重组Bacillus subtilis产B.stearothermophilus环糊精葡萄糖基转移酶的发酵优化

利用本研究室已构建的重组菌Bacillus subtilis/pBSMuL3-α/β-CGTase对产B.stearothermophilus环糊精葡萄糖基转移酶的发酵产酶进行了优化,考察了培养基中重要成分:碳源、有机氮源、无机氮源、有机与无机氮源质量比、碳源与氮质量比、金属离子种类等单因素对该重组菌产α/β-CGTase的影响,并采用正交实验对发酵培养基进行优化,对优化结果分析可知,重组菌B.subtilis/pBSMuL3-α/β-CGTase发酵产α/β-CGTase的最优培养基成本为:葡萄糖5 g/L,氮源(鱼骨蛋白胨∶NH4Cl=3∶1)25 g/L,1 mmol/L Mg^2+。在最优条件下发酵培养,α/β-CGTase的酶活由原来TB发酵培养基的9.20 U/mL提高至20.32 U/mL,是优化前酶活的2.2倍,为α/β-环糊精葡萄糖基转移酶的工业应用提供了理论支持。     

UDP-Galactose:Ceramide Galactosyl Transferase of Isolated Oligodendroglia

Abstract: The activity of UDP-galactose:ceramide galactosyl transferase (CGalT) has been studied in isolated oligodendroglia from bovine brain white matter and myelinating rat brain. The specific activity and activity per mg DNA are 4- and 10-fold higher in rat oligodendroglia compared with neuronal perikarya from rat brain, and is higher in oligodendroglia from myelinating rat brain compared with bovine oligodendroglia. In membranes isolated from oligodendroglia, the specific activity decreased in the order endoplasmic reticulum > plasma membrane > myelin.     

Purification and Characterization of Enterotoxin Produced by Aeromonas sobria

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.     

Purification and Properties of Lysophospholipase Produced by Corticium centrifugum

Lysophospholipase (EC 3. 1. 1. 5) from the culture broth of Corticium centrifugum was purified 92-fold in specific activity by DEAE-Sephadex and hydroxylapatite column chromatography. The isoelectric point was at about pH 3.9, and the molecular weight was about 130,000. The optimal pH was about 3.5~5.0. The stable pH range was from 7.0 to 8.0. Lysophospholipase activity was inhibited by Fe³⁺, Hg²⁺ and Al³⁺, but stimulated by various organic solvents. Diazobenzene p-sulfonic acid, N-bromosuccinimide and diisopropyl-fluorophosphate also inhibited the activity. This enzyme did not hydrolyze mono-, di-or tripalmitin or phosphatidylcholine. Apparent Michaelis constants of lysophospholipase activity for 1-acyl-LPC, 1-palmitoyl-LPC and 1-oleoyl-LPC were 0.35, 0.16 and 0.09 mm, respectively. The effect of detergents on the enzyme activity was observed to differ with the fatty acid composition of substrate.     

Purification and Characterization of an Endo-d-arabinase Produced by Cellulomonas

An extracellular endo-d-arabinase enzyme produced by the bacterial strain of Cellulomonas was purified 77.1-fold with 0.20% recovery for protein by DEAE Sepharose anion exchange, Sephacryl S-300 gel filtration and blue Sepharose affinity chromatography, and designated as CEDAase. The apparent molecular mass of CEDAase was 45 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CEDAase is an endoenzyme for arabinogalactan with the main and specific product of hexa-arabinofuranoside. It reacts optimally with its substrate, arabinogalactan, at approximately pH 8.0 and at 40 °C. CEDAase shows stability in the pH range of 6.0–9.0 and at the temperature below 50 °C. The K_m measured for the CEDAase was 55.6 μM, with an apparent V_max of 0.083 μmol/min. To our knowledge, for the first time, the current work obtains an extracellular Cellulomonas endo-d-arabinase enzyme that might be potentially served as a tool enzyme for hydrolyzing specific cell wall such as Mycobacterium cell. It is purified as an important potential initial material basis for mass spectrometric sequencing and chemical gene synthesis. It may make it possible to clone and express this valuable endo-d-arabinase and make it available to the mycobacteria scientific community.     

