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Purification of valuable engineered proteins and enzymes can be laborious, costly, and generating large amount of chemical waste. Whilst enzyme immobilization can enhance recycling and reuse of enzymes, conventional methods for immobilizing engineered enzymes from purified samples are also inefficient with multiple-step protocols, regarding both the carrier preparation and enzyme binding. Nickel ferrite magnetic nanoparticles (NiFe2O4 MNPs) offer distinct advantages in both purification and immobilization of enzymes. In this work, we demonstrate the preparation of NiFe2O4 MNPs via a one-step solvothermal synthesis and their use in direct enzyme binding from cell lysates. These NiFe2O4 MNPs have showed an average diameter of 8.9 ± 1.7 nm from TEM analysis and a magnetization at saturation (Ms) value of 53.0 emu g–1 from SQUID measurement. The nickel binding sites of the MNP surface allow direct binding of three his-tagged enzymes, d-phenylglycine aminotransferase (d-PhgAT), Halomonas elongata ω-transaminase (HeωT), and glucose dehydrogenase from Bacillus subtilis (BsGDH). It was found that the enzymatic activities of all immobilized samples directly prepared from cell lysates were comparable to those prepared from the conventional immobilization method using purified enzymes. Remarkably, d-PhgAT supported on NiFe2O4 MNPs also showed similar activity to the purified free enzyme. By comparing on both carrier preparation and enzyme immobilization protocols, use of NiFe2O4 MNPs for direct enzyme immobilization from cell lysate can significantly reduce the number of steps, time, and use of chemicals. Therefore, NiFe2O4 MNPs can offer considerable advantages for use in both enzyme immobilization and protein purification in pharmaceutical and other chemical industries. [ABSTRACT FROM AUTHOR]

• Designed the RESCOT software for restriction enzyme (RE) usage in ribose-seq. • Defined and calculated the rNMP-captured region for reference genomes. • Optimized the RE usage for ribose-seq library preparation. • Validated the high rNMP-capture performance of optimized RE sets and combinations. • Expanded RESCOT usage to all existing rNMP-capture techniques. The incorporation of ribonucleoside monophosphates (rNMPs) in genomic DNA is a frequent phenomenon in many species, often associated with genome instability and disease. The ribose-seq technique is one of a few techniques designed to capture and map rNMPs embedded in genomic DNA. The first step of ribose-seq is restriction enzyme (RE) fragmentation, which cuts the genome into smaller fragments for subsequent rNMP capture. The RE selection chosen for genomic DNA fragmentation in the first step of the rNMP-capture techniques determines the genomic regions in which the rNMPs can be captured. Here, we designed a computational method, Restriction Enzyme Set and Combination Optimization Tools (RESCOT), to calculate the genomic coverage of rNMP-captured regions for a given RE set and to optimize the RE set to significantly increase the rNMP-captured-region coverage. Analyses of ribose-seq libraries for which the RESCOT tools were applied reveal that many rNMPs were captured in the expected genomic regions. Since different rNMP-mapping techniques utilize RE fragmentation and purification steps based on size-selection of the DNA fragments in the protocol, we discuss the possible usage of RESCOT for other rNMP-mapping techniques. In summary, RESCOT generates optimized RE sets for the fragmentation step of many rNMP capture techniques to maximize rNMP capture rate and thus enable researchers to better study characteristics of rNMP incorporation. [ABSTRACT FROM AUTHOR]

• Immobilization of Bacillus subtilis lipase A (BSLA) is reported for the first time. • Several surface modifications of silica supports were used for immobilization. • A selective immobilization of BSLA from microbial culture supernatant was observed. • Silica glyoxyl had the highest performance showing a stabilization factor of 436. • The reported method can be applied in immobilized biocatalyst engineering. Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50 °C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns. [ABSTRACT FROM AUTHOR]

Abstract The present study identified at least two novel angiotensin I-converting enzyme (ACE) inhibitory peptides in horse gram flour after hydrolysis with alcalase. The ACE inhibitory peptides from horse gram hydrolysate (HGH) were purified by ultrafiltration and different chromatography techniques followed by identification using nano liquid chromatography-mass spectrometry (nano LC-MS/MS). Further, based on in silico analysis, twelve potential ACE inhibitory peptides from HGH were synthesized and assayed for their ACE inhibitory activity. Two peptides TVGMTAKF and QLLLQQ exhibited the highest ACE inhibitory activity (IC 50 values of 30.3 μM and 75.0 μM, respectively) and were characterized kinetically to show K i values of 0.01 μM and 0.18 μM, respectively. In addition, these two novel peptides exhibited competitive mode of inhibition as well as retained their ACE inhibitory activity after incubation with gastrointestinal proteases (pepsin, trypsin, and chymotrypsin). Thus, the ACE inhibitory peptides identified from HGH shows promise to be used as a functional food ingredient for controlling hypertension. Highlights • Two novel peptides TVGMTAKF and QLLLQQ were identified from horse gram hydrolysate. • TVGMTAKF and QLLLQQ showed IC 50 values of 30.3 μM and 75.0 μM, respectively. • Inhibitory mechanism of both the peptides is competitive. • These peptides were highly stable to gastrointestinal proteases. • Both peptides have potential to be used as functional food ingredients. [ABSTRACT FROM AUTHOR]

