Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Studies on Chrysanthemic Acid

Ribose-5-phosphate ketol-isomerase, an enzyme isomerizing ribose-5-phosphate to ribulose-5-phosphate, is isolated from Candida utilis which is grown in a medium containing xylose. The enzyme is also purified by means of fractionation with ammonium sulfate, acetone, and by DEAE-cellulose column chromatography.

The enzyme has its optimum pH at 7.5 and optimum temperature at 50°C.

Michaelis-Menten constant for d-ribose-5-phosphate is 7.38 × 10^?4 M and activation energy of the enzyme reaction is 10,525 calories.

The enzyme activity is inhibited by p-CMB, EDTA and sodium pyrophosphate, and activated by the addition of magnesium ion.

Extract of Candida utilis contains polyol: NAD oxidoreductase which catalyzes the conversion of polyols to the corresponding ketoses.

By fractionation with ammonium sulfate and on DEAE-cellulose column chromatography, the purity of enzyme has been increased about 14-fold.

The relatively high activity with both xylitol and sorbitol suggests that they may be the natural substances for the enzyme.

Evidence suggests that this enzyme relates to the metabolism of d-xylose in Candida utilis.     

Isolation and Characterization of Nα-Benzyloxycarbonyl Amino Acid Urethane Hydrolase II from Lactobacillus fermenti 36 ATCC 9338

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of N^α-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co²⁺, is essential for its activity.     

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K _m and K _cat of the enzyme were 0.31 mM and 3.01 s⁻¹, respectively. Fe³⁺ could enhance the enzyme activity, whereas Ag⁺, Hg²⁺, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement of uric acid.     

Enhancement of the thermostability and catalytic activity of stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution

-Amino-acid amidases, which catalyze the stereospecific hydrolysis of -amino-acid amide to yield -amino acid and ammonia, have attracted increasing attention as catalysts for stereospecific production of -amino acids. We screened for the enzyme variants with improved thermostability generated by a directed evolution method with the goal of the application of evolved enzyme to the production of -amino acids. Random mutagenesis by error-prone PCR and a filter-based screening was repeated twice, and as a result the most thermostable mutant BFB40 was obtained. Gene analysis of the BFB40 mutant indicated that the mutant enzyme had K278 M and E303 V mutations. To compare the enzyme characteristics with the wild-type enzyme, the mutant enzyme, BFB40, was purified from the Escherichia coli (E. coli) transformant. Both the thermostability and apparent optimum temperature of the BFB40 were shifted upward by 5 °C compared with those of the wild-type enzyme. The apparent K_m value for -phenylalaninamide of BFB40 enzyme was almost the same with that of the wild-type enzyme, whereas V_max value was enhanced about three-fold. Almost complete hydrolysis of -phenylalaninamide was achieved in 2 h from 1.0 M of racemic phenylalaninamide–HCl using the cells of E. coli transformant expressing BFB40 enzyme, the conversion of which was 1.7-fold higher than the case using cells expressing wild-type enzyme after the same reaction time.     

Optimum Culture Conditions for the Production of N-Substituted Formamide Deformylase by Arthrobacter pascens F164

We investigated the optimum culture conditions for the production of a novel enzyme, N-substituted formamide deformylase, which acts mainly on N-benzylformamide, in Arthrobacter pascens F164. The highest enzyme activity was obtained when this strain F164 was cultivated in a synthetic medium with N-benzylformamide as sole nitrogen source. This deformylase was found to be an inducible enzyme depending on N-benzylformamide.     

Non-thermal Effects of a Ceramics Heater Radiation on Xanthine Oxidase Activity

The non-thermal effects of ceramics heater radiation on xanthine oxidase activity have been investigated using the enzyme, substrate, and competitive inhibitors which were irradiated on cooling. The K_m and V_max in the irradiated enzyme system were reduced to 51% and 85%, of the non-irradiated control, respectively. The K_i for a competitive inhibitor, folic acid, in the irradiated enzyme system decreased to 22% of the non-irradiated control. A steady-state molecular kinetic analysis for the reaction estimates that the irradiated enzyme may be kept in a folding state, and the formation of a Michaelis complex has been accelerated, and the activated Michaelis complex has been stabilized, and that a solvation or an electrostriction of xanthine, folate, and an active center of the enzyme with water may be promoted by irradiating the components in an aqueous solution, by which modification of the enzyme activity has been regulated.     