Purification and Properties of Lysozyme Produced by Staphylococcus aureus

A method based on cold ethyl alcohol fractionation at different pH levels and ionic strengths and on gel filtration on a Sephadex G-200 column was used to concentrate and purify lysozyme from the culture supernatant fluid of Staphylococcus aureus strain 524. The final, nondialyzable product exhibited a 163-fold rise in specific activity over that of the starting material. Staphylococcal lysozyme is a glycosidase which splits N-acetylamino sugars from the susceptible substrate. Staphylococcal lysozyme was shown to be similar to egg white lysozyme in its optimal temperature for reaction, optimal pH, activation by NaCl and Ca(++) ions, inhibition by sodium citrate and ethylenediaminetetraacetate, and inactivation by Cu(++) ions and sodium dodecyl sulfate. It differs from the egg white lysozyme in its temperature susceptibility range (staphylococcal lysozyme is inactivated at 56 C). It acts on whole cells and cell walls of Micrococcus lysodeikticus, murein from S. aureus 524, and cell walls of S. epidermidis Zak. The last substrate was not susceptible to the action of egg white lysozyme in the test system used. The mechanism of action of staphylococcal lysozyme seems to be analogous to that of egg white lysozyme; however, the biological specificity of the two enzymes may be different.     

啤酒酵母生产的重组水蛭素的纯化及脱色

对啤酒酵母生产的重组水蛭素变异体1(rHV1)进行多步骤的纯化。首先将分泌到培养上清液中的水蛭素进行硫酸铵沉淀和SephadexG-50凝胶过滤,再用Q-SepharoseHP阴离子交换层析分离,最后用HPLCSP-5PW阳离子交换柱脱色及HPLCC8柱反相层析。真空干燥后得到的水蛭素蛋白经SPS-PAGE、N端氨基酸序列分析、抗凝血酶活力分析鉴定,证明已获得高纯度的重组水蛭素HV1制剂,为利用基因工程方法生产重组水蛭素的规模化生产及临床应用提供了依据     

Purification and Characterization of Lipid Bioflocculant Produced by Rhodococcus erythropolis

The bioflocculant produced by Rhodococcus erythropolis S-1 was found to exist as huge assemblies, the molecular mass of which is over one million daltons, composed of many polypeptides and lipids in aqueous solution. We have isolated and purified this lipid bioflocculant by ultracentrifugation, extracting with 90% acetone, and two successive silica gel chromatographies from the culture broth. It was homogeneous on silica gel thin-layer chromatography. ¹H-NMR and HPLC studies showed that it was a kind of glycolipid that contained a C₁₆ methylene chain on the average and glucose in its chemical structure. The flocculating activity against kaolin clay suspension was dependent on the Ca²⁺ concentration.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and Characterization of Anti-Listeria Compounds Produced by Geotrichum candidum

Geotrichum candidum can produce and excrete compounds that inhibit Listeria monocytogenes. These were purified by ultrafiltration, centrifugal partition chromatography, thin-layer chromatography, gel filtration, and high-pressure liquid chromatography, and analyzed by liquid chromatography-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation. Two inhibitors were identified: d-3-phenyllactic acid and d-3-indollactic acid.

