Molecular cloning of the arylsulfate sulfotransferase gene and characterization of its product from Enterobacter amnigenus AR-37

The gene encoding the Enterobacter amnigenus AR-37 arylsulfate sulfotransferase (ASST) was cloned, sequenced, and expressed in Escherichia coli NM522. Sequencing led to the identification of three contiguous open reading frames (ORFs) on the same strand. Based on amino acid sequence homology, ORF1, ORF2, and ORF3 are designated astA, dsbA, and dsbB, respectively. A multiple sequence alignment revealed conserved regions in ASST. An N-terminal amino acid sequence analysis of the purified ASST from E. coli NM522 (pEAST72) showed that it is subject to N-terminal processing. The specific activity of purified ASST is 436.5 U/mg of protein. The enzyme is a monomeric protein with a molecular mass of 64 kDa. Using phenol as an acceptor substrate, 4-methylumbelliferyl sulfate is the best donor substrate, followed by beta-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. For PNS, alpha-naphthol is the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine, and tyrosine. The enzyme has a different acceptor specificity than the enzyme purified from Eubacterium A-44. It is similar to Klebsiella K-36 and Haemophilus K-12. The apparent K(m) values for PNS using phenol as an acceptor and for phenol using PNS as a donor are 0.163 and 0.314 mM, respectively. The pI and optimum pH are 6.1 and 9.0, respectively.     

Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase

An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol.     

Characterization of free and alginate-polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate, and phenol into L-tyrosine

The whole cell tyrosine phenol-lyase (TPL, E.C. 4.1.99.2) activity of Erwinia herbicola (ATCC 21434) was microen-capsulated. We studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 degrees C to 32 degrees C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 degrees C the cells grow rapidly, but they also enter the death phase rapidly. There is only limited growth of E. herbicola at 42 degrees C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 +/- 100 mum, mean +/- std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of-the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K(M) of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K(M) for phenol is 0.5 mM. The apparent K(M) values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme. (c) 1995 John Wiley & Sons, Inc.     

Human Platelet Phenol Sulfotransferase: Partial Purification and Detection of Two Forms of the Enzyme

To begin to study the usefulness of platelet phenol sulfotransferase (PST) as a possible measure of the enzyme activity in other organs such as the brain, we purified human platelet PST 36-120-fold. Activity toward 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine, 5-hydroxytryptamine (5-HT), and phenol eluted in the same Sephadex G-100 and Affi-Gel Blue column fractions. Specific activities of the enzyme with MHPG, dopamine, 5-HT, and phenol as substrates were 1198, 1068, 401, and 408 units/mg protein, respectively. Optimal assay conditions were established for each substrate. Apparent Km values were 598 microM, 21 microM, 19 microM, and 500 microM for MHPG, dopamine, phenol, and 5-HT, respectively. Apparent Km values for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) with the same four substrates ranged from 0.11 to 0.25 microM. The pH optima were 6.3 for phenol, 6.8 for dopamine, and 7.0 for MHPG and 5-HT. An additional pH optimum at 8.6 was present for 5-HT. A thermolabile form of the enzyme measured with dopamine and 5-HT, as well as a thermostable form measured with phenol, were present. Dichloronitrophenol (10(-5) M) noncompetitively inhibited the thermostable enzyme activity by 96% but decreased the thermolabile activity by only 36%. These studies provide the basis for a more accurate comparison of human platelet PST with the enzyme in the human brain and in other tissues.     

Studies of the mechanism of phenol hydroxylase: effect of mutation of proline 364 to serine

An active site residue in phenol hydroxylase (PHHY), Pro364, was mutated to serine to investigate its role in enzymatic catalysis. In the presence of phenol, the reaction between the reduced flavin of P364S and oxygen is very fast, but only 13% of the flavin is utilized to hydroxylate the substrate, compared to nearly 100% for the wild-type enzyme. The oxidative half-reaction of PHHY using m-cresol as a substrate is similarly affected by the mutation. Pro364 was suggested to be important in stabilizing the transition state of the oxygen transfer step by forming a hydrogen bond between its carbonyl oxygen and the C4a-hydroperoxyflavin [Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J. (2000) J. Am. Chem. Soc. 122, 8728-8738]. The P364S mutation may weaken this interaction by increasing the flexibility of the peptide chain; hence, the transition state would be destabilized to result in a decreased level of hydroxylation of phenol. However, when the oxidative half-reaction was studied using resorcinol as a substrate, the P364S mutant form was not significantly different from the wild-type enzyme. The rate constants for all the reaction steps as well as the hydroxylation efficiency (coupling between NADPH oxidation and resorcinol consumption) are comparable to those of the wild-type enzyme. It is suggested that the function of Pro364 in catalysis, stabilization of the transition state, is not as important in the reaction with resorcinol, possibly because the position of hydroxylation is different with resorcinol than with phenol and m-cresol.     