Continuous enzymatic production of galactosyl-oligosaccharides (GOS) from a lactose substrate in an ultrafiltration membrane reactor (UMR) coupled with a nanofiltration separation system (CPNSS) was investigated. The overall rate of production over 4 h of continuous production was 80–104 mg GOS formed/U with an average residence time of 66 min, an initial lactose concentration of 300 g/L, an inlet pressure of 2.0 bar inlet pressure, and an outlet pressure of 1.5 bar. In the continuous diafiltration (CD) process, the concentration of various sugars and the relationship between the yield and purity of oligosaccharides was well predicted by mathematical models, the increased rate of sugar rejections was less than 10%, and the decreased rate of concentrations in the tank was less than 15%. In the CPNSS, 33.4 wt.% GOS was obtained in the UMR, and 1.24 kg of high-purity GOS (specifically, GOS purity of 57.2% and lactose content less than 20%) was achieved. This final yield was 80.1% GOS, which meets industry standards. [ABSTRACT FROM AUTHOR]

Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyllide and phytol. [ABSTRACT FROM AUTHOR]

Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed. [ABSTRACT FROM AUTHOR]

L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples. [ABSTRACT FROM AUTHOR]

Abstract: We show for the first-time Ganoderma lucidum laccase enzyme production using medium containing 3% (v/v) ethanol, which enhanced the enzyme production up to 14.1 folds. A more than 400-folds increase could be achieved if grown in the presence of the novel lignocellulosic biomass tamarind shell plus ethanol (3%, v/v), CuSO4 (0.4mM) and gallic acid (1mM). A 38.3kDa laccase enzyme was purified from the initial protein preparation with an overall yield of 32% using Sephadex G-100 and DEAE-cellulose column chromatography. The enzyme was identified through MALDI-TOF/TOF tandem mass spectrometry (MS/MS) as G. lucidum laccase-3. This enzyme exerted its optimal activity at a pH of 5 and a temperature of 55°C with ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as an ideal substrate. The catalytic efficiencies (k cat/K m) determined for ABTS and guaiacol were 11.5×105 and 3.9×105 s−1 M−1, respectively. The G. lucidum laccase decolorized various textile dyes and industrial textile dye effluent up to 90% and 97%, respectively. [Copyright &y& Elsevier]

In this article, I reflect on research on two ATPases. The first is F1F0-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization. [ABSTRACT FROM AUTHOR]

Abstract: Chitosanase (EC3.2.1.132) catalyzes the hydrolysis of β-1,4-glycosidic bonds in chitosan, converting it into chitooligosaccharides, which exhibit versatile application potentials in food, pharmaceutical, and agricultural areas. In this paper we present a new inducible chitosanase, isolated, and purified from a bacterial culture medium of Streptomyces roseolus DH by precipitation with ammonium sulfate and combined column chromatographies. The SDS–PAGE results show its molecular mass is around 41kDa, with a purity of more than 95%. The purified chitosanase exhibits optimum activity at 50°C, pH 5.0. It is stable between 30 and 60°C and at pH values between 5 and 7. It shows the highest activity towards colloidal chitosan and breaks down glycol chitosan and glycol chitin weakly. The enzyme is significantly inhibited by Cu2+, Co2+, Mn2+, Zn2+, and EDTA, but slightly activated by Mg2+. Further action mode analysis based on chitosan oligomers and a polymer reveals that the chitosanase could split chitooligosaccharides with degree of polymerization (DP) >4 and chitosan in an endolytic manner. The resultant hydrolytes are mainly chitotrisaccharides, indicating it is suitable for the uniform bioconversion of chitosan and its derivatives with high efficiency. [Copyright &y& Elsevier]