Contamination by Listeria has become a problem over the past 20 years in many parts of the world. The ubiquitous nature of Listeria monocytogenes, its capacity to multiply at refrigeration temperatures, its thermal tolerance (11), and its resistance to relatively low pH (it can multiply at pH 5.3 and 4°C and at pH 4.39 and 30°C) (5), together with its tolerance of high salt concentrations (4, 18), make controlling this potentially pathogenic microorganism in food products difficult. This bacterium has been incriminated in several cases of food poisoning (2, 10, 19). At risk are the immunodepressed, the old, pregnant women, fetuses, and newborn babies. Several groups have worked on biological control. As a result, many bacteriocins, which inhibit the growth of L. monocytogenes, have been isolated, purified, and characterized (12, 13, 16, 18). We have worked with Geotrichum candidum, a yeast-like member of the natural milk flora that is used as a maturing agent for soft and hard cheeses. In an extensive study carried out in 1984 (7), the interactions between G. candidum and the microflora in cheeses were examined. G. candidum inhibited the growth of gram-negative bacteria, gram-positive bacteria, and fungi (6). We recently showed (3) that G. candidum inhibits the growth of L. monocytogenes on both solid and liquid media (a bacteriostatic effect). The inhibitors are stable over a wide pH range and can be heated to 120°C for 20 min. The present report describes the purification and characterization of compounds responsible for this antibacterial action.Microorganisms, culture conditions, and detection of inhibitory activity.The strain of G. candidum used came from the collection of the Caen University Food Microbiology Laboratory, Caen, France (UCMA G91) and was initially isolated from a cheese, Pont l’Evêque. One percent of a preculture (optical density at 620 nm [Milton Roy Spectronic 301; Bioblock Scientific, Illkirch, France] of 0.7 [10⁷ arthrospores or hyphae/ml]) of G. candidum was grown in a fermentor (20 liters; Biolafitte type PI) in 15 liters of Trypticase soy broth (30 g/liter; Biomerieux, Marcy l’Etoile, France) with yeast extract (6 g/liter; AES, Combourg, France) (TSBYE) buffered to pH 6.3 with 0.1 M citrate-0.2 M phosphate. The culture was stirred at 300 rpm for 64 h at 25°C under a pressure of 0.2 bar and was then filtered through a 1,000-Da cut-off membrane by tangential ultrafiltration (Sartorius, Palaiseau, France) under a pressure of 2 bars. The resulting ultrafiltrate was sterilized by passage through a capsule (Sartorius) containing 0.45-μm- and 0.2-μm-pore-size membranes.The inhibition of L. monocytogenes was checked at each purification step by the agar diffusion well assay (3). Antimicrobial activity was estimated by measuring the diameter of the inhibitory halo on two right-angle axes (average of two plates). The strain of L. monocytogenes (UCMA L205) (serovar 1/2a; Centre National de Référence des Listeria, Nantes, France) and lysovar 1652 (Institut Pasteur, Paris, France) came from the laboratory collection and was isolated from milk. The initial lyophilized ultrafiltrate (900 mg/ml) gave a halo diameter of 36 ± 0.7 mm in the inhibition assay.Purification.Samples of ultrafiltrate (20 μl) were spotted on thin-layer chromatography (TLC) plates (silica gel, 10 by 5 cm, 0.25 mm thick, 60 F₂₅₄; Merck, Darmstadt, Germany), with 20 μl of TSBYE for controls, and eluted by vertical chromatography with a butanol-acetic acid-water (40:10:20 [vol/vol/vol]) solvent system. The bands were examined under UV light (254 nm) or after treatment with Ehrlich’s reagent. Four well-separated bands were found (R_fs, 0.11 ± 0.04; 0.41 ± 0.04; 0.7 ± 0.03; and 0.86 ± 0.03), but only the band with an R_f of 0.7 ± 0.03 differed from that of control preparations (TSBYE not containing G. candidum). The microbiological bioautography test (1) confirmed the presence of the inhibitor in the band with an R_f of 0.7. Lyophilized ultrafiltrate was subjected to centrifugal partition chromatography (Sanki 1000 Engineering Ltd.; EverSeiko, Tokyo, Japan) in butanol-acetic acid-water (40:10:50 [vol/vol/vol]). Partitioning was carried out under the following conditions: ascending mode, 1,200 rpm; flow rate, 3 ml/min; pressure, 40 bars. An aliquot of material (3 g) previously equilibrated with the solvent system was injected into the separatus via a 12-ml injection loop. Fractions (10 ml each) were collected and evaporated to dryness in a SpeedVac (Jouan RC 1022, Saint Herblain, France). The dried extracts of certain fractions were taken up in 800 μl of water and brought to pH 5.6 with 0.2 M NaOH. A total of 10 mg of pooled fractions with an R_f close to 0.7 and showing L. monocytogenes inhibitory activity (36 ± 0.7 mm for a solution of 38 mg/ml) was taken up in 250 μl of methanol-water (50:50 [vol/vol]) and automatically deposited (Camag Linomat) on a 10- by 20-cm TLC plate (silica gel, 0.25 mm thick, 60 F₂₅₄; Merck). The plate was developed with butanol-acetic acid-water (40:10:20 [vol/vol/vol]) and examined under UV light. Bands with an R_f of 0.7 were scraped off and placed in methanol. The silica was washed several times and removed by centrifugation and filtration through a 0.2-μm-pore-size filter. An aliquot (20 μl) was spotted on a small silica TLC plate to confirm elution of the solute by the methanol solvent. The purity of the band with an R_f of 0.7 was confirmed by high-pressure liquid chromatography (HPLC) coupled with a photodiode array detector at 206 and 222 nm (solvent A: 0.1% formic acid in water; solvent B: CH₃CN-H₂O [95:5] plus 0.1% formic acid) on a C₁₈ Grom-Sil ODS2 column (4.6 by 30 mm; particle size, 1.5 μm; Grom Analytic, Herrenberg, Germany) at the flow rate of 1 ml/min. Inhibitory activity was assessed as above. Preparative TLC indicated that the band with an R_f of 0.7 contained two components, one eluting at 4.5 min on HPLC (peak 1) and the other at 5.5 min (peak 2). The latter fraction gave an inhibitory halo of 36 ± 0.7 mm at a concentration of 20 mg/ml (a 45-fold purification over the ultrafiltrate). The material from preparative TLC (40 mg in 200 μl of methanol-water) was placed on a column of Sephadex LH20 (1 m by 1 cm; Pharmacia), and the column was eluted with methanol-water at 12 ml h⁻¹. Fractions (1 ml each) were collected and examined by HPLC to determine the material in each fraction. This final purification on Sephadex LH20 gave two peaks, with two-thirds of the eluate at peak 1 and one-third at peak 2 (Fig. ​(Fig.1).1). As the concentration for peak 2 was very low, only the inhibitory activity for peak 1 was assayed. A concentration of 20 mg/ml gave a halo diameter of 26 ± 0.7 mm. Open in a separate windowFIG. 1Reverse-phase liquid chromatography (HPLC) of inhibitory compounds of G. candidum after purification by centrifugal partition chromatography, preparative TLC, and Sephadex LH20 gel filtration. Column, C₁₈ Grom-Sil ODS2 column (4.6 by 30 mm; particle size, 1.5 μm; Grom Analytic). Eluent: solvent A (0.1% formic acid in water), solvent B (CH₃CN-H₂O [95:5] plus 0.1% formic acid). Flow rate, 1 ml/min. (a) Product 1 (detection at 206 nm); (b) product 2 (detection at 222 nm).

