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1.
Tyrosinase, purified from the cap flesh tissue of portabella mushrooms, was characterized with regard to its physical and biochemical properties. A native molecular size of 41 kDa for the enzyme was obtained by size exclusion chromatography, whereas SDS-PAGE indicated that the enzyme contained a single subunit with a size of approximately 48 kDa under reduced and nonreduced conditions. The purified enzyme showed a single immunological cross-reacting protein after Western blotting when probed with antibodies against Agaricus bisporus tyrosinase. Isoelectric focusing demonstrated that the enzyme preparation, apparently homogeneous by electrophoresis, still contained three isoforms of pI 5.1, 5.2, and 5.3. The purified enzyme was able to oxidize a variety of mono-, di-, and triphenolic compounds. An apparent K(m) of 5 mM was obtained using catechol as the substrate, and an apparent K(m) of 9 mM was found using L-Dopa as a substrate. Ascorbic acid, kojic acid, tropolone, mercaptobenzothiazole, and salicylhydroxamic acid inhibited the enzyme severely at 100 microM.  相似文献   

2.
Polyphenol oxidase (PPO) has been extracted from beet root, in both soluble and membrane fractions. In both cases, the enzyme was in its latent state, and it was activated by sodium dodecyl sulfate. PPO was purified to apparent homogeneity. The soluble PPO purification was achieved by hydrophobic interaction chromatography and gel filtration chromatography, with apparent molecular mass of 55 kDa. The membrane PPO purification was achieved by anion exchange chromatography and gel filtration with apparent molecular mass of 54 kDa. A totally denaturing SDS-PAGE indicated the presence of a single polypeptide with an apparent molecular mass of 60 kDa for both fractions, with the band also revealed by Western blot. A partially denaturing SDS-PAGE stained a single active 36 kDa band for both fractions. Under native isoelectric focusing, a major acidic band of pH 5.2 was detected in both fractions. Kinetic characterization of PPO on the natural substrate l-dopa was carried out.  相似文献   

3.
In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH(4))(2)SO(4) precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V(max)/K(m) values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic acid, gallic acid, d,L-dithiothreitol, tropolone, glutathione, sodium azide, benzoic acid, salicylic acid, and 4-aminobenzoic acid using 4-methylcatechol, pyrogallol, and catechol as substrate. The presence of EDTA, 4-aminobenzoic acid, salicylic acid, gallic acid, and benzoic acid did not cause the inhibition of artichoke PPO. A competitive-type inhibition was obtained with sodium azide, L-cysteine, and d,L-dithiothreitol inhibitors using 4-methylcatechol as substrate; with L-cysteine, tropolone, d,L-dithiothreitol, ascorbic acid, and sodium azide inhibitors using pyrogallol as substrate; and with L-cysteine, tropolone, d,L-dithiotreitol, and ascorbic acid inhibitors using catechol as a substrate. A mixed-type inhibition was obtained with glutathione inhibitor using 4-methylcatechol as a substrate. A noncompetitive inhibition was obtained with tropolone and ascorbic acid inhibitors using 4-methylcatechol as substrate, with glutathione inhibitor using pyrogallol as substrate, and with glutathione and sodium azide inhibitors using catechol as substrate. From these results, it can be said that the most effective inhibitor for artichoke PPO is tropolone. Furthermore, it was found that the type of inhibition depended on the origin of the PPO studied and also on the substrate used.  相似文献   

4.
A partial characterization of polyphenol oxidase (PPO) activity in Ocimum basilicum L. is described. PPO in O. basilicum L. was extracted and purified through (NH4)2SO4 precipitation, dialysis, and a Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. The samples obtained from (NH4)2SO4 precipitation and dialysis were used for the characterization of PPO. At the end of purification by affinity chromatography, 11.5-fold purification was achived. The purified enzyme exhibited a clear single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be approximately 54 kDa. The contents of total phenolic and protein of O. basilicum L. extracts were determined. The total phenolic content of O. basilicum L. was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 280 mg 100 g(-1) on a fresh weight basis. The protein content was determined according to the Bradford method. The enzyme showed activity to 4-methylcatechol, catechol, and pyrogallol substrates, but not to tyrosine. Therefore, of these three substrates, 4-methylcatecol was the best substrate due to the highest V(max)/K(m) value, followed by pyrogallol and catechol. The optimum pH was at 6, 8, and 9 for 4-methylcatechol, catechol, and pyrogallol, respectively. The enzyme had an optimum temperature of 20, 40, and 50 degrees C for 4-methylcatechol, catechol, and pyrogallol, respectively. It was found that optimum temperature and pH were dependent on the substrates studied. The enzyme activity with increasing temperature and inactivation time for 4-methylcatechol, catechol, and pyrogallol substrates decreased due to heat denaturation of the enzyme.  相似文献   

