Characterization of CenC, an enzyme from Cellulomonas fimi with both endo- and exoglucanase activities

The cenC gene, encoding beta-1,4-glucanase C (CenC) from Cellulomonas fimi, was overexpressed in Escherichia coli with a tac-based expression vector. The resulting polypeptide, with an apparent molecular mass of 130 kDa, was purified from the cell extracts by affinity chromatography on cellulose followed by anion-exchange chromatography. N-terminal sequence analysis showed the enzyme to be properly processed. Mature CenC was optimally active at pH 5.0 and 45 degrees C. The enzyme was extremely active on soluble, fluorophoric, and chromophoric glycosides (4-methylumbelliferyl beta-glycosides, 2'-chloro-4'-nitrophenyl-beta-D-cellobioside, and 2'-chloro-4'-nitrophenyl-lactoside) and efficiently hydrolyzed carboxymethyl cellulose, barley beta-glucan, lichenan, and, to a lesser extent, glucomannan. CenC also hydrolyzed acid-swollen cellulose, Avicel, and bacterial microcrystalline cellulose. However, degradation of the latter was slow compared with its degradation by CenB, another C. fimi cellulose belonging to the same enzyme family. CenC acted with inversion of configuration at the anomeric carbon, in accordance with its classification as a family 9 member. The enzyme released mainly cellobiose from soluble cellodextrins and insoluble cellulose. Attack appeared to be from the reducing chain ends. Analysis of carboxymethyl cellulose hydrolysis suggests that CenC is semiprocessive enzyme with both endo- and exoglucanase activities.     

Characterization of recombinant E. coli ATCC 11303 (pLOI 297) in the conversion of cellulose and xylose to ethanol

This work describes the characterization of recombinant Escherichia coli ATCC 11303 (pLOI 297) in the production of ethanol from cellulose and xylose. We have examined the fermentation of glucose and xylose, both individually and in mixtures, and the selectivity of ethanol production under various conditions of operation. Xylose metabolism was strongly inhibited by the presence of glucose. Ethanol was a strong inhibitor of both glucose and xylose fermentations; the maximum ethanol levels achieved at 37 degrees C and 42 degrees C were about 50 g/l and 25 g/l respectively. Simultaneous saccharification and fermentation of cellulose with recombinant E. coli and exogenous cellulose showed a high ethanol yield (84% of theoretical) in the hydrolysis regime of pH 5.0 and 37 degrees C. The selectivity of organic acid formation relative to that of ethanol increased at extreme levels of initial glucose concentration; production of succinic and acetic acids increased at low levels of glucose (< 1 g/l), and lactic acid production increased when initial glucose was higher than 100 g/l.     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase (M(r) 31,000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 degrees C, respectively. pH stability ranged from 5 to 9.5 and the t1/2 at 50 degrees C was 490 min. It had a Km of 4.88 mg ml-1 and a Vmax of 588 mumol min-1 mg-1. The activity was inhibited (95%) by AlCl3 (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.     

Purification and properties of a xylan-binding endoxylanase from Alkaliphilic bacillus sp. strain K-1

An alkaliphilic bacterium, Bacillus sp. strain K-1, produces extracellular xylanolytic enzymes such as xylanases, beta-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium. One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan. The enzyme bound to insoluble xylan but not to crystalline cellulose. The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa. The enzyme was stable at alkaline pHs up to 12. The optimum temperature and optimum pH of the enzyme activity were 60 degrees C and 5.5, respectively. Metal ions such as Fe2+, Ca2+, and Mg2+ greatly increased the xylanase activity, whereas Mn2+ strongly inhibited it. We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively. The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.     

