Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp

Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

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.     

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli

A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45 degrees C, respectively, and the enzyme maintained high activity at pH 5.0 and 60 degrees C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (k(cat)/K(M)) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached approximately 71% at 20 degrees C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for alpha-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.     

Lipoxygenase from banana leaf: purification and characterization of an enzyme that catalyzes linoleic acid oxygenation at the 9-position

The objective of the present study was to purify and characterize the lipoxygenase (LOX) from banana leaf (Giant Cavendishii, AAA), an unutilized bioresource. LOX was extracted, isolated, and purified 327-fold using 25-50% saturation of ammonium sulfate fractionation, hydroxyapatite column separation, and gel filtration on Superdex 200. The molecular mass of the purified LOX was 85 kDa, K(m) was 0.15 mM, and V(max) was 2.4 microM/min.mg using linoleic acid as substrate. Triton X-100 was required in the extraction medium; otherwise, no LOX activity was detected. LOX activity increased with the concentration of Triton X-100 with an optimum at 0.1%. The optimal pH of the purified LOX from banana leaf was 6.2, and optimal temperature was 40 degrees C. The LOX showed the highest reactivity toward 18:2 followed by 18:3 and 20:4. A very low reaction rate was observed toward 20:5 and 22:6. On the basis of retention time in normal phase HPLC, the products of 18:2 or 18:3 catalyzed by purified LOX were hydroperoxyoctadecadienoic acid or hydroperoxyoctadecatrienoic acid. It seems that 9-LOX is the predominant enzyme in banana leaf. Banada leaf dried at 110 degrees C for 2 h developed algal aroma. Banana leaf extract stored at 10 degrees C for 12 h formed an oolong tea-like flavor. Banana leaf extract reacted with 18:2 or soybean oil pretreated with bacterial lipase produced green and melon-like aroma, whereas the same reaction with 18:3 produced a sweet, fruity, cucumber-like flavor note.     

Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste

Bacillus sp. nov. SK006 producing four extracellular fibrinolytic enzymes was isolated from fermented shrimp paste, a traditional and popular Asian seasoning. One fibrinolytic enzyme was purified to homogeneity with a molecular mass of 43-46 kDa by SDS-PAGE and gel filtration chromatography. The specific activity was determined to be 11.2 units/mg using plasmin as a standard. The enzyme displayed optimal activity at 30 degrees C and pH 7.2. It was stable below 40 degrees C for 4 h between pH 5.0 and pH 11.0. Zinc ion stimulated the enzyme activity whereas Cu2+, Ca2+, Fe3+, and Hg2+ caused its inhibition. The fibrinolytic activity was strongly inhibited by PMSF and moderately inhibited by EDTA as well as PCMB. The enzyme exhibited a higher affinity toward N-Succ-Ala-Ala-Pro-Phe-pNA and was able to degrade fibrin clots either by forming active plasmin from plasminogen or by direct fibrinolysis. The N-terminal amino acid sequence was found to be AQSVPYEQPHLSQ, which is different from that of other known fibrinolytic enzymes.     

Effect of intrinsic and extrinsic factors on the interaction of plant pectin methylesterase and its proteinaceous inhibitor from kiwi fruit

A proteinaceous pectin methylesterase inhibitor (PMEI) was isolated from kiwi fruit (Actinidia chinensiscv. Hayward) and purified by affinity chromatography on a cyanogen bromide (CNBr) Sepharose 4B-orange PME column. The optimal pH of banana PME activity was 7.0, whereas that for carrot and strawberry PME activity was 9.0. The optimal pH for the binding between kiwi fruit PMEI and these PMEs was 7.0. The kiwi fruit PMEI has a different affinity for PME depending on the plant source. The inhibition kinetics of kiwi fruit PMEI to banana and strawberry PME followed a noncompetitive type, whereas that to carrot PME followed a competitive type. The kiwi fruit PMEI was mixed with banana, carrot, and strawberry PME to obtain PMEI-PME complexes, which were then subjected to thermal (40-80 degrees C, atmospheric pressure) or high-pressure (10 degrees C, 100-600 MPa) treatment. Experimental data showed that the PMEI-PME complexes were easily dissociated by both thermal and high-pressure treatments.     