Polylactic acid has received much attention because of its biodegradable characteristics. However, to produce the polylactic acid, enormous energy is required at the current purification processes developed up to now. In this work, in order to reduce energy consumption, we proposed a novel purification method which combines lactic acid extraction and enzyme esterification instead of the dehydration and the chemical esterification which are conventionally used. As a result, the new purification method is found to be excellent as the solvents used in this work. It revealed that some solvents (diethyl ether, tert-butyl methyl ether, and methy isobuthyl ketone) have excellent abilities for both lactic acid extraction and enzymatic esterification. [ABSTRACT FROM AUTHOR]

Extraction of bromelain from pineapple fruit in an aqueous two phase system (ATPS) composed of polyethylene glycol (PEG) 1500 and potassium phosphate has been studied using response surface methodology. The various process variables such as PEG, potassium phosphate and NaCl concentration, and pH were optimized using a central composite rotatable design (CCRD) of response surface methodology (RSM) based on the partition coefficient, % yield, and purification factor of an enzyme. An optimized ATPS composed of 14% (w/w) PEG 1500, 17.66% (w/w) potassium phosphate and 1 mM sodium chloride at pH 7.5 was used to purify bromelain from a pineapple fruit. With this system, a maximum enzyme partition coefficient of 12.62 and %yield of 90.33 in the top PEG-rich phase with a purification factor of 2.4 was predicted. The enzyme partition coefficient, % yield, and purification factor obtained from experimentation are 12.22, 89.65, and 2.8, respectively, in the top PEG phase. The response model is validated by the closeness between the predicted and experimental results. [ABSTRACT FROM AUTHOR]

Abstract: Nowadays, the use of enzymes for the pharmaceutical, cosmetic or food industry steadily increases. Although the specification and production of enzymes are steadily enlarged, the downstream processing strategies lag behind. Thus, the development or improvement of enzyme purification methods to efficient and competitive techniques is a main issue. Adsorptive bubble separation e.g. foam fractionation represents such a improvable separation process. In this study, foam fractionation was used to separate an extracellular esterase from submerged cultures of the basidiomycete Coprinopsis cinerea. Parameters such as pH-value and column design affect the transport of active enzymes into the foam phase and thus the efficiency of the foam fractionation process. The influence and interactions of various parameters regarding the performance of the foam fractionation process were analyzed using Design of Experiments (DoE). The use of Design of Experiments allowed a fast identification of impact parameters and a direct optimization of the foam fractionation process. [Copyright &y& Elsevier]

Two alkaline keratinases-I and II secreted by Bacillus halodurans PPKS-2 were purified and characterized. Both the keratinases were purified using ammonium sulfate, DEAE-Sephadex followed by Sephadex G-200 column chromatography. The purification was 21.5-fold and 11.17% yield for keratinase-I and 23.7-fold with yield 18.46 for keratinase-II and its molecular weights 30 and 66 kDa. Both purified enzymes were relatively stable over a broad pH range 7.0–13.0 and optimally active at pH 11.0 and 60–70 °C. Keratinase-II was found to be more stable at 70 °C for 3 h and retained 100% of its activity, whereas keratinase-I lost 10% activity. Keratinase-I had high keratin disulfide reductase activity with low keratinase activity whereas keratinase-II had high keratinase activity with low keratin disulfide reductase activity. Keratinase activities of both the enzymes were completely inhibited by PMSF at 1 mM, whereas keratin disulfide reductase activity of keratinase-I was not affected. Enzymes were active and stable in the presence of the surfactants, bleaching agents (20% H2O2), commercial detergents (1%), and SDS (20%). Both the enzymes were partially sequenced and found that keratinase-I and II had a homology with disulfide reductases and serine type of proteases, respectively. [ABSTRACT FROM AUTHOR]

Abstract: The demand for enantiopure substances, e.g. for pharmaceutical applications or fine chemical production, continues to increase. This has led to the development of numerous stereoselective synthesis methods. Nevertheless a large number of chemical syntheses still result in racemic mixtures making a subsequent enantioseparation step necessary and thus are restricted to a maximum yield of 50%. Our work focuses on strategies to overcome this limitation by combining physicochemical separation processes with enzymatic racemization of the unwanted enantiomer in order to produce enantiopure amino acids. This paper deals with the production and characterization of a suitable amino acid racemase with broad substrate specificity (EC 5.1.1.10) from Pseudomonas putida which we cloned into Escherichia coli. Two enzyme lyophilizates of different purity were obtained from which the crude (CL) was sufficient for the racemization of methionine (Met) and the pure (PL) was used for asparagine (Asn). Racemization reactions of D-/L-Asn in H2O and D-/L-Met in 95vol.% 100mM KPi-buffer, 5vol.% methanol (MeOH) at different pH values and temperatures were characterized. The studied range of reaction parameters was chosen in dependency on planned enantioseparation processes. We found increasing V max values when temperature was risen stepwise from 20 to 40°C for both systems and when pH was shifted from 6 to 8 for the Met system. The presented results provide the basis for engineering enzyme-assisted physicochemical enantioseparation processes. [Copyright &y& Elsevier]

