Enzymatic Synthesis of the Crithidia Factors in Cell-free Extracts of Serratia indica

The concomitant production of formic acid and pterin compounds from guanosine-5′-triphosphate (GTP) has been found in cell-free extracts of Serratia indica. Among the pterin compounds, l-threo-neopterin–the major Crithidia factor in S. indica–, a cyclic phosphate of neopterin (cNP), d-erythro-neopterin and 6-hydroxymethyl pterin were detected and isolated. Formate-¹⁴C elimination from GTP-8-¹⁴C was quantitatively distributed in the ethyl acetate layer in the ehyl acetate-hydrochloric acid partition system. Carbon 8 of GTP was released as formic acid. Enzymatic production of formate and cNP was linear for 2 hr at 37°C. Formate production was proportional to the enzyme concentration. The optimum pH for formate elimination was observed around pH 8.6. Optimum temperature for the production of formate and cNP was 50°C. The apparent Km value of GTP for formate production was 6.2×10^?bm. Formate eliminating activity was activated by disodium phosphate but was inhibited by Mg²⁺ or AMP. Incorporation of GTP-U-¹⁴C into pterin compounds was also regulated with disodium phosphate. Effective incorporation into cNP and d-erythro-neopterin occurred in the presence of phosphate. When phosphate was omitted from the system, however, effective incorporation into 6-hydroxymethyl pterin was observed. The biosynthetic process of the Crithidia factors, i.e. l-threo-neopterin and cNP, from GTP in S. indica is also discussed.     

Isolation and Characterization of a Cyclic Phosphate of Neopterin and Other 6-Alkylpterin Compounds Enzymatically Synthesized from GTP by Cell-free Extracts of Serratia indica

A cell-free system for the biosynthesis of l-threo-neopterin, a growth factor for protozoan, Crithidia fasciculata from guanosine-5′-triphosphate (GTP) was obtained from extracts of Serratia indica IFO 3759. This preparation catalyzed the production of a specific pteridine from GTP, which was isolated and characterized as a cyclic phosphate of neopterin (cNP). Among the other products, l-threo-neopterin, as the Crithidia factor, 6-hydroxymethylpterin, and erythro-neopterin were tentatively identified. Requirements for the synthesis of these products include GTP, Mg²⁺, and disodium phosphate. Fluorescence formation was inhibited by purine nucleotides.

When a disodium phosphate was included in the reaction system, cNP and erythro-neopterin were effectively synthesized from GTP. On the other hand, when the phosphate was omitted 6-hydroxymethylpterin was formed.

The possible biosynthetic process of l-threo-neopterin was discussed.     

Isolation and Identification of Crithidia Factors from Bacillus cereus IFO 3131

Bacillus cereus IFO 3131 produces the largest amount of Crithidia factors of 27 bacterial species tested (J. Bacteriol., 104, 197, 1970). The factors in the culture fluid and cell were isolated with a Florisil column and reversed-phase high-performance liquid chromatography (HPLC). They were identified by HPLC, fluorescence and ultraviolet spectra, thin-layer chromatography and bioassay. The major factor in the cell was d-erythro-neopterin and that in fluid was 6-hydroxymethylpterin and pterin.     

A Novel Reaction of Sugars with Anion Radical of Carbon Dioxide Produced from Formate

Radiolysis of some monosaccharides (fructose, glucose and ribose) in air-free condition was markedly enhanced by the addition of formate at concentrations above 20 mm, while it was inhibited at concentrations below 20 mm. The following compounds were detected in the irradiated sugar solutions containing excess formate (100mm): 1-Deoxy-d-arabinohexulose (1, G=4.4) and 1,3- dideoxy-d-erythrohexulose (2, G= 1.3) from fructose; 2-deoxy-d-ribose (3, G=2.3) and 2-deoxyribitol (4, G =0.6) from ribose; and 2-deoxy-d-glucose (5, G=0.5) and 2-deoxy-d-glucitol (6, G=0.4) from glucose. A mechanism for radiolytic formation of the products was proposed, based on interaction of - formed from formate with sugars.     

Fukiic Acid Isolated from the Hydrolysate of a Polyphenol in Petasites japonicus

d-Glucose-isomerizing enzyme was purified in a crystalline form with a good yield from the cells of Bacillus coagulans, strain HN-68, and some phsicochemical properties were investigated.

