Hydrolysis of isoflavone glycosides by a thermostable β-glucosidase from Pyrococcus furiosus

The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purified with a specific activity of 330 U/mg for genistin by His-trap chromatography. The specific activity of the purified enzyme followed the order genistin > daidzin > glycitin> malonyl glycitin > malonyl daidzin > malonyl genistin. The hydrolytic activity for genistin was highest at pH 6.0 and 95 °C with a half-life of 59 h, a K(m) of 0.5 mM, and a k(cat) of 6050 1/s. The enzyme completely hydrolyzed 1.0 mM genistin, daidzin, and glycitin within 100, 140, and 180 min, respectively. The soybean flour extract at 7.5% (w/v) contained 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin. Genistin, daidzin, and glycitin in the soybean flour extract were completely hydrolyzed after 60, 75, and 120 min, respectively. Of the reported β-glucosidases, P. furiosusβ-glucosidase exhibited the highest thermostability, k(cat), k(cat)/K(m), yield, and productivity for hydrolyzing genistin. These results suggest that this enzyme may be useful for the industrial hydrolysis of isoflavone glycosides.     

Heterologous expression and characterization of an N-acetyl-β-D-hexosaminidase from Lactococcus lactis ssp. lactis IL1403

The lnbA gene of Lactococcus lactis ssp. lactis IL1403 encodes a polypeptide with similarity to lacto-N-biosidases and N-acetyl-β-D-hexosaminidases. The gene was cloned into the expression vector pET-21d and overexpressed in Escherichia coli BL21* (DE3). The recombinant purified enzyme (LnbA) was a monomer with a molecular weight of approximately 37 kDa. Studies with chromogenic substrates including p-nitrophenyl N-acetyl-β-D-glucosamine (pNP-GlcNAc) and p-nitrophenyl N-acetyl-β-D-galactosamine (pNP-GalNAc) showed that the enzyme had both N-acetyl-β-D-glucosaminidase and N-acetyl-β-D-galactosaminidase activity, thus indicating that the enzyme is an N-acetyl-β-D-hexosaminidase. K(m) and k(cat) for pNP-GlcNAc were 2.56 mM and 26.7 s(-1), respectively, whereas kinetic parameters for pNP-GalNAc could not be determined due to the K(m) being very high (>10 mM). The optimal temperature and pH of the enzyme were 37 °C and 5.5, respectively, for both substrates. The half-life of activity at 37 °C and pH 6.0 was 53 h, but activity was completely abolished after 30 min at 50 °C, meaning that the enzyme has relatively low temperature stability. The enzyme was stable in the pH 5.5-8 range and was unstable at pH below 5.5. Studies with natural substrates showed hydrolytic activity on chito-oligosaccharides but not on colloidal chitin or chitosan. Transglycosylation products were not detected. In all, the data suggest that LnbA's role may be to degrade chito-oligosaccharides that are produced by the previously described chitinolytic system of L. lactis.     

Characterization of a GH3 family β-glucosidase from Dictyoglomus turgidum and its application to the hydrolysis of isoflavone glycosides in spent coffee grounds

A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 31 U/mg by His-Trap affinity chromatography. D. turgidum β-glucosidase was identified as a memmber of the glycoside hydrolase (GH) 3 family on the basis of its amino acid sequence. The native enzyme existed as an 86 kDa monomer with an activity maximum at pH 5 and 85 °C with a half-life of 334 min. The hydrolytic activity of the enzyme with aryl-glycoside substrates was the highest for p-nitrophenyl (pNP)-β-D-glucopyranoside (with a K(m) of 1.3 mM and a k(cat) of 13900 1/s), followed by oNP-β-D-glucopyranoside, pNP-β-D-xylopyranoside, pNP-β-D-fucopyranoside, and pNP-β-D-galactopyranoside. However, no activity was observed for oNP-β-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-mannopyranoside, pNP-β-L-arabinopyranoside, and pNP-α-L-rhamnopyranoside. The hydrolytic activity of the β-glucosidase for coffee isoflavones followed the order genistin (with a K(m) of 0.67 mM and a k(cat) of 5750 1/s) > daidzin > ononin > glycitin. The concentrations of daidzin in ground coffee and spent coffee grounds were 160 and 107 μg/g, respectively, but other isoflavones were present at low concentrations or absent. The enzyme completely hydrolyzed 1.2 mM daidzin in spent coffee grounds after 2 h, with a productivity of 0.6 mM/h. This is the first report concerning the enzymatic hydrolysis of isoflavone glycosides in spent coffee grounds.     