5.
The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.  相似文献   

6.
Acetic acid esterase (EC 3.1.1.6) cleaves the acetyl groups substituted at O-2/O-3 of the xylan backbone of arabinoxylans and is known to modulate their functional properties. To date, this enzyme from cereals has not received much attention. In the present study, acetic acid esterase from 72 h ragi malt was isolated and purified to apparent homogeneity by a four-step purification, i.e., ammonium sulfate precipitation, DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose column chromatography, with a recovery of 0.36% and a fold purification of 34. The products liberated from alpha-NA and PNPA by the action of purified ragi acetic acid esterase were authenticated by ESI-MS and 1H NMR. The pH and temperature optima of the enzyme were found to be 7.5 and 45 degrees C, respectively. The enzyme is stable in the pH range of 6.0-9.0 and temperature range of 30-40 degrees C. The activation energy of the enzymatic reaction was found to be 7.29 kJ mol-1. The apparent Km and Vmax of the purified acetic acid esterase for alpha-NA were 0.04 microM and 0.175 microM min-1 mL-1, respectively. The molecular weight of the native enzyme was found to be 79.4 kDa by GPC whereas the denatured enzyme was found to be 19.7 kDa on SDS, indicating it to be a tetramer. EDTA, citric acid, and metal ions such as Fe+3 and Cu+2 increased the activity while Ni+2, Ca+2, Co+2, Ba+2, Mg+2, Mn+2, Zn+2, and Al+3 reduced the activity. Group-specific reagents such as eserine and PCMB at 25 mM concentration completely inhibited the enzyme while iodoacetamide did not have any effect. Eserine was found to be a competitive inhibitor.  相似文献   

7.
Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius) was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The enzyme had a molecular weight of 45 490+/-3500 Da and Km values of 0.23, 1.14, 1.34, and 5.0 mM for the substrates caffeic acid, chlorogenic acid, 4-methylcatechol, and catechol, respectively. When assayed with resorcinol, DL-DOPA, pyrogallol, protocatechuic, p-coumaric, ferulic, and cinnamic acids, catechin, and quercetin, the PPO showed no activity. The optimum pH varied from 5.0 to 6.6, depending on substrate. PPO activity was inhibited by various phenolic and nonphenolic compounds. p-Coumaric and cinnamic acids showed competitive inhibition, with Ki values of 0.017 and 0.011 mM, respectively, using chlorogenic acid as substrate. Heat inactivation from 60 to 90 degrees C showed the enzyme to be relatively stable at 60-70 degrees C, with progressive inactivation when incubated at 80 and 90 degrees C. The Ea (apparent activation energy) for inactivation was 93.69 kJ mol-1. Sucrose, maltose, glucose, fructose, and trehalose at high concentrations appeared to protect yacon PPO against thermal inactivation at 75 and 80 degrees C.  相似文献   

8.
A lipoxygenase from Terfezia claveryi Chatin ascocarp, a mycorrhizal hypogeous fungus, is described for the first time. The higher proportion of PUFA in T. claveryi ascocarps makes lipid rancidity the main factor limiting its storage life. Thus, the studies on LOX from T. claveryi are important because this enzyme, among other roles, may be involved in an alteration of lipids leading to consumer rejection. The enzyme has been purified to apparent homogeneity by phase partitioning in the presence of Triton X-114, followed by two steps of cation-exchange chromatography. The purified T. claveryi LOX preparation consisted of a single major band with an apparent molecular mass of 66 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymic activity exhibited a strong specificity toward linoleic and linolenic acids as substrates, while only 32% activity was observed using gamma-linolenic acid. The pH optimum of this enzyme was pH 7.0. When the enzyme reacted with linoleic acid, it produced a single peak, which comigrated with standard 13-hydroperoxy-octadecadienoic acid; 13-hydroperoxy-octadecatrienoic acid was produced during the reaction with linolenic acid.  相似文献   