Abstract  A facile and fast approach for the purification of deoxycytidine kinase (dCK) from calf thymus was developed using a fast performance liquid chromatography system. A 73-fold enrichment of the enzyme was noted compared to unfractionated dCK. Characterization studies demonstrated that dCK had a molecular mass of 31 kDa using SDS–PAGE, an optimum pH of 7.0 and the enzyme maintained stability between 30 and 40°C. The rapid preparation of dCK demonstrated here will be valuable in the synthesis of nucleotide analogs. [ABSTRACT FROM AUTHOR]

Abstract: A thermostable endoglucanase was purified to homogeneity from culture supernatants of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight of the enzyme was 46.4kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was at pH 4.9. The temperature for maximum activity was 70°C, with 85% of its maximum activity retained after 150min of incubation at 50°C, but was rapidly inactivated at 70°C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0–7.0 at 50°C. The enzyme was significantly inhibited by Hg2+, Cu2+, and Fe2+, and stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT, and EDTA. The enzyme also hydrolyzed filter paper, and Avicel® PH-101 at rates of 25.8%, and 7.3%, respectively when compared with carboxymethyl-cellulose. The enzyme did not hydrolyze soluble starch, oat spelt xylan, birch wood xylan, or locust bean gum. The enzyme catalyzed the hydrolysis of carboxymethyl-cellulose with a K m of 1.74mg/ml and a V max of 0.63U/min/mg protein. This enzyme was competitively inhibited by glucose and cellobiose with K i values of 0.67 and 0.45M, respectively. TLC showed that the endoglucanase produces cellotetraose, cellotriose, cellobiose, and a small amount of glucose. The deduced internal amino acid sequences of the D. eschscholzii endoglucanase showed similarity to the sequences of the glucosyl hydrolase family 5. [Copyright &y& Elsevier]

Growth on a wheat bran media induced production of an extracellular β-glucanase by Rhizomucor miehei (DSM 1330). The enzyme was purified to homogeneity. Substrate specificity studies coupled with protein database similarity searching using mass spectrometry-derived sequence data indicate it to be an endo-1,3(4)-β-glucanase (EC 3.2.1.6). The enzyme was characterised in terms of potential suitability for use in animal (poultry) feed. Significant activity was observed over the entire pH range typical of the avian upper digestive tract (pH 2.6–6.5). The enzyme was also found to be more thermostable than current commercialized β-glucanases, particularly when heated at a high enzyme concentration, and retained twice as much residual activity as the latter upon exposure to simulated avian digestive tract conditions. There are no previous reports of the production, purification or characterization of a β-glucanase from a Rhizomucor, and the enzyme’s application-relevant physicochemical characteristics render it potentially suited for use in animal feed. [ABSTRACT FROM AUTHOR]

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified {18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg"1) at 45°C and pH 8.O. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thio- phen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as byproducts was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of D-sorbitol or xylitol (20% wfv each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme. [ABSTRACT FROM AUTHOR]

TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of α2 and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret. [ABSTRACT FROM AUTHOR]

The characteristics of polyphenol oxidase (PPO) from Chinese water chestnut (CWC) and its potential inhibitors for browning reactions were investigated. PPO was isolated from fresh-cut CWC and was purified on a Sephadex G-100 column, with a yield of total activity close to 10%. The molecular weight, Michaelis constant (K m ), substrate specificity, optimal pH and temperature of CWC PPO were examined. Kinetic studies indicated that the K m and V max values of CWC PPO for catechol were 10.32 mmol/L and 6.452 × 10 4 U/min, respectively. The optimal pH and temperature for CWC PPO was 6.5 and 40C, respectively. Among the browning inhibitors tested, 4-hexylresorcinol, at a concentration of 0.3 mmol/L, showed the strongest inhibition (70%) against the PPO activity of CWC, followed by 3.0 mmol/L N-acetyl-L-cysteine with an inhibition of 53%. [ABSTRACT FROM AUTHOR]

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH4)2SO4 fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant ( k cat/ K m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of ( R)-(−)-mandelic acid from readily available mandelonitrile. [ABSTRACT FROM AUTHOR]