Influence of ethylenediaminetetraacetic acid (EDTA) on the structural stability of endoglucanase from Aspergillus aculeatus

The effect of the chelating agent ethylenediaminetetraacetic acid (EDTA) on the structure and function of endoglucanase is studied. In the presence of 2 mM EDTA, endoglucanase showed an enhanced enzymatic activity of 1.5-fold compared to control. No further change in activity was observed with increase in the concentration of EDTA to 5 mM. The K(m) values for control and in the presence of EDTA are 0.060 and 0.044%, respectively, and K(cat) was 1.9 min(-1) in the presence of EDTA. The kinetic parameters indicated a decrease in the K(m) with an increase in the K(cat). Far-ultraviolet circular dichroism (far-UV-CD) results showed a 20% decrease in ellipticity values at 217 nm in the presence of EDTA compared to native enzyme. The apparent T(m) shifted from a control value of 57 ± 1 to 76 ± 1 °C in the presence of EDTA (5 mM). The above results suggested that the enhanced activity in the presence of EDTA is due to an increase in the K(cat) and flexible conformation of the enzyme. The stability of endoglucanase increased in the presence of EDTA.     

Functional proteomic analysis of rice bran esterases/lipases and characterization of a novel recombinant esterase

An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 °C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 μM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) μM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.     

Purification and characterization of lipoxygenase from Pleurotus ostreatus

Lipoxygenase was purified homogeneously from cups of Pleurotus ostreatus by Sephacryl S-400 HR gel filtration, Dyematrex Green A affinity, and DEAE-Toyopearl 650M ion-exchange chromatographies. The molecular weight of the enzyme was estimated to be 67,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 66,000 by gel filtration; the isoelectric point was pH 5.1. The optimum pH and temperature of the enzymatic activity were 8.0 and 25 degrees C, respectively. The enzyme contained non-heme iron, and a thiol group seemed to be involved in its activity. The K(m), V(max), and k(cat) values of the enzyme for linoleic acid were 0.13 mM, 23.4 micromol.min(-1).mg(-1), and 25.7 s(-1), respectively. The enzyme showed high specificity toward linoleic acid. When linoleic acid was incubated with the enzyme, 13-hydroperoxy-9Z,11E-octadecadienoic acid was found to be the main oxidative product.     

Characterization of a thermophilic L-rhamnose isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494

L-Rhamnose isomerase (EC 5.3.1.14, l-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-rhi gene encoding L-RhI was PCR-cloned from Caldicellulosiruptor saccharolyticus ATCC 43494 and then expressed in Escherichia coli. A high yield of active L-RhI, 3010 U/g of wet cells, was obtained after 20 °C induction for 20 h. The enzyme was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 90 °C. The enzyme was stable at pH values ranging from 4 to 11 and retained >90% activity after a 6 h incubation at 80 °C and pH 7-8. Compared with other previously characterized L-RhIs, the L-RhI from C. saccharolyticus ATCC 43494 has a good thermostability, the widest pH-stable range, and the highest catalytic efficiencies (k(cat)/K(M)) against L-rhamnose, L-lyxose, L-mannose, D-allose, and D-ribose, suggesting that this enzyme has the potential to be applied in rare sugar production.     

Comparison of three thermostable β-glucosidases for application in the hydrolysis of soybean isoflavone glycosides

A novel thermostable β-glucosidase (Te-BglA) from Thermoanaerobacter ethanolicus JW200 was cloned, characterized and compared for its activity against isoflavone glycosides with two β-glucosidases (Tm-BglA, Tm-BglB) from Thermotoga maritima. Te-BglA exhibited maximum hydrolytic activity toward pNP-β-d-glucopyranoside (pNPG) at 80 °C and pH 7.0, was stable for a pH range of 4.6-7.8 and at 65 °C for 3 h, and had the lowest K(m) for the natural glycoside salicin and the highest relative substrate specificity (k(cat)/K(m))((salicin))/(k(cat)/K(m))((pNPG)) among the three enzymes. It converted isoflavone glycosides, including malonyl glycosides, in soybean flour to their aglycons more efficiently than Tm-BglA and Tm-BglB. After 3 h of incubation at 65 °C, Te-BglA produced complete hydrolysis of four isoflavone glycosides (namely, daidzin, genistin and their malonylated forms), exhibiting higher productivity of genistein and daidzein than the other two β-glucosidases. Our results suggest that Te-BglA is preferable to Tm-BglA and Tm-BglB, but all three enzymes have great potential applications in converting isoflavone glycosides into their aglycons.     

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.)

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).     

Characterization of a heterodimeric GH2 β-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.     

