Hydrolytic enzyme substrates. 3. Phosphomonoesterase substrates

This report documents the use of a new and sensitive colorimetric method for measuring phosphomonoesterase activity. The substrates are the phosphate esters of 4-(p-nitrophenoxy)-1,2-butanediol (PNB), 4-(2,4-dinitrophenoxy)-1,2-butanediol (DNB) and 3-(p-nitrophenoxy)-1,2-propanediol (PNG). The key intermediate in the assay is the nitrophenoxy diol which is obtained by enzyme hydrolysis of its phosphate ester. Periodate oxidation of this substance in solution containing methylamine quantitatively yields its nitrophenolate ion whose concentration is determined colorimetrically. The amount of nitrophenolate ion is thus equivalent to the amount of nitrophenoxy diol whose concentration is a function of the phosphomonoesterase activity in the assay sample. The unhydrolyzed phosphomonoester is completely stable to periodate and the hydrolytic conditions used in the assay. The enzymes used to test the substrates were E. coli alkaline phosphomonoesterase and wheat germ phosphomonoesterase. These new esters were all better substrates than the glycerol phosphate esters. Their Michaelis-Menten constants were determined for E. coli phosphomonoesterase.     

Hydrolytic enzyme substrates. II. A new method for measuring periodate

This report describes an accurate and sensitive method for quantitatively measuring periodate concentration. The substances used to determine periodate are 4(p-nitrophenoxy)1,2-butanediol and 4(2,4-dinitrophenoxy)1,2-butanediol. These substances are readily oxidized by periodate yielding β-nitrophenoxy aldehydes which undergoes a facile β-elimination in base to yield the colored nitrophenolate ion. The concentration of the nitrophenolate ion is thus equivalent to the concentration of periodate. This report documents the validity of this reaction as an analytical method. The method was shown to be capable of accurately measuring periodate in concentrations as low as 10^?8M. Its value in biochemical analyses was demonstrated by quantitatively measuring the amount of periodate used to oxidize small quantities of adenosine 5′-phosphate, d-arabitol and d-glucose. Its accuracy, sensitivity and ease of use was shown by its utility in estimating the molecular weight of yeast transfer RNA using about 6 A₂₆₀ units of this material.     

Availability of Energy in Ester and Ether Derivatives of Glycols by Growing Chicks

Biological availability of 33 esters, 17 ethers and 2 acetals of ethanediol, 1,2-propanediol, 1,3-butanediol and 1,4-butanediol was compared by mini-test with chicks. Chicks can utilize esters of ethanediol, 1,2-propanediol and 1,3-butanediol with acetic acid and fatty acids of carbon chain length from 5 to 12 with more improved palatability than that of free acids, while availability of esters of these glycols with propionic and butyric acids was low. Esters of 1,4-butanediol and ether derivatives of these glycols was not available, except ethyl ether of di-ethanediol which was partially available. Acetacetal of ethanediol was partially available but n-butyracetal was not.     

Glass-forming tendency and stability of the amorphous state in the aqueous solutions of linear polyalcohols with four carbons. I. Binary systems water-polyalcohol

All the aqueous solutions of linear saturated polyalcohols with four carbons have been investigated at low temperature. Only ice has been observed in the solutions of 1,3-butanediol and 1,2,3- and 1,2,4-butanetriol. For same solute concentration, the glass-forming tendency on cooling is highest with 2,3-butanediol, where it is comparable to that with 1,2-propanediol, the best solute reported to date. However, the quantity of ice and hydrate crystallized is particularly high on slow cooling or on subsequent rewarming. The highest stability of the amorphous state is observed on rewarming the 1,2-butanediol and 1,3-butanediol solutions. With respect to this property, these compounds come just after 1,2-propanediol and before all the other compounds studied so far. They are followed by dimethylsulfoxide and 1,2,3-butanetriol. The glass-forming tendency of the 1,3-butanediol solutions is also very high; it is third only to that of 1,2-propanediol and 2,3-butanediol. The glass-forming tendency is a little smaller with 1,2-butanediol, but it is cubic instead of ordinary hexagonal ice which crystallizes on cooling rapidly with 35% 1,2-butanediol. Cubic ice is thought to be innocuous. A gigantic glass transition is observed with 45% of this strange solute. 1,4-Butanediol, 45% also favors cubic ice greatly. Therefore, 1,2- and 1,3-butanediol with comparable physical properties are perhaps as interesting as 1,2-propanediol for cryopreservation of cells or organs by complete vitrification. Together with 1,2-propanediol, 1,2- and 1,3-butanetriol, 1,2,3-butanetriol, and perhaps 2,3-butanediol provide an interesting battery of solutions for cryopreservation by vitrification.     

