Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS-PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine7-amido-4-methylcoumarin with K(m) (69.92 microM) and k(cat) (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 degrees C. It showed thermal stability below 40 degrees C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.     

Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum

An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60 degrees C, respectively. In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity. The alpha-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).     

Purification and characterization of myrosinase from horseradish (Armoracia rusticana) roots.

Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.     

Purification and characterization of a novel (R)-hydroxynitrile lyase from Eriobotrya japonica (Loquat)

A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH(4))(2)SO(4) fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a K(m) of 161 microM and a k(cat)/K(m) of 348 s(-1) mM(-1) for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40 degrees C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO(4), HgCl(2), AgNO(3), FeCl(3), beta-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (k(cat)/K(m)) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one.     

Purification and properties of glucose 6-phosphate dehydrogenase from turkey erythrocytes

Glucose 6-phosphate dehydrogenase (G6PD) was purified from turkey erythrocytes by ammonium sulphate precipitation and followed by ADP Sepharose affinity gel chromatography. The yield was 49.71% and specific activity of the enzyme was found to be 44.16 EU/mg protein. By gel filtration the molecular mass was found to be 75 kDa. The enzyme had an optimum pH at 9.0, and optimum temperature at 50 degrees C. Km and Vmax for NADP(+) and glucose 6- phosphate (G6-P) as substrates were also determined and effects of inhibitors such as ATP, NADH and NADPH were examined.     

Purification, characterization and subcellular localization of pig liver alpha-L-iduronidase

alpha-L-Iduronidase was purified about 100,000-fold from pig liver by employing column chromatography on cellulose phosphate (P11), concanavalin A-Sepharose 4B, heparin-Sepharose 4B, Toyopearl HW-55, Sephadex G-100 and chelating Sepharose 6B charged with cupric ions. The molecular mass of the purified enzyme was estimated to be 70 kDa by Sephadex G-100 column chromatography. The purified enzyme gave a single band on disc polyacrylamide gel electrophoresis without using sodium dodecyl sulfate. However, two separate components of 70 kDa and 62 kDa appeared when it was analyzed by SDS/polyacrylamide gel electrophoresis. These 70-kDa and 62-kDa components were confirmed as alpha-L-iduronidase immunochemically. The isoelectric points of these enzymes were both 9.1 as measured by isoelectric focusing in a polyacrylamide gel containing ampholine and sucrose. The optimal pH and Km values were 3.0-3.5 and 65 microM 4-methylumbelliferyl-alpha-L-iduronide, respectively. The purified enzyme was stable in the pH range 3.5-6.0 under conditions with or without 0.5 M NaCl. However, in the presence of 0.5 M NaCl, it was unstable at pH 3.0. Moreover, it was conversely stabilized at pH 7.0 in the presence of 0.5 M NaCl. Immunohistochemically, the enzyme was found in the Kupffer cells and was abundant on their lysosomal membranes. In liver cells, however, the immunohistochemical reaction was weak.     

Purification and characterization of glutathione reductase from bovine erythrocytes

Glutathione reductase (E.C.1.8.1.7; GR) was purified from bovine erythrocytes and some characteristics properties of the enzyme were investigated. The purification procedure was composed of preparation of the hemolysate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose 4B, and gel filtration chromatography on Sephadex G-200. As a result of four consecutive procedures, the enzyme was purified 31,250-fold with a yield of 11.39%. Specific activity at the final step was 62.5 U (mg proteins)(-1). For the enzyme, optimum pH, optimum temperature, optimum ionic strength, and stable pH were found to be 7.3, 55 degrees C, 435 mM, 7.3, respectively. The molecular weight of the enzyme was found to be 118 kDa by Sephadex G-200 gel filtration chromatography and the subunit molecular weight was found to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Km and Vmax values were determined for glutathione disulfide (GSSG) and NADPH. Ki constants and inhibition types were established for glutathione (GSH) and NADP+. Also, effects of NADPH and GSSG were investigated on the enzyme activities.     

