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     

Streptomyces rimosus extracellular proteases

Summary A trypsin-like proteinase was isolated from Streptomyces rimosus culture filtrates obtained from an oxytetracycline production process. The isolation procedure includes ultrafiltration, chromatography on CM-Sephadex, AH-Sepharose and CM-cellulose and gives a homogeneous protein with 19% yield. The enzyme is an anionic trypsin (M_r 28 000, pI 4.5), is stable from pH 4.5 to 9 and up to 40°C, and contains three disulphide bridges, three histidines and three methionines per molecule. At its pH optimum (pH 8.4–8.8) it splits peptide, ester and arylamide bonds of arginine in the endo-position and, to a smaller extent, in the exo-position. Like other streptomycete trypsins, it is a more efficient catalyst than bovine trypsin and has a relative preference for peptide-arylamides, N-benzyloxycarbonyl-l-norleucyl-l-prolyl-l-arginine-p-nitroanilide being by far its best substrate.     

Comparative study on the proteases from fish pyloric caeca and the use for production of gelatin hydrolysate with antioxidative activity

Proteases from pyloric caeca extract of three fish species including brownstripe red snapper (Lutjanus vitta), bigeye snapper (Priacanthus tayenus) and threadfin bream (Nemipterus marginatus) were comparatively studied. The extracts from bigeye snapper and threadfin bream exhibited the highest hydrolytic activities toward casein, α-N-benzoyl-dl-arginine-p-nitroanilide and α-N-ρ-tosyl-l-arginine methyl ester at pH 8.0 and 60 °C and pH 8.5 and 55 °C, respectively. The extract of brownstripe red snapper showed the optimal pH and temperature of 8.0 and 60 °C with all substrates used except the optimal temperature was 65 °C when casein was used. All proteases were strongly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-l-lysine chloromethylketone (TLCK) and partially inhibited by N-tosyl-l-phenylalanine chloromethylketone for all substrates tested, suggesting that trypsin-like proteases were the major enzymes. Substrate-gel activity staining of 40–60% ammonium sulfate (AS) fraction revealed that major activity bands were observed with molecular mass of 24, 22 and 20 kDa for brownstripe red snapper, bigeye snapper and threadfin bream, respectively. Those activity bands were partially inhibited by SBTI and TLCK. AS fraction was further used to produce gelatin hydrolysate from the skin of brownstripe red snapper with different degrees of hydrolysis (DH). Hydrolysate with DH of 15% exhibited the highest DPPH and ABTS radical scavenging activities and ferric reducing antioxidant power (p < 0.05). Therefore, the extract from pyloric caeca could be used to produce the gelatin hydrolysates possessing antioxidative activities.     

Improvement of a useful enzyme (subtilisin BPN′) by an experimental evolution system

In order to improve a natural enzyme so as to fit industrial purposes, we have applied experimental evolution techniques comprised of successive in vitro random mutagenesis and efficient screening systems. Subtilisin BPN, a useful alkaline serine protease, was used as the model enzyme, and the gene was cloned to an Escherichia coli host-vector system. Primary mutants with reduced activities of below 80% of that of the wild type were first derived by hydroxylamine mutagenesis directly applied to subtilisin gene DNA, followed by screening of clear-zone non-forming transformant colonies cultured at room temperature on plates containing skim-milk. Then, secondary mutants were derived from each primary mutant by the same mutagenic procedure, but screened by detecting transformant colonies incubated at 10°C with clear zones that were greater in size than that of the wild type. One such secondary mutant, 12–12, derived from a primary mutant with 80% activity, was found to gain 150% activity (k _cat/K _m value) of the wild-type when the mutant subtilisin gene was subcloned to a Bacillus subtilis host-vector system, expressed to form secretory mutant enzyme in the medium, and the activity measured using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate. When N-succinyl-l-Ala-l-Ala-l-Pro-l-Leu-p-nitroanilide was used, 180% activity was gained. Genetic analysis revealed that the primary and secondary mutations corresponded to D197N and G131D, respectively. The activity variations found in these mutant subtilisins were discussed in terms of Ca²⁺-binding ability. The thermostability was also found to be related to the activity.     

Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60

Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M _r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K _m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co²⁺, Mn²⁺ and Fe²⁺ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH₂-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%.     