Inhibition of ruminal cellulose fermentation by extracts of the perennial legume cicer milkvetch (Astragalus cicer)

Cicer milkvetch (Astragalus cicer L.) is a perennial legume used as a pasture or rangeland plant for ruminants. A study was undertaken to determine whether reported variations in its ruminal digestibility may be related to the presence of an antinutritive material. In vitro fermentation of neutral detergent fiber (NDF) of cicer milkvetch by mixed rumen microflora was poorer than was the fermentation of NDF in alfalfa (Medicago sativa L.). Fermentation of cicer milkvetch NDF was improved by preextraction of the ground herbage with water for 3 h at 39 degrees C. Such water extracts selectively inhibited in vitro fermentation of pure cellulose by mixed ruminal microflora and by pure cultures of the ruminal bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85. Inhibition of the cellulose fermentation by mixed ruminal microflora was dependent upon the concentration of cicer milkvetch extract and was overcome upon prolonged incubation. Pure cultures exposed to the extract did not recover from inhibition, even after long incubation times, unless the inhibitory agent was removed (viz., by dilution of inhibited cultures into fresh medium). The extract did not affect the fermentation of cellobiose by R. flavefaciens but did cause some inhibition of cellobiose fermentation by F. succinogenes. Moreover, the extracts did not inhibit hydrolysis of crystalline cellulose, carboxymethyl cellulose, or p-nitrophenylcellobioside by supernatants of these pure cultures of cellulolytic bacteria or by a commercial cellulase preparation from the fungus Trichoderma reesei. The agent caused cellulose-adherent cells to detach from cellulose fibers, suggesting that the agent may act, at least in part, by disrupting the glycocalyx necessary for adherence to, and rapid digestion of, cellulose.     

Purification and characterization of an esterase involved in poly(vinyl alcohol) degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45 degrees C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K(m) and Vmax of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 microM, and 6.52 and 12.6 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Hydrolysis of phosphodiesters through transformation of the bacterial phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10(8) times faster than the uncatalyzed reaction (kcat = 0.06 s-1 and Km = 38 mM). Upon the addition of various alkylamines to the reaction mixture, the kcat/Km for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (kcat = 0.34 s-1 and Km = 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wild-type enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.     

Simultaneous determination of maltose and glucose using a screen-printed electrode system

A screen-printed sensor system consisting of a glucose oxidase (GOD) electrode and an amyloglucosidase/glucose oxidase (A/G) electrode was constructed to determine maltose and glucose simultaneously in a mixture. Sensor construction was optimised so that it contained 20 units of GOD/40 units of amyloglucosidase and 0.2 mM 1,1'-ferrocenedimethanol. These components were deposited onto a screen-printed carbon electrode and an outer membrane was printed from 3.5% hydroxyethyl cellulose (HEC) solution. The optimum pH was 4.8. The linear range of the system was up to 40 mM glucose or 20 mmol/L maltose with coefficients of variation (CVs) ranging from 3.5% to 5.29%. The results obtained by using the enzyme electrode system agreed well with those obtained by the Fehling titration method. When stored dry, especially at 4 degrees C, the enzyme electrodes showed good stability over four months.     

HIV testing for HIV prevention: a comparative analysis of policies in Britain, Hungary and Sweden

Growth of vegetative cells and outgrowth of spores of enterotoxigenic psychrotrophic Bacillus cereus in refrigerated minimally processed food products is a public health concern. A study was undertaken to determine the combined effects of pH, nisin, and temperature on growth and survival of 20 strains of B. cereus. The minimum growth temperatures in tryptic soy broth (pH 7.3) and brain heart infusion broth (BHI broth, pH 7.4) were 5 degrees C for two strains and 8 degrees C for five other strains. Vegetative cells of four of eight strains grew at 8 degrees C in BHI broth (pH 6.01 and 6.57) containing 10 micrograms of nisin per ml. At 15 degrees C, all strains grew at pH 5.53 to 6.57; three strains tolerated nisin at 50 micrograms/ml (pH 6.57), whereas two other strains had a maximum tolerance of 10 micrograms of nisin per ml. Tolerance of vegetative cells of B. cereus to nisin increased as the pH of the broth was increased from 5.53 to 6.01 and again to pH 6.57. Outgrowth of spores (six of six strains tested) was inhibited by 5 and 50 micrograms of nisin per ml at 8 and 15 degrees C, respectively. At 15 degrees C, outgrowth of spores of two strains occurred at pH 6.52 in BHI broth containing 10 micrograms of nisin per ml. The effectiveness of nisin in controlling the growth of psychrotrophic strains of B. cereus capable of causing human illness was more pronounced at 8 degrees C than at 15 degrees C and as the pH was decreased from 6.57 to 5.53. Studies to determine the effectiveness of nisin in controlling growth of psychrotrophic B. cereus in nonpasteurized foods held at refrigeration temperatures are warranted.     

Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana

Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95 degreesC, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106 degreesC. Both enzymes are characterized by a high level of activity (high Vmax value and low apparent Km value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the Vmax values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106 degreesC and 26 min at 110 degreesC. A single clone encoding the celA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA and celB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.     

Properties of alpha-mannosidase partially purified from the apple snail, Pomacea canaliculata

Pomacea canaliculata alpha-mannosidase (260 kDa), composed of at least two isoforms with different pI, was partially purified. The activity was maximum at pH 4 and unaltered after incubation at 60 degrees C for 60 min. ZnCl2, CaCl2, NaCl, and SH-reagents increased the activity, while MnCl2 and EDTA inhibited it. The enzyme catalyzed the hydrolysis of alpha 1-2, alpha 1-3, and alpha 1-6 mannosidic linkages.     

Syntheses and beta-adrenergic binding affinities of (R)- and (S)-fluoronaphthyloxypropanolamines

Hydrogenase from the marine green alga, Chlorococcum littorale, was purified 1485-fold, resulting in a specific activity for hydrogen evolution of 75.7 micromol/min/mg of protein at 25 degrees C, using reduced methyl viologen as an electron donor. The K(m) value for methyl viologen was 0.5 mM. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be 55 kDa by SDS-PAGE, and 57 kDa by gel filtration. The optimum temperature and pH value for hydrogen evolution were 50 degrees C and 7.5, respectively. The partially purified hydrogenase catalyzed hydrogen evolution from ferredoxin that had been isolated from the same cells, but not from NADH or NADPH. The K(m) value for ferredoxin was 0.68 microM. The enzyme was extremely oxygen sensitive, losing over 95% of its activity upon exposure to air within minutes, even at 4 degrees C. Two peptide fragments were obtained from the hydrogenase protein digested enzymatically, and their amino acid sequences were determined. No significant homology was found to any other known sequences of hydrogenases.     

Isolation of the 36-kD German (Blattella germanica) cockroach allergen using fast protein liquid chromatography

The carotenogenic enzyme phytoene dehydrogenase has been purified from the C9carR21(-) (lycopene-accumulating) mutant of the filamentous fungus Phycomyces blakesleeanus. Solubilization of the membrane-bound enzyme with 1% Tween-60 was followed by a 250-fold purification to homogeneity using polyethylene glycol precipitation, CM-Sepharose, gel filtration and isoelectric focusing. Multiple peaks of enzymic activity were found in eluates from ion-exchange and gel filtration chromatography, with the lowest molecular weight fraction having an apparent molecular mass of approx. 14 kDa. All active fractions catalyzed the dehydrogenation of 15-cis phytoene into all-trans lycopene, with a cis-trans isomerization occurring at phytofluene. Both NADP+ and FAD were required for the dehydrogenation reaction. The presence of > 0.5% Tween-60 was necessary to maintain enzymic activity, although in its absence lipids restored some activity. The enzyme could be stored for at least 6 weeks at -70 degrees C in the presence of 20% (v/v) glycerol.     

Hydrophobic interactions of peptides with membrane interfaces

Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase-glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity-chromatography, while jejunal sucrase-isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl-Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M(r) 145,000, 125,000 and 115,000. Although the N-terminal amino acid sequences bear no homology to SI, the M(r) 115,000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5. The glycohydrolases were most active at 58 degrees C, but were quickly denatured above 60 degrees C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters (K(m), kcat and kcat/K(m)) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.     