Characterization.

The pooled fractions were run on HPLC with a Grom-Sil ODS2 column coupled to a mass detector (Sciex Api III, triple quadrupole; Thornhill, Canada). Product 1, analyzed by desorption and chemical ionization, gave a signal at an m/z of 184 for (M⁺ NH₄)⁺ on desorption and chemical ionization and thus had a mass of 166. Product 2 was analyzed by ion spray and gave a signal at an m/z of 297 (M + 4 Na)⁺ for a mass of 205. Spectra were determined in a Nicolet model 60 SXR FT-IR. Samples were dissolved in dimethyl sulfoxide, and the ¹H and ¹³C resonances were measured in a Brucker spectrometer at 200 and 400 Hz, respectively. The purified material was taken up in methanol, and the isomeric form of the substance(s) inhibiting L. monocytogenes was determined in a Perkin-Elmer model 341 polarimeter. Two inhibitors were identified (Fig. ​(Fig.2);2); product 1 was 2-hydroxy-3-phenylpropanoic acid (phenyllactic acid, mass 166), and product 2 was 2-hydroxy-3-indolpropanoic acid (indollactic acid, mass 205). The rotation of polarized light showed that the phenyllactic acid produced by G. candidum was the d form. The spectrum properties of the isolated compounds are identical to those of authentic commercial compounds (Sigma Chemical Co., St. Louis, Mo.). d-Phenyllactic acid can be purchased from Aldrich (product no. 37 690-6), and dl-indollactic acid is available from Sigma (catalog no. I2875). Inhibitory activity with commercial compounds showed that dl-phenyllactic acid (Sigma catalog no. P7251) was a stronger inhibitor of Listeria than dl-indollactic acid (34 and 26 ± 0.7 mm for 187 mM, respectively) and that the d form of phenyllactic acid was more active (38 mm for 120 mM) than the l form (Aldrich 11, 306-9, 30 mm for 120 mM). The samples were taken up in methanol-water (50:50 [vol/vol]) and brought to pH 5.6 (Table ​(Table1).1). Open in a separate windowFIG. 2Structure of two inhibitory compounds of G. candidum characterized by LC-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation.TABLE 1Anti-Listeria activity of phenyllactic acid and indollactic acid^a

