Inactivation of kanamycin, neomycin, and streptomycin by enzymes obtained in cells of Pseudomonas aeruginoa

Ten strains of Pseudomonas aeruginosa were disrupted and centrifuged. The supernatant fluids from centrifugation at 105,000 x g contained enzymes inactivating kanamycin, neomycin, and streptomycin in the presence of adenosine triphosphate. Kanamycin-inactivating enzyme was precipitated with ammonium sulfate at 66% of saturated concentration, and the inactivated kanamycin was shown to be kanamycin-3'-phosphate in which the C-3 hydroxyl group of 6-amino-6-deoxy-d-glucose moiety was phosphorylated. This is identical with kanamycin inactivated by Escherichia coli carrying R factor. Streptomycin-inactivating enzyme was precipitated with ammonium sulfate at 33% of saturated concentration.     

Solubilization and partial purification of n,n'-dicyclohexylcarbodiimide-sensitive ATPase from pea cotyledon mitochondria

The N,N′-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.

The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F₀F₁ preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis.

     

Inactivation and Phosphorylation of Kanamycin by Drug-resistant Staphylococcus aureus

The cell-free system of clinical isolates of drug-resistant Staphylococcus aureus inactivated kanamycin, and the inactivated product was identified with kanamycin-3′-phosphate, in which the C-3-OH of the 6-amino-6-deoxy-d-glucose moiety of kanamycin was phosphorylated.     

Polyphenol oxidase isoenzymes in avocado

Avocado polyphenol oxidase (PPO) was precipitated mainly in the 30–90% saturated ammonium sulfate fraction. The 40–75% saturated ammonium sulfate fraction (the partially purified enzyme) had the highest specific activity in the cultivars Lerman, Horeshim and Fuerte. The PPO was active towards o-dihydroxyphenols. Six active enzymes (a–f) were detected with D,L-DOPA, 4-methylcatechol, catechol, caffeic acid or chlorogenic acid. Band e was the most active in all cases. More isoenzyme bands (fast-moving) were observed with caffeic acid than with 4-methylcatechol. Furthermore, the isoenzyme patterns of the partially purified extracts of the cultivars could be distinguished with respect to caffeic acid.     

Isolation, Purification, and Some Properties of Penicillium chrysogenum Tannase

Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The K_m value was found to be 0.48 × 10⁻⁴ M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels.     

Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus

Summary The extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium.Direct concentration and separation from liquid media, omitting ammonium sulfate treatment, was also found to be possible, although prolonged flushing with buffer was necessary to obtain satisfactory separation.During purification from the ammonium sulfate fractions, amylase activity was found to decrease but could be restored by Ca-ions. At 70°C, a final concentration of 0.5 mM CaCl₂, was sufficient for full restoration, while three times that amount was necessary at 80°C. Determination of the K _m-values for Ca at different temperatures resulted in an asymptotically increasing curve at temperatures beyond 75°C. Addition of Ca had a pronounced effect on the stability of the amylase at 80°C but not at 90°C. Protease activity and stability was not affected by Ca-ions.     

Purification,Crystallization and Some Properties of Intracellular Pullulanase from Aerobacter aerogenes

Intracellular pullulanase was entirely extracted with sodium dodecylsulfate from the cells and was purified by means of ammonium sulfate fractionation and DEAE-cellulose and Sephadex chromatography. Crystalline pullulanase was precipitated with saturated ammonium sulfate solution. Intracellular pullulanase was purified over 150 fold in 17% yield to a final specific activity of 7000 per mg protein from the enzyme solution obtained by SDS-extraction. On ultracentrifugation analysis, the enzyme showed a symmetrical peak. The sedimentation coefficient, s_{20, w} was 6.29 S. Polyacrylamide disc electrophoresis gave a main band and a sub-band, and both showed activity. Molecular weight of intracellular pullulanase was estimated to be (8±1) × 10,000 from gel filtration with Sephadex G-200 and to be (9±1) × 10,000 from sedimentation equilibrium. These values were higher than that (6~7 × 10,000) of extracellular pullulanase. Both enzymes differed slightly in thermal- and pH-stabilities.     

GTP Cyclohydrolase I: Purification, Characterization, and Effects of Inhibition on Nitric Oxide Synthase in Nocardia Species

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH₄), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a K_m for GTP of 6.5 μM. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56°C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH₄ inhibited enzyme activity by 25% at a concentration of 100 μM. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a K_i of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO₂⁻ plus NO₃⁻. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH₄.     

A (1-->3)-beta-d-Glucan Isolated From Zea Shoot Cell Wall Preparations

A small quantity of (1→3)-β-d-glucan was extracted with a (1→3),(1→4)-β-d-glucan by hot water after treatment of the insoluble fraction of a buffer homogenate of Zea shoots with 3 molar LiCl. An ammonium sulfate precipitation procedure effected a separation of the (1→3)-β-d-glucan from the more prevalent (1→3),(1→4)-β-d-glucan. The minor component polysaccharide precipitated at a concentration of 20% ammonium sulfate (w/v) and was, as a consequence of precipitation, rendered insoluble in water. The insoluble products were dissolved in 1 normal NaOH followed by neutralization with CH₃COOH. The purified polysaccharide accounted for approximately 0.3% of total hot water extract. It consisted mostly of glucose and its average mol wt was estimated to be about 7.0 × 10⁴, based on elution from a calibrated Sepharose CL-4B column. Methylation analysis and enzymic hydrolysis or partial acid-hydrolysis of the polysaccharide followed by analysis of the hydrolysate showed that the polysaccharide consisted of (1→3)-β-linked glucose residues.     