Isolation and properties of tyrosine phenol-lyase from Citrobacter intermedius

A method for preparation of homogeneous tyrosine phenol lyase (EC 4.199.2) from Citrobacter intermedius has been developed. The cells were cultivated in the media with a view to obtain a cell culture with a high activity of tyrosine phenol lyase. The isoelectric point for the enzyme lies at pH 4.9. Tyrosine phenol lyase is strictly stereospecific: it catalyzes the formation of pyruvate only from L-tyrosine, but not from D-tyrosine. Kinetic studies showed that K+ and NH4+ cations are non-competitive activators of the enzyme (Ka = 3.57 X 10(-3) and 1.34 X 10(-4) M, respectively).     

Characterization of an extracellular salicyl alcohol oxidase from larval defensive secretions of Chrysomela populi and Phratora vitellinae (Chrysomelina)

Larvae of a number of chrysomelid leaf beetles sequester phenol glucosides such as salicin from their food plants, i.e. Salix and Populus spp. Salicin is hydrolyzed in the glandular reservoir of the defensive glands. The resulting salicyl alcohol (saligenin) is oxidized by an extracellular oxidase. The product salicylaldehyde accumulates as major defensive compound. The secretions from Chrysomela populi and Phratora vitellinae were preserved in saturated ammonium sulfate solution and subjected to micro-purification of the oxidase by means of electrophoretic methods. The enzyme from P. vitellinae has a native M(r) of 334,000 and a subunit M(r) of 79,000 indicating a tetrameric enzyme. The isoelectric points of the enzymes from C. populi and P. vitellinae are at pH 5.4 and 5.2, respectively. In the oxidation of salicyl alcohol oxygen functions as electron acceptor yielding hydrogen peroxide as product. Hydrogen peroxide does not accumulate in native secretions but appears to be degraded most likely by a catalase. The oxidases from the two species show broad pH optima in the range 5.5 to 6.5, they oxidize salicyl alcohol as main substrate. Minor substrates are several ortho-substituted and to a lesser extent meta- but not para-substituted benzyl alcohols. In the presence of 8-hydroxygeraniol only trace amounts of the respective aldehyde are formed. The Km values of salicyl alcohol are 132 mM (C. populi) and 63 mM (P. vitellinae). The extracellular enzyme, which is functionally related to fungal aryl alcohol oxidase (EC 1.1.3.7) and vanillyl alcohol oxidase (EC 1.1.3.38) was named salicyl alcohol oxidase. The continuous formation of salicylaldehyde in the glandular reservoir can be compared to the operation of an enzyme reactor. Due to its low aqueous solubility the produced aldehyde steadily leaves the aqueous reaction fluid and builds up an organic phase which may account for 15% of the total liquid volume of the secretion.     

Characterization of an inducible chlorophenol O-methyltransferase from Trichoderma longibrachiatum involved in the formation of chloroanisoles and determination of its role in cork taint of wines

A novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of Trichoderma longibrachiatum CECT 20431. The enzyme could be identified in partially purified protein preparations by direct photolabeling with [methyl-(3)H]SAM, and this reaction was prevented by previous incubation with S-adenosylhomocysteine. Gel filtration indicated that the M(r) was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500. The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28 degrees C, with a pI of 4.9. The K(m) values for 2,4,6-trichlorophenol and SAM were 135.9 +/- 12.8 and 284.1 +/- 35.1 micro M, respectively. S-Adenosylhomocysteine acted as a competitive inhibitor, with a K(i) of 378.9 +/- 45.4 micro M. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu(2+), Hg(2+), Zn(2+), and Ag(+), and to a lesser extent by p-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents. The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures. Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated.     

