Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

Purification and Characterization of β-N-Acetylhexosaminidase from the Liver Prawn,Penaeus japonicus

β-N-Acetvlhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeus japonicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS–PAGE. The apparent molecular weight was 64,000 and 110,000 by SDS–PAGE and gel filtration, respectively. The pI was less than 3.2 by chromatofocusing. The aminoterminal amino acid sequence was NH₂-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-VaI-?-Val-Lys-Gly-Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50°C, respectively. The enzyme was stable from pH 4 to 11, and below 55°C. It was 39% inhibited by 10mM HgCl₂.

Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAc_n, n = 2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-β-GlcNAc_n, n= 1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the non-reducing end of the substrate. The parameters of K_m and k_cat at 25°C and pH 5.5 were 0.137 mM and 598s^–1 for pNp-β-GlcNAc, 0.117 mM and 298s^–1 for GlcNAc₂, 0.055 mM and 96.4s^–1 for GlcNAc₃, 0.044 mM and 30.1 s^–1 for GlcNAc_4, 0.045 mM and 14.7 s^–1 for GlcNAc₅, and 0.047 mM and 8.3 s^–1 for GlcNAc₆, respectively. These results suggest that this β-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.     

Purification and Some Properties of Acid Invertase of Persimmon Fruits

Single cells were prepared from mesocarp tissue of ripe persimmon (Diospyros kaki cv. Fuyu) fruits, and inter- or intracellular localization of acid invertase (AI, EC 3.2.1.26) was studied. AI was localized in the intercellular fraction (cell wall fraction). AI was isolated and purified from the cell wall fraction of ripe persimmon fruits by column chromatography on SE-53 cellulose and Toyopearl HW 55F. The specific activity of purified AI was 570 units per mg protein at 30°C. The molecular mass of AI was estimated to be 44 kDa by gel filtration over Sephacryl S-200 and 70 kDa by SDS–PAGE. The optimum pH of the activity for sucrose was 4.25. The purified enzyme hydrolyzed sucrose and raffinose but not melibiose. The enzyme had a K_m of 3.2 mM for sucrose and a K_m of 2.6 mM for raffinose. Silver nitrate (5 μM), HgCI₂ (2 μM), p-chloromercuribenzoate (100mM), pyridoxamine (10mM), and pyridoxine (2.5mM) inhibited AI activity by 95, 85, 100, 41, and 300%, respectively.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

Reactivity of sorbose dehydrogenase from Sinorhizobium sp. 97507 for 1,5-anhydro-d-glucitol

Purified recombinant sorbose dehydrogenase from Sinorhizobium sp. 97507 exhibited high reactivity for 1,5-anhydro-d-glucitol (1,5-AG) and l-sorbose, but little activity for the other sugars or sugar alcohols tested. Kinetic analysis revealed that its catalytic efficiency (k_cat/K_m) for l-sorbose and 1,5-AG is 1.8 × 10² and 1.5 × 10² s^?1·M^?1, respectively.     

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme

ABSTRACT

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5–10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi–Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The k_cat(app)/K_m(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23–12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.     

On the Fermenting Ability of Sand Cultures of Acetone-Butanol Organisms Stored for Long Years

β-N-Acetyl-D-hexosaminidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purifted by making an acetone-dried preparation of the mid-gut gland, extracting with 50 mM citrate-phosphate buffer (pH 4.0) (about 13% of the extracted proteins was β-N-acetyl-D-hexosaminidase), ammonium sulfate fractionation, and column chromatographies on CM-Sepharose and DEAE-Sepharose. The purifted β-N-acetyl-D-hexosaminidase was homogeneous on SDS–PAGE, and sufficiently free from other exo-type glycosidases. The molecular weight was 56,000 by SDS–PAGE. The enzyme hydrolyzed both p-nitrophenyl β-N-acetyl-D-glucosaminide and p-nitrophenyl β-N-acetyl-D-galactosaminide. For p-nitrophenyl β-N-acetyl-D-glucosaminide, the pH optimum was 3.7, the optimum temperature was 45°C, and the K_m was 0.24 mM. For p-nitrophenyl β-N-acetyl-D-galactosaminide, these were pH 3.4, 45°C, and 0.15 mM, respectively. The enzyme liberated non-reducing terminal β-Iinked N-acetyl-D-glucosamine or N-acetyl-D-galactosamine from various 2-aminopyridyl derivatives of oligosaccharides of N-glycan or glycolipid type except of G_M2-tetrasaccharide. As the enzyme was stable around pH 3.5–5.5, it may be useful for long time reactions around the optimum pH.     

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.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.     

