The role of formylmethanofuran: tetrahydromethanopterin formyltransferase in methanogenesis from carbon dioxide

Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified to electrophoretic homogeneity from cells of Methanobacterium thermoautotrophicum. The enzyme is a tetramer of similar or identical subunits (Mr = 41,000). The equilibrium favors transfer of the formyl group to tetrahydromethanopterin (H4MPT) at physiological pH. The product of formyl transfer by the purified enzyme was shown by a number of criteria to be 5-formyl-H4MPT, as opposed to 10-formyl-H4MPT or 5,10-methenyl-H4MPT. Reconstitution of a portion of the methanogenic C1 cycle was effected by combining purified formyltransferase, methenyl-H4MPT cyclohydrolase, formylmethanofuran, and H4MPT to give methenyl-H4MPT. Additional reconstitution experiments established that the formyltransferase is an essential enzyme for the conversion of carbon dioxide to methane. In conjunction with previously published data (Donnelly, M.I., Escalante-Semerena, J.C., Rinehart, K. L., Jr., and Wolfe, R.S. (1985) Arch. Biochem. Biophys. 242, 430-439), these data substantiate the role of 5-formyl-H4MPT as an intermediate of methanogenesis.     

5-Formyl-5,6,7,8-tetrahydromethanopterin is the intermediate in the process of methanogenesis in Methanosarcina barkeri.

Formylmethanofuran:tetrahydromethanopterin (H4MPT) formyltransferase and 5,10-methenyl-H4MPT cyclohydrolase purified from Methanosarcina barkeri catalyze a formyl group transfer and the hydrolysis of the methenyl function, respectively. The results from UV spectroscopy and HPLC analyses, and comparison with results obtained with the enzymes isolated from Methanobacterium thermoautotrophicum showed 5-formyl-H4MPT to be the product of the formyltransferase and cyclohydrolase reactions in M. barkeri. The findings disagree with an earlier report in which 10-formyl-H4MPT was identified as the product of the cyclohydrolase in the latter organism. In addition, it was observed that 10-formyl-H4MPT, which is non-enzymically formed from 5,10-methenyl-H4MPT at alkaline pH, becomes rapidly converted into the 5-formyl derivative. The latter finding explains why the nature of the formyl species previously had been improperly assigned.     

Tetrahydromethanopterin, a carbon carrier in methanogenesis

Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.     

The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri.

BACKGROUND: The reduction of carbon dioxide to methane in methanogenic archaea involves the tetrahydrofolate analogue tetrahydromethanopterin (H(4)MPT) as a C(1) unit carrier. In the third step of this reaction sequence, N(5)-formyl-H(4)MPT is converted to methenyl-H(4)MPT(+) by the enzyme methenyltetrahydromethanopterin cyclohydrolase. The cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri (Mch) is extremely thermostable and adapted to a high intracellular concentration of lyotropic salts. RESULTS: Mch was crystallized and its structure solved at 2.0 A resolution using a combination of the single isomorphous replacement (SIR) and multiple anomalous dispersion (MAD) techniques. The structure of the homotrimeric enzyme reveals a new alpha/beta fold that is composed of two domains forming a large sequence-conserved pocket between them. Two phosphate ions were found in and adjacent to this pocket, respectively; the latter is displaced by the phosphate moiety of the substrate formyl-H(4)MPT according to a hypothetical model of the substrate binding. CONCLUSIONS: Although the exact position of the substrate is not yet known, the residues lining the active site of Mch could be tentatively assigned. Comparison of Mch with the tetrahydrofolate-specific cyclohydrolase/dehydrogenase reveals similarities in domain arrangement and in some active-site residues, whereas the fold appears to be different. The adaptation of Mch to high salt concentrations and high temperatures is reflected by the excess of acidic residues at the trimer surface and by the higher oligomerization state of Mch compared with its mesophtic counterparts.     

A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1.

Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units. In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U x mg-1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U x mg-1). Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N-terminal amino acid sequence. The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U x mg-1 (Vmax = 1500 U x mg-1; Km = 30 microm) was found to be composed of two identical subunits of molecular mass 33 kDa. Its sequence was approximately 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea. The methenyl H4F cyclohydrolase with a specific activity of 100 U x mg-1 (Vmax = 330 U x mg-1; Km = 80 microm) was found to be composed of two identical subunits of molecular mass 22 kDa. Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases. Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl-H4F cyclohydrolase an anabolic function in the C1-unit metabolism of M. extorquens AM1.     