Pichia pastoris β-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate, gel filtration, and ion-exchange chromatography with Q-Sepharose and CM-Sepharose. The enzyme is a tetramer (275 kD) made up of four identical subunits (70 kD). The pH optimum is 7.3, and it is fairly stable in the pH range 5.5–9.5. The temperature optimum is 40°C. The purified β-glucosidase is effectively active on p-/ o-nitrophenyl-β-D-glucopyranosides ( p-/ o-NPG) and 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) with K m values of 0.12, 0.22, and 0.096 mM and V max values of 10.0, 11.7, and 6.2 µmol/min per mg protein, respectively. It also exhibits different levels of activity against p-nitrophenyl-1-thio-β-D-glucopyranoside, cellobiose, gentiobiose, amygdalin, prunasin, salicin, and linamarin. The enzyme is competitively inhibited by gluconolactone, p-/ o-nitrophenyl-β-D-fucopyranosides ( p-/ o-NPF), and glucose against p-NPG as substrate. o-NPF is the most effective inhibitor of the enzyme activity with K i value of 0.41 mM. The enzyme is more tolerant to glucose inhibition with K i value of 7.2 mM for p-NPG. Pichia pastoris has been employed as a host for the functional expression of heterologous β-glucosidases and the risk of high background β-glucosidase activity is discussed. [ABSTRACT FROM AUTHOR]

Describes the purification and characterization of hydantoin racemase (HRase) from Microbacterium liquefaciens AJ 3912 (MHR). Enzymatic characteristics of the purified MHR; Comparison of the N-terminal amino acid sequence of MHR with those of other HRases.

Discusses the purification and characterization of inducible cephalexin synthesizing enzyme (CSE) in Gluconobacter oxydans. Microorganism and cultivation conditions; Inducer of CSE; Activity of CSE; Assay of enzyme activity.

Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. γ-CD yield of CGTase from alkalophilic Bacillus species is usually much lower than β-CD, while from alkalophilic Bacillus sp. 7-12. γ-CD yield is close to β-CD. A CGTase from alkalophilic Bacillus sp. 7-12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and Sepharose CL-6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS-PAGE. The enzyme showed a K m of 1.24 mg/mL and V max 0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag + , Cu 2+ , Mg 2+ , Al 3+ , Co 2+ , Zn 2+ , Fe 2+ and slightly inhibited by Sn 2+ , Mn 2+ . The ions Ca 2+ and K + , EDTA and DTT had no influence on the enzyme activity. [ABSTRACT FROM AUTHOR]

Discusses research on the structure of macrophomate synthase (MPS) enzyme compared with structure of 2-dehydro-3-deoxygalactarate (DDG) aldolase. Ability of MPS crystal structure to catalyze chemical transformations oxalacetate and 2-pyrone; Purification and crystallization of MPS; Instruments used in the study; Analysis of MPS crystals.

Acetyl esterase (acetic-ester acetylhydrolase, EC 3.1.1.6) from citrus peel, whose natural role is not well known, catalyses, in vitro, the hydrolysis of acetyl groups from a wide range of substrates. This enzyme was extracted from Mediterranean orange peel, largely available in Italy, and purified 190-fold by a single chromatographic step on Sepabeads FP-HG. SDS polyacrylamide gel electrophoresis of the purified enzyme showed a major protein band, corresponding to a molecular mass of 45 kDa. Both free and immobilised enzyme were used in biotransformations. The enzyme removed the acetyl group in the 3 position of β-lactamic antibiotics, such as cephalosporin C and the intermediate 7-aminocephalosporanic acid with ≥98% conversion and 91-93% product yield. [ABSTRACT FROM AUTHOR]

cis,cis-Muconate cycloisomerase (MC) was purified to homogeneity from benzamide-assimilating Arthrobacter sp. BA-5-17. The purified enzyme showed high activities for cis,cis-muconate and 3-methyl-cis,cis-muconate, and preferred the 3-substituted derivatives over the derivatives with the same substituent at the 2 position as a substrate. A gene encoding MC of strain BA-5-17 was cloned and named catB. The catB gene was clustered with catR encoding a putative LysR-type regulator, catC encoding a putative muconolactone isomerase, and catA-II encoding the catechol 1,2-dioxygenase isozymes CD-III-1 and III-2. These genes showed the same orientation in transcriptional direction and the organization of cloned genes was catRBCA-II. In the phylogenetic analysis of MCs and chloro-MCs, the BA-5-17 and Streptomyces setonii MCs formed a subfamily, clearly distinguished from those of other MCs. [Copyright &y& Elsevier]

The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, α-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50±4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic α-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 °C to 60 °C. The results also implicated several prototropic groups in the catalytic reaction. [ABSTRACT FROM AUTHOR]

