Selenium-containing tRNAs from Clostridium sticklandii. Cochromatography of seleno-tRNA I with l-valyl-tRNA

It has been previously shown that Clostridium sticklandii specifically synthesized three readily separable ⁷⁵Se-labeled tRNAs, designated seleno-tRNAs I, II and III, and the partially purified seleno-tRNA II cochromatographed with l-prolyl-tRNA on DEAE-Sephadex A-50 (Chen, C.S. and Stadtman, T.C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1403–1407). In the present study a highly purified ⁷⁵Se-labeled tRNA I was obtained by chromatography on benzoylated DEAE-cellulose, DEAE-Sephadex A-50 and Sepharose 4B. The ⁷⁵Se-labeled tRNA I cochromatographed with an l-valine-accepting species on DEAE-Sephadex A-50 and Sepharose 4B. Addition of a 285-fold molar excess of unlabeled l-valine to the l-valine acceptor activity assay mixture markedly decreased the amount of l-[¹⁴C]valine bound to seleno-tRNA I.     

Ubiquity of selenium-containing tRNA in plants

Sodium[⁷⁵Se]selenite supplemented culture of Chlamydomonas, wild carrot, tobacco, bamboo, and rice cells as well as mung bean and soybean seedlings incorporated, without exception, ⁷⁵Se into tRNAs. The content of ⁷⁵Se-labeled tRNAs ranged from 0.04 to 1.89% of the total tRNAs in these seven plant species. [⁷⁵Se]tRNA samples of wild carrot and mung bean were fractionated into six or seven seleno-tRNA species by chromatography on RPC-5 column. Samples of tobacco, bamboo and Chlamydomonas each exhibited only a single seleno-tRNA species with a close interspecific resemblance in the elution position among the three samples. All these [⁷⁵Se]tRNAs contained a new, not yet identified ⁷⁵Se-labeled nucleoside, whose retention time on HPLC was distinctly different from that of the previously reported bacterial selenonucleosides. [⁷⁵Se]tRNA samples of rice, tobacco, bamboo, mung bean and Chlamydomonas also contained one or two minor ⁷⁵Se-labeled nucleosides. These results suggest that (1) selenium-containing tRNAs appear to be widespread in the plant kingdom and (2) a new, not yet characterized selenonucleoside might be universal in plants.     

Neutral Proteinases I and II of Aspergillus sojae

Epimerization of (?)-isodihydrocarvone (I) to (?)-dihydrocarvone (II) by Pseudomonas fragi IFO 3458 was studied. I was easily epimerized to II by the growing cells, the resting cells or the cell-free extracts.

An epimerase catalyzing the conversion of I to II was partially purified from the bacterial extracts about 56-fold with heat-treatment, ammonium sulfate precipitation and DEAE-Sephadex A-50 column chromatography. By the action of this epimerase, the ratio of I to II becomes about 25: 75 (K=3). It appeared that the epimerase is very stable to heat; the activity of epimerization remains 66 and 36% after treatment at 97°C for 60 and 120 min, respectively.     

Gewinnung und Charakterisierung von Prolidase aus Escherichia coli B,II. Charakterisierung,Reinigung und Trägerfixierung des Enzyms

Prolidase, a specific exopeptidase, is isolated from Escherichia coli B. The enzyme being present in the raw extract is purified and enriched by fractionated ammonium sulfate precipitation, ion exchange chromatography on DEAE-Sephadex A 50 as well as by gel filtration on Sepharose 4 B. Total yield of prolidase amounts to 19% with a 67fold enrichment Substrate specifity of the enzyme mainly corresponds to that of the animal prolidase. It is able to hydrolize the imido linkage at the N-terminal end of prolin in the case of di- and tripeptides. The temperature optimum of prolidase from E. coli B is 37 °C, the pH-optimum from pH 7.6 to 9.0. Storage stability at pH 8.6 and a temperature of 4 °C is optimal. The enzyme is only active in presence of Mn²⁺-ions. This metal cannot be replaced by Mg^2-- or Zn²⁺-ions A high enzyme activity and storage stability in presence of Mn²⁺-ions can be reached by immobilization of the prolidase, by covalent binding on Sepharose 6 B, adsorption on DEAE-Sephadex as well as by combination with glutar dialdehyde on DEAE-Sephadex.     

Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver

The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [⁷⁵Se]selenocysteine and [⁷⁵Se]selenite as substrates. [⁷⁵Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [⁷⁵Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [³⁵S]cysteme, the most highly purified ⁷⁵Se-fractions were > 100-fold purified relative to ³⁵S. These fractions contained < 0.7% of the [³⁵S]cysteine originally present in the total tRNA. When [³⁵Se]selenocysteyl tRNA was purified from a mixture of ¹⁴C-labeled amino acids, over 97% of the [¹⁴C]aminoacyl tRNA was removed. The [⁷⁵Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [⁷⁵Se]selenocysteine, reaction with hydroxylamine and recovery of [⁷⁵Se]selenocysteyl hydroxamic acid and release of ⁷⁵Se by ribonuclease. The specificity of [⁷⁵Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.     

Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21,000 and 36,000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10-12 (protease I) and pH 10.5 (protease II). The optimum temperture for the activity of protease I was 70 degrees C and that for protease II was 60 degrees C. Protease I was stable in the range of pH 4.0-8.0 up to 60 degrees C and protease II was stable in the range of pH 6.0-12.0 up to 50 degrees C.     

Properties of Purine Nucleoside Phosphorylase from Enterobacter cloacae

Purine nucleoside phosphorylase from Enterobacter cloacae KY3074 was partially purified by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and DEAE-Sephadex A-50, and gel filtration on Sephadex G-100 and Sepharose 4B. The molecular weight of the enzyme was calculated to be about 87,000 by a gel filtration method on Sephadex G-200. The enzyme was found to be most active at pH 7.5 to 8.5 and 50°C, stable between pH 7.0 and 7.3, and the activity was nearly lost above 70°C. The enzyme split 2´-deoxyinosine and ribonucleosides. Lineweaver-Burk plots for phosphate were non-linear, showing substrate activation. The break-down of inosine approached an equilibrium when approximately 14% of inosine was phosphorylated.     

Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

T sujibo , H., M iyamoto , K., H asegawa , T. & I namori , Y. 1990. Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete. Journal of Applied Bacteriology 69 , 520–529.
Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21000 and 36000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10–12 (protease I) and pH 10.5 (protease II). The optimum temperature for the activity of protease I was 70°C and that for protease II was 60°C. Protease I was stable in the range of pH 4.0–8.0 up to 60°C and protease II was stable in the range of pH 6.0–12.0 up to 50°C.     

Chemical composition and immunological properties of glycoproteins of Sporothrix species

Glycoproteins of 11Sporothrix species were purified from their respective culture filtrates by use of DEAE-Sephadex A-50 and QAE-Sephadex A-25 column chromatography and investigated for their chemical and immunological properties. On the basis of sugar composition, the glycoproteins of the 11Sporothrix species could be divided into two groups, i.e., rhamnose containing (i.e., Rha⁺), and non rhamnose containing (i.e., Rha^–) groups. The species in the former group wereS. curviconia, S. inflata, S. schenckii andS. schenckii var. luriei, and those in the latter group wereS. cyanescens, S. foliorum, S. fungorum, S. ghanensis, S. imectorum, S. luteoalba andS. ramosissima. The glycoproteins of four of the (Rha⁺) species were relatively similar in elution patterns of DEAE-Sephadex A-50 chromatograms, sugar and amino acid compositions, serological reactivity with rabbit andS. schenckii serum and rabbit antiKlebsiella pneumoniae K47 serum, and cutaneous delayed hypersensitivity. In the case of the (Rha^–) species, the glycoproteins of five species cross-reacted with rabbit antiS. schenckii serum and all, but theS. cyanescens, glycoprotein were reactive to some degree in skin tests in sporotrichotic patients. These results strongly suggest that the chemical and immunological properties of these glycoproteins correspond with the morphological observations amongSporothrix species.     

Studies on Barley and Malt Amylases

From the 2 m urea extract of ground barley two zymogen β-amylase (Z-β-A) fractions (Fractions A and B) and one active β-amylase (A-β-A) fraction (Fraction C) were isolated by Sephadex G-75 gel filtration and purified by DEAE-Sephadex A-50 column chromatography. The molecular weights of Fractions A, B and C were estimated to be approximately 280,000, 160,000 and 56,000, respectively.

Both the Z-β-A fractions which were ultracentrifugally and electrophoretically homogeneous were found to be accompanied by a small amount of saccharogenic activities. From the estimation of Km values for these saccharogenic activities and the behavior in their activation with 2-mercaptoethanol and papain, it seems reasonable to conclude that Fraction A is a heteropolymer type of Z-β-A composed of both A-β-A and barley reserve proteins; that Fraction B is a homopolymer type of Z-β-A composed of A-β-A alone; and that two different activation mechanisms, proteolytic activation and disulfide bond cleaving activation, are necessary for full activation of Z-β-A in barley.     