Tetrahydromethanopterin-dependent methanogenesis from non-physiological C1 donors in Methanobacterium thermoautotrophicum.

Methanogenesis from the non-physiological C1 donors thioproline, thiazolidine, hexamethylenetetramine, formaldehyde (HCHO), and HOCH2-S-coenzyme M (CoM) was catalyzed by cell extracts of Methanobacterium thermoautotrophicum under a hydrogen atmosphere. Tetrahydromethanopterin (H4MPT) and HS-CoM were required in the reaction mixture. The non-physiological compounds were found to be in chemical equilibrium with HCHO, which has been shown to react spontaneously with H4MPT to form methylene-H4MPT, an intermediate of the methanogenic pathway at the formaldehyde level of oxidation. Highfield (360 MHZ) 1H and 13C nuclear magnetic resonance studies performed on the interaction between HCHO and HS-CoM showed that these compounds are in equilibrium with HOCH2-S-CoM and that the equilibrium is pH dependent. When methanogenesis from the non-physiological donors was followed under a nitrogen atmosphere, the C1 moiety from each compound underwent a disproportionation, forming methenyl-H4MPT+ and methane. The compounds tested served as substrates for the enzymatic synthesis of methenyl-H4MPT+.     

Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5-aminolevulinic acid

4,5-Dioxovaleric acid (DOVA) was synthesized from 5-bromolevulinic acid via formation of the pyridinium bromide of 5-bromolevulinic acid, followed by nitrone formation with p-nitrosodimethylaniline, and hydrolysis of the nitrone to yield DOVA. Partial purification of DOVA was obtained by passage of the reaction mixture through a cation exchange column. DOVA was identified by paper electrophoresis and by a specific fluorometric assay. DOVA was nonenzymatically transaminated to 5-aminolevulinic acid (ALA) with glycine serving as the amino donor. Other compounds tested were less effective amino donors. Glyoxylic acid was identified as a reaction product by paper electrophoresis and a specific calorimetric test. ALA was identified by paper electrophoresis, paper chromatography of a pyrrole derivative, reaction with Ehrlich reagent, and by its enzymatic conversion by a barley extract to porphobilinogen and uroporphyrin. The nonenzymatic transamination was inhibited by Tris and was stimulated by high pH. The existence of this nonenzymatic activity is discussed in relation to previous reports of dova transaminase activity in cell extracts.     

Triazene drug metabolites. Part 17: Synthesis and plasma hydrolysis of acyloxymethyl carbamate derivatives of antitumour triazenes

A series of 3-acyloxymethyloxycarbonyl-1-aryl-3-methyltriazenes 5 was synthesised by the sequential reaction of 1-aryl-3-methyltriazenes with (i) chloromethyl chloroformate, (ii) NaI in dry acetone, and (iii) either the silver carboxylate or the carboxylic acids in the presence of silver carbonate. The hydrolysis of these compounds was studied in pH 7.7 isotonic phosphate buffer and in human plasma. Triazene acyloxycarbamates demonstrated their ability to act as substrates for plasma enzymes. For compound 5f, a pH-rate profile was obtained which showed the hydrolysis to involve acid-base catalysis. The reaction is also buffer catalysed. Thus, at pH 7.7, pH-independent, base-catalysed and buffer-catalysed processes all contribute to the hydrolysis reaction. The sensitivity of the hydrolysis reaction to various structural parameters in the substrates indicates that hydrolysis occurs at the ester rather than the carbamate functionality. In plasma, the rates of hydrolysis correlate with partition coefficients, the most lipophilic compounds being the most stable. An aspirin derivative suffers two consecutive enzymatic reactions, the scission of the aspirin acetyl group being followed by the scission of the acyloxy ester group. These results indicate that triazene acyloxymethyl carbamates are prodrugs of the antitumour monomethyltriazenes. They combine chemical stability with a rapid enzymatic hydrolysis, and are consequently good candidates for further prodrug development. Moreover, this type of derivative allowed the synthesis of mutual prodrugs, associating the antitumour monomethyltriazenes with anti-inflammatory NSAIDs as well as with the anticancer agent butyric acid.     