Purification and characterization of a prolidase from Aureobacterium esteraromaticum

An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440,000 by gel permeation chromatography, and about 40,000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro. Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45 degrees C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60 degrees C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn2+ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.     

Immobilization of whole-cell penicillin G acylase by entrapping within polymethacrylamide beads

Escherichia coli ATCC 11105 containing the periplasmic penicillin G acylase was entrapped within a copolymer of methacrylamide and N,N'-methylenebisacrylamide. A solution of monomer that was made up from methacrylamide and N,N'-methylenebisacrylamide dissolved in buffer was mixed with lyophilized cells and ammonium persulfate. This suspension was then pumped drop by drop into in soybean oil supplemented with 0.06% (v/v) 3-(dimethylamino)-propionitril. During submerging in the oil phase, the droplets were hardened and induced to polymerize within the droplets. Particles with a volume ranging from 0.013-0.017 mL per bead containing a biomass concentration up to 38.0 g/L were prepared. The optimal condition for the deacylation of penicillin G to 6-aminopencillanic acid (6-APA) catalyzed by the immobilized whole-cell penicillin G acylase was found to be 45 degrees C and pH 8.0. Product inhibition of this enzyme by 6-APA could be eliminated by controlling pH value at 8 during the course of penicillin G hydrolysis using a pH-stat. Conversion determined by the pH-stat method were 0.3% higher than that by p-dimethylaminobenzaldehyde method. Cell concentration in the matrix was found to be an important factor influencing the maximum velocity and the specific activity retained in the matrix. A kinetic model, in which the mass transfer resistances as a result of external film mass transfer and pore diffusion were assumed to be negligible, could properly describe the hydrolysis of penicillin G by the cells entrapped within the polymethacylamide beads.     

Immobilization of the tetrameric and monomeric forms of pigeon liver malic enzyme on Sepharose beads

Pigeon liver malic enzyme was chemically attached to Sepharose 4B-CL beads. The enzyme lost approximately 50% of its original activity when immobilization was carried out with 5 mg CNBr/ml gel. Immobilization performed at pH 8.0 or pH 4.5 resulted in the formation of matrix-bound tetramer and monomer, respectively. Matrix-bound reconstituted tetramer was derived from matrix-bound monomer by mixing the latter with soluble enzyme at pH 4.5, then raised the pH of the solution to 8.0. The matrix-bound monomer was demonstrated to be enzymically fully active in terms of specific activity. The pH profile for the enzymic reaction was similar for both soluble and immobilized enzymes. However, the latter had a broader range for the optimum pH (pH 6.8-7.8). The Arrhenius plots for all immobilized enzyme forms were biphasic with inflection at approximately 27 degrees C. The apparent Michaelis constants for the substrates increased about 2-3-fold after immobilization. All immobilized enzyme forms, including the matrix-bound monomer, showed substrate inhibition at high concentrations of L-malate. Both high-affinity and low-affinity binding sites for Mn2+ existed for all immobilized enzyme forms. These results are consistent with an existing asymmetric model, but are not compatible with a sequential model for the enzyme tetramer. The immobilized enzyme was stable for at least four months at 4 degrees C. As compared to soluble enzyme, the immobilized enzyme was less inhibited by (NH4)2SO4 or NaCl. It was also resistant to inactivation with periodate-oxidized aminopyridine adenine dinucleotide phosphate, an affinity label for malic enzyme. Incubation of the immobilized enzyme (1.25 microM) with the reagent (5.6 mM) resulted in pseudo-first-order inactivation with a rate constant of 0.0108 min-1 that was at least an order of magnitude smaller than that for the soluble enzyme.     

