Purification and characterization of intracellular and extracellular α‐glucosidases from Geobacillus toebii strain E134

Two different α‐glucosidase‐producing thermophilic E134 strains were isolated from a hot spring in Kozakli, Turkey. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, the strain was proposed to be a species of G. toebii. Its thermostable exo‐α‐1,4‐glucosidases also were characterized and compared, which were purified from the intracellular and extracellular fractions with estimated molecular weights of 65 and 45 kDa. The intracellular and extracellular α‐glucosidases showed optimal activity at 65 °C, pH 7·0, and at 70 °C, pH 6·8, with 3·65 and 0·83 K_m values for the pNPG substrate, respectively. Both enzymes remained active over temperature and pH ranges of 35–70 °C and 4·5–11·0. They retained 82 and 84% of their activities when incubated at 60 °C for 5 h. Their relative activities were 45–75% and 45–60% at pH 4·5 and 11·0 values for 15 h at 35 °C. They could hydrolyse the α‐1,3 and α‐1,4 bonds on substrates in addition to a high transglycosylation activity, although the intracellular enzyme had more affinity to the substrates both in hydrolysis and transglycosylation reactions. Furthermore, although sodium dodecyl sulfate behaved as an activator for both of them at 60 °C, urea and ethanol only increased the activity of the extracellular α‐glucosidase. By this study, G. toebii E134 strain was introduced, which might have a potential in biotechnological processes when the conformational stability of its enzymes to heat, pH and denaturants were considered. Copyright © 2011 John Wiley & Sons, Ltd.     

Amino acid esterification by alpha-chymotrypsin immobilized in spiropyran membrane.

α-Chymotrypsin was immobilized in a collagen membrane modified with a spiropyran compound. The immobilized chymotrypsin was used for the esterification of N-acetyl-l-tyrosine (AT). N-Acetyl-l-tyrosine ethyl ester (ATEE) was synthesized from AT and ethanol by immobilized chymotrypsin under visible light. The optimum pH for the esterification was 7. An increase of the chymotrypsin content in the spiropyran-collagen membrane increased the rate and the yield of ATEE. The yield of ATEE reached 40% under visible light. Initially, ATEE was synthesized in the dark. However, the ATEE synthesized was gradually hydrolyzed in the dark. The amount of ATEE in the reaction mixture increased with irradiation by visible light and decreased in the dark. Therefore, the esterification of N-acetyl-l-tyrosine was controlled by light irradiation.     

Forssman hapten N-acetyl-alpha-D-galactosaminidase in rat brain and kidney

—Forssman hapten (N-acetyl-α-galactosaminosyl-N-acetyl-β-galactosaminosyl-α-galactosyl-β-galactosyl-glucosylceramide), prepared from sheep erythrocytes was specifically labelled with tritium at the terminal N-acetyl-α-galactosamine moiety by the galactose oxidase-sodium [³H]borohydride method. Activities to cleave the terminal N-acetyl-α-galactosamine from Forssman hapten were detected in the high-speed supernatant of the frozen-thawed and sonicated crude mitochondrial fraction from adult rat brain and kidney. The optimal pH of the reaction was approximately 4·4. The reaction was linear for at least 1 h for the kidney enzyme and up to 3 h for the brain enzyme. Taurocholate was required for the activity. The optimal concentration was 1·5-2 mg/ml. Several other detergents and bile salts tested could not replace taurocholate. The apparent K_m of the brain and kidney enzymes were 1·0×10^?4M and 3·5×10^?4m , respectively. During development, Forssman hapten-cleaving activities of both brain and kidney gradually declined in specific activity as the animal matured. These changes were similar to those of nonspecific p-nitrophenyl N-acetyl-α-galactosaminidase. Several rat organs examined all showed detectable activities to cleave Forssman hapten.     

Use of an IgG fragment prepared with particulate plasmin to study the C1 binding and activation.