Characterization of polyphenol oxidase from litchi pericarp using (-)-epicatechin as substrate

Polyphenol oxidase (PPO) from litchi (Litchi chinensis Sonn.) pericarp was characterized using (-)-epicatechin, which was the major endogenous polyphenol in litchi pericarp as a substrate. The optimum pH for PPO activity with (-)-epicatechin was 7.5, and the enzyme was unstable below pH 4.5 and stable in the pH range of 6.0-8.0. Residual activities of PPO were 86.25, 86.31, and 80.17% after 67 days of incubation at 4 degrees C at pH 6.0, 7.5, and 8.0, respectively. From thermostability studies, the Ki value increased with temperature and the results suggested that the enzyme was unstable above 45 degrees C. Moreover, the results also provided strong evidence that the denaturalization temperature of PPO was near 70 degrees C. The inhibition studies indicated that l-cysteine and glutathione were strong inhibitors even at low concentrations while NaF inhibited moderately. In addition, the results also indicated that the inhibition mechanisms of thiol groups were different from those of halide salts.     

Preparation of neoglycoprotein from carp myofibrillar protein and alginate oligosaccharide: improved solubility in low ionic strength medium

Alginate oligosaccharide (AO) was conjugated with carp myofibrillar protein (Mf) by using the controlled Maillard reaction, and the change in the solubility of Mf in low ionic strength media as affected by the glycosylation was investigated. AO was prepared by degrading sodium alginate using alginate lyase, which was purified from the culture supernatant of Pseudoalteromonas elyakovii. When a lyophilized Mf and AO mixture was incubated at 40 degrees C and 65% relative humidity, the conjugation of AO was confirmed at myosin heavy chain, actin, and tropomyosin. When >30 microg/mg of AO was conjugated to Mf, the protein solubility in a low ionic strength medium was greatly improved without significant loss of available lysine. These results indicate that the conjugation with AO is a superior manner for improving the water solubility of Mf in view of its nutritional aspect.     

Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous)

Three gelatinolytic proteases (A1, A2, and B) were purified using a synthetic substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, from the hepatopancreas of Northern shrimp (Pandalus eous) by several chromatographic steps involving hydroxyapatite column chromatography, gel filtration on Superdex75, and ion-exchange chromatography on a MonoQ column. Collagenolytic proteases A2 and B, but not protease A1, were demonstrated to digest native porcine type I collagen at 25 degrees C and pH 7.5. Further characterizations of these two collagenolytic proteases showed that the pH optimum of enzyme A2 against DNP-peptide was found to be 11, whereas that of enzyme B was 8.5. The optimum temperature ranged between 40 and 45 degrees C for both enzymes, although enzyme B appeared to be thermally more stable than enzyme A2 at pH 7.5. Both enzymes were strongly inhibited by PMSF and antipain, which suggests that they belong to collagenolytic serine proteases.     

29 kDa Trypsin from the pyloric ceca of Atlantic Bonito (Sarda sarda): recovery and characterization

Trypsin from the pyloric ceca of Atlantic bonito (Sarda sarda) was purified and characterized with respect to its purity; molecular weight; sensitivity to temperature, pH, and inhibition; and N-terminal sequence. The purified trypsin had a molecular weight of 29 kDa as per sodium dodecyl sulfate polyacrylamide gel electrophoresis, and optimal activity was observed at pH 9 and 65 degrees C with BAPNA as a substrate. The enzyme was stable to heat treatment up to 50 degrees C and within the pH range of 7-12. It was stabilized by calcium ions, but its activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone, and phenyl methyl sulfonyl fluoride. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration (0-30%). The N-terminal 20 amino acid residues of Atlantic bonito trypsin were determined as IVGGYECQAHSQPWQPVLNS and were homologous with other trypsins.     

三七根际链霉菌Streptomyces sp. YNUCC0233木聚糖酶性质研究

从三七根际分离到一株产木聚糖酶链霉菌Streptomycessp.YNUCC0233。该菌所产生的木聚糖酶的最适反应温度为67℃,最适pH为6.0,在pH5.0~9.0和60℃以下时相对稳定。Ba2+和K+对该木聚糖酶有较强的促进作用,Hg2+、Cu2+、Mn2+、Co2+、Ni2+和EDTA对该酶有较强的抑制作用。对Streptomycessp.YNUCC023316SrDNA的1105bp片段的序列分析结果表明,Streptomycessp.YNUCC0233的16SrDNA片段与GenBank中所有已注册的链霉菌分类单位均不同。     

Partial purification and kinetic characterization of a carotenoid cleavage enzyme from quince fruit (Cydonia oblonga)

For the first time, a cytosolic carotenoid cleavage enzyme isolated from quince (Cydonia oblonga) fruit is described. The enzyme was partially purified by using centrifugation, acetone precipitation, ultrafiltration (300 kD, 50 kD), isoelectric focusing (pH 3-10), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained that contained three similar proteins, all exhibiting molecular weights in the range of 20 kD. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at a wavelength of 505 nm. The time constant of the reaction was 8.2 min, the Michaelis constant (K(m)) was 11.0 micromol x L(-1), and the maximum velocity (v(max)) was 0.083 micromol x L(-1) x min(-1) x mg(protein)(-1). The optimum temperature was above 50 degrees C.     