An overview of the purification of an oligomeric enzyme, an extramitochondrial acetyl-coenzyme A hydrolase from rat liver, is presented. The enzyme has been purified to homogeneity using two successive size-exclusion chromatography runs, first for the monomeric and second for the oligomeric form of the enzyme. The sequential gel-filtration steps efficiently removed the contaminants of any molecular size, first of different size from that of the monomeric form of the enzyme (Kav=0.47 on Superdex 200) and second of different size from that of the oligomeric form (Kav=0.33), allowing us to purify the enzyme in high purity. This strategy provides an excellent model for purifying many other oligomeric proteins including key enzymes or allosteric enzymes regulating metabolism. [Copyright &y& Elsevier]

Arsenate reductase (ArsC) from Staphylococcus aureus pI258 is extremely sensitive to oxidative inactivation. The presence of oxidized ArsC forms was not that critical for NMR, but kinetics and crystallization required an extra reversed-phase purification to increase sample homogeneity. The salt ions observed in the X-ray electron density of ArsC were investigated. Carbonate was found to have the lowest dissociation constant for activation (Ka=1.1 mM) and potassium was stabilizing ArsC (ΔTm=+6.2 °C). Also due to the use of these salt ions, the final yield of the purification had improved with a factor of four, i.e. 73 mg/l culture. [Copyright &y& Elsevier]

The cytoplasmic pyrophosphatase of the photosynthetic bacterium Rhodospirillum rubrum was purified to electrophoretic homogeneity. The enzyme is a homohexamer of 20-kDa monomers. The gene was cloned and sequenced. Alignment of the deduced 179-amino-acid protein with known bacterial pyrophosphatases revealed conservation of all residues in the active site. Attempts to obtain an insertion mutant of the cytoplasmic pyrophosphatase gene did not yield any cell completely devoid of cytoplasmic pyrophosphatase activity. The mutants obtained showed 50% of the enzymatic activity and grew in twice the generation time of wild-type cells. This suggests that the membrane-bound pyrophosphatase of Rsp. rubrum is not sufficient for a normal growth rate, whereas the cytoplasmic enzyme is essential for growth. The characteristics of the gene and the encoded protein fit those of prokaryotic family I pyrophosphatases. [ABSTRACT FROM AUTHOR]

Describes the structure of hyperthermophilic 2-deoxy-D-ribose-5-phosphate aldolase (DERA) and of aldolase from the Archaea domain. Expression of the gene APE2347 that encodes a DERA homolog; Purification and characterization of Aeropyrum pernix DERA.

Membrane-bound L-(+)-pantoyl lactone dehydrogenase, an enzyme that catalyzes the formation of ketopantoyl lactone from L-(+)-pantoyl lactone, was solubilized with Brij 35 and purified 78-fold to apparent homogeneity, with a 3.7% overall recovery, from Nocardia asteroides through purification procedures including successive ammonium sulfate fractionation, and DEAE-Sephacel, Sepharose CL-6B and Cellulofine GC-700-m column chromatography in the presence of Brij 35. The relative molecular mass of the native enzyme, as estimated on high-performance gel-permeation chromatography, is at least more than 600 kDa and its subunit molecular mass is 42 kDa. The enzyme shows high specificity for L-(+)-pantoyl lactone as a substrate (Km = 26.8 mM; Vmax = 4.22 µmol · min-1 · mg protein-1). Brij 35 acts as a stabilizer and also as an efficient activator of the enzyme. The prosthetic group of L-(+)-pantoyl lactone dehydrogenase was identified as noncovalently bound FMN. [ABSTRACT FROM AUTHOR]

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (α subunit 26 kDa, β subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the β subunit. However, the amino acid sequence of the α subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes. [ABSTRACT FROM AUTHOR]

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/ mg). The enzyme is an α4 homotetramer of four 25-kDa subunits. It has a Km of 2 µM and a kcat/Km of 1.8 × 108 M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyI-CoA above 10 µM. K is 10-10 M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase. [ABSTRACT FROM AUTHOR]

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The highmolecular-mass band occurred in Iow quantity and consisted of, probably eight, apparently identical chains of Mr = 33 000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with Mrα = 63 000-65 000 and Mrβ = 61 000-62 000. The N termini of both chains were threonine, whereas their C-terminal sequences were different: α, -(Val)-(Ser)-(Ala)-Leu; β, -(His)-(Asp)-(Ala)-Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different. In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (αβ)2 structure, the apoenzyme αβ. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase. [ABSTRACT FROM AUTHOR]

Reports on the purification and characterization of brain pyridoxal kinase from sheep brain. Use of ammonium sulphate fractionation, diethylaminoethanol-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration; Molecular mass of the native enzyme.