Facb, fragment antigen and complement binding (this last property is shown when the fragment is involved in an immune complex); Fc, C-terminal half of the heavy-chain dimer; pFc′, major C-terminal fragment released from IgG by pepsin or plasmin digestion; DFP, diisopropylphosphofluoridate; ATEE, N-acetyl-L-tyrosine ethyl ester; BAEE, N-benzoyl-L-arginine ethyl ester; TAME, p-toluene sulfonyl-L-arginine methyl ester; SDS, sodium dodecyl sulfate; SBTI, soybean trypsin inhibitor.The nomenclature of the complement components (C1, C1q, C1r, C1s) follows the W.H.O. recommendations. Enzymatic activities are expressed in nanokatals (nkat.) as recommended in the Enzyme Nomenclature (1973).     

Effect of glycerol on thermolysin-catalyzed peptide bond synthesis

The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl-l-aspartic acid with l-phenylalanine methyl ester to form N-benzyloxycarbonyl-l-aspartyl-l-phenylalanine methyl ester (Z · Asp · Phe · OMe), the precursor to the sweet-tasting compound l-aspartyl-l-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl-l-glycyl-l-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z · Asp · Phe · OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.     

THE BIOSYNTHESIS OF OCTOPAMINE-CHARACTERIZATION OF LOBSTER TYRAMINE β-HYDROXYLASE

—Tyramine β-hydroxylase catalyzes the biosynthesis of octopamine in the lobster nervous system. This enzyme has been characterized and a rapid microassay, based on the enzymic release of tritiated water from [1,2-(side chain) ³H] tyramine, has been developed. Lobster tyramine β-hydroxylase resembled mammalian dopamine β-hydroxylase. The most conspicuous differences were that the lobster enzyme was inhibited by anions, particularly fumarate, and had a higher affinity for substrates. Tyramine β-hydroxylase activity was present in both particulate and soluble fractions of homogenates of the lobster nervous system. Bound activity, extracted by repeated freezing and thawing, was partially purified. The enzyme had the following properties: (1) The optimum pH for the conversion of tyramine to octopamine was 7·4. (2) The apparent Michaelis constant for tyramine was 0·15 mm and for ascorbic acid was 0·2 mm at pH 6·6. (3) The purified enzyme was inhibited by salts; the degree of inhibition was sensitive to the anion and decreased in the order chloride ? fumarate > sulphate > acetate. (4) Tyramine β-hydroxylase was inhibited by metal chelating agents and by cupric sulphate at concentrations greater than 10^?4m ; N-ethylmaleimide had no significant effect on activity in concentrations up to 3 mm . (5) The purified enzyme also β-hydroxylated dopamine to form norepinephrine, with an apparent Michaelis constant of 0·24 mm . This activity co-purified with tyramine β-hydroxylase, suggesting that a single enzyme catalyzed both reactions.     

Comparison of the catalytic properties of alpha- and beta-forms of thrombin

18-25-fold purified alpha-thrombin, having high esterase activity and coagulating ability of 2500 NIH u per 1 mg of protein, was isolated using chromatography of commercial thrombin through SP-Sephadex C-50. Limited proteolysis of alpha-thrombin on the column with immobilized trypsin resulted in the appearance of beta-thrombin with alpha-thrombin-like esterase activity and tracing coagulating activity (2-5 NIH u per 1 mg of protein). Molecular weight analysis of alpha- and beta-thrombin forms suggests that a peptide (or peptides) with Mr of 1100 is splitted off under proteolysis. Some similarity is revealed in kinetic parameters (Km(app) and kkat) of TAME and BAME hydrolysis by alpha- and beta-thrombin, although Km(app) is somewhat low (approximately 2-fold) for alpha-thrombin. Investigation of TAME hydrolysis kinetics by both thrombin forms at a wide range of substrate concentrations has revealed the effect of substrate activation. Kinetic constants Ks and beta for high substrate concentrations are calculated. It is suggested that the similarity of alpha- and beta-thrombin action on arginine esters and sharp differences in their effect on fibrinogen may be a result of a disturbance of substrate-binding region of beta-thrombin active site.     