Purification and Characterization of a Xylanase Produced by Chaetomium thermophile NIBGE

Summary Xylanase was produced by growing Chaetomium thermophile NIBGE in a submerged liquid culture using wheat straw and urea as carbon and nitrogen sources respectively. The xylanase was purified to electrophoretic homogeneity after ammonium sulphate precipitation, anion exchange chromatography by FPLC and gel filtration. The molecular mass of this xylanase BII was 50 kDa. The pH and temperature optima were 6.5 and 70 °C respectively. The xylanase BII showed reasonable stability at high pH and 65 °C temperature. Some metal ions and EDTA caused little inhibition at low concentrations but complete inhibition was observed at concentrations higher than 2 mM. The K_m and V_max values with oat spelt xylan as the substrate were found to be 12.5 mg/ml and 83.3 IU/mg protein, respectively. Liberation of reducing sugars from commercial paper pulp samples suggest the feasibility of a biopulping process using this xylanase.     

Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced by Vibrio tubiashii

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.     

Purification and Characterization of a Low-Molecular-Weight Xylanase Produced by Acrophialophora nainiana

A low-molecular-weight xylanase activity (XynI) was isolated from the fungus Acrophialophora nainiana after growth in a solid medium containing wheat bran. XynI was purified to apparent homogeneity by ultrafiltration and gel filtration chromatography. The purified enzyme had a molecular weight value of approx. 17 kDa, as determined by SDS-PAGE. This enzyme was most active at 50°C and pH 6.0. At 50°C the half-life was 150 min. The apparent K _m value for birchwood xylan was much lower than the K _m value for oat spelt xylan. XynI was activated by L-cysteine, DTE, β-mercaptoethanol, and L-tryptophan. XynI did not show significant sequence homology with other xylanases. The analysis of hydrolysis products of xylans and wood pulps showed that XynI was able to release xylooligomers ranging from X2 to X3 and X2 to X6, respectively. The enzyme was not active against acetylated xylan. A small amount of xylose was released from deacetylated, birchwood, and oat spelt xylans. The results obtained with enzymatic treatment of Kraft pulp indicated a reduction in the amount of chlorine compounds required for the process and enhanced brightness gain. Received: 6 May 1998 / Accepted: 29 July 1998     

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45°C. Activities of both enzymes were inhibited by Hg²⁺, Zn²⁺, Cu²⁺, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn²⁺ and Co²⁺. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.     

Purification and Properties of a Phytotoxic Polysaccharide Produced by Corynebacterium sepedonicum

A polysaccharide possessing a capacity to wilt plant cuttings has been isolated and purified from cultures of Corynebacterium sepedonicum. The molecular weight, based on the average of molecular weights determined by 3 physical methods, is 21,450. The empirical formula of the polysaccharide is C₄₈H₉₆O₄₈N. It is antigenic and the borate complex migrates in an electric field. It has an intrinsic viscosity of 0.125 and an S_20w of 0.76. A hydrolysate of the compound yields glucose, mannose, 2 unidentified reducing compounds and 1 unidentified non-reducing compound. The nitrogen in the toxin can be accounted for in 6 amino acids. Although the toxin is primarily polysaccharide it might more aptly be termed a glycopeptide.     

Partial Purification and Properties of the Pyruvate-Uridine Diphospho-N-Acetylglucosamine Transferase from Staphylococcus epidermidis

A method for the partial purification of the pyruvate-uridine-diphospho-N-acetylglucosamine transferase from Staphylococcus epidermidis is presented. Some properties of the enzyme are discussed.     