Isolation of a bacterium assimilating (R)-3-chloro-1,2-propanediol and production of (S)-3-chloro-1,2-propanediol using microbial resolution

A bacterium capable of assimilating 3-chloro-1,2-propanediol was isolated from soil by enrichment culture. The strain was identified as Alcaligenes sp. by taxonomic studies. The crude extracts of the cells had dehalogenating activities and converted various halohydrins to the corresponding epoxides. 3-Chloro-1,2-propanediol was degraded stereospecifically by the strain, liberating chloride ion. The residual isomer was found to be the (S)-form (99.4% enantiomeric excess). (S)-3-Chloro-1,2-propanediol was obtained from the racemate by use of this strain in 38% yield, and (S)-glycidol (99.4% enantiomeric excess) was subsequently synthesized from the obtained (S)-3-chloro-1,2-propanediol by alkaline treatment.     

Evaluation of Nutritive Value of Glycol Esters as Energy Source for Growing Chicks and Rats

Nutritive value of 4 glycol esters, i.e. ethanediol diacetate, 1,2-propanediol diacetate, 1,3-butanediol diacetate and 1,3-butanediol dioctylate, was estimated biologically by feeding the esters to growing chicks and rats. Energy in the esters taken by both chicks and rats was well utilized, though feed intake of the diets containing the esters at high level tended to decrease. Bitter taste of the esters was suspected to be related to low appetite. The acetates were somewhat volatile and released free acetic acid in the diet during storage. These properties of the acetates makes their use for dietary energy source difficult in practical condition.     

2,3-Butanediol Metabolism in the Acetogen Acetobacterium woodii

The acetogenic bacterium Acetobacterium woodii is able to reduce CO₂ to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD⁺-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO₂ to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.     

Bacterial 2,3-butanediol dehydrogenases

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidized only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was partially purified (30-fold) with a specific activity of 24.5. Except NAD and NADH no other cofactors were required. Optimum pH-values for oxidation and reduction were pH 9 and pH 7, respectively. The optimum temperature was about 60°C. The molecular weight was 100000 to 107000. The K _m-values were 3.3 mM for D(-)-butanediol, 6.25 mM for meso-butanediol, 0.53 mM for acetoin, 0.2 mM for NAD, 0.1 mM for NADH, 87 mM for diacetyl, 38 mM for 1,2-propanediol; 2,3-pentanedion was not a substrate for this enzyme. The L(+)-butanediol dehydrogenase from Serratia marcescens was purified 57-fold (specific activity 22.3). Besides NAD or NADH no cofactors were required. The optimum value for oxidation was about pH 9 and for reduction pH 4.5. The optimum temperature was 32–36°C. The molecular weight was 100000 to 107000. The K _m-values were 5 mM for meso-butanediol, 10 mM for racemic butanediol, 6.45 for acetoin, 1 mM for NAD, 0.25 mM for NADH, 2.08 mM for diacetyl, 16.7 mM for 2,3-pentanedion and 11.8 mM for 1,2-propanediol.Abbreviations Bud 2,3-butanediol - DH dehydrogenase     

Oxidation of primary aliphatic alcohols by Acetobacterium carbinolicum sp. nov., a homoacetogenic anaerobe