A rate-limiting step of enteropeptidase hydrolysis

It has been shown for the first time that deacylation is the rate-limiting step in the enteropeptidase-catalyzed hydrolysis of highly effective oligopeptide substrates containing four Asp residues in positions P2–P5. On the other hand, the rate-limiting step in the hydrolysis of low-efficiency peptide substrates containing less than four Asp or Glu residues in positions P2–P5 is acylation, as it has previously been suggested for all amide and peptide substrates of serine proteases on the basis of classical works of Bender et al. The method of introduction of an additional nucleophile or another effector that selectively affects the deacylation step was used to determine the rate-limiting step in the enteropeptidase hydrolysis of N ^α-benzyloxycarbonyl-L-lysine thiobenzyl ester, the highly efficient amide substrate GlyAsp₄-Lys β-naphthyl amide, and the low-efficiency peptide substrate VLSAADK-GNVKAAWG (where a hyphen denotes the hydrolysis site).     

Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis–Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37 °C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis–Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays.     

The assay of the bromelains using N alpha-CBZ-L-lysine p-nitrophenyl ester and N-CBZ-glycine p-nitrophenyl ester as substrates

Two new esterolytic assays of the pineapple stem bromelains are described. They use as substrates the p-nitrophenyl esters of N^α-CBZ-l-lysine (CLN) and N-CBZ-glycine (CGN). The activity is monitored by the direct spectrophotometric measurement of the enzyme-catalyzed hydrolysis of these esters at 340 nm. The bromelains are rapidly activated with 1 mm l-cysteine at pH 4.6 for the CLN assay and pH 6.1 for the CGN assay. EDTA has no measurable effect. The sensitivities of the assays approach 10 μg/ml in a reaction time of 3 min.     

The synthesis and hydrolysis of a series of deoxyfluoro-d-glucopyranosyl phosphates

The synthesis of all four deoxyfluoro-α-d-glucopyranosyl phosphates is described. Rate constants for their acid-catalyzed hydrolysis were determined, and fluorine substitution was shown to have a significant effect in lowering the rate, particularly when the substitution is adjacent to the anomeric center. Relative rate-constants measured in m HClO₄ at 25° are 60.30:1.00:7.05:3.97:16.5 for α-d-glucopyranosyl phosphate and the 2-, 3-, 4- and 6-deoxyfluoro derivatives, respectively. The hydrolysis of 2-deoxy-2-fluoro-α-d-glucopyranosyl phosphate was studied in more detail, and an activation entropy and enthalpy of 4.1 e.u. (m reactant) and 113.5 kJ.mol⁻¹, respectively, were determined for hydrolysis in m HClO₄ at 60° The pH dependence of its hydrolysis was investigated, and rate constants for hydrolysis of the monoanion (k_M = 1.88 × 10⁻⁶ s⁻¹) and neutral (k_N = 6.23 × 10⁻⁵ s⁻¹) species were thus extracted. Hydrolysis of the monoanion is not significantly affected by fluorine substitution, as expected. The ability or inability of several mechanistically distinct enzymes to utilize these fluorinated substrates is rationalized in the light of these findings.     

MEMBRANE-BOUND NEURAMINIDASE IN THE BRAIN OF DIFFERENT ANIMALS: BEHAVIOUR OF THE ENZYME ON ENDOGENOUS SIALO DERIVATIVES AND RATIONALE FOR ITS ASSAY