Biochemical characterization of a dihydromethanopterin reductase involved in tetrahydromethanopterin biosynthesis in Methylobacterium extorquens AM1

During growth on one-carbon (C1) compounds, the aerobic alpha-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H4MPT) derivative dephospho-H4MPT as a C1 carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H4MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H4MPT biosynthesis is the reduction of dihydromethanopterin (H2MPT) to H4MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H2MPT reductase gene (dmrA). In the present work, we describe the biochemical characterization of H2MPT reductase (DmrA), which is encoded by dmrA. The gene was expressed with a six-histidine tag in Escherichia coli, and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H2MPT with a specific activity of 2.8 micromol of NADPH oxidized per min per mg of protein at 30 degrees C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H2MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F420, used, respectively, in bacteria and methanogenic archaea.     

Identification and biochemical properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells

The folate compound 10-formyldihydrofolate (H2folate) has not been found as a component of intracellular folates in normal tissues but has been identified in the cytosol of methotrexate (MTX)-treated MCF-7 breast cancer cells and normal human myeloid precursor cells. Its identity was verified by coelution of this compound with a synthetic marker on high pressure liquid chromatography, its reduction to 10-formyltetrahydrofolate (H4folate) in the presence of dihydrofolate reductase, and its enzymatic deformylation to dihydrofolate in the presence of aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. Chemically synthesized monoglutamated or pentaglutamated 10-formyl-H2folate was examined for its interaction with three folate-dependent enzymes: AICAR transformylase, glucinamide ribotide (GAR) transformylase, and thymidylatesynthase. 10-Formyl-H2folate-Glu5 was a competitive inhibitor of thymidylate synthase (Ki = 0.16 microM with 5,10-methylene-H4folate-Glu1 as substrate and 1.6 microM with 5,10-methylene-H4folate-Glu5) and inhibited GAR transformylase (Ki = 2.0 microM). It acted as a substrate for AICAR transformylase (Km = 5.3 microM), and its efficiency was equal to that of the natural substrate 10-formyl-H4folate-Glu5. The inhibition of thymidylate synthase by 10-formyl-H2folate was highly dependent on the inhibitor's polyglutamation state, the -Glu5 derivative having a 52-85-fold greater affinity as compared to the affinity of -Glu1. Polyglutamation of 10-formyl-H2folate did not affect its inhibition of GAR transformylase. While the actual role of 10-formyl-H2folate contributing to the cytotoxicity of MTX has not been determined, this compound has the potential to enhance inhibition of GAR transformylase and thymidylate synthase, and at the same time provides additional substrate for AICAR transformylase. The MTX-induced intracellular accumulation of 10-formyl-H2folate and H2folate may play a role in the drug-related cytotoxicity through the contribution of these folates to the inhibition of thymidylate synthase and de novo purine synthesis.     

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.     

Purification and characterization of the methylene tetrahydromethanopterin dehydrogenase MtdB and the methylene tetrahydrofolate dehydrogenase FolD from Hyphomicrobium zavarzinii ZV580

Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H(4)F)- and tetrahydromethanopterin (H(4)MPT)-dependent enzymes. Here we report on the purification of two methylene tetrahydropterin dehydrogenases from the methylotroph Hyphomicrobium zavarzinii ZV580. Both dehydrogenases are composed of one type of subunit of 31 kDa. One of the dehydrogenases is NAD(P)-dependent and specific for methylene H(4)MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H(4)MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H(4)F (specific activity: 180 U/mg) and also exhibits methenyl H(4)F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H(4)F dehydrogenases/cyclohydrolases (FolD) found in many bacteria and eukarya. Apparently, the occurrence of methylene tetrahydrofolate and methylene tetrahydromethanopterin dehydrogenases is not uniform among different methylotrophic alpha-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H(4)MPT dehydrogenase MtdA was present in the bacterium that also shows H(4)F activity.     