Stable enzyme biosensors based on chemically synthesized Au-polypyrrole nanocomposites

This work describes development and optimization of a generic method for the immobilization of enzymes in chemically synthesized gold polypyrrole (Au-PPy) nanocomposite and their application in amperometric biosensors. Three enzyme systems have been used as model examples: cytochrome c, glucose oxidase and polyphenol oxidase. The synthesis and deposition of the nanocomposite was first optimized onto a glassy carbon electrode (GCE) and then, the optimum procedure was used for enzyme immobilization and subsequent fabrication of glucose and phenol biosensors. The resulting nanostructured polymer strongly adheres to the surface of the GCE electrode, has uniform distribution and is very stable. The method has proved to be an effective way for stable enzyme attachment while the presence of gold nanoparticles provides enhanced electrochemical activity; it needs very small amounts of pyrrole and enzyme and the Au-PPy matrix avoids enzyme leaking. The preparation conditions, Michaelis-Menten kinetics and analytical performance characteristics of the two biosensors are discussed. Optimization of the experimental parameters was performed with regard to pyrrole concentration, enzyme amount, pH and operating potential. These biosensors resulted in rapid, simple, and accurate measurement of glucose and phenol with high sensitivities (1.089 mA/M glucose and 497.1 mA/M phenol), low detection limits (2 x 10(-6)M glucose and 3 x 10(-8)M phenol) and fast response times (less than 10s). The biosensors showed an excellent operational stability (at least 100 assays) and reproducibility (R.S.D. of 1.36%).     

Inhibition and affinity chromatography of chicken lung angiotensin I-converting enzyme with captopril.

1. Angiotensin I-converting enzyme (EC 3.4.15.1) has been purified to electrophoretic homogeneity from chicken lung by using a facile two-step protocol which included affinity chromatography on Sepharose-bound captopril. 2. Captopril was a potent inhibitor of chicken lung angiotensin I-converting enzyme with Ki values of 2.0 nmol/l and 1.6 nmol/l for detergent-extracted and trypsin-extracted angiotensin I-converting enzymes, respectively. 3. Molecular weight comparison of trypsin-extracted (M(r)270,000) and detergent-extracted (M(r)690,000) angiotensin I-converting enzyme indicated that membrane-binding sequence contributed to a large extent to the enzyme molecule. 4. Kinetic properties of both forms of the enzyme suggested that the membrane-bound sequence contributed to an increase of the enzyme-substrate affinity.     

Effects of glucose on phenol biodegradation by heterogeneous populations

The effect of the presence of more easily degradable alternative carbon sources on the biodegradation of toxic waste components is of great practical importance. In this work, a mixed phenol/glucose waste was fed to two heterogeneous populations acclimated to different conditions: one was acclimated to phenol as a sole source of carbon and one to a mixed phenol/glucose substrate. Batch substrate utilization experiments were performed under both growth and nonproliferating (no medium nitrogen source) conditions in order to assess substrate removal patterns at the levels of enzyme production and enzyme function. The results indicated that the substrate removal pattern exhibited by the cells was significantly influenced by the acclimation characteristics of the culture. The phenol acclimated cells showed an initial preference for phenol, but the presence of glucose hindered phenol removal rate under both growth and nonproliferating conditions. The cells acclimated to the mixed phenol/glucose waste demonstrated rapid initial glucose removal with a slower concomitant utilization of phenol; acclimation to the mixed waste evidently had a significant impact on the substrate removal pattern for this mixed substrate system.     

Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol.     

Cooxidation of phenol and 4-aminoantipyrin, catalyzed by polymers and copolymers of horseradish root peroxidase and Penicillium funiculosum 46.1 glucose oxidase

Polymers and copolymers of horseradish root peroxidase (HRP) and Penicillium funiculosum 46.1 glucose oxidase (GO) have been synthesized and their catalytic properties have been characterized (free and immobilized forms of each enzyme were studied). The cooxidation reaction of phenol and 4-aminoantipyrin (4-AAP), performed in an aqueous medium in the presence of equimolar amounts of GO and HRP, was characterized by effective K(M) and k(cat) of 0.58 mM and 20.9 s(-1) (for phenol), and 14.6 mM and 18.4 s(-1) (glucose), respectively. The catalytic efficiency of polymerization products (PPs) of GO (GO-PPs) depended on the extent of their aggregation. The combinations GO + HRP-PP and HRP + GO-PP, as well as the copolymer HRP*-GO-PP, proved promising as reagents for enzyme-based analytical systems. When adsorbed on aluminum hydroxide gels, GO-PPs exhibited higher catalytic activity than the non-polymeric enzyme. Maximum retention of GO-PP activity on the inorganic carrier was observed in the case of GO-PP copolymers with an activated HRP. Polymerization of HRP in the presence of a zinc hydroxide gel, paralleled by HRP-PP immobilization onto the gel, increased both the activity of the enzyme and its operational stability.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.     

