl) nornicotine and 1 '-(7-Hydroxyoctanoyl) nornico-tine,Two New Alkaloids from Japanese Domestic Tobacco

A Streptomyces-pepsin inhibitor (S-PI or pepstatin Ac)-insensitive carboxyl proteinase was found in a still culture filtrate of Ganoderma lucidum (Mannen-take). The new carboxyl proteinase was purified, and about 15 mg of the purified enzyme was obtained from 15 liters of culture filtrate, with 13% recovery. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis.

The enzyme was most active at pH 3.2 toward hemoglobin, and at pH 2.0 toward casein, and stable only in the narrow pH range of 3.5 to 5.2 even under mild treatment (37°C for 3hr). The molecular weight and isoelectric point were 36,000 and pH 5.3, respectively. The enzyme did not contain methionine.

The enzyme was characterized by the following points: (1) the proteolytic activity was not inhibited by carboxyl proteinase inhibitors such as S-PI, DAN, and EPNP; (2) the enzyme was very unstable; (3) the caseinolytic activity was very low compared with the hydrolysis of hemoglobin (about 15%); (4) the enzyme split preferentially the Phe(24)–Phe(25) bond of oxidized insulin B-chain at the rate of 50% for total hydrolysis. These characteristics were compared with the carboxyl proteinases isolated from Scytalidium lignicolum and Lentinus edodes, which were reported to be SPI- and DAN-insensitive carboxyl proteinases.     

Carboxyl Proteinase from the Wood Deteriorating Basidiomycete Pycnoporus coccineus :Substrate Specificity with Oxidized Insulin Peptide B1 ~ B16 and B15 ~ B24, Angiotensin and Proangiotensin

The specificity of highly purified carboxyl proteinase from Pycnoporus coccineus (formerly designated Trametes sanguined) was investigated with oligopeptides at pH 2.7. Hydrolysis of oxidized insulin peptide Bl ~ B16 was observed at two peptide bonds (His¹⁰-Leu¹¹ and Ala¹⁴-Leu¹⁵) during 3-hr incubation. The enzyme did not hydrolyze oxidized insulin peptide B15 ~ B24. Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotensin (formerly designated angiotensin I) was also at the Tyr⁴-Ile⁵ bond. In conclusion, peptide bonds which have a hydrophobic amino acid in the P′₁ position (as defined by Schechter and Berger, Biochem. Biophys. Res. Commun., 27, 157 (1967)) are preferentially cleaved by the trypsinogen activating carboxyl proteinase of Pycnoporus coccineus.     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.     

Studies on an Alkaline Proteinase of Aspergillus sydowi

An alkaline proteinase of Aspergillus sydowi (Bainier et Sartory) Thom et Church has been purified approximately 4.5-fold from a culture filtrate by fractionation with ammonium sulfate, treatment with acrynol and Alumina gel C_γ, and DEAE-Sephadex column chromatography. The purified proteinase obtained as needle crystals was monodisperse in both the ultracentrifuge and the electrophoresis on polyacrylamide gel.

The optimum pH and temperature for the activity were 8.0 and 40°C, respectively. Fifty per cent of the activity was lost at 45°C within ten minutes and 95% at 50°C. At 5°C, the enzyme was highly stable at the range of pH 6 to 9. None of metallic salts tested promoted the activity, but Zn⁺⁺, Ni⁺⁺ and Hg⁺⁺ were found to be inhibitory. Sulfhydryl reagent, reducing and oxidizing reagents tested except iodine had no effect on the activity, but potato inhibitor, DFP and NBS caused a marked inhibition.

The alkaline proteinase from Aspergillus sydowi was markedly protected from inactivation by the presence of Ca⁺⁺ in the enzyme solution. The protective effect of Ca⁺⁺ was influenced remarkably by the pH values of the enzyme solution, i.e., optimum concentrations of Ca⁺⁺ for the protective effect at pH 7.1, 7.5 and 7.8 were 10^?2, 10^?3 and 10^?4 M, respectively. Conversely, at higher pH values such as 9.0, Ca⁺⁺ accelerated the rate of inactivation. There was a parallelism between the loss in activity and the increase in ninhydrin-positive material in the enzyme solution.