A processing mannosidase acting on (Man)9(GlcNAc)2 oligosaccharides, Man9 mannosidase, has been purified 2190-fold from calf liver crude microsomes by a four-step procedure involving (a) differential salt/detergent extraction, (b) affinity chromatography on AH-Sepharose 4B with N-5-carboxypentyl-l-deoxymannojirimycin as ligand, (c) ConA-Sepharose and (d) DEAE-Sephacel chromatography. (Man)9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA-Sepharose, indicating the absence of high-mannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca2+ being most effective. It is inhibited by 1-deoxymannojirimycin (dMM), N-methyl-dMM and N-5-carboxypentyl-dMM with Ki = 7 μM, 75 μM, and 140 μM, respectively. Man9 mannosidase cleaves three of the four αl,2-1inked mannose residues from the (Man)9(GlcNAc)2 oligosaccharide, does not hydrotyse the remaining (Man)6(GlcNAc)2 structure and is not active against aryl α-mannosides. This pronounced substrate specificity points to the participation of Man9 mannosidase in the N-linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Glc)3(Man)9(GlcNAc)2 intermediate, we assume that the enzyme is located in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

A membrane-bound α-L-fucosyltransferase, which is involved in the synthesis of a developmentally regulated carbohydrate antigen, SSEA-1, was purified about 2000-fold from F9 embryonal carcinoma cells. The procedures used were solubilization with Triton X-100, column chromatography on SP-Sephadex, DEAE-Sephadex, RCA-agarose and on G DP-agarose. Upon sodium dodecyl sulfate gel electrophoresis, the purified preparation gave a protein band with a relative molecular mass of 65000. The optimum pH of the enzyme was between 6.0 and 7.0 and the Km toward N-acetyllactosamine was 0.55 mM. The enzyme was active with asialofetuin, but not with intact fetuin. Susceptibility of the product to α-L-fucosidase I from almond emulsin verified that the enzyme transferred fucose to C-3 hydroxyl of N-acetylglucosamine in the N-acetyllactosamine structure. Activities of βgalactoside α1 → 2-fucosyltransferase and N-acetylglucosaminide α1 → 4-fucosyltransferase acting on synthetic substrates were not detected in the purified enzyme nor in the crude extract of F9 cells. PYS-2 parietal endoderm cells lacked all the fucosyltransferases mentioned above. [ABSTRACT FROM AUTHOR]

A diamine oxidase has been purified lo homogeneity from the latex of an herbaceous shrub, Euphorbia characias. This enzyme has a relative molecular mass of 144000 and is composed of two identical subunits. It contain two Cu(II) and two carbonyl-like groups per dinner. The purified enzyme is pink and shows a broad absorption in the visible region centered at 480 nm, which is modified by the addition of phenylhydrazine or semicarbazide. The electron paramagnetic resonance spectrum is typical of copper(II) in a tetragonal symmetry. This enzyme oxidizes putrescine and cadaverine at fairly high rate and, less efficiently a few related compounds, but not histamine, spermine or spermidine. [ABSTRACT FROM AUTHOR]

Acetoacetyl-CoA synthetase was purified to electrophoretic homogeneity from Zoogloea ramigera 1-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium, which lacks 3-ketoacid CoA-transferase. The purified enzyme had a specific activity of 52.2 μmol acetoacetyl-CoA formed min-1 mg protein-1, which constituted a 680-fold purification compared to the crude extract, with a 5.1% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH4+) and a divalent cation (Mg2+, Mn2+, Ca2+ or Ni2+) for the activation of acetoacetate, yielding acetoacetyl-CoA, AMP and pyrophosphate in equimolar amounts. The pH optimum of the enzyme reaction was 8.4. The molecular weight of the enzyme was approximately 70 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and 72 000 by Sephadex G-200 gel filtration. The enzyme was active only on acetoacetate and to a lesser extent on L(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L(-+-)-3-hydroxybutyrate, ATP and CoA were 7.6 x 10-5 M, 1.4 x 10-3 M, 3.3 x 10-5 M and 9.1 x 10-5 M respectively. [ABSTRACT FROM AUTHOR]