—The activity of brain membrane-bound neuraminidase on endogenous and exogenous substrates was comparatively studied in various animals (rat, chicken, rabbit, pig, calf and human). The maximum rate of hydrolysis of endogenous substrates by membrane-bound neuraminidase (using a crude preparation of the enzyme) was different in the various animals (from 0·05 to 0·73 units, referred to 1 mg protein) and was obtained under similar but not identical optimum conditions (pH from 4·1 to 5·1; requirement or not of Triton X-100). The maximum degree of hydrolysis of endogenous substates was also different (from 15 to 27 nmol released NeuNAc/mg protein) and was obtained within different incubation periods (from 2 to 18 h). It corresponded (in rabbit, calf, human brain only), or not, to the actual exhaustion of the endogenous substrates. The endogenous substrates were recognized as both gangliosides and sialoglycoproteins. The extent of hydrolysis of sialoglycoproteins varied from 1·5% in rabbit to 15·6% in chicken brain; the hydrolysis of gangliosides (ranging from 14·1% in pig to 53·7% in rabbit brain) reached only in some animals (rabbit, calf, human) the complete transformation of major oligosialogangliosides into the neuraminidase resistant monosialoganglioside GMI. Upon addition of exogenous substrates (sialyl-lactose, ganglioside GD1a, brain sialopeptides, ovine submaxillary mucin) the actual rate of liberation of total NeuNAc (from both endogenous and exogenous substrates) considerably exceeded, although at a different extent (depending on the animal and on the added substrate used) the rate of hydrolysis of sole endogenous substrates. The possibility of an accurate assay of brain membrane-bound neuraminidase in a crude enzyme preparation is evaluated and guidelines for the assay procedure suggested.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.     

MEMBRANE THIAMINE TRTPHOSPHATASE FROM RAT BRAIN: INHIBITION BY ATP AND ADP

—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10^?5m and 7·5 × 10^?5m respectively. Nucleoside monophosphates and free nuclcosides as well as P_i have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10^?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed K_i, of 4 × 10^?5m for the ATP analog adenosine-PPCP and 9 × 10^?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg²⁺. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed K_m and pH and V_max are not shifted by the presence of similar concentrations of inhibitor.     

The allosteric effect of salt on human mast cell tryptase

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.     

Interactions of five- and six-membered cyclic esters with α-chymotrypsin: Kinetic evidence for covalent enzyme intermediates

Interactions of α-chymotrypsin with 2-coumaranone (I), 3,4-dihydrocoumarin (II), o-hydroxy-α-toluenesulfonic acid sultone (III), and β-o-hydroxyphenylethanesulfonic acid sultone (IV) were studied in the presence of 14% acetonitrile at pH 7.0 by means of the proflavin displacement technique and by inhibition of N-acetyl-l-tryptophan ethyl ester (ATrEE) hydrolysis. Under saturating conditions of either I, II, or III, an enzyme intermediate was shown to accumulate using either the proflavin displacement technique or the ATrEE activity assay. The intermediates have characteristics of covalent enzyme-substrate compounds and are believed to decompose simultaneously by two pathways, one to give free enzyme and hydrolyzed cyclic ester, and the other to give the original cyclic ester and free enzyme. With α-chymotrypsin and III the observed first-order rate constant for decomposition of the intermediate by the two pathways was 0.19 ± 0.04 min^?1, while the rate constant for the hydrolytic pathway alone was 0.013 ± 0.0009 min^?1. These results indicate that the covalent-like intermediate with this sultone is not only capable of reverting to starting cyclic ester but prefers this pathway over hydrolysis. Sultone IV was found to bind to enzyme; but in contrast to the behavior of esters I–III, the binding did not result in accumulation of a covalent-like intermediate.     

A two-step procedure for purification of papain from extract of papaya latex

A method is presented for purifying papain from extracts of papaya latex. The procedure involves precipitation of the extract of papaya latex with sodium chloride followed by affinity chromatography of the redissolved precipitate. Precipitation of the protein from the latex extract is necessary to separate the papain from material which interferes with the binding of papain to the affinity column. During affinity chromatography, the affinity column is overloaded to insure absence in the final product of impurities which are capable of binding to the affinity column.The papain prepared by this procedure yielded an amino acid analysis and an N-terminal amino acid analysis expected for a sample of pure papain. No Met was detected on amino acid analysis nor was the presence of N-terminal residues other than He detected. On polyacrylamide disc gel electrophoresis at pH 4.3, papain prepared by the method described in this work was indistinguishable from crystalline papain which was prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Both disc gel patterns consisted of a single band and a trailing shadow which was less than 5% of the main band. In routine spectrophotometric assays, the specific activity toward N,α-benzoyl-l-arginine ethyl ester of papain prepared by the procedure described in this work was indistinguishable from crystalline papain prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Values of 24 sec^?1' and 15 mm were obtained from the turnover number and K_m for the papain-catalyzed hydrolysis of N,α-benzoyl-l-arginine ethyl ester at 25 °C, pH 6.00, $\text{Γ}\text{2 0.30}$ using a pH stat.     