Purification and Characterisation of Rat Kidney Glutathione Reductase

Glutathione reductase [GR, E.C.1.8.1.7] catalyses NADPH dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH). Thus, it is the crucial enzyme to maintain high [GSH]/[GSSG] ratio and physiological redox status in cells. Kidney and liver tissues were considered as a rich source of GR. In this study, rat kidney GR was purified and some of its properties were investigated. The enzyme was purified 2,356 fold with a yield of 16% by using heat-denaturation and Sephadex G25 gel filtration, 2′,5′-ADP Agarose 4B, PBE94 column chromatographies. The purified enzyme had a specific activity (Vm) of 250 U/mg protein and the ratio of absorbances at wavelengths of A ₂₇₃/A _463, A ₂₈₀/A ₄₆₀, A ₃₆₅/A ₄₆₀, and A ₃₇₉/A ₄₆₃, were 7.1, 6.8, 1.2 and 1.0, respectively. Each mol of GR subunit bound 0.97 mol of FAD. NADH was used as a coenzyme by rat kidney GR but with a lower efficiency (32.7%) than NADPH. Its subunit molecular weight was estimated as 53 kDa. An optimum pH of 6.5 and optimum temperature of 65 °C were found for rat kidney GR. Its activation energy (Ea) and temperature coefficient (Q₁₀) were calculated as 7.02 kcal/mol and 1.42, respectively. The Km_(NADPH) and kcat/Km _(NADPH) values were found to be 15.3 ± 1.4 μM and 1.68 × 10⁷ M⁻¹ s⁻¹ for the concentration range of 10-200 μM NADPH and when GSSG is the variable substrate, the Km_(GSSG) and the kcat/Km_(GSSG) values of 53.1 ± 3.4 μM and 4.85 × 10⁶ M⁻¹ s⁻¹ were calculated for the concentration range of 20–1,200 μM GSSG.     

Further Purification of Amylase Inhibitor Produced by Streptomyces sp.

The amylase inhibitor produced by Streptomyces sp. consisted of, at least, three kinds of inhibiting materials (inhibitor A, B and C). These inhibitors were separated by the Chromatographic techniques on Amberlite CG–120 and Dowex 1 × 2, and by paper chromatography etc.

Inhibitor A, B and C seemed to contain both carbohydrates and amino acids, but the specific activities (I. U./mg glucose) of the inhibitors A, B and C were different, that is, 18,800, 45,700 and 59,000, respectively. Also their ratios of amino acids to neutral sugars (expressed by moles of leucine per those of glucose) were approximately 0.09 (inhibitor A), 2.77 (inhibitor B) and 0.58 (inhibitor C).     

Purification and Properties of a Polygalacturonic Acid Trans-Eliminase Produced by Bacillus pumilus

A strain of Bacillus pumilus produced an extracellular pectic enzyme with polygalacturonic acid as the substrate. This enzyme, with optimal activity at pH 8.0 to 8.5, produced reaction products that strongly absorbed light at 232 nm, indicating the presence of a pectic acid trans-eliminase (PATE). Neither pectin esterase nor polygalacturonase was detected in the cell-free culture fluid. Chromatographic examination of the end products revealed the presence of large quantities of unsaturated oligouronides unlike those found with B. polymyxa. It was found that the PATE was produced extracellularly during the negative logarithmic death phase of the organism. The filtrate from sonically treated cells did not show any activity for PATE or hydrolases for lower oligogalacturonides at any time during the growth cycle. The enzyme was inducible. Pectin, National Formulary (NF) was the best inducer, followed by polygalacturonic acid and galacturonic acid. Enzyme activity was markedly stimulated by calcium and other divalent ions. Copper and cobalt ions were inhibitory. The partially purified enzyme showed no significant activity on pectin containing a high methoxyl content (96% esterified). However, pectin NF with a lower methoxyl content (68% esterified) was attacked to a degree by the partially purified and crude enzyme preparations. The initial rate of PATE activity increased up to 60 C, about 16-fold higher than that observed at room temperature. The activation energy was calculated as 12,183 cal/mole. A protective action of calcium chloride against heat inactivation of the PATE was observed. Degradation of polygalacturonic acid by this enzyme produced several unsaturated oligouronides soon after its addition to the substrate. The major endproduct was thought to be different from that of other known PATE enzymes. Paper chromatographic studies and viscosity measurements disclosed the random cleaving nature of the enzyme an endo-PATE.