Residues involved in the mechanism of the bifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase: the roles of glutamine 100 and aspartate 125

The human bifunctional dehydrogenase-cyclohydrolase domain catalyzes the interconversion of 5,10-methylene-H(4)folate and 10-formyl-H(4)folate. Although previous structure and mutagenesis studies indicated the importance of lysine 56 in cyclohydrolase catalysis, the role of several surrounding residues had not been explored. In addition to further defining the role of lysine 56, the work presented in this study explores the functions of glutamine 100 and aspartate 125 through the use of site-directed mutagenesis and chemical modification. Mutants at position 100 are inactive with respect to cyclohydrolase activity while preserving significant dehydrogenase levels. We succeeded in producing a K56Q/Q100K double mutant, which has no cyclohydrolase yet retains more than two-thirds of wild type dehydrogenase activity. Neither activity is detectable in aspartate 125 mutants with the exception of D125E. The results indicate that the function of glutamine 100 is to activate lysine 56 for cyclohydrolase catalysis and that aspartate 125 is involved in the binding of the H(4)folate substrates. In highlighting the importance of these residues, catalytic mechanisms are proposed for both activities as well as an explanation for the differences in channeling efficiency in the forward and reverse directions.     

Lipid-mediated glycosylation in human liver. Characterization of the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine and mannose from GDP-mannose to dolichyl phosphate

The enzymatic transfer of GlcNAc from UDP-GlcNAc and Man from GDP-Man to Dol-P has been characterized in human liver preparations. The presence of low concentrations of detergent, divalent cation and exogenous Dol-P are required for both enzymatic activities. The pH optimum of both reactions is broad with maximal activity near pH 7.8. The majority of N-acetylglucosaminyltransferase (90%) and mannosyltransferase (85%) activities is particulate but approximately 90% of both activities can be released into supernatant fluids by using Triton X-100 in the homogenizing buffer. The supernatant fluid enzymes have properties similar to those of the particulate enzymes although their activities are considerably less stable. Preliminary characterization of the enzymatic reaction products gave the following evidence for formation of GlcNAc and Man derivatives of Dol-P: (1) radiolabelled products are soluble in organic solvents; (2) for each reaction no detectable product is found without addition of exogenous Dol-P and increasing amounts of product are found with increasing amounts of this lipid; (3) acid and base hydrolysis of the glycolipid product (from the N-acetylglucosaminyltransferase reaction) result in radioactive, water-soluble compounds which comigrate with authentic GlcNAc and GlcNAc-1-P, respectively; (4) acid and base hydrolysis of the glycolipid product (from the mannosyltransferase reaction) result in radioactive, water-soluble compounds which comigrate with authentic Man and Man-1-P, respectively.     

Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498

Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosics ethanol. In this study, we used acid pretreatment, delignification and enzymatic hydrolysis to evaluate the pretreatment to produce more sugar, to be fermented to ethanol. Dilute H(2)SO(4) (3.0%,v/v) treatment resulted in hydrolysis of hemicelluloses from lignocellulosic complex to pentose sugars along with other byproducts such as furfural, hydroxymethyl furfural (HMF), phenolics and acetic acid. The acid pretreated substrate was delignified to the extent of 93.2% by the combined action of sodium sulphite (5.0%,w/v) and sodium chlorite (3.0%,w/v). The remaining cellulosic residue was enzymatically hydrolyzed in 0.05 M citrate phosphate buffer (pH 5.0) using 3.0 U of filter paper cellulase (FPase) and 9.0 U of beta-glucosidase per mL of citrate phosphate buffer. The maximum enzymatic saccharification of cellulosic material (82.8%) was achieved after 28 h incubation at 50 degrees C. The fermentation of both acid and enzymatic hydrolysates, containing 18.24 g/L and 37.47 g/L sugars, with Pichia stipitis and Saccharomyces cerevisiae produced 7.13 g/L and 18.52 g/L of ethanol with corresponding yield of 0.39 g/g and 0.49 g/g, respectively.     

Characterization of two methanopterin biosynthesis mutants of Methylobacterium extorquens AM1 by use of a tetrahydromethanopterin bioassay

An enzymatic assay was developed to measure tetrahydromethanopterin (H(4)MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1. H(4)MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H(4)MPT biosynthesis. The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5'-phosphate synthase, the first committed step of H(4)MPT biosynthesis. These results provide the first biochemical evidence for H(4)MPT biosynthesis genes in bacteria.     