A chymotrypsin-like enzyme has been purified from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogenous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 18500-19000. This enzyme hydrolyses N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyse N-benzoyl-L-arginine ethyl ester, N-α-toluenesulfonyl-L-arginine methyl ester, N-α-benzoyl-DL-arginine-p-nitroanilide, hippuryl-L-arginine or hippuryl-L-phenylalanine. The Michaelis constants for AcTyrOEt and BzTyrOEt are 0.05 mM and 0.0106 mM, respectively. The enzyme activity is inhibited completely by phenylmethylsulfonyl fluoride (PhMeSO2F), chymostatin and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCH2Cl), and partially by soybean trypsin inhibitor and N-α-p-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl). The enzyme is activated by CaCl2, MgCl2, NaCl and KCl, and loses its activity in 5 min at 67 °C. It digests the jelly coat and vitelline layer, not the fertilization membrane. The microvilli of unfertilized eggs elongate and decrease in number as the vitelline layer lyses. The vitelline layer lytic activity is inhibited completely by PhMeSO2F, TosPheCH2Cl, and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2Cl, and α1-antitrypsin. We have confirmed by transmission electron microscopy that our chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported. [ABSTRACT FROM AUTHOR]

Reports on the purification of an alpha-amanitin-resistant DNA-dependent RNA polymerase II from the lower eukaryote Aspergillus nidulans to apparent homogeneity. Use of extraction of the enzyme at low salt concentration, polymin P fractionation, binding to ion-exchangers and density gradient centrifugation; Resolution of the subunit composition by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate.

Glucose-6-phosphate dehydrogenase has been purified 192-fold from Methylomonas M15, grown on methanol as sole source for carbon and energy. The purification procedure involved N-cetyl- N,N,N,-trimethylammonium bromide precipitation, ion-exchange chromatography and affinity chromatography on blue-dextran-Sepharose. The final enzyme preparations were homogeneous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weight of the enzyme was calculated to be 108000 ± 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels indicates that the enzyme is a dimer composed of two probably identical subunits each having a molecular weight of 55000. The enzyme exhibits activity with either NAD or NADP as electron acceptor. Mg2+ or an other bivalent cation is not essential for enzyme activity. The Km values were found to be 0.2 mM for NAD, 0.014 mM for NADP, 0.29 mM for glucose 6-phosphate with NAD and 0.11 mM for glucose 6-phosphate with NADP as electron acceptor. The reduced coenzymes NADPH and NADH as well as ATP are powerful inhibitors of the enzyme. The intracellular levels of glucose 6-phosphate, ATP, NAD and NADP were measured and found to be approximately 2.5 mM, 1.5 mM, 1 mM and 2 mM respectively, during exponential growth. From 3 consideration of the substrate pool sizes and types of inhibitors, it is concluded th3.t this enzyme may function in two roles m the cell: NADH production for energetics and NADPH production for reductive biosynthesis. [ABSTRACT FROM AUTHOR]

Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Natl Acad. Sci. U.S.A. 72, 1589]. Although no function has been assigned to δ protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SP01-coded regulatory subunits. This report describes the purification of δ and σ proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified σ polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template. In contrast, purified δ protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of δ protein on poly(dA-dT) transcription was strongly dependent on the presence of KCI in the RNA synthesis reaction mixture. [ABSTRACT FROM AUTHOR]

A new method for discovering the minimum degree of rate equations using only the experimental graphs and a straight-edge (transparent ruler) is presented. This method is then illustrated by an analysis of the wavy υ vs [S] curves and non-linear double-reciprocal plots reproducibly given by NAD-dependent 15-hydroxyprostaglandin dehydrogenase for which a new improved purification is described. It is shown by this analysis that the enzyme has a complex mechanism involving cooperatively linked dependent sites and requiring a rate equation of at least fourth degree in prostaglandin and NAD+ with no kinetically significant dead-end complexes. [ABSTRACT FROM AUTHOR]

An inducible membrane-bound L-4-hydroxymandelate oxidase (decarboxylating) from Pseudomonas convexa has been solubilized and partially purified. It catalyzes the conversion of L-4-hydroxymandelic acid to 4-hydroxybenzaldehyde in a single step with the stoichiometric consumption of 02 and liberation of CO2. The enzyme is optimally active at pH 6.6 and at 55 °C. It requires FAD and Mn2+ for its activity. The membrane-bound enzyme is more stable than the solubilized and purified enzyme. After solubilization it gradually loses its activity when kept at 5 °C which can be fully reactivated by freezing and thawing. The Km values for DL-4-hydroxymandelate and FAD are 0.44 mM and 0.038 mM respectively. The enzyme is highly specific for DL-4-hydroxymandelic acid. DL-3,4-Dihydroxymandelic acid competitively inhibited the enzyme reaction. From the Dixon plot the Ki for DL-3,4-dihydroxymandetic acid was calculated to be 1.8 × 10-4 M. The enzyme is completely inactivated by thiol compounds and not affected by thiol inhibitors. The enzyme is also inhibited by denaturing agents, heavy metal ions and by chelating agents. [ABSTRACT FROM AUTHOR]