The purified enzyme was homogeneous on both ultracentrifugal and disc-electrophoretical analyses. The molecular weight of the enzyme was determined to be 175,000 and 160,000 from the sedimentation-viscosity method and the gel filtration method, respectively.

The sedimentation coefficient , partial specific volume, at 280 mμ, and the nitrogen content of the enzyme were determined to be 10.2×10^?13 sec, 0.705 cm³g^?1, 10.6 and 16.2%, respectively. The integral numbers of amino acid residues per molecule calculated on the basis of 160,000 were as follows; Lys₁₂₀, His₄₉, Arg₆₁, Asp₁₈₂, Thr₈₇, Ser₇₀, Glu₁₃₆, Pro₄₄, Gly₁₀₆, Ala₁₄₀, Half-Cys₀, Val₅₃, Met₂₇, Ileu₅₁, Leu₁₃₄, Tyr₅₈, Phe₉₆, Try₁₃, and amide-ammonia₈₀.

Purified enzyme preparation obtained from Bacillus coagulans, strain HN-68 requires Co²⁺ for d-glucose- and d-ribose-isomerizing activities and Mn²⁺ for d-xylose-isomerizing activity. The values of Km for d-glucose, d-xylose and d-ribose were 9×10^?2, 1.1×10^?3, 7.7×1O^?m and of the relative V_max were 0.52, 1.1 and 0.25 mg/min at 40°C, respectively. d-Glucose-isomerizing activity was inhibited by d-xylose and d-ribose. However, there was not a difference among three activities of the enzyme with respect to following properties: Activation energy was 14,600 cal per mol. The enzyme was inhibited in a competitive manner by tris(hydroxymethyl)aminomethane, d-xylitol, d-sorbitol and d-mannitol, and the Ki values for these inhibitor were 3×10^?4, 2.5×10^?3, 2.9×10^?2 and 7×10^?2m, respectively. The ratio of three activities did not change by heat- and pH-treatments. Mn²⁺, Co²⁺ and Ni²⁺ protected strongly the enzyme from heat denaturation. The enzyme can isomerize d-glucose, d-xylose and d-ribose to their corresponding ketose, but the kinetic constants and induction studies indicated that ^d-xylose is the natural substrate for the enzyme.     

Enzymatic Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

Some enzymatic properties of Malbranchea β-xylosidase were investigated. The β- xylosidase activity was inhibited by Hg²⁺, Zn²⁺, Cu²⁺, N-bromosuccinimide, p-chloromercuribenzoate and sodium laurylsulfate, while this activity was activated by Ca²⁺. The enzyme released xylose as the end product even from 10% xylobiose solution without forming any xylooligosaccharides. The enzyme well acted on aryl-β-d-xylosides, but showed no activity on alkyl-β-d-xylosides, and it was practically free from glucosidase activity. The Km and V_max values of this enzyme for xylobiose were calculated to be 2.86 × 10^?8 m and 34.5 μmoles/mg/min, respectively, and these values determined for phenyl-β-d-xyloside were 3.01 × 10^?8 m and 16.2 μmoles/mg/min, respectively.     

Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate

An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-erythro-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium Pseudomonas sp. N99. The enzyme exhibited ammonia-lyase activity toward l-threo-3-hydroxyaspartate and d-erythro-3-hydroxyaspartate, but not toward other 3-hydroxyaspartate isomers. The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of l-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.16) from Pseudomonas sp. T62 (74%) and Saccharomyces cerevisiae (64%) and serine racemase from Schizosaccharomyces pombe (65%). These results suggest that the enzyme is similar to l-threo-3-hydroxyaspartate ammonia-lyase from Pseudomonas sp. T62, which does not act on d-erythro-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in Escherichia coli to produce optically pure l-erythro-3-hydroxyaspartate and d-threo-3-hydroxyaspartate from the corresponding dl-racemic mixtures. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure l-erythro-3-hydroxyaspartate.     

Identification of d-Erythro-Dihydroneopterin Triphosphate as a Product of Guanosine Triphosphate Cyclohydrolase from Serratia indica

Guanosine triphosphate cyclohydrolase (EC 3.5.4.16) was previously shown to exist in two forms (GTP cyclohydrolase D-I and D-II) in Serratia indica IFO 3759, and they were homogeneously isolated. The present study deals with the characterization of their reaction products. A fluorescent product formed from guanosine triphosphate by GTP cyclohydrolase D-II was identified as 7,8-dihydroneopterin triphosphate by its absorption spectra, phosphate analysis and gas chromatography-mass spectrometry of the dephosphorylated trimethylsilyl derivative. After oxidation and dephosphorylation, the d-erythro configuration of the side chain was made clear by the elution profile on ECTEOLA-cellulose chromatography, Rf values on thin-layer chromatography and by biological activity to Crithidia fasciculata ATCC 12857. The fluorescent products from GTP cyclohydrolase D-I and D-II were indistinguishable.     