Modelling of the enzymatic kinetically controlled synthesis of cephalexin: influence of diffusion limitation

In this study the influence of diffusion limitation on enzymatic kinetically controlled cephalexin synthesis from phenylglycine amide and 7-aminodeacetoxycephalosporinic acid (7-ADCA) was investigated systematically. It was found that if diffusion limitation occurred, both the synthesis/hydrolysis ratio (S/H ratio) and the yield decreased, resulting in lower product and higher by-product concentrations. The effect of pH, enzyme loading, and temperature was investigated, their influence on the course of the reaction was evaluated, and eventually diffusion limitation was minimised. It was found that at pH >or=7 the effect of diffusion limitation was eminent; the difference in S/H ratio and yield between free and immobilised enzyme was considerable. At lower pH, the influence of diffusion limitation was minimal. At low temperature, high yields and S/H ratios were found for all enzymes tested because the hydrolysis reactions were suppressed and the synthesis reaction was hardly influenced by temperature. The enzyme loading influenced the S/H ratio and yield, as expected for diffusion-limited particles. For Assemblase 3750 (the number refers to the degree of enzyme loading), it was proven that both cephalexin synthesis and hydrolysis were diffusion limited. For Assemblase 7500, which carries double the enzyme load of Assemblase 3750, these reactions were also proven to be diffusion limited, together with the binding-step of the substrate phenylglycine amide to the enzyme. For an actual process, the effects of diffusion limitation should preferably be minimised. This can be achieved at low temperature, low pH, and high substrate concentrations. An optimum in S/H ratio and yield was found at pH 7.5 and low temperature, where a relatively low reaction pH can be combined with a relatively high solubility of 7-ADCA. When comparing the different enzymes at these conditions, the free enzyme gave slightly better results than both immobilised biocatalysts, but the effect of diffusion limitation was minimal.     

N10-Formyltetrahydrofolate is the formyl donor for glycinamide ribotide transformylase in Escherichia coli.

Glycinamide ribotide transformylase from Escherichia coli was obtained free of N5,N10-methenyltetrahydrofolate cyclohydrolase activity by DEAE-cellulose chromatography. In reaction mixtures containing this enzyme preparation in potassium maleate buffer, pH 7.2, no detectable interconversion of N5,N10-methenyltetrahydrofolate occurred. Upon addition of glycinamide ribotide, N-formylglycinamide ribotide was formed when N10-formyltetrahydrofolate was present; no formylation occurred in the presence of N5,N10-methenyltetrahydrofolate. A method for the synthesis and purification of glycinamide ribotide is presented.     

A sensitive enzyme-catalytic nanogold-resonance scattering spectral assay for alkaline phosphate

In pH 8.9 Tris-HCl buffer solutions, alkaline phosphatase (ALP) catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP) substrate to form ascorbic acid. Then H(3)PO(4) was added to stop the enzymatic reaction and HAuCl(4) was used to react with ascorbic acid to generate gold nanoparticles that exhibited a resonance scattering (RS) peak at 600 nm. Under the selected conditions, when the activity of ALP increased, the formed ascorbic acid and gold nanoparticles also increased. Thus, the RS intensity at 600 nm enhanced linearly. The linear range was 0.06-22 U/L, with a detection limit of 0.03 U/L. The ALP in serum was analyzed, and the results were in agreement with those of the fluorescence method.     

A MEMBRANE SEPARATOR/BIOREACTOR COUPLED WITH ABSORBANCE MEASUREMENT FOR DETECTION OF Escherichia coli O157:H7

A membrane separator/bioreactor system was developed for rapid detection of Escherichia coli O157:H7. The system consisted of a membrane separator/bioreactor (0.45 μm of the pore size) to separate the-complexes of E. coli O157:H7 and alkaline phosphatase-conjugated anti-E. coli O157:H7 antibodies from the sample and to produce p-nitrophenol through the enzymatic reaction (p-nitrophenyl phosphate hydrolysis), and an optical detector for measuring the p-nitrophenol absorbance at 400 nm. The membrane material and the flow rate of the substrate for the enzymatic hydrolysis had great effects on the absorbance of p-nitrophenol. The optimum conditions for the enzymatic reaction were determined as 1.0 M Tris buffer, pH 8.0, and 0.1 M MgCl₂ for this system. The detection range was 10⁴± 10⁷ CFU/mL with a relative standard deviation of 4.3 ± 14.2%, and whole procedure could be completed in 50 min without any enrichment and culture. Other bacteria such as Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes had no significant interference with the detection of E. coli O157:H7.