Proteolytic activity of gut bacteria isolated from the velvet bean caterpillar Anticarsia gemmatalis

The development of proteinase inhibitors as potential insect control agents has been constrained by insect adaptation to these compounds. The velvet bean caterpillar (Anticarsia gemmatalis) is a key soybean pest species that is well-adapted to proteinase inhibitors, particularly serine-proteinase inhibitors, which are abundant in the caterpillar host. The expression of diverse proteolytic enzymes by gut symbionts may allow the velvet bean caterpillar to circumvent proteinase inhibitors produced by the host plant. In this study, we characterized the proteolytic activity of the four nonpathogenic species of gut bacteria isolated from the velvet bean caterpillar—Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii and Staphylococcus xylosus. Two proteinase substrates, N-α-benzoyl-l-Arg-p-nitroanilide (l-BApNA) and N-α-p-tosyl-l-Arg methyl ester (l-TAME) and five proteinase inhibitors [aprotinin, E-64, ethylenediamine tetraacetic acid (EDTA), pepstatin and N-α-tosyl-l-lysine chloromethyl ketone (TLCK)] as well as CaCl₂, pH and temperature profiles were used to characterize the expressed proteolytic activity of these bacterial strains in vitro. Kinetic parameters for proteolytic activity were also estimated. The results of these experiments indicated that serine- and cysteine-proteinase activities were expressed by all four gut bacteria symbionts of the velvet bean caterpillar. The cysteine- and serine-proteinase activities of these gut symbionts were distinct and different from that of gut proteinases of the caterpillar itself. This finding provides support for the potential involvement of gut symbionts in the mitigation of the negative effects of serine-proteinase inhibitors in the velvet bean caterpillar.     

Temperature adaptation and the contractile properties of live muscle fibres from teleost fish

Summary The contractile properties of swimming muscles have been investigated in marine teleosts from Antarctic (Trematomus lepidorhinus, Pseudochaenichthys georgianus), temperate (Pollachius virens, Limanda limanda, Agonis cataphractus, Callionymus lyra), and tropical (Abudefduf abdominalis, Thalassoma duperreyi) latitudes. Small bundles of fast twitch fibres were isolated from anterior myotomes and/or the pectoral fin adductor profundis muscle (m. add. p). Live fibre preparations were viable for several days at in vivo temperatures, but became progressively inexcitable at higher or lower temperatures. The stimulation frequency required to produce fused isometric tetani increased from 50 Hz in Antarctic species at 0°C to around 400 Hz in tropical species at 25°C. Maximum isometric tension (P_o) was produced at the normal body temperature (NBT) of each species (Antarctic, 0–2°C; North Sea and Atlantic, 8–10°C; Indo-West Pacific, 23–25°C). P₀ values at physiological temperatures (200–300 kN·m^–2) were similar for Antarctic, temperate, and tropical species. A temperature induced tension hysteresis was observed in muscle fibres from some species. Exposure to <0°C in Antarctic and <2°C in temperate fish resulted in the temporary depression of tension over the whole experimental range, an effect reversed by incubation at higher temperatures. At normal body temperatures the half-times for activation and relaxation of twitch and tetanic tension increased in the order Antarctic>temperate>tropical species. Relaxation was generally much slower at temperatures <10°C in fibres from tropical than temperate fish. Q₁₀ values for these parameters at NBTs were 1.3 2.1 for tropical species, 1.7–2.6 for temperate species, and 1.6–3.5 for Antarctic species. The forcevelocity (P-V) relationship was studied in selected species using iso-velocity releases and the data below 0.8 P₀ iteratively fitted to Hill's equation. The P-V relation at NBT was found to be significantly less curved in Antarctic than temperate species. The unloaded contraction velocity (V_max) of fibres was positively correlated with NBT increasing from about 1 muscle fibre length·s^–;1 in an Antarctic fish (Trematomus lepidorhinus) at 1°C to around 16 muscle fibre lengths·s^–1 in a tropical species (Thalassoma duperreyi) at 24°C. It is concluded that although muscle contraction in Antarctic fish shows adaptations for low temperature function, the degree of compensation achieved in shortening speed and twitch kinetics is relatively modest.Abbreviations ET environmental temperature - m. add. p major adductor profundis - m. add. s. major adductor superficialis - NBT normal body temperature - P ₀ maximum isometric tension - P-V force velocity - SR sarcoplasmic reticulum - T 1/2 a half activation time - T 1/2 r half relaxation time - V _max unloaded contraction     

Biotransformation of alkyl and aryl carbonates: enantioselective hydrolysis

Summary N-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl₂ with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h ^–1.Offprint requests to: K. Nakanishi     