Purification,Crystallization and Some Properties of β-Galactosidase from Saccharomyces fragilis

Crystalline β-galactosidase was prepared from the cell extract of Saccharomyces fragilis KY5463, by procedures including protamine sulfate treatment and DEAE-cellulose, hydroxylapatite and DEAE-Sephadex column chromatographies. Crystals were formed when solid ammonium sulfate was added to solutions of the purified enzyme. This procedure resulted in a 55-fold purification with an over-all yield of l5.4%. The crystalline enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis.

The sedimentation coefficient, , was determined to be 10.0 S. The molecular weight was estimated to be approximately 203,000 by the sedimentation equilibrium method of Yphantis. Electrolysis with carrier ampholytes revealed that this enzyme has an isoelectric point at around pH 4.4.

The enzyme was activated by K⁺ in addition to bivalent cations, such as Mn²⁺, Mg^2? and Co²⁺. The Km values for o-NPG and lactose were 4.0×10^?3m and 21.0×10^?3m, respectively. The enzyme is sulfhydryl dependent and was completely inactivated by mercuric ions or p-chloromercuribenzoate.     

Studies on Bacterial Protease

A crystalline alkaline protease was prepared from B. amylosacchariticus, which was isolated as a strain of saccharogenic α-amylase-producing Bacillus subtilis. The enzyme was most active at pH values between 10.3 and 10.7 towards casein and was stable at pH values from 6 to 11 on twenty hour incubation at 30°C. Calcium ions were effective to stabilize the enzyme especially at higher temperatures. The enzyme was markedly inactivated by DFP as well as protease inhibitor from potato and slightly by surface active agents, but not affected by sulfhydryl reagents and divalent metal ions except Hg⁺⁺ .Hemoglobin was the best substrate for the enzyme and more than 20% of the peptide bonds were hydrolyzed. Of numerous synthetic peptides tested, only the two compounds, and , were found to be hydrolyzed. A cyclic peptide, gramicidin S, was split by the enzyme only at the peptide bond of -l-valyl-l-ornithyl-. Methyl n-butyrate and tributyrin were also good substrates for the alkaline protease obtained here.     

Characterization of α-Glucosyltransferase from Pseudomonas mesoacidophila MX-45

α-Glucosyltransferase was purified from Pseudomonas mesoacidophila MX-45. The molecular weight was estimated to be 63,000 by SDS–PAGE, and the isoelectric point was pi 5.4. For enzyme activity based on sucrose decomposition, the optimum pH and the optimum temperature were pH 5.8 and 40°C, respectively. The ranges of stable pH and temperature were pH 5.1–6.7 and below 40°C, respectively. The purified enzyme of MX-45 converted sucrose into trehalulose (1-O-α-d-glucopyranosyl-d-fructose) and isomaltulose (palatinose, 6–O-α-d-glucopyranosyl-d-fructose) simultaneously, and the ratio of trehalulose to isomaltulose increased at lower reaction temperatures. Therefore, optimum conditions for trehalulose production were pH 5.5–6.5 at 20°C. The yield of trehalulose from sucrose (20–40% solution) was 91%. The K_m for sucrose was 19.2 ± 3.3 mm estimated by the Hanes–Woolf plot. Product inhibition was observed, and the product inhibition constant was 0.17 m. Hg²⁺, Fe³⁺, Cu²⁺, Mg²⁺, Ag⁺, Pb²⁺, glucono-1,5-lactone, and Tris(hydroxymethyl)aminomethane inhibited the reaction.     

Purification and Characterization of an Intracellular α-D-Xylosidase II from Aspergillus flavus MO-5

α-D-Xylosidase II activity from Aspergillus flavus MO-5 was increased roughly 5- to 10-fold by use of xylose instead of methyl α-D-xylopyranoside (α-MX) as a carbon source.