9.
An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the pure acid phosphatase resolved a single protein band that migrated to approximately 60 kDa. Nondenaturing SDS-PAGE electrophoresis revealed a single protein band around 120 kDa after staining with Coomassie Brilliant blue. Quantitative gel filtration chromatography estimated a native molecular mass of this enzyme to be 120 kDa. Thus, this acid phosphatase likely functions as a homodimer, consisting of two similar 60 kDa subunits. An electrophoretic technique using the flourogenic substrate 4-methylumbelliferyl phosphate enabled visualization of an acid phosphatase activity that corresponded to the protein band at 120 kDa on a nondenaturing PAGE gel. It was determined that the acid phosphatase had a pH optimum of 6.0 at 25 degrees C. The enzyme activity appeared to be stable over a broad range of temperatures (10-40 degrees C) and in the presence of the metals Zn2+, Mn2+, and Mg2+ as well as the chelating agents ethylenedinitrilotetraacetic acid and ethylene glycol tetraacetic acid. It was shown that this acid phosphatase could hydrolyze a variety of physiological organophosphate compounds including beta-glycerophosphate, phosphoserine, adenosine triphosphate, adenosine diphosphate, adenosine monphosphate, and pyrophosphate. Furthermore, analysis using capillary electrophoresis demonstrated that this hydrolytic enzyme could transform a wide array of organophosphate pesticides including S-2-ethylthioethyl O,O-dimethylphosphorothioate (demeton-S-methyl); S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethylphosphorodithioate (malathion); O,O-dimethyl O-4-nitrophenyl (paraoxon); O,O,O,O-tetraethyldithiopyrophosphate (sulfatep); O-2-chloro-4-nitrophenyl O,O-dimethylphosphorothioate (dicapthon); and 2,2-dichlorovinyl dimethylphosphate (dichlorvos).  相似文献   

10.
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.  相似文献   

11.
A preliminary survey demonstrated activity for alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-arabinosidase, beta-D-glucosidase, beta-D-xylosidase, and beta-D-galactosidase in orange fruit flavedo and albedo tissue. alpha-L-Rhamnosidase was not detected. Subsequently, a beta-glucosidase was purified from mature fruit rag tissue (composed of intersegmental septa, squeezed juice sacs, and fruit core tissue) of Citrus sinensis var. Valencia. The beta-glucosidase exhibited low levels of activity against p-nitrophenyl-beta-D-fucopyranoside (13.5%) and p-nitrophenyl-alpha-D-glucopyranoside (7.0%), compared to its activity against p-nitrophenyl-beta-D-glucopyranoside (pNPG, 100%). The enzyme was purified by a combination of ion exchange (anion and cation) and gel filtration (Superdex and Toyopearl HW-55S) chromatography. It has an apparent molecular mass of 64 kDa by denaturing electrophoresis or 55 kDa by gel filtration chromatography (BioGel P-100). Hydrolysis of pNPG demonstrated a pH optimum between 4.5 and 5.5. At pH 5.0 the temperature optimum was 40 degrees C. At pH 5.0 and 40 degrees C the K(m) for pNPG was 0.1146 mM and it had a V(max) of 5.2792 nkatal x mg(-1) protein (katal = 0.06 International Units = the amount of enzyme that produces, under standard conditions, one micromol of product per min). Of the substrates tested, the enzyme was most active against the disaccharide cellobiose (1-->4), but was not active against p-nitrophenyl-beta-D-cellobioside. High levels of activity also were observed with the disaccharides laminaribiose (1-->3), gentiobiose (1-->6), and sophorose (1-->2). Activity greater than that observed with pNPG was obtained with the flavonoids hesperetin-7-glucoside and prunin (naringenin-7-glucoside), salicin, mandelonitrile-beta-D-glucoside (a cyanogenic substrate), and sinigrin (a glucosinolate). The enzyme was not active against amygdalin, coniferin, or limonin glucoside.  相似文献   

12.
Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.  相似文献   

13.
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).  相似文献   

14.
A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.  相似文献   

15.
An isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) from endophytic bacterium, Pseudomonas ZD-8 was purified to homogeneity by successive ammonium sulfate precipitation, gel filtration on SephadexG-100, DEAE-sephrose CL-6B and DEAE-Sephacel chromatography. The enzyme was a monomeric protein with an apparent molecular mass of 33 kDa as determined by SDS-PAGE and gel filtration. It was optimally active at pH 6.0 and 40 degrees C and had a specific activity of 1485 U mg of protein(-1) against genistin. The ICHG readily hydrolyzed rho-nitrophenyl-beta-glucoside, rho-nitrophenyl-beta-galactoside, genistin, daidzin, with Km values of 1.64, 1.87, 0.012, 0.014 mM, respectively. The ICHG showed a pronounced specificity for glucose in the 7-position of isoflavone and flavone conjugates and hydrolyzed effectively malonyl isoflavone glucosides as well as isoflavone glucosides with similar kinetics. Glucose and glucono-delta-lactone inhibited the enzyme competitively with Ki values of 84 mM and 23 mM, respectively. The enzyme did not require divalent cations for activity, and its activity was strongly inhibited by Hg2+, Ag+, rho-chloromercuribenzoate, iodoacetic acid, and N-ethylmaleimide while reducing agents such as beta-mercaptoethanol, dithiothreitol, dithioerythritol, glutathione slightly activated the enzyme.  相似文献   