Four strains of new homoacetogenic bacteria were enriched and isolated from freshwater sediments and sludge with ethanol, propanol, 1,2-propanediol, or 1,2-butanediol as substrates. All strains were Gram-positive nonsporeforming rods and grew well in carbonate-buffered defined media under obligately anaerobic conditions. Optimal growth occurred at 27° C around pH 7.0. H₂/CO₂, primary aliphatic alcohols C₃–C₅, glucose, fructose, lactate, pyruvate, ethylene glycol, 1,2-propanediol, 2,3-butanediol, acetoin, glycerol, and methyl groups of methoxylated benzoate derivates and betaine were fermented to acetate or, in case of primary alcohols C₃–C₅ and 1,2-propanediol, to acetate and the respective fatty acid. In coculture with methanogens methane was formed, probably due to interspecies hydrogen transfer. Strain WoProp 1 is described as a new species, Acetobacterium carbinolicum. It had a DNA base composition of 38.5±1.0% guanine plus cytosine, and contained murein of crosslinkage type B similar to A. woodii.     

Reconstitution of the Z-Disk

Methanol-utilizing bacteria, Klebsiella sp. No. 101 and Microcyclus eburneus could grow aerobically and statically on 1,2-propanediol. The authors examined the presence of enzyme activity of adenosyl-B₁₂ dependent diol dehydratase as well as NAD dependent diol dehydroagenase. Adenosyl-B₁₂ dependent diol dehydratase activity was not detected in these organisms, even if these grown statically.

The dehydrogenase activity was found in the extract from these methanol-utilizing bacteria cells grown on various carbon sources. The partially purified enzyme preparation from the cells of Mic. eburneus grown aerobically on 1,2-propanediol dehydrogenated 1,2-propanediol, 1,2-butanediol and 2,3-butanediol. The enzyme activity was separated into two fractions, namely enzyme I and II on DEAE-Sephadex A-25 column chromatography. The enzyme I was different from the enzyme II in the ratio of enzyme activity to 1,2-propanediol and 2,3-butanediol, heat stability, pH stability and pH optimum, and effect of 2-mercaptoethanol.     

Glycerol Dehydrogenase from Cellulomonas sp. NT3060: Purification and Characterization

NAD⁺-dependent glycerol dehydrogenase from Cellulomonas sp. NT3060 was purified by a procedure of 10 steps involving crystallization. Dihydroxyacetone was identified as the oxidation product of glycerol with the enzyme. The purified enzyme did not lose activity on heating below 60°C. The enzyme oxidized other alcohols such as 1,2-propanediol, 2,3-butanediol and glycerol-α-monochlorohydrin, beside glycerol. The enzyme activity was inhibited by p-chloromercuribenzoate, Zn²⁺, Cu²⁺ and Cd²⁺. Oxidation of glyberol was activated by Na⁺ and reduction of dihydroxyacetone was activated by K⁺ at pH 7.5.     

Cryoprotection of red blood cells by a 2,3-butanediol containing mainly the levo and dextro isomers.

A 2,3-butanediol containing 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl) has been used for the cryoprotection of red blood cells. The erythrocytes were cooled to -196 degrees C at rates between 2 and 3500 degrees C/min, followed by slow or rapid warming. Up to 20% (w/w) of this polyalcohol, only the classical peak of survival is observed, as with up to 20% (w/w) 1,2-propanediol or 1,3-butanediol. Twenty percent 2,3-butanediol 97% dl can protect red blood cells very efficiently. The maximum survival, of 90%, as with 20% glycerol, is a little lower than with 20% 1,2-propanediol and higher than with 20% 1,3-butanediol. Fifteen percent 2,3-butanediol protects fewer red blood cells than 15% glycerol or 1,2-propanediol, with a maximum survival of about 80%. The best cryoprotection by 30% 2,3-butanediol 97% dl is obtained at the slowest cooling and warming rates, where survival approaches 90%. After a minimum, an increase of survival is observed at the fastest cooling rates, which would correspond to complete vitrification. These rates are lower than with 30%, 1,2-propanediol or 1,3-butanediol, in agreement with the higher glass-forming tendency of 2,3-butanediol 97% dl solutions. In agreement with the remarkable physical properties of its aqueous solutions, the present experiments also suggest that 2,3-butanediol containing mainly the levo and dextro isomers could be a very useful cryoprotectant for organ cryopreservation. However, it would perhaps be better to use it in combination with other cryoprotectants, since it is a little more toxic than glycerol or 1,2-propanediol at high concentrations.     