Decay of the 4a-hydroxy-FAD intermediate of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase involves the formation of three spectrally distinct intermediates (Detmer, K.M., and Massey, V. (1985) J. Biol. Chem. 260, 5998-6005). Addition of an aerobic NADPH-regenerating system, phenol, and azide quantitatively converted oxidized enzyme to the third intermediate, a 4a-hydroxy-FAD species (Detmer, K.M., and Massey, V. (1984) J. Biol. Chem. 259, 11265-11272). This intermediate was isolated in the presence of azide and a wide variety of phenolic ligands. Decay rates were followed for the dehydration of 4a-hydroxy-FAD enzyme resulting in the original oxidized form. Deviation from the rate observed in the absence of phenolic ligands was presumed to be indicative of a binding interaction. Several phenols displayed further stabilization of the 4a-hydroxyflavin species. These ligands exhibited saturation kinetics with respect to the decay half-lives, consistent with a mechanistic model in which both free and bound 4a-hydroxy-FAD enzyme may be directly dehydrated to produce the oxidized species. The lack of stabilization by catechol, the natural product, suggests that product is released from the enzyme during turnover by the time that this intermediate is formed. A pH profile, generated for the decay rates in the absence and presence of phenolic ligand, suggests both acid and base catalysis by hydronium ion and hydroxide, respectively.     

Fractionation of rat ventricular nuclei.

Myocardial cells were isolated after treatment with collagenase (0.05%) and hyaluronidase (0.1%) by discontinuous-gradient centrifugation on 3% Ficoll. Nuclei derived from these myocardial cells were then fractionated on a discontinuous sucrose density gradient with the following steps: (I) 2.0M/2.3M, (II) 2.3M/2.4M, (III) 2.4M/2.5M, (IV) 2.5M/2.6M, and (V) 2.6M/2.85M. The myocardial nuclei were sedimented in the interfaces of gradient fractions (II) and (III). Nuclei from whole ventricles that had been treated with the enzymes before isolation sedimented into five major subsets of nuclei. These findings suggest that nuclei sedimented in the isopycnic gradient at fractions (II) and (III) are most probably derived from myocardial cells. However, this procedure is laborious and lengthy, and the recovery of myocardial-cell nuclei is low. An alternative method was developed to isolate an enriched fraction of myocardial-cell nuclei from whole ventricular tissue without exposing the tissues to enzyme digestion. These ventricular nuclei could be fractionated into five nuclear subsets by using the same discontinuous sucrose density gradient as that described above. The content of DNA, RNA and protein per nucleus for each band was determined. Although the DNA content per nucleus was constant (10pg), that of RNA varied from 1.5 to 4.5pg and that of protein from 16 to 24pg. Nuclei from each band were examined by light-microscopy: large nuclei occurred in the ligher regions whereas smaller nuclei were found in the denser regions of the gradient. From the size distribution pattern of myocardial-cell nuclei compared with that of total ventricular nuclei, it was found that nuclear subsets (II), (III), and (IV) were similar to myocardial nuclei. Electrophoretic analyses of the proteins solubilized in sodium dodecyl sulphate/phenol or Tris/EDTA/2-mercaptoethanol/phenol obtained from each nuclear subset indicate that these fractions are similar, with limited qualitative differences. These findings indicate that isolation of an enriched fraction of myocardial-cell nuclei could be achieved by discontinuous-sucrose-density-gradient centrifugation.     

Purification and characterization of a protein phosphatase from winged bean.

A protein phosphatase (WbPP) has been purified from the soluble fraction of the winged bean (Psophocarpus tetragonolobus) shoot extract. The preparation is essentially homogenous as shown by the constant specific activity of the enzyme across the peak fractions, eluted from the thiophosphorylated histone-Sepharose affinity column, the last step of purification and by single protein bands on polyacrylamide gel electrophoresis (PAGE) in the presence as well as absence of denaturating agents. The monomeric nature of WbPP is revealed by an M(r) of 92,000 and 85,000, respectively, as estimated by SDS-PAGE and gel permeation chromatography under non-denaturating conditions. Autophosphorylated calmodulin-like domain protein kinase (P-WbCDPKI) [Saha, P., & Singh, M. (1995). Biochem. J., 305, 205] and phosphohistone H1 (P-hisH1), prepared by using the other homologous CDPK, i.e. WbCDPKII [Ganguly, S., & Singh, M. (1998). Phytochemistry, 48(1), 61], are good substrates of the purified enzyme, while P-hisH1 and phosphocasein prepared by using heterologous cAMP-dependent protein kinase, are respectively very poor and totally inactive as substrate. WbPP is adjudged to be a protein phosphoserine phosphatase since phosphoserine is the only phosphorylated amino acid residue detected in our earlier analysis of P-WbCDPKI and P-hisH1. The enzyme is strongly stimulated by a combination of Mg2+ and Ca2+, without being dependent on either of them and is also unaffected by calmodulin and fluphenazine. Orthovanadate strongly inhibits the enzyme while okadaic acid is a poor inhibitor.     