Hemolymph factor in armyworm larvae infected with a nuclear-polyhedrosis virus toxic to Apanteles militaris

The parasitoid, Apanteles militaris, was killed or failed to pupate when its armyworm host was infected with the hypertrophic strain of a nuclear-polyhedrosis virus. Virion-free plasma from diseased larvae killed the parasitoids when injected into uninfected larvae. The toxic factor was partially precipitated from plasma that was 50% saturated with ammonium sulfate and was completely precipitated in 50% ethanol or methanol. Parasitoids in larvae infected with the typical strain of nuclear-polyhedrosis virus seemed unaffected.     

Milk-clotting Enzyme from Microorganisms: V. Purification and Crystallization of Mucor Rennin from Mucor pusillus var. Lindt

A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg²⁺, Ag⁺, Zn²⁺, and KMnO₄.     

Milk-clotting Enzyme from Microorganisms: V. Purification and Crystallization of Mucor Rennin from Mucor pusillus var. Lindt.1

A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg²⁺, Ag⁺, Zn²⁺, and KMnO₄.     

De novo synthesis of 3'-nucleotidase in germinating wheat embryo

The enzyme 3′-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up ³⁵SO₄ between 40 and 46 hours of germination synthesized ³⁵S-labeled 3′-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3′-nucleotidase activity is a consequence of de novo synthesis of the enzyme.     

Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.)

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg²⁺ ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a K_m of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO₃⁻ with S_0.5 value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, α-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca²⁺ and Mn²⁺. Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate.     

Purification and Characterization of the NAD-Dependent Glutamate Dehydrogenase in the Ectomycorrhizal FungusLaccaria bicolor(Maire) Orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) fromLaccaria bicolorwas purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE–Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at −75°C, 4 days at 4°C, and 1 h at 50°C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at −75°C. NAD-GDH activity was stimulated by Ca²⁺and Mg²⁺but strongly inhibited by Cu²⁺and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 μM, 89 μM, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and itsK_mvalue increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid.     

Glyceraldehyde-3-Phosphate Dehydrogenase (NADP) from Sinapis alba L: Reversible Association of the Enzyme with a Protein Factor as Controlled by Pyridine Nucleotides in Vitro

Aggregation of glyceraldehyde-3-P dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings during gel filtration on Sepharose 6B is dependent on the presence of a fraction (“binding fraction”) which can be separated from the enzyme by precipitation with 55% ammonium sulfate. Association of the enzyme with this binding fraction is NAD-dependent whereas NADP⁺ causes release. Dithioerythritol (2 mM) has no influence on these reversible processes.

Binding fractions, partially purified by ammonium sulfate and acetone fractionation, were submitted to dodecylsulfate-polyacrylamide gel electrophoresis. They always contain one or two dominant polypeptides with apparent molecular weights 42,000 and 58,000. The 42,000 polypeptide comigrates during dodecylsulfate electrophoresis with the corresponding subunit of the enzyme. It comprises up to 70% of the total protein in partially purified binding fractions and can be regarded as a major protein in seedling extracts.

The differential transport behavior of glyceraldehyde-3-P dehydrogenase (NADP) on Sephadex G-200 in the presence of NAD⁺ and NADP⁺ can be used as a simple and effective purification procedure. The enzyme isolated in this way has an isoelectric point of about 4.5 and maintains under all tested conditions a heterogeneous subunit composition of at least three different polypeptide chains (apparent molecular weights, 39,000, 42,000, 43,000).

The present data suggest that NAD(P)-controlled aggregation of glyceraldehyde-3-P dehydrogenase (NADP) from Sinapis alba L. is due primarily to enzyme association with a separate binding fraction rather than to enzyme polymerization. It is possible that a major component of this binding fraction, the 42,000 polypeptide, consists of “surplus” nonactive enzyme subunits, which self-associate and interact with the NAD-conformer of the enzyme.

     

Production and recovery of recombinant protease inhibitor α1-antitrypsin

Human α₁-antitrypsin (AAT) was produced in the recombinant yeast Saccharomyces cerevisiae ATCC 20699 grown in batch and fed-batch culture. The final biomass concentration and antitrypsin concentration attained were 55 g·L⁻¹ and 1.23 g·L⁻¹, respectively, in the fed-batch. The maximum productivities of biomass and antitrypsin were 1.6 and > 0.04 g L⁻¹h⁻¹, respectively, or substantially greater than the highest productivity values reported in the past. For recovering the antitrypsin, the cell slurry was concentrated 4-fold (231 g·L⁻¹ biomass, 122 min of processing) by cross-flow microfiltration and the cells were disrupted by bead milling (3 passes of 3 min total retention time). The cell homogenate was treated with aluminum chloride or PBS (pH 7) to aid separation of the cell debris by flocculation and sedimentation. The clarified cell homogenate was subjected to ammonium sulfate fractionation to precipitate the recombinant antitrypsin. The AAT precipitated at 45–75% saturation of ammonium sulfate, depending on the age of the homogenate. The crude AAT in the homogenate degraded at room temperature (25°C), with a zero order deactivation rate of 1.815 × 10⁻³ ± 3.43 × 10⁻⁴ g AAT L⁻¹h⁻¹.     

What is pea legumin — Is it glycosylated?

Since there is some question as to whether or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of Pisum sativum L. supplied with [¹⁴C]-labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387–400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509–520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40–70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263–279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Urease of Spirulina maxima

Urease (EC 3.5.1.5) was purified from Spirulina maxima by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-200. The enzyme had maximum activity at pH 8.7, a K_m for urea of 0.12 mM and a MW of ca 232 000. A MW of 38 000 was determined for the subunits. The enzyme was inactivated by p-hydroxymercuribenzoate.