Ligula intestinalis (Cestoda: Pseudophyllidae): Studies on the properties of proteolytic and protease inhibitor activities of plerocercoid larvae

The somatic extract of L. intestinalis plerocercoids reveals hydrolytic activity against N-Benzoyl-l-tyrosine ethyl ester (BTEE) and Azocoll, and inactivates the esterolysis by mammalian trypsin and chymotripsin. The proteolytic enzyme activity and the inhibitory effect were completely separated by Sephadex G-100 column chromatography. Gel chromatography of the somatic extract revealed two peaks of proteolytic activity : one is bound to macromolecular substances, the other appears to be in free form and has a molecular weight of approx 60,000–65,000. The proteolytic activity showed the following characteristics : Tris-HCl buffer provided the highest activity against BTEE, the pH optimum was 7·4–7·8; the enzyme was activated by 10^?5m-Ca²⁺, Mg²⁺ or Mn²⁺, it was inhibited by 10^?5m-Cu²⁺, but not by 10^?5m-Zn²⁺. 0.001% soybean trypsin inhibitor, 2 × 10^?3m-EDTA, 1 mm-tosyl-l-phenylalanyl chloromethane, 1000 KIU/ml Trasylol did not inhibit the proteolytic activity, but it was inhibited by 1 mm-phenylmethyl-sulphonyl fluoride. The enzyme activity completely ceased upon 5 % TCA treatment or incubation at 56°C for 30 min. The trypsin and chyrnotrypsin inhibitor activities were eluted from the Sephadex G-100 column in a single peak with an estimated molecular weight of 6700–7200. The inhibitory effect was not sensitive to pH changes, and treatment by 5% TCA or incubation at 80°C for 15 min was ineffective. The proteolytic activity of plerocercoid extract was not effected ‘in vitro’ by the inhibitors isolated from this parasite.     

Isolation and characterization of five serine proteases with trypsin-, chymotrypsin- and elastase-like characteristics from the gut of the lugworm Arenicola marina (L.) (Polychaeta)

Summary Five proteases were isolated from the digestive fluid of the lugworm, Arenicola marina L. The enzymes (molecular weight 24.0–24.6 kDa) were classified as serine proteases. Three enzymes showed a cleavage specificity corresponding to mammalian trypsin (E.C. 3.4.21.4). One protease possessed a chymotrypsin-like cleavage pattern (E.C. 3.4.21.1), and the fifth preferred cleavage behind short-chain amino acids like an elastase (E.C. 3.4.21.36). Detailed investigations revealed differences in molecular characteristics and cleavage patterns compared to mammalian proteases, especially in the chymotrypsin- and the elastase-like enzymes.Abbreviations APNE N-acetyl-d/l-Phe -naphthyl ester - BANA N-benzoyl-d/l-Arg -naphthylamide - BAPNA N-benzoyl-d/l-Arg-4-nitroanilide - BIGGANA N-benzoyl-l-Ile-l-Glu-Gly-l-Arg-4-nitroanilide - BLPNA N-benzoyl-d/l-Lys-4-nitroanilide - BTEE N-benzoyl-l-Tyr ethyl ester - enzyme T1/T2/T3 trypsin-like enzyme - enzyme ChT chymotrypsin-like enzyme - enzyme E elastase-like enzyme - GPANA N-glutaryl-l-Phe-4-nitroanilide - MUF 4-methylumbelliferryl - MW molecular weight - PMSF phenylmethylsulphonyl fluoride - SAAPPNA N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-4-nitroanilide - SBTI soybean trypsin inhibitor - SPPNA N-succinyl-l-Phe-4-nitroanilide - TAME N-tosyl-l-Arg methyl ester - TFA trifluoracetic acid - TLCK N-tosyl-l-Lys chloromethyl ketone - TPCK N-tosyl-l-Phe chloromethyl ketone - TRIS tris(hydroxymethyl)aminomethane