An electron paramagnetic resonance study on the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydrase by glycerol and other substrates

Adenosylcobalamin-dependent diol dehydrase undergoes mechanism-based inactivation by glycerol or other substrates during catalysis. X-band electron paramagnetic resonance spectra of holoenzyme were measured at −130°C after reaction with such substrates. After short time of incubation, broad signals assigned to low-spin Co(II) of cob(II)alamin and doublet signals assigned to an organic radical intermediate derived from each substrate were observed with 1,2-propanediol, 1,2-ethanediol, glycerol and meso-2,3-butanediol with the magnitude of their exchange interaction (J-value) decreasing in this order. A substrate with the smaller magnitude of exchange interaction between low-spin Co(II) and an organic radical intermediate seems to be an efficient mechanism-based inactivator. Since the magnitude of exchange interaction decreases with the distance between radical species in a radical pair, these results suggest that a stabilizing effect of holoenzyme on radical intermediates during reactions decreases with the distance between Co(II) and a radical.     

Effects of introducing heterologous pathways on microbial metabolism with respect to metabolic optimality

Although optimality of microbial metabolism under genetic and environmental perturbations is well studied, the effects of introducing heterologous reactions on the overall metabolism are not well understood. This point is important in the field of metabolic engineering because heterologous reactions are more frequently introduced into various microbial hosts. The genome-scale metabolic simulations of Escherichia coli strains engineered to produce 1,4-butanediol, 1,3-propanediol, and amorphadiene suggest that microbial metabolism shows much different responses to the introduced heterologous reactions in a strain-specific manner than typical gene knockouts in terms of the energetic status (e.g., ATP and biomass generation) and chemical production capacity. The 1,4-butanediol and 1,3-propanediol producers showed greater metabolic optimality than the wild-type strains and gene knockout mutants for the energetic status, while the amorphadiene producer was metabolically less optimal. For the optimal chemical production capacity, additional gene knockouts were most effective for the strain producing 1,3-propanediol, but not for the one producing 1,4-butanediol. These observations suggest that strains having heterologous metabolic reactions have metabolic characteristics significantly different from those of the wild-type strain and single gene knockout mutants. Finally, comparison of the theoretically predicted and ¹³C-based flux values pinpoints pathways with non-optimal flux values, which can be considered as engineering targets in systems metabolic engineering strategies. To our knowledge, this study is the first attempt to quantitatively characterize microbial metabolisms with different heterologous reactions. The suggested potential reasons behind each strain’s different metabolic responses to the introduced heterologous reactions should be carefully considered in strain designs.     

The reaction of ammonia with acylated disaccharides : XI. The wohl reaction with octa-O-acytylmelibiononitrile

Octa-O-acetylmelibiononitrile (1) was prepared from melibiose oxime. The reaction of aqueous ammonia with 1 gave 1,1-bis(acetamido)-1-deoxy-5-O-α-D-galactopyranosyl-D-arabinitol (2), N-acetyl-5-O-α-D-galactopyranosyl-α-D-arabinofuranosylamine (3), and the anomeric pair of 5-O-α-D-galactopyranosyl-D-arabinofuranoses (4 and 5). The hepta-O-acetyl derivative of 2 was prepared, and the acyclic structure of the nitrogen-containing moiety was established by oxidation with periodate. The α anomeric configuration of 3 was demonstrated by periodate oxidation and subsequent reduction with sodium borohydride and hydrolysis.     

The structure of Klebsiella serotype 11 capsular polysaccharide

Using periodate oxidation, methylation analysis, the characterization of oligosaccharides obtained by partial acid hydrolysis, p.m.r. spectroscopy, and analytical ultracentrifugation, the structure of the (mildly alkali-treated) Klebsiella serotype 11 capsular polysaccharide has been elucidated. The tetrasaccharide repeating-unit comprises the sequence ?3)-β-D-Glcp-(1?3)-β-D-GlcUAp-(1?3)-α-D-Galp-(1→ with a 4,6-O-(1-car?yethylidene)-α-D-galactosyl residue linked to O-4 of the glucuronic acid residue. The structural basis for some serological cross-reactions of the Klebsiella K11 antigen is discussed, and it is shown that rabbit antisera against the Klebsiella K11 test-strain predominantly contain K agglutinins specific for branch-terminal 4,6-O-(1-car?yethylidene)-D-galactose.     