Key enzymes of the kallikrein-kinin system in Antarctic teleosts

In this study, some of the mammalian kallikrein-kinin system (KKS)-like components were identified in two species of Antarctic notothenioid [Chionodraco hamatus (Channichthydae) and Trematomus bernacchii (Nothothenidae)]. The kidney and heart were assayed for kallikrein-like activity using the synthetic substrate d-Val-Leu-Arg-paranitroanilide. Values expressed as nmol p-nitroanilide/mg proteins, were in C. hamatus 15.5±1.5 and 15.2±1.4 in kidney and heart, respectively, and 15.8±2.2 and 15.9±1 in kidney and heart of T. bernacchii. Kallikrein-like activity was inhibited bvy aprotinin and phenylmethylsulfonyl fluoride (PMSF). The assay was stable at 20°C. Kininase II-like activity was performed on kidney, gills and heart using the substrate hippuryl-lhistidyl-l-leucine. The activity was inhibited by captopril, and in kidney and gills by high temperatures (20°C and 37°C); in the heart the enzymatic activity was measurable also at 20°C. Bradykinin-like immunoreactive cells were localized by immunohistochemistry in the nephron, in the gills, and in the arterial walls of the heart. These results show that Antarctic teleosts possess elements comparable to those of the KKS, including kallikrein-like, and kininase II-like activities, and the end product of the enzymatic cascade, bradykinin. The enzymatic cascade appears to be fully active only at low temperatures. Received: 19 April 1996/Accepted: 30 June 1996     

Deacetylation ofN-acetylthienamycin to thienamycin by a cell-free extract ofStreptomyces cattleya,the thienamycin producer

Summary A cell-free extract from the thienamycin producer,Streptomyces cattleya, has been found to deacetylate the co-product,N-acetylthienamycin. The pH optimum of the reaction is 7.5. Due to the lability ofN-acetylthienamycin, we used thed andl forms of the synthetic substrateN-chloroacetylvaline. We found that the enzyme is anl-deacetylase, has a molecular weight of 58 000, is stable up to 40°C, acts optimally at 45°C, is stable at pH 5–8, is not activated by divalent metal ions and is inhibited by Hg⁺⁺, Cu⁺⁺ andp-chloromercuribenzoate. This is the first report of an extract from a carbapenem producer which carries out the deacetylation ofN-acetylthienamycin, suggesting that the acetylated derivative is a precursor of thienamycin.Abbreviations THM thienamycin - N-AcTHM N-acetylthienamycin - CFE cell-free extract - N-Cl-Ac-l-Val N-chloroacetyl-l-valine - N-Cl-Ac-d-Val N-chloroacetyl-d-valine     

Purification and Characterization of Leu-Proteinase, the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves

The leucine specific serine proteinase present in the soluble fraction of leaves from Spinacia oleracea L. (called Leu-proteinase) has been purified by acetone precipitation and a combination of gel-filtration, ion exchange, and adsorption chromatography. This enzyme shows a molecular weight of 60,000 ± 3,000 daltons, an isoelectric point of 4.8 ± 0.1, and a relative electrophoretic mobility of 0.58 ± 0.03. The Leu-proteinase catalyzed hydrolysis of p-nitroanilides of N-α-substituted(-l-)amino acids as well as of chromogenic macromolecular substrates has been investigated between pH 5 and 10 at 23 ± 0.5°C and I = 0.1 molar. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 7.5, reflecting the acid-base equilibrium of groups with pK_a values of 6.8 ± 0.1 and 8.2 ± 0.1 (possibly the histidyl residue present at the active site of the enzyme and the N-terminus group). Among the substrates considered, N-α-benzoyl-l-leucine p-nitroanilide shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 1 × 10⁻⁹ molar. In agreement with the enzyme specificity, only N-α-tosyl-l-leucine chloromethyl ketone, di-isopropyl fluorophosphate and phenylmethylsulfonyl fluoride, among compounds considered specific for serine enzymes, strongly inhibit the Leu-proteinase. Accordingly, the enzyme activity is insensitive to cations, chelating agents, sulfydryl group reagents, and activators.     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Enzyme kinetics in cold water: characteristics of N-acetyl-β-d-glucosaminidase activity in the Antarctic krill,Euphausia superba,compared with other crustacean species