Oxidation of pentagalloylglucose to the ellagitannin,tellimagrandin II,by a phenol oxidase from Tellima grandiflora leaves

A new enzyme has been isolated from leaves of the weed Tellima grandiflora (fringe cups, Saxifragaceae) that catalyzed the O(2)-dependent oxidation of 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose to tellimagrandin II, the first intermediate in the (4)C(1)-glucose derived series of ellagitannins. CD-spectra revealed that the 4,6-O-HHDP-residue of the in vitro product had the (S)-stereoconfiguration characteristic of tellimagrandin II from natural sources. The enzyme, for which a M(r) of ca. 60,000 was determined, was purified to apparent homogeneity. It had a pH-optimum at pH 5.0, an isoelectric point at pH 6.3 and was most stable at pH 4.2. Inhibition studies suggested that this new enzyme, for which the systematic name 'pentagalloylglucose: O(2) oxidoreductase' is proposed, belongs to the vast group of laccase-type phenol oxidases (EC 1.10.3.2).     

Mediator-free phenol sensor based on titania sol-gel encapsulation matrix for immobilization of tyrosinase by a vapor deposition method

A novel amperometric phenol sensor was constructed by immobilizing tyrosinase in a titania sol-gel matrix. The tyrosinase entrapped sol-gel film was obtained with a vapor deposition method, which simplified the traditional sol-gel process and avoided the shrinkage and cracking of conventional sol-gel-derived glasses. This matrix provided a microenvironment for retaining the native structure and activity of the entrapped enzyme and a very low mass transport barrier to the enzyme substrates. Phenol could be oxidized by dissolving oxygen in presence of immobilized tyrosinase to form a detectable product, which was determined at -150 mV without any mediator. The phenol sensor exhibited a fast response (less than 5 s) and sensitivity as high as 103 microA/mM, which resulted from the porous structure and high enzyme loading of the sol-gel matrix. The linear range for phenol determination was from 1.2x10(-7) to 2.6x10(-4) M with a detection limit of 1.0x10(-7) M. The apparent Michaelis-Menten constant of the encapsulated tyrosinase was calculated to be (0.29+/-0.02) mM. The stability of the biosensor was also evaluated.     

Inhibition of beta-carotene-15,15'-dioxygenase activity by dietary flavonoids

Beta-carotene-15,15'-dioxygenase is an enzyme responsible for providing vertebrates with vitamin A by catalyzing oxidative cleavage of beta-carotene at its central double bond to two molecules of retinal in intestinal cells. However, little data have been reported regarding regulation of the enzyme activity. We have evaluated the effects of antioxidants and dietary flavonoids on the beta-carotene dioxygenase activity in vitro using a pig intestinal homogenate as the enzyme source. 2,6-Di-tert-butyl-4-methylphenol (BHT), a synthetic antioxidant, strongly inhibited the activity at the level of 10(-6) M (a mixed-type inhibition), whereas butylated hydroxyanisole, nor-dihydroguaiaretic acid, n-propyl gallate, and curcumin were moderately inhibitory. Flavonoids such as luteolin, quercetin, rhamnetin, and phloretin remarkably inhibited the dioxygenase activity noncompetitively, whereas flavanones, isoflavones, catechins, and anthocyanidins were less inhibitory. The structure-activity relationship indicated that catechol structure of ring B and a planar flavone structure were essential for inhibition. The enzyme inhibition was also indicated in the cultured Caco-2 cells by the significantly reduced conversion of beta-carotene to retinol when incubated with BHT and rhamnetin at 2 microM and 5 microM, respectively. The results suggest that some dietary antioxidants derived from food sources modulate conversion of beta-carotene to vitamin A in intestinal cells.