Screening of postharvest agricultural wastes as alternative sources of peroxidases: characterization and kinetics of a novel peroxidase from lentil ( Lens culinaris L.) stubble

Aqueous crude extracts of a series of plant wastes (agricultural, wild plants, residues from sports activities (grass), ornamental residues (gardens)) from 17 different plant species representative of the typical biodiversity of the Iberian peninsula were investigated as new sources of peroxidases (EC 1.11.1.7). Of these, lentil (Lens culinaris L.) stubble crude extract was seen to provide one of the highest specific peroxidase activities, catalyzing the oxidation of guaiacol in the presence of hydrogen peroxide to tetraguaiacol, and was used for further studies. For the optimum extraction conditions found, the peroxidase activity in this crude extract (110 U mL(-1)) did not vary for at least 15 months when stored at 4 °C (k(inact) = 0.146 year(-1), t(1/2 inact) = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL(-1)) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k(inact) = 2.2 × 10(-3) day(-1), t(1/2 inact) = 315 days). Using guaiacol as an H donor and a universal buffer (see above), all crude extract samples exhibited the highest peroxidase activity in the pH range between 4 and 7. Once semipurified by passing the crude extract through hydrophobic chromatography on phenyl-Sepharose CL-4B, the novel peroxidase (LSP) was characterized as having a purity number (RZ) of 2.5 and three SDS-PAGE electrophoretic bands corresponding to molecular masses of 52, 35, and 18 kDa. The steady-state kinetic study carried out on the H(2)O(2)-mediated oxidation of guaiacol by the catalytic action of this partially purified peroxidase pointed to apparent Michaelian kinetic behavior (K(m)(appH(2)O(2)) = 1.87 mM; V(max)(appH(2)O(2)) = 6.4 mM min(-1); K(m)(app guaicol) = 32 mM; V(max)(app guaicol) = 9.1 mM min(-1)), compatible with the two-substrate ping-pong mechanism generally accepted for peroxidases. Finally, after the effectiveness of the crude extracts of LSP in oxidizing and removing from solution a series of last-generation dyes present in effluents from textile industries (1) had been checked, a steady-state kinetic study of the H(2)O(2)-mediated oxidation and decolorization of Green Domalan BL by the catalytic action of the lentil stubble extract was carried out, with the observation of the same apparent Michaelian kinetic behavior (K(m)(appGD) = 471 μM; V(max)(appGD)= 23 μM min(-1)). Further studies are currently under way to address the application of this LSP crude extract for the clinical and biochemical analysis of biomarkers.     

Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10

Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 μmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.     

Dehydroascorbate reductase cDNA from sweet potato (Ipomoea batatas [L.] Lam): expression, enzyme properties, and kinetic studies

A cDNA encoding a putative dehydroascorbate reductase (DHAR) was cloned from sweet potato. The deduced protein showed a high level of sequence homology with DHARs from other plants (67 to approximately 81%). Functional sweet potato DHAR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 12% native PAGE. The protein's half-life of deactivation at 50 degrees C was 10.1 min, and its thermal inactivation rate constant K(d) was 6.4 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 6.0-11.0 and in the presence of 0.8 M imidazole. The K(m) values for DHA and GSH were 0.19 and 2.38 mM, respectively.     

Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 degrees C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 degrees C. The k(cat) values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s(-1), respectively, whereas the k(cat) values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s(-1), respectively. The K(M) values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.     

Modeling the kinetics of whey protein hydrolysis brought about by enzymes from Cynara cardunculus

The purpose of this research work was to study the proteolytic activity of aqueous crude extracts of flowers of the plant Cynara cardunculus on the major whey proteins, namely, beta-lactoglobulin (beta-Lg) and alpha-lactalbumin (alpha-La). These extracts, containing a mixture of cardosins A and B (i.e., two distinct aspartic proteases), have been employed for many years in traditional cheese-making in Portugal and Spain. Cow's milk sweet whey was incubated for up to 24 h at various ratios of addition of crude enzyme extract, under controlled pH (5.2 and 6.0) and temperature (55 degrees C). The samples collected were assayed by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A mechanistic model was proposed for the kinetics of the hydrolysis process, which is basically a double-substrate, double-enzyme Michaelis-Menten rate expression; the kinetic parameters were estimated by multiresponse, nonlinear regression analysis. The best estimates obtained for the specificity ratio (i.e., k(cat)/K(m)) of each cardosin within the mixture toward each whey protein indicated that said aspartic proteases possess a higher catalytic efficiency for alpha-La (0.42-4.2 mM(-1).s(-1)) than for beta-Lg (0-0.064 mM(-1).s(-1)), at least under the experimental conditions used. These ratios are below those previously reported for caseins and a synthetic hexapeptide. Cardosins are more active at pH 5.2 than at pH 6.0 and (as expected) at higher enzyme-to-substrate ratios.     

Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L

Polyphenol oxidase (PPO) was purified and characterized from Chinese cabbage by ammonium sulfate precipitation and DEAE-Toyopearl 650M column chromatography. Substrate staining of the crude protein extract showed the presence of three isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be approximately 65 kDa by gel filtration on Toyopearl HW-55F. On SDS-PAGE analysis, this enzyme was composed of a subunit molecular weight of 65 kDa. The optimum pH was 5.0, and this enzyme was stable at pH 6.0 but was unstable below pH 4.0 or above pH 7.0. The optimum temperature was 40 degrees C. Heat inactivation studies showed temperatures >40 degrees C resulted in loss of enzyme activity. PPO showed activity to catechol, pyrogallol, and dopamine (K(m) and V(max) values were 682.5 mM and 67.6 OD/min for catechol, 15.4 mM and 14.1 OD/min for pyrogallol, and 62.0 mM and 14.9 OD/min for dopamine, respectively). The most effective inhibitor was 2-mercaptoethanol, followed in decreasing order by ascorbic acid, glutathione, and L-cysteine. The enzyme activity of the preparation was maintained for 2 days at 4 degrees C but showed a sudden decreased after 3 days.     

Characterization of novel β-glucosidases with transglycosylation properties from Trichosporon asahii

Two novel β-glucosidases from Trichosporon asahii, named BG1 and BG2, were purified to electrophoretic homogeneity using ammonium sulfate precipitation, hydrophobic interaction, ion exchange, and gelfiltration chromatography. The molecular weight of BG1 and BG2 were estimated as 160 kDa and 30 kDa, respectively. The K(m), V(max), K(cat), and K(cat)/K(m) values of the two β-glucosidases for p-nitrophenyl-β-D-glucopyranoside were determined. Both enzymes showed relatively high affinity to p-nitrophenyl-β-D-glucopyranoside in 4-nitrophenol glycosides and gentiobiose in saccharide substrates. The enzymes exhibited optimum activity at pH 6.0 and pH 5.5, respectively. Their respective optimum temperatures were 70 and 50 °C. Metal ions and inhibitors had different effects on the enzymes activities. Circular dichroism (CD) spectroscopy demonstrated that the purified BG1 exhibited a β-sheet-rich structure and that BG2 displayed a high random coil conformation. HPLC analysis of transglycosylation and reverse hydrolysis assays revealed that only BG1 possessed transglycosylation activity and synthesized cello-oligosaccharides by the addition of glucose. This suggested that BG1 could be used to produce complex bioactive glycosides and could be considered as a potential enzyme for industrial application.     

Purification and characterization of cathepsin L from the muscle of silver carp (Hypophthalmichthys molitrix)

Cathepsin L in silver carp musle was purified to 48.4-fold by acid-heat treatment and ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 30 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64 and insensitive to PMSF and pepstatin A, suggesting that the purified enzyme belongs to a family of cysteine proteinase. Consistent with this conclusion, Zn2+, Cu2+, Co2+, Ni2+, and Fe2+ could strongly inhibit the activity of this enzyme. The optimal pH and temperature were 5.0 and 55 degrees C, respectively. The enzyme catalyzed the hydrolysis of Z-Phe-Arg-MCA with a parameter of K(m) (8.27 microM) and K(cat) (28.7 s(-1)) but hardly hydrolyzed Z-Arg-Arg-MCA, Arg-MCA, and Boc-Val-Leu-Lys-MCA. The microstructure analysis by scanning electron microscopy showed that this proteinase is capable of destroying the network structure of silver carp surimi gels. The enzyme exhibited a higher hydrolytic activity on surimi protein at 65 degrees C than at 40 degrees C.     

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.     

Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution

A leucine aminopeptidase was purified for the first time from marine fish red sea bream ( Pagrus major) skeletal muscle to homogeneity with 4850-fold and a yield of 7.4%. The purification procedure consisted of ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, and phenyl-Sepharose. The enzyme was approximately 96 kDa as estimated by SDS-PAGE and gel filtration and preferentially hydrolyzed substrate Leu-MCA. The enzymatic activity was optimal at 45 degrees C and pH 7.5. The K m and k cat values of the enzyme for Leu-MCA were 1.55 microM and 26.4 S (-1) at 37 degrees C, respectively. Activation energy ( E a) of the enzyme was 59.6 kJ M (-1). The enzyme was specifically inhibited by metal-chelating agents, and Zn (2+) and (or) Mn (2+) seemed to be its metal cofactor(s). In addition, bestatin strongly inhibited its activity, and K i was 1.44 microM. Using a highly specific polyclonal antibody, the location of enzyme was demonstrated intracellularly and distributed in different tissues.