Purification of a flavoprotein having NADPH-cytochrome c reductase and transhydrogenase activities from Nitrobacter winogradskyi and its molecular and enzymatic properties

A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD⁺ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP⁺ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP dichlorophenolindophenol - Tris trishydroxy-methylaminomethane - Mops 3-(N-morpholino) propanesulfonic acid - SDS sodium dodecylsufate     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2442 isolated from inclusion bodies

Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni²⁺-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.     

Biochemical Studies on Ricin Part X Effect of the Two Constituent Polypeptide Chains of Ricin D toward Mouse Sarcoma Ascite Tumor Cells

A maltotetraose-forming amylase from Pseudomonas stutzeri was highly purified by adsorption on starch granules and by chromatographies on Sephadex G-100 and DEAE-cellulose. The purified enzyme showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. The optimum pH for enzyme action on starch was 6.0-6.5, and the optimum temperature was 45°C. The purified enzyme attacked starch from the non-reducing end to produce α-anomer oligosaccharides. This indicated that the enzyme was an exo-α-amylase which had not hitherto been found. The enzyme activity was markedly inhibited by the addition of Cu²⁺, Hg²⁺, N-bromosuccinimide and 2,3-butanedione. The molecular weight of the enzyme determined by the method of Weber and Osborn was about 5.7 × 10⁴. The isoelectric point of the enzyme was estimated to be 5.3 by polyacrylamide gel electrofocusing. The Km and k₀ values of this enzyme for starch, glycogen, short chain amylose and some maltooligosaccharides were calculated from Lineweaver-Burk plots.     

A novel fibrinolytic enzyme from <Emphasis Type="Italic">Cordyceps militaris</Emphasis>, a Chinese traditional medicinal mushroom

A novel fibrinolytic enzyme from Cordyceps militaris was purified and partially characterized for the first time, which was designated C. militaris fibrinolytic enzyme (CMase). This extracellular enzyme from C. militaris was isolated by ammonium sulphate fraction, and purified to electrophoretic homogeneity using gel filtration chromatography. The apparent molecular mass of the purified enzyme was estimated to be 27.3 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 6.0 and 25 °C, respectively. In the presence of metal ions such as Mg²⁺ and Fe²⁺ ions the activity of the enzyme increased, whereas EDTA and Cu²⁺ ion inhibited the enzyme activity. Interestingly the N-terminal amino acid sequences of the enzyme is extremely similar to those of the trypsin proteinases from insects, and has no significant homology with those of the fibrinolytic enzyme from other medicinal mushroom. In conclusion, C. militaris produces a strong fibrinolytic enzyme CMase and may be considered as a new source for thrombolytic agents.     

Enzymatic synthesis of (+)- and (-)-bisdechlorogeodin with sulochrin oxidase from Penicillium frequentans and Oospora sulphurea-ochracea

Sulochrin oxidase is a blue copper-containing glycoenzyme that catalyzes a stereospecific formation of bisdechlorogeodin from sulochrin. The enzyme has been isolated from Penicillium frequentans and Oospora sulphureaochracea which catalyzes the formation of (+)-form and (-)-form of bisdechlorogeodin respectively. The Penicillium enzyme has a molecular weight of 157,000 and contains 19.5% of carbohydrates. Amino acid and carbohydrate compositions are given. The enzyme has probably a dimeric structure containing 6 Cu-atoms. Apparent K _m-values of various substrates are presented. The Oospora enzyme has a molecular weight of 128,000 and except for its stereospecificity its properties are very similar to those of the Penicillium enzyme.     