Effect of Dry Heating with Ionic Gums at Controlled pH on Starch Paste Viscosity

Waxy maize and potato starches were dispersed in pH 6.0 and 8.0 aqueous solutions (1%) of an ionic gum (sodium alginate, sodium carboxymethylcellulose, and xanthan). The mixture was dried at 45°C overnight and then heat‐treated 2 hr at 130°C. Effects on the paste viscosity of the products in a pH 7.0 buffer were examined. Heating with sodium alginate or sodium carboxymethylcellulose (CMC) increased the paste viscosity of waxy maize starch but reduced that of potato starch. In both starches, xanthan effected greater viscogram changes than did sodium alginate or CMC. Use of xanthan in the treatment produced products with restricted granular swelling and increased shear stability of the pastes. The pH of the starch‐gum mixtures affected the thermally induced viscosity changes. Mild acidity (pH 6.0) effected a viscosity decrease for the heat‐treated starch product, whereas alkalinity (pH 8.0) raised the viscosity regardless of the presence of gum. But pH 6 before heat treatment was favored for viscosity increase by sodium alginate, whereas pH 8 gave a greater increase in viscosity when xanthan was used. By using gum mixtures such as xanthan‐alginate and xanthan‐CMC, both viscosity increase and good shear‐stability were achieved.     

A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: purification and biochemical properties

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.     

Survival of freeze-dried Lactobacillus bulgaricus KFRI 673 in chitosan-coated calcium alginate microparticles

The aim of this study was to investigate the effect of alginate microparticles coated with three kinds of chitosans of different molecular weights on the survival of Lactobacillus bulgaricus KFRI 673 in simulated gastric (SGJ) and intestinal juices (SIJ) and on their stability during storage at 4 and 22 degrees C. L. bulgaricus KFRI 673 loaded in alginate microparticles was prepared by spraying the mixture of sodium alginate and cell culture into the calcium chloride solution using an air-atomizing device. When L. bulgaricus KFRI 673 was exposed to SGJ of pH 2.0 for 60 min, none of the microorganism survived. Contrary to this result, microbiological analysis indicated that microencapsulation in alginate microparticles improved the survival of acid-sensitive L. bulgaricus KFRI 673 in SGJ and that high molecular weight chitosan coating resulted in the highest survival in SGJ. To study storage stability of free and microencapsulated cells, in vitro studies were conducted at 4 and 22 degrees C during a 4 week period. Both free and microencapsulated cells showed similar stabilities during 4 weeks of storage at 4 degrees C. However, the stability of Lactobacillus at 22 degrees C was appreciably improved when loaded in high molecular weight chitosan-coated alginate microparticles. In conclusion, microencapsulation of lactic acid bacteria with alginate and chitosan coating offers an effective way of delivering viable bacterial cells to the colon and maintaining their survival during refrigerated storage.     

Changes in molecular weight of transacylated pectin catalyzed by tomato and citrus pectinesterases as determined by gel permeation chromatography

The changes in molecular masses of pectin in 0.5% pectin-pectinesterase (PE) mixtures (2 units/mL) incubated at various temperatures, pH values, and NaCl levels for 30 min were observed by a Toyopearl TSK HW-65 (F) gel permeation chromatography. The molecular mass of pectin was remarkably increased from 103 to 266 kDa when the incubation temperature of pectin-tomato PE was increased from 25 to 45 degrees C. A further increase in molecular mass was observed when a pectin-citrus PE mixture was incubated at 65 degrees C. The values of pH and NaCl levels were also crucial to the transacylation activity of PEs. Reaction at pH 7.5 with tomato PE and citrus PE remarkably expanded the molecular mass of pectin to 410 and 670 kDa, respectively. The NaCl level of 0.3-0.5 and 0.3 M was favorable for the transacylation reaction of tomato PE and citrus PE, respectively. Only high methoxylpectin was the suitable substrate for PE to conduct the transacylation reaction.     

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.     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.