Purification and Properties of a Novel Enzyme,Maltooligosyl Trehalose Synthase,from Arthrobacter sp. Q36

Arthrobacter sp. Q36 produces a novel enzyme, maltooligosyl trehalose synthase, which catalyzes the conversion of maltooligosaccharide into the non-reducing saccharide, maltooligosyl trehalose (α-maltooligosyl α-D-glucoside) by intramolecular transglycosylation. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on Sepabeads FP-DA13, DEAE-Sephadex A-50, Ultrogel AcA44, and Butyl-Toyopearl 650M. The enzyme was specific for maltooligosaccharides except maltose, and catalyzed the conversion to form maltooligosyl trehalose. The K_m of the enzyme for maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were 22.9mM, 8.7mM, 1.4mM, and 0.9mM, respectively. The enzyme had a molecular mass of 81,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal and C-terminal amino acids of the enzyme were methionine and serine, respectively. The enzyme showed the highest activity at pH 7.0 and 40°C, and was stable from pH 6.0 to 9.5 and up to 40°C. The enzyme activity was inhibited by Hg²⁺ and Cu2⁺.     

Theanine Formation by Tea Suspension Cells

The theanine (THE: γ-glutamylethylamide) content and the growth rate of cultured cells of tea (Camellia sinensis L.) were increased greatly to 22.3%, in dry wt. with a medium containing 60 mM nitrate and 25 mM ethylamine as a nitrogen source. The optimum concentrations of nitrate, Mg²⁺, and K⁺ for the growth and formation of THE in suspension cells were 40mM, 3mM, and 104mM, respectively. The yield of THE accumulated in the cultured cells with the medium modified for THE formation was increased greatly due to a great increase of the growth rate.     

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.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

A Highly N-Glycosylated Chitin Deacetylase Derived from a Novel Strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2–4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1–10 mM Co²⁺ and strongly inhibited by 10 mM Cu²⁺. It required at least two GlcNAc residues for catalysis. When (GlcNAc)₆ was used as substrate, K _m and V _max were determined to be 1.1 mM and 54.6 μmol min^?1 respectively.     

Substrate Specificity of an α-Glucosidase in Sugar Beet Seed

The substrate specificity of sugar beet α-giucosidase was investigated. The enzyme showed a relatively wide specificity upon various substrates, having α-1,2-, α-1,3-, α-1,4- and α-l,6-glucosidic linkages.

The relative hydrolysis velocity for maltose (G2), nigerose (N), kojibiose (K), isomaltose (I), panose (P), phenyl-a-maltoside (?M) and soluble starch (SS) was estimated to be 100:130: 10.7: 22.6: 54.6: 55.8: 120 in this order; that for malto-triose (G3), -tetraose (G4), -pentaose (G5), -hexaose (G6), -heptaose (G7), -octaose (G8), amyloses (G13) and (G17), 91: 91: 91: 91: 80: 57: 75: 73. The Km values for N, K, I, P, and SS were 16.7 mM, 1.25 mM, 10.8 mM, 8.00 mM, 4.12 mM and 1.90 mg/ml, respectively; that for G2, G3, G4, G5, G6, G7, G8, G13 and G17 were 20.0 mM, 3.67 mM, 2.34 mM, 0,64 mM, 0.42 mM, 0.32 mM, 0.23 mM, 0.36 mM and 0.26 mM, respectively.

The enzyme, though showed higher affinity and activity toward soluble starch than toward maltose, was considered essentially to be an α-glucosidase.     

Synthesis of the Enantiomers of (Z)-5-(1-Octenyl)oxacyclopentan-2-one,a Sex Pheromone of the Cupreous Chafer Beetle,Anomala cuprea Hope

Glutaminase (EC 3.5.1.2) was isolated from Pseudomonas nitroreducens IFO 12694 grown on 0.6% sodium glutamate as a nitrogen source (325-fold purification, 13% yield). The molecular weight of the enzyme was estimated to be 40,000 by gel filtration and SDS-gel electrophoresis. The enzyme hydro-lyzed glutamine optimally at pH 9, and its K_m was 6.5 mm. d-Glutamine, γ-glutamyl p-nitroanilide, γ-glutamylmethylamide, γ-glutamylethylamide (theanine), and glutathione showed respectively 107, 85, 78, 74, and 82% reactivity of glutamine. Zn²⁺, Ni²⁺, Cd²⁺, Co²⁺, Fe²⁺, and Cu²⁺ repressed the enzyme activity strongly.

Glutaminase formed γ-glutamylhydroxamate in the reaction mixture containing glutamine and hydroxylamine (transferring reaction). The optimum pH of the transferring reaction was 7–8, and the K_m for glutamine and hydroxylamine were 4 mm and 120 mm, respectively. γ-Glutamyl derivatives hydrolyzable by glutaminase showed reactivity for the transferring reaction. Methylamine or ethylamine was replaceable for hydroxylamine with 3 or 8% reactivity. The effect of divalent cations was not so striking as in the hydrolyzing reaction.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Purification and Characterization of Chlorophyllase from Alga (Phaeodactylum tricornutum) by Preparative Isoelectric Focusing

Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′ showed specific activities of 15.2 × 10^?4, 226.7 ×10^?4 and 33.8 × 10^?4 µmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII′. The optimum pH for chlorophyllase activity was 8.0 for FI′ and 8.5 for both FII′ and FIII′. Apparent K_m values for enzyme fractions FI′, FII′, and FIII′ were 2.1nM, 2.3nM, and 2.0 nM, respectively. Enzyme fractions FII′ and FIII′ showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI′ had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29mM, 2.14mM, and 0.71mM for FI′. FII′, and FIII′, respectively.