The proteinase acted on various denaturated proteins, but not on native proteins. In digestion of casein by the proteinase, 92% of nitrogen was turned into soluble form in 0.2 m trichloroacetic acid solution, with 14~17% of peptide bonds being hydrolyzed. Casein hydrolyzed with the Asp. sydowi proteinase was further hydrolyzed by Pen. chrysogenum, B. subtilis or St. griseus proteinases, which further increased the free amino residues in the reaction mixtures. On the contrary, the Asp. sydowi proteinase reacted only slightly on casein hydrolyzed by the above-mentioned proteinases.     

Purification and Properties of an S-PI(Pepstatin Ac)-insensitive Carboxyl Proteinase from a Xanthomonas sp. Bacterium

A S-PI(Pepstatin Ac)-insensitive carboxyl proteinase was found in culture filtrate of a Xanthomonas sp. bacterium. The carboxyl proteinase was highly purified and about 100 mg of the enzyme was obtained from 601 of culture filtrate, with a recovery of 25%. The optimum condition for the action of the purified enzyme toward casein was approx. pH 2.7 and its activity was not inhibited by any of such carboxyl proteinase inhibitors as Pepstatin, S-PI, and DAN but EPNP inhibited it. Such behavior of the enzyme against inhibitors resembles that of Pseudomonas sp. carboxyl proteinase, the first found from a bacterium. Some differences were observed, however, in their properties such as optimum pH, isoelectric point, and amino acid composition.     

Enzymatic Properties of Alkaline Proteinase from Aspergillus candidus

Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg²⁺ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae.     

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°C. The enzyme cleaved the oxidized insulin B chain initially at Leu¹⁵-Tyr¹⁶ and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH₄)₂SO₄-saturated acetate buffer (pH 5.0) as plank-like cyrstals.     

Purification and Some Properties of Carboxyl Proteinase in Mycelium of Lentinus edodes

The effect of Streptomyces-pepsin inhibitor (S-PI) on fruit-body formation of Lentinus edodes (Berk.) Sing, was studied. The addition of S-PI to the culture medium (5 ~ 10 µg/ml) shortened the time required for mature fruit-bodies, and increased the fruiting-percentage and the overall yield 3.4 times compared to the control.

The intracellular proteinase in the mycelium was investigated. Proteinases having an optimal pH of 2.7 and 7.0 were found in the vegetative mycelial extract. When S-PI was added to the culture medium, their activities were strikingly changed; the carboxyl proteinase activity was remarkably decreased, and, in the contrary, the metal proteinase activity was increased to 1.5 times that of the control.

The carboxyl proteinase was purified. This enzyme was strongly inhibited by S-PI and synthetic pepsin inhibitors such as DAN and EPNP. The molecular weight and isoelectric point were 43,000 and pH 3.4, respectively.     

Isolation and Characterization of Two Momordins,Ribosome-Inactivating Proteins from the Seeds of Bitter Gourd (Momordica charantia)

Cathepsins L and L-like (58 kDa) proteinase from mackerel were purified to electrophoretical homogeneity by Concanavalin A-Sepharose and Econo-Pac S chromatographies. The molecular weights of cathepsins L and L-like proteinase were 30,000 and 58,000, and the optimal pH for the hydrolysis of Z-Phe-Arg-MCA (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-[4-methyl] coumarylamide) were 5.0 and 5.5, respectively. The stability of both purified proteinases at various pHs was low, when the pH was above 7.0. According to the substrate specificity analysis, these proteinases hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA, but did not hydrolyze Z-Arg-MCA and L-Arg-MCA. The activities of these two proteinases were effectively activated by cysteine and dithiothreitol. Their thiol-dependent proteolytic activity against Z-Phe-Arg-MCA was strongly inhibited by E-64 (trans-epoxysuccinyl-L-leucylamido[4-guanidino]butane), antipain, chymostatin, iodoacetic acid, and leupeptin, but not inhibited by pepstatin or phenylmethane sulfonyl floride. The inactivation rate constants (K_D) of cathepsins L and L-like proteinases at 50°C were 5.1 × 10^?5 and 6.9 × 10^?4 s^?1, respectively. K⁺, Na⁺, Mg⁺, and Sr⁺ did not affect them, while Zn²⁺, Cd²⁺, Co²⁺, Ni²⁺, Cu²⁺, Hg²⁺, Fe²⁺, and Fe³⁺ inhibited the activity of the purified cathepsins L and L-like proteinase.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