Characterization of an intracellular oligopeptidase from Lactobacillus paracasei.

An intracellular oligopeptidase from Lactobacillus paracasei Lc-01 has been purified to homogeneity by Fast Flow Q Sepharose, hydroxyapatite, and Mono Q chromatography. The molecular mass of the enzyme was determined to be 140 kDa by gel filtration and approximately 30 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis. The pI of the enzyme was at pH 4.5. The enzyme expressed maximum activity at pH 8.0 and 40 degrees C. Oligopeptidase activity on bradykinin was inhibited strongly by 1,10-phenantroline and EDTA and partly by p-chloromercuribenzoic acid but not by phosphoramidon or phenylmethylsulfonyl fluoride. Marked inhibition by beta-casein fragment 58 to 72 was demonstrated. The enzyme showed neither general aminopeptidase nor caseinolytic activity, and it degraded only oligopeptides between 8 and 13 amino acids. The enzyme readily hydrolyzed the Phe-Ser and Pro-Phe bonds of bradykinin; the Phe-His bond of angiotensin I; the Pro-Gln, Gln-Phe, and Phe-Gly bonds of substance P; and the Pro-Tyr bond of neurotensin. Weak activity toward the Ala-Tyr and Pro-Ser bonds of alpha(s1)-casein fragment 157 to 164, was observed. The N-terminal amino acid sequence of the oligopeptidase showed a high degree of homology to the lactacin B inducer from Lactobacillus acidophilus.     

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.     

Purification and characterization of protein methylase II from Helicobacter pylori

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes.

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.     

Cloning, expression, and characterization of a cold-adapted lipase gene from an antarctic deep-sea psychrotrophic bacterium, Psychrobacter sp 7195

A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at 30 degrees C, and was unstable at temperatures higher than 30 degrees C, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24 h incubation at 4 degrees C. The addition of Ca2+ and Mg2+ enhanced the enzyme activity of LipA1, whereas the Cd2, Zn2+, Co2+, Fe3+, Hg2+, Fe2+, Rb2+, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate (C14 acyl groups).     

A thermostable alpha-arabinofuranosidase from xylanolytic Bacillus pumilus: purification and characterisation

Bacillus pumilus PS213 secretes an alpha-L-arabinofuranosidase (AF) when grown in the presence of arabinogalactan or oat meal. The enzyme has been purified to homogeneity and characterised. Its molecular mass, as determined by gel filtration, is 220 kDa, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of approximately 60 kDa. According to the result of the mass spectrometry analysis showing a molecular mass of 56 kDa, the enzyme should be a homotetramer. The isoelectric point was found to be 5.2, the enzyme activity was optimal at 55 degrees C and pH 7.0. The enzyme retained 80% of its activity after 2 h at 65 degrees C and lost 50% of activity at 75 degrees C after 135 min. The Michaelis constant K(m) and V(max) for p-nitrophenylarabinofuranoside at 37 degrees C were 1.7 mM and 52.9 U mg(-1), respectively. N-terminal sequence analysis and internal peptide fragments showed homology with glycosyl hydrolases of family 51.     

Purification and properties of a nonheme bromoperoxidase from Streptomyces aureofaciens

The first bacterial nonheme type bromoperoxidase has been purified to homogeneity from the chlorotetracycline-producing actinomycete Streptomyces aureofaciens Tü 24. Purification was accomplished by (NH4)2SO4 precipitation, DEAE-cellulose chromatography at different pH-values, and molecular sieve chromatography. The purified enzyme has a molecular mass of 90 to 95 kDa based on ultracentrifugation and gel filtration. The enzyme is composed of three subunits of identical molecular mass (m = 31 kDa). Bromoperoxidase catalyses the bromination of monochlorodimedone, but not its chlorination, and has no peroxidase or catalase activity. The optimum pH is 4.5. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metals in equimolar amounts. Bromoperoxidase is stable in a pH range from pH 4.0 to pH 10.0 at 4 degrees C for weeks and does not loose any activity when incubated at 80 degrees C for 2 h.     