The enzyme was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SWXL. The purified enzyme hydrolyzed isoprimeverose [α-D-xylopyranosyl-(1→6)-D-glucopyranose] and p-nitrophenyl α-D-xylopyranoside (α-p-NPX), but not α-MX or xyloglucan oligosaccharide. The apparent K_m and V_max of the enzyme for α-p-NPX and isoprimeverose were 0.97 mM and 28.0 µmol/min/mg protein, and 47.62 mM and 2.0 µmol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 67,000 by SDS-polyacrylamide gel electrophoresis and 180,000 by gel filtration chromatography (TSK-gel G3000SWXL).

The enzyme showed the highest activity at pH 6.0 and 40°C, and was stable in the pH range from 6.0 to 7.0 and at the temperatures up to 40°C. The activity was inhibited by Cu²⁺, Zn²⁺, Hg²⁺, p-CMB, SDS, Fe³⁺, and N-ethylmaleimide.

This enzyme had nothing in common with α-D-xylosidase I and four α-D-xylosidases reported already.     

Substrate Specificity of α-Glucosidase II in Rice Seed

The substrate specificity of rice α-glucosidase II was studied. The enzyme was active especially on nigerose, phenyl-α-maltoside and maltooligosaccharides. The actions on isomaltose and phenyl-α-glucoside were weak, and on sucrose and methyl-α-glucoside, negligible. The α-glucans, such as soluble starch, amylopectin, β-limit dextrin, glycogen and amylose, were also hydrolyzed.

The ratio of the maximum velocities for hydrolyses of maltose (G₂), nigerose (N), kojibiose (K), isomaltose (I), phenyl-α-maltoside (?M) and soluble starch (SS) was estimated to be 100: 94.4: 14.2: 7.1: 89.5: 103.1 in this order, and that for hydrolyses of malto-triose (G₃), -tetraose (G₄), -pentaose (G₅), -hexaose (G₆), -heptaose (G₇), -octaose (G₈), and amyloses ( and ), 113: 113: 113: 106: 113: 100: 106: 106. The Km values for N, K, I, ?M and SS were 2.4 mm, 0.58 mm, 20 mm, 1.6 mm and 5.0 mg/ml, respectively; those for G₂, G₃, G₄, G₅, G₆, G₇, G₈, and , 2.4 mm, 2.2 mm, 2.1 mm, 1.5 mm, 1.0 mm, 1.1 mm, 0.95 mm, 1.5 mm and 1.1 mm.

Rice α-glucosidase II is considered an enzyme with a preferential activity on maltooligosaccharides.     

Guanosine Diphosphate Mannose in Conformational Relation to Other Suger Nucleotides and Phosphates in Solution

Molecular conformational transition of GDPMan and solution conformation of α-d- mannopyranose moiety in Man-l-P and GDPMan were examined in relation to other sugar nucleotides and phosphates. GDPMan and other sugar nucleotides examined revealed changes in the optical rotation in sigmoidal curve in water by addition of urea. The change was reversible without significant decomposition and is attributable to dissociation of an ordered form into a random form. Optical conformational values in 8m urea solution were+116° for GDPMan, +58°~+79° for UDPGlc, +79° for UDPGal, +135°~+143° for UDPGlcNAc, and +138°~ +155° for UDPGIcA.

NMR analysis and periodate oxidation study revealed the ⁴C₁ conformation of α-d-hexopyranose moieties in Man-1-P, Glc-l-P, GDPMan, UDPGlcNAc and UDPGalNAc.     

Leupeptins Produced by the Marine Alteromonas sp. B-10–31

Endo-1,4-β-D-mannanase (1,4-β-D-mannanohydrolase, EC 3.2.1.78) was purified from viscera of a mud snail, Pomacea insularus (de Ordigny). The purified enzyme gave a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was estimated to be 44,000. The amino-terminal sequence was H· Gly-X-Leu-Arg-Arg-Gln-Gly-Thr-Asn-Ile-Val-Asp-Ser-His-Gly-His-Lys-Val-Phe-Leu-Ser-Gly-Ala-Asn-Thr-Ala-Trp-Val-Ala-Tyr-Gly-Tyr-Asp-. The enzyme was stable from pH about 5.0 to about 10.5 and had its maximum activity at pH about 5.5. The purified enzyme produced M2, M3, M4,and M5 from β-1,4-mannan. Enzyme activity was greatly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and dithiothreitol at 1 mM concentration. In addition, N-bromosuccinimide completely inhibited the enzyme activity.     