Characterization and Engineering of a Novel Pyrroloquinoline Quinone Dependent Glucose Dehydrogenase from <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> So ce56

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized. The recombinant protein has a relative molecular mass of 63 kDa and shows 43% homology to PQQ GDH-B from Acinetobacter calcoaceticus. In the presence of PQQ and CaCl₂ the enzyme has dehydrogenase activity with the substrate glucose as well as with other mono- and disaccharides. The thermal stability and its pH activity profile mark the enzyme as a potential glucose biosensor enzyme. In order to decrease the activity on maltose, which is unwanted for a potential application in biosensors, the protein was rationally modified at three specified positions. The best variant showed a 59% reduction in activity on maltose compared to the wild type enzyme. The catalytic efficiency (k _cat/K _M) was reduced fivefold but the specific activity still amounted to 63% of the wild type activity.     

Comparative Kinetic Effects of Mn (II), Mg (II) and the ATP/ADP Ratio on Phosphoenolpyruvate Carboxykinases from <Emphasis Type="Italic">Anaerobiospirillum succiniciproducens</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The kinetic affinity for CO₂ of phosphoenolpyruvate PEP⁵ carboxykinase from Anaerobiospirillum succiniciproducens, an obligate anaerobe which PEP carboxykinase catalyzes the carboxylation of PEP in one of the final steps of succinate production from glucose, is compared with that of the PEP carboxykinase from Saccharomyces cerevisiae, which catalyzes the decarboxylation of oxaloacetate in one of the first steps in the biosynthesis of glucose. For the A. succiniciproducens enzyme, at physiological concentrations of Mn²⁺ and Mg²⁺, the affinity for CO₂ increases as the ATP/ADP ratio is increased in the assay medium, while the opposite effect is seen for the S. cerevisiae enzyme. The results show that a high ATP/ADP ratio favors CO₂ fixation by the PEP carboxykinase from A. succiniciproducens but not for the S. cerevisiae enzyme. These findings are in agreement with the proposed physiological roles of S. cerevisiae and A. succiniciproducens PEP carboxykinases, and expand recent observations performed with the enzyme isolated from Panicum maximum (Chen et al. (2002) Plant Physiology 128: 160–164).     

Esterase polymorphisms in Microtus ochrogaster: interaction and linkage

Three polymorphic loci have been identified in the prairie vole, Microtus ochrogaster. Together they control a group of plasma esterases which can be separated using starch gel electrophoresis. A structural locus, Es-1, produces an enzyme which from genetic evidence appears to be a dimer. The allele Es-1 ^a produces a wholly active subunit, and homozygotes give a single enzyme band. The product of the second allele, Es-1 ^o, cannot form active enzyme on its own but will dimerize with the Es-1 ^a subunit, giving a hybrid enzyme with a slower electrophoretic mobility than the pure Es-1 ^a enzyme. The third allele, Es-1 ^–, has no detectable product. A second structural locus, Es-2, is linked to Es-1. The allele Es-2 ^a produces a single enzyme band, but the second allele Es-2 ^– has no detectable product. A modifier locus, Me, changes the mobility of the Es-1 enzymes. Me ^f is dominant over me ^s, and in homozygotes for me ^s the mobility is reduced.This work was supported by National Science Foundation Grant GB6273.This is contribution No. 869 from that Department.     

DOPA Production with Enterobacter cloacae NB 320 by Transamination Reaction

Taxonomical investigation was performed on the bacterium, strain NB 320 isolated from soil, and it was identified as Enterobacter cloacae. This bacterium produced the enzyme which catalyzed the transamination reaction between 3,4-dihydroxyphenyl pyruvate and an amino acid to form l-Dopa.

The optimum culture conditions for the enzyme production were studied along with the characteristics of the enzyme. The enzyme of the strain was different in some properties from that of Alcaligenes faecalis IAM 1015 which had been already studied. The former utilized glutamate as an amino donor best among the amino acids tested for transamination and was induced by the addition of glutamine and asparagine. Intact cells of the strain did not catalyze the reaction unless they were treated with sonication or with a detergent.     

Leucine Dehydrogenase of a Thermophilic Anaerobe,Clostridium thermoaceticum: Gene Cloning,Purification and Characterization

The leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli- pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15 min; at 55°C and various pH’s between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD⁺ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin- amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].     

Purification and characterization of fibrinolytic alkaline protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. BLB

Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH₄)₂SO₄ and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus.     

Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.