16.
The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.  相似文献   

17.
Phenyllactic acid (PLA) is a novel antimicrobial compound synthesized by lactic acid bacteria (LAB), and its production from phenylpyruvic acid (PPA) is an effective approach. In this work, a lactate dehydrogenase (LDH), which catalyzes the reduction of PPA to PLA, has been purified to homogeneity from a cell-free extract of Lactobacillus sp. SK007 by precipitation with ammonium sulfate, ion exchange, and gel filtration chromatography. The purified enzyme had a dimeric form with a molecular mass of 78 kDa (size exclusion chromatography) or 39 kDa (SDS-PAGE). The ratio of enzyme activity with PPA to that with pyruvate being almost invariable at every purification step indicated that, in Lactobacillus sp. SK007, LDH is responsible for the conversion of PPA into PLA. HPLC profiles of PPA transformation into PLA by growing cells, cell-free extract, and purified LDH of Lactobacillus sp. SK007 were also investigated. Results showed that the presence of NADH was found to be necessary for the enzymatic production of PLA from PPA. The purified LDH displayed optimal activity for PPA at pH 6.0 and 40 degrees C. The Km values of the enzyme for PPA and pyruvate were 1.69 and 0.32 mM, respectively. Moreover, because other screened LAB strains exhibiting relatively high LDH activity toward PPA produced also considerable amounts of PLA, LDH activity for PPA could be therefore used as a screening marker for PLA-producing LAB.  相似文献   

18.
To isolate and characterize novel angiotensin I-converting enzyme (ACE) inhibitory peptide from loach (Misgurnus anguillicaudatus), six proteases, pepsin, α-chymotrypsin, bromelain, papain, alcalase, and Neutrase, were used to hydrolyze loach protein. The hydrolysate (LPH) generated by bromelain [ratio of enzyme to substrate, 3:1000 (w/w)] was found to have the highest ACE inhibitory activity (IC(50), 613.2 ± 8.3 μg/mL). Therefore, it was treated by ultrafiltration to afford fraction of LPH-IV (MW < 2.5 kDa) with an IC(50) of 231.2 ± 3.8 μg/mL, having higher activity than the other fractions. Then, LPH-IV was isolated and purified by consecutive purification steps of gel filtration chromatography and reverse-phase high-performance liquid chromatography to afford a purified peptide with an IC(50) of 18.2 ± 0.9 μg/mL, an increase of 33.7-fold in ACE inhibitory activity as compared with that of LPH. The purified peptide was identified as Ala-His-Leu-Leu (452 Da) by Q-TOF mass spectrometry and amino acid analyzer. An antihypertensive effect in spontaneously hypertensive rats revealed that oral administration of LPH-IV could decrease systolic blood pressure significantly.  相似文献   

19.
Latent polyphenol oxidase (LPPO), an enzyme responsible for the browning reaction of sago starches during processing and storage, was investigated. The enzyme was effectively extracted and partially purified from the pith using combinations of nonionic detergents. With Triton X-114 and a temperature-induced phase partitioning method, the enzyme showed a recovery of 70% and purification of 4. 1-fold. Native PAGE analysis of the partially purified LPPO revealed three activity bands when stained with catechol and two bands with pyrogallol. The molecular masses of the enzymes were estimated by SDS-PAGE to be 37, 45, and 53 kDa. The enzyme showed optimum pH values of 4.5 with 4-methylcatechol as a substrate and 7.5 with pyrogallol. The LPPO was highly reactive toward diphenols and triphenols. The activity of the enzyme was greatly enhanced in the presence of trypsin, SDS, ethanol, and linoleic acid.  相似文献   

20.
Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5'-OH group of arabinosyl residues of arabinoxylans and is known to modulate their functional properties. In this study, ferulic acid esterase from 96 h finger millet malt was purified to apparent homogeneity by three-step purification with a recovery of 3% and a fold purification of 22. The substrate p-nitrophenylferulate (PNPF) was synthesized and used to assay this enzyme spectrophotometrically. The products liberated from ragi and wheat water-soluble polysaccharides by the action of purified ragi ferulic acid esterase were identified by ESI-MS. The pH and temperature optima of the enzyme were found to be 6.0 and 45 degrees C, respectively. The pH and temperature stabilities of the enzyme were found to be in the range of 5.5-9.0 and 30 degrees C, respectively. The activation energy of the enzymatic reaction was found to be 4.08 kJ mol(-1). The apparent K m and V max of the purified ferulic acid esterase for PNPF were 0.053 microM and 0.085 unit mL(-1), respectively. The enzyme is a monomer with a molecular mass of 16.5 kDa. Metal ions such as Ni(2+), Zn(2+), Co(2+), and Cu(2+) and oxalic and citric acids enhanced the enzyme activity. The enzyme was completely inhibited by Fe(3+). Group specific reagents such as p-chloromercuric benzoate and iodoacetamide inhibited the enzyme, indicating the possible presence of cysteine residues in the active site pocket.  相似文献   

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