Species differences in the hydrolysis of 2-cyanoethylene oxide, the epoxide metabolite of acrylonitrile

The carcinogenic effects of acrylonitrile in rats are believed to be mediated by its DNA-reactive epoxide metabolite, 2-cyanoethylene oxide (CEO). Previous studies have shown that conjugation with glutathione is the major detoxication pathway for both acrylonitrile and CEO. This study investigated the role of epoxide hydrolase in the hydrolysis of CEO by HPLC analysis of the products from [2,3-14C]CEO. CEO is a relatively stable epoxide with a half-life of 99 min at 37 degrees C in sodium phosphate buffer (0.1 M), pH 7.3. Incubation with hepatic microsomes or cytosols from male F-344 rats or B6C3F1 mice did not enhance the rate of hydrolysis of CEO (0.69 nmol/min). Human hepatic microsomes significantly increased the rate of hydrolysis of CEO, whereas human hepatic cytosols did not. Human hepatic microsomal hydrolysis activity was heat-sensitive and potently inhibited by 1,1,1-trichloropropene oxide (IC50 of 23 microM), indicating that epoxide hydrolase was the catalyst. The hydrolysis of CEO catalyzed by hepatic microsomes from six individuals exhibited normal saturation kinetics with KM ranging from 0.6 to 3.2 mM and Vmax from 8.3 to 18.8 nmol hydrolysis products/min/mg protein. Pretreatment of rodents with phenobarbital or acetone induced hepatic microsomal hydrolysis activity toward CEO, whereas treatment with beta-naphthoflavone, dexamethasone or acrylonitrile itself was without effect. These data show that humans possess an additional detoxication pathway for CEO that is not active in rodents (but is inducible). The presence of an active epoxide hydrolase hydrolysis activity toward CEO in humans should be considered in assessments of cancer risk from acrylonitrile exposure.     

Characterization of a fourth type of 2-keto acid-oxidizing enzyme from a hyperthermophilic archaeon: 2-ketoglutarate ferredoxin oxidoreductase from Thermococcus litoralis

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. It is known to contain three distinct ferredoxin-dependent, 2-keto acid oxidoreductases, which use pyruvate, aromatic 2-keto acids such as indolepyruvate, or branched-chain 2-keto acids such as 2-ketoisovalerate, as their primary substrates. We show here that T. litoralis also contains a fourth member of this family of enzymes, 2-ketoglutarate ferredoxin oxidoreductase (KGOR). In the presence of coenzyme A, KGOR catalyzes the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A and CO2 and reduces T. litoralis ferredoxin. The enzyme was oxygen sensitive (half-life of approximately 5 min) and was purified under anaerobic conditions. It had an M(r) of approximately 210,000 and appeared to be an octomeric enzyme (alpha2beta2gamma2delta2) with four different subunits with M(r)s of 43,000 (alpha), 29,000 (beta), 23,000 (gamma), and 10,000 (delta). The enzyme contained 0.9 mol of thiamine PPi and at least four [4Fe-4S] clusters per mol of holoenzyme as determined by metal analyses and electron paramagnetic resonance spectroscopy. Significant amounts of other metals (Cu, Zn, Mo, W, and Ni) were not present (<0.1 mol/mol of holoenzyme). Pure KGOR did not utilize other 2-keto acids, such as pyruvate, indolepyruvate, or 2-ketoisovalerate, as substrates, and the apparent Km values for 2-ketoglutarate, coenzyme A, T. litoralis ferredoxin, and thiamine PPi were approximately 250, 40, 8, and 9 microM, respectively. The enzyme was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.0 (at 80 degrees C). KGOR was quite thermostable, with a half-life at 80 degrees C (under anaerobic conditions) of about 2 days. An enzyme analogous to KGOR has been previously purified from a mesophilic archaeon, but the molecular properties of T. litoralis KGOR more closely resemble those of the other oxidoreductases from hyperthermophiles. In contrast to these enzymes, however, KGOR appears to have a biosynthetic function rather than a role in energy conservation.