A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization

High levels of an extracellular alpha-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH(4))(2)SO(4) fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric alpha-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5-5.5 and 65 degrees C, respectively. The purified enzyme retains more than 90% of its activity at 45 degrees C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-alpha-galactopyranoside, raffinose and stachyose and very similar K(m) values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn(++) ions activates enzyme activity, whereas inhibitory effects can be observed with Ca(++), Zn(++) and Hg(++). Five min incubation at 65 degrees with 10 mM Ag(+) results in complete inactivation of the purified alpha-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the alpha-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.     

Purification and properties of a carboxymethylcellulase from phytopathogenic fungus Macrophomina phaseolina

From the culture filtrate of Macrophomina phaseolina, two forms of carboxymethylcellulase were separated by ion-exchange chromatography and designated as CMCase-I and CMCase-II. CMCase-I was purified following a four-step procedure involving gel filtration on Sephadex G-75, Con-A Sepharose 4B affinity chromatography, fast protein liquid chromatography on mono Q anion-exchanger and on Superose 12 gel filtration. The final preparation was homogeneous by SDS-PAGE, isoelectric focussing in thin layers of polyacrylamide gels and immunoelectrophoresis. The enzyme showed optimum activity at pH 5.5 and 65 degrees C, was stable to heating at 65 degrees C for 10 min, and retained 31% of original activity after heating at 80 degrees C for 10 min. The molecular weight of the enzyme was 3.5 x 10(4) Da. A Km of 0.25 mg/ml was determined using carboxymethyl-cellulose as the substrate.     

Purification and partial characterization of alpha-N-acetylglucosaminidase from human liver.

A deficiency in alpha-N-acetylglucosaminidase is known as mucopolysaccharidosis IIIB or Sanfilippo B syndrome. We purified this enzyme almost 39,000-fold from liver to homogeneity with 3% recovery. Use of concanavalin A (Con A)-Sepharose and heparin-Sepharose resulted in 13.4-fold and 11.6-fold purifications of the enzymatic activity, respectively. The molecular mass was estimated to be 300 kDa by gel filtration and 80 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The isoelectric point was 5.1, optimal pH was 4.5, and the Km for p-nitrophenyl alpha-N-acetylglucosamine was 0.13-0.20 mM. The purified enzyme was stable at 50 degrees C for 1 h and within the pH range of 6.5-8.5. Anti-serum against the purified enzyme raised in BALB/c mice inhibited the activities of alpha-N-acetylglucosaminidase.     

Isolation of ADP-ribosyltransferase by affinity chromatography

An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described.     

Purification of an active gelatinase from collagen fiber fraction of granulation tissue in rats

Gelatinase was extracted at 60 degrees C from the collagen fiber-rich fraction of granulation tissue induced by carrageenin in rats. A large part of the extracted gelatinase was unbound to Zn-chelating Sepharose. The unbound gelatinase gave a single band corresponding to a molecular mass of 57 kDa on SDS-substrate PAGE, but showed a much higher molecular mass (greater than 200 kDa) on Sephadex G-150 gel filtration. In addition, that unbound fraction contained gelatin fragments was revealed by SDS-PAGE. When the unbound fraction of Zn-chelating Sepharose was incubated at 37 degrees C, the gelatin fragments disappeared and the apparent molecular mass of gelatinase in gel filtration decreased. This gelatin degradation of the unbound fraction was enhanced by treatment with a 4-aminophenylmercuric acetate (APMA). The results suggest that the gelatinase is bound to gelatin fragments in the unbound fraction. After the treatment with APMA, the gelatinase was purified to to homogeneity; the purified gelatinase gave a single band corresponding to a molecular mass of 57 or 67 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The purified gelatinase is a metalloproteinase, and extensively degraded gelatin, but showed no proteolytic activity toward alpha-casein or types I and IV collagens. The results suggest that the 67-kDa active gelatinase is bound to collagen fibers and plays an important role in a rapid degradation of collagen fibers in granulation tissue.