Microbial production of (R)-3-halolactic acid from (±)-3-halo-1,2-propanediol

Microbial oxidation of (±)-3-halo-1,2-propanediol was studied and it was found that several microorganisms accumulated (R)-3-halolactic acid. Geotrichum loubieri CBS 252.61 produced the most and gave optically pure (R)-3-chlorolactic acid and (R)-3-bromolactic acid from the corresponding diols.     

Utilization of Alcohols by Hansenula miso

The ability of Hansenula miso IFO 0146 to utilize various alcohols and acidic salts as sole sources of carbon and the ability of resting cells to oxidize various alcohols and glucose were studied. Growing cells could utilize only ethanol, glycerol, acetate and lactate, while resting cells grown on ethanol medium could oxidize various alcohols such as 1,2-ethanediol, DL-1,2-propanediol, 1,3-propanediol, meso-2,3-butanediol, DL-1,3-butane-diol, and 1,4-butanediol. From 2 g of 1,2-ethanediol and DL-l,3-butanediol, 1.3 g of glycolic acid and 0.5 g of β-hydroxybutyric acid respectively were produced. The organism formed d-arabinitol from glycerol and glucose, respectively. From 100 ml of culture in medium containing 6 ml of ethanol and 3.0 g of (NH₄)₂HPO₄ as carbon and nitrogen sources 3.40 g of dried cells were obtained.     

Structure of an l-arabino-d-xylan from the bark of Cinnamomum zeylanicum

An arabinoxylan isolated from the bark of Cinnamomum zeylanicum was composed of l-arabinose and d-xylose in the molar ratio 1.6:1.0. Partial hydrolysis furnished oligosaccharides which were characterised as α-d-Xylp-(1→3)-d-Ara, β-dXylp-(1→4)-d-Xyl, β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xyl, β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-β-d-Xylp-Xylp-(1→4)-d-Xyl, xylopentaose, and xylohexaose. Mild acid hydrolysis of the arabinoxylan gave a degraded polysaccharide consisting of l-arabinose (8%) and d-xyolse (92%). Methylation analysis indicated the degraded polysaccharide to be a linear (1→4)-linked d-xlan in which some xylopyranosyl residues were substituted at O-2 or O-3 with l-arabinofuranosyl groups. These data together with the results of methylation analysis and periodate oxidation of the arabinoxylan suggested that it contained a (1→4)-linked β-d-xylan backbone in which each xylopyranosyl residue was substituted both at O-2 and O-3 with l-arabinofuranosyl, 3-O-α-d-xylopyranosyl-l-arabinofuranosyl, and 3-O-l-arabinofuranosyl-l-arabinofuranosyl groups.     

Structural studies of an acidic polysaccharide from the mucin secreted by Drosera capensis

The polysaccharide of the mucin secreted by the leaves of Drosera capensis is composed of l-arabinose, d-xylose, d-galactose, d-mannose, and d-glucuronic acid in the molar ratio of 3.6:1.0:4.9:8.4:8.2. For structural elucidation, methylation analysis using g.l.c. and g.l.c.-m.s. was performed on the native, the carboxyl-reduced, and the degraded polysaccharides. Partial hydrolysis, periodate oxidation, chromium trioxide oxidation, and uronic acid degradation were also performed on the native and carboxyl-reduced polysaccharides. Partial hydrolysis of the native and carboxyl-reduced polysaccharides gave various oligosaccharides that were characterized and suggest a structure containing a d-glucurono-d-mannan backbone having a repeating unit → 4)-β-d-GlcpA-(1 → 2)-α-d-Manp-(1 →. l-Arabinose and d-xylose are present as nonreducing furanosyl and pyranosyl end-groups, respectively, both attached to O-3 of d-glucuronic acid residues of the backbone. d-Galactose is present as non-reducing pyranosyl end-group linked to O-3 of d-mannose residues.