N-acetyl--d-glucosaminidase, a chitin-degrading enzyme, is highly active in the integument and digestive tract of euphausiids. The enzyme was used as a model to compare temperature-dependent enzymatic parameters of Antarctic krill, Euphausia superba, with those of a euphausiid species (Meganyctiphanes norvegica) found in both the Scandinavian Kattegat and the Mediterranean. Other species examined were an Antarctic isopod, Serolis polita, and a tropical crab, Ocypode ryderi. Enzyme isoforms of NAGase were isolated chromatographically. Temperature optimum (between 30 and 53 °C) and activation-energy (47–59 kJ·mol^-1) of isoenzymes were generally unrelated to genotypic cold adaptation. Although pH profiles were temperature-dependent, there was no apparent temperature-related control of activities by pH in the experienced physiological range. In contrast, apparent Michaelis constants showed minima at ambient water temperatures (total range: 0.1–0.6 mol·l^-1). Potentially, enzyme variants play a role in acclimatisation regulated by Michaelis constants. Apparently, the rate-limiting effects of polar temperatures are partly compensated in the Antarctic crustaceans by construction of enzymes with substrate affinities similar to those of species from warmer climates. The significance of apparent Michaelis constants in evaluating mechanisms of metabolic cold compensation is discussed. Necessary additional experimental approaches are highlighted.Abbreviations CPB citrate-phosphate buffer - FPLC fast protein liquid chromatography - K _m ^app apparent Michaelis constant - LC liquid chromatography - MW molecular weight - NAGase N-acetyl--d-glucosaminidase     

Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix)

Trypsin was purified from the pyloric caeca of bluefish (Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) were 9.5 and 55 °C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 °C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N-p-tosyl-l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl₂ partially protected trypsin against activity loss at 40 °C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

Substrate regulation of ascorbate transport activity in astrocytes

Astrocytes possess a concentrativel-ascorbate (vitamin C) uptake mechanism involving a Na⁺-dependentl-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellularl-ascorbate on the activity of this transport system. Initial rates ofl-ascorbate uptake were measured by incubating primary cultures of rat astrocytes withl-[¹⁴C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake (V _max) increased rapidly (<1 h) when cultured cells were deprived ofl-ascorbate. In contrast, there was no change in the apparent affinity of the transport system forl-[¹⁴C]ascorbate. The increase inV _max was reversed by addition ofl-ascorbate, but notD-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures withl-ascorbate did not affect uptake of 2-deoxy-D-[³H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.     

Purification and characterization of 5-oxo-l-prolinase from Paecilomyces varioti F-1, an ATP-dependent hydrolase active with l-2-oxothiazolidine-4-carboxylic acid

An enzyme cleaving l-2-oxothiazolidine-4-carboxylic acid to l-cysteine was purified 75-fold with 8% recovery to near homogeneity from crude extracts of Paecilomyces varioti F-1, which had been isolated as a fungus able to assimilate l-2-oxothiazolidine-4-carboxylic acid. The molecular mass was estimated to be 260 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 140 kDa upon SDS-PAGE. The maximum activity was observed at a range of pH 7.0–8.0 and at 50 °C. The enzyme activity was completely inhibited by SH-blocking reagents such as AgNO₃, p-chloromercuribenzoic acid, N-ethylmaleimide, and N-bromosuccinimide. The enzyme required ATP, Mg²⁺, and KCl for the cleavage of l-2-oxothiazolidine-4-carboxylic acid. The enzyme also cleaved 5-oxo-l-proline to l-glutamic acid and is considered to be 5-oxo-l-prolinase. Received: 23 March 1999 / Accepted: 22 June 1999     

Characterization of an alpha-L-rhamnosidase from Aspergillus kawachii and its gene

An α-l-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on l-rhamnose as the sole carbon source. The α-l-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 °C. Further, it was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60 °C for 1 h. Its T ₅₀ value was determined to be 72 °C. The enzyme was able to hydrolyze α-1,2- and α-1,6-glycosidic bonds. The specific activity of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii α-l-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the α-l-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enantioselective synthesis ofN-acetyl-l-methionine with aminoacylase in organic solvent

We have developed an enzymatic procedure for the enantiospecific synthesis ofN-acetyl-l-methionine with aminoacylase in an organic solvent.N-Acetyl-l-methionine was most effectively synthesized with a yield of about 90% (on the basis of thel-methionine used) when the reaction mixture, composed of 100 mm sodium acetate, 20 MMdl-methionine and aminoacylase (1000 units) immobilized on celite in 1 ml ethyl acetate saturated with 32 l 140mm sodium phosphate buffer (pH 7.0) containing 0.1 mm CoCl₂, was incubated at 30°C for 24 h.N-Acetyl-l-methionine was isolated from the reaction mixture and the enantiomeric excess was 100%.d-Methionine was also isolated from the mixture with a yield of about 95% and 90% enantiomeric excess. The method is applicable to the synthesis of otherN-acetyl-l-amino acids.