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

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 (k_cat, 5.41 s⁻¹; K_m, 32 μM) and Met-Gly-Pro-Arg^*Gly-Phe-Pro-Gly-Ser (k_cat, 351 s⁻¹; K_m, 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.     

A neutral proteinase produced by Bacillus cereus with high sequence homology to thermolysin: Production,isolation and characterization

Summary Extracellular neutral proteinase was produced in 10 l and 240 l batch cultivations of Bacillus isolate X-3, identified as B. cereus and deposited as DSM 3101. The enzyme concentration was about 37–47 mg/l in the fermentation broth. The enzyme was extracted from the medium by adsorption chromatography with Amberlite XAD-7-resin, and further purified by acetone precipitation and affinity chromatography. The mol. wt. is 35 000 Da. The enzyme is thermostabilized by calcium, inhibited by EDTA and o-phenanthrolin and has its pH-optimum at pH 6.8. The specific activity is 4.36·10^-4 kat·mg^-1 at 35°C and the k _cat/K _m on FAGLA (furylacryloyl-glyleu-NH₂) is 2.25·10⁴ M^-1 s^-1 at 30°C, pH 6.8. The proteinase is stable up to 60°C. The N-terminal amino acid sequence exhibits a high sequence homology (63%) to thermolysin and a low homology (18%) to B. subtilis neutral protease A. The enzyme may therefore be suitable for structural comparison with thermolysin in order to study factors affecting thermostability.     

Trypanosoma cruzi: Isolation and characterization of a proteinase

Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K_m = 2.5 mg/ml) as well as bovine and human hemoglobins (K_m = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO^2?₃, mercaptoethanol, cysteine) and was inhibited by sulfhydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60×) of the proteinase was carried out as follows: (1) precipitation at ?20 C, pH 4.5, with 80% acetone, (2) gel filtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents.     

Phylogenetic analysis of alkaline proteinase producing fluorescent pseudomonads associated with green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> L.) rhizosphere

Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099^T), Pseudomonas aureofaciens (NCIM2026^T), and Pseudomonas aeruginosa (MTCC2582^T). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH₄)₂SO₄ precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn²⁺, Cu²⁺, and Ni²⁺ ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.     

Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

Subsite preferences of pepstatin-insensitive carboxyl proteinases from prokaryotes: kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase

Kumamolysin, a carboxyl proteinase from Bacillus novosp. MN-32, is characterized by its thermostability and insensitivity to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidated using two series of synthetic chromogenic substrates: P(5)-P(4)-P(3)-P(2)-Phe*Nph (p-nitrophenylalanine: *cleavage site)-P(2)'-P(3)', in which the amino acid residues at the P(5)-P(2), P(2)' and P(3)' positions were systematically substituted. Among 74 substrates, kumamolysin was shown to hydrolyze Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this peptide were K(m) = 41+/-5 microM, k(cat) = 176+/- 10 s(-1), and k(cat)/K(m) = 4.3+/-0.6 mM(-1) x s(-1). These systematic analyses revealed the following features: (i) Kumamolysin had a unique preference for the P(2) position. Kumamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P(2) position; this was also observed for the pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al. (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomonas sp. 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas sp. T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P(2) position. (ii) Kumamolysin preferred such charged amino acid residues as Glu or Arg at the P(2)' position, suggesting that the S(2)' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4. In general, the S(2)' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydrophilic nature of the S(2)' subsite was confirmed to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases from prokaryotes.