6-Azathymidine-4′-thionucleosides: synthesis and antiviral evaluation

The synthesis of dideoxy-6-azathymidine 4′-thionucleoside 1-(2,3-dideoxy-4-thio-β-D-erythro-pentofuranosyl)-(6-azathymidine) (2), and the L-nucleoside, 1-(4-thio-β-L-erythro-pentofuranosyl)-(6-azathymidine) (3) and their evaluation against a wide panel of antiviral assays are described. The L-thionucleoside (3) was devoid of antiviral activity. The dideoxy-thionucleoside (2) was moderately active against vaccinia virus (VV) and the herpes simplex virus strains HSV-1 (strain KOS) and HSV-2 (strain G) (MIC 12 μM) and retained inhibitory activity vs a thymidine kinase-deficient strain HSV-1/TK^–, suggesting that (2) is not dependent on viral TK-catalysed phosphorylation for antiviral activity and/or may use an alternative metabolic activation pathway.     

Adaptive Use of N2-Dimethyl-Substituted Pterins by Cultures of Crithidia fasciculata1

The compound 6-(L-erythro-1,2′,3′-trihydroxypropyl)pterin, at a concentration of 50 pg/ml (“L-erythro-neopteria”), supports half-maximal growth of Crithidia fasciculata; biopterin at a concentration of 30 pg/ml is shown to yield similar growth. N²-dimethyl-6-(L-erythro-1′,2′,3′-trihydroxypropyl)pterin (A) was inactive even at 100 ng/ml. Synergism was observed with the N²-dimethylamino derivative (A) in the presence of suboptimal biopterin, its activity then being of the order of L-erythro-neopterin. In contrast, the stereoisomeric N²-dimethyl-6-(D-erythro-1′,2′,3′-trihydroxypropyl)pterin (“dimethyl-D-erythro-neopterin”) and its 3′-mono-phosphate only slightly enhanced growth under similar conditions but both threo-isomers had no supplementary activity. Biopterin-induced growth was slowed by 6-(D-erythro1′,2′,3′-trihydroxypropyl)pterin (D-neopterin); the threo-isomers had no such effect. An adaptive demethylation capacity by growing cultures and competition of biopterin uptake by D-neopterin seems likely. The report of the occurrence in Euglena of N₂-dimethyl-6-(L-threo-1′,2′,3′-trihydroxypropyl)pterin and its 3′-mono-phosphate adds further interest to our observations.     

Same Advantages of Using Low Speed Centrifugation for the Purification of Rat Liver Mitochondria

Twelve strains of lactose-fermenting yeast isolated from raw milk were evaluated on β-galactosidase producing ability. The enzymes from the four strains (Tolulopsis versatilis M6, Tolulopsis sphaerica J28, Candida pseudotropicalis B57 and A60), selected by high productivity, showed very similar properties and were characterized by a pH optimum of 7.0 or 7.5 and a relatively low optimal temperature of 30°C. The molecular weights were estimated by gel filtration to be 200,000-233,000. The Km values for o-nitrophenyl-β-d-galactopyranoside were 3.45 mm, 2.09 mm, 3.45 mm and 2.82 mm for enzymes from M6, J28, B57 and A60, respectively. All enzymes were activated by Mn²⁺ and inhibited by Mg²⁺, Zn²⁺ and Ca²⁺. The enzymes are sulfhydryl dependent and were completely inhibited by Hg²⁺ and sulfhydryl reagents. The yeasts may be a potential source for the enzyme for industrial use.     

Studies on Alginase

Chemical investigations were made on a new unsaturated crystalline diuronide isolated from alginase hydrolysate of alginic acid. This uronide has (in water), and m.p. 135.5~136.5°C (decomp.). The presence of an α/β-unsaturated carboxylic acid formulation is supported by the following evidences: (a) an ultraviolet absorption band at 232 m/μ, (b) infrared absorption bands at 1648 cm^-1 due to double bond and at 1720 cm^-1 due to conjugated carboxylic group, (c) the consumption of about 1 mole of bromine per mole of the compound, (d) the production of oxalic acid on oxidation with ozone, (e) the formation of a substance that shows absorption maximum at 550 mμ, caused by the addition of thiobarbituric test. After hydrolysis, crystalline mannuronic lactone was obtained from the unsaturated diuronide. Occurrence of mannuronic moiety in the reducing unit was observed by paper chromatography of the hydrolysate of borohydride-reduced unsaturated compound. From these results it can be seen that the possible structure of this unsaturated diuronide is 4-O- (β-d-Δ4,5 mannoseenpyranosyluronic acid) -d-mannuronic acid.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.