An Arylamidase from Flavobacterium meningosepticum

An arylamidase was purified from Flavobacterium meningosepticum by a series of chromatographies on CM-cellulose, DEAE-Sephadex A-50 and Sephadex G-150. The purified enzyme appeared homogeneous on SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be more than 500,000 dalton by using a column of Sepharose 4B and to be 62,000 when checked by SDS-gel electrophoresis. The enzyme was most active at pH 7.5 toward Leu-β-naphthylamide (Leu-β-NA). It catalyzed the hydrolysis of not only various amino acid-β-naphthylamides but also some peptides, but the hydrolysis rate of the latter substrates was quite low. Cys-di-β-naphthylamide was split by this enzyme at an optimal pH of 6.2. Incubation of oxytocin with the enzyme resulted in a decrease in the biological activity, indicating that this arylamidase possesses an oxytocinase (cystyl aminopeptidase)-like activity.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

Purification and properties of two ribonucleases and a nuclease from barley seeds

Three enzymes possessing RNAase activity were isolated from barley seeds. These enzymes were further purified by ammonium sulphate precipitation DEAE-cellulose chromatography, gel filtration on Sephadex G-75 and DEAE-Sephadex A-50 chromatography. These enzymes have been characterized and classified as: 1. Plant RNAase I (EC 3.1.27.1). It has a pH optimum at 5.7 and molecular weight of 19 000. 2. Plant RNAase II (EC 3.1.27.1). It has a pH optimum at 6.35 and molecular weight of 19 000. 3. Plant nuclease I (EC 3.1.30.2). It has a pH optimum at 6.8 and molecular weight of 37 000. Two RNAases were purified to homogeneity by means of affinity chromatography on poly(G)-Sepharose 4B, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Aggregated Form of Dextransucrases from Leuconostoc mesenteroides NRRL B-512F and Its Constitutive Mutant

Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively, DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS–PAGE were all 170kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V₀) on Sepharose 6B, while the DSM-G was eluted at 1.2 × V₀ volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.     

Ester Formation by Alcohol Acetyltransferase from Brewers’ Yeast

Alcohol acetyltransferase responsible for the formation of acetate esters during beer fermentation was found to be localized at the cell membrane of brewers’ yeast. This cell membrane-bound enzyme was purified 120-fold by solubilization with Triton X-100, gel filtration on a Sepharose 6B column and chromatography on a DEAE-Sephadex A-50 column. The enzyme was most active at 30°C at pH 7 ? 8. It was least active against C₃ alcohol among C₁ ? C₆ alcohols, and slightly more active against straight-chain alcohols than against branched-chain alcohols with the same carbon number. The enzyme was strongly inhibited by unsaturated fatty acids, heavy metal ions and sulfhydryl reagents.     

Comparative study of reactor performance for the resolution of d,l-amino acids

The racemic resolution of l-valine and l-serine by fungal aminoacylase has been evaluated by comparing the performance of various reactor configurations including an anion exchange nylon tangential flow membrane reactor, a tubular reactor with aminoacylase adsorbed onto DEAE-Sephadex as support and a continuous stirred tank reactor with enzyme recycling using a flat ultrafiltration module (CSTR/UF). Among the substrates tested, the N-chloroacetyl-d,l-amino acids were the preferred substrates, showing the highest catalytic efficiency (V_m/K_m).Optimum reactor operational conditions obtained in discontinuous assays were selected to study the behaviour of the reactors in a continuous mode. DEAE-Sephadex loaded six-fold more enzyme than anion exchange nylon (60 and 10 gE/litre, respectively, related to reactor volume), whereas enzyme concentration within the CSTR/UF reactor was limited only by enzyme solubility.The tangential flow membrane reactor configuration with a 10 g/litre enzyme concentration produced higher productivity values (0·35 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 161 days) than the CSTR/UF reactor (0·24 kg l-valine/litre per day, and 80% conversion degree) performing with the same enzyme concentration. The tubular reactor with the enzyme adsorbed onto DEAE-Sephadex (60 g/litre enzyme load) showed higher productivity values (1·9 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 70 days) than the CSTR/UF reactor (1·05 kg l-valine/litre per day, and 80% conversion degree). However, the CSTR/UF reactor was the preferred configuration, as it had the highest enzyme load and productivity (1·95 kg l-valine/litre per day of reactor volume, and 80% conversion degree), a half-life of 55 days at 50°C, and the possibility of easy continuous enzyme addition.     

Purification and Properties of Dihydrostreptomyein-Phosphorylating Enzyme from Pseudomonas aeruginosa

The distribution of the dihydrostreptomycin (DHSM)-phosphorylating enzyme was investigated using DHSM-resistant strains of Pseudomonas aeruginosa, indicating that this enzyme was demonstrated from all of 7 DHSM-resistant strains examined but not from a DHSM-sensitive one. The DHSM-phosphorylating enzyme was isolated from P. aeruginosa TI-13 and purified about 205-fold using Sephadex G-75 and DEAE-Sephadex A-50 column chromatography. The optimal pH for the DHSM-inactivation was around 10.0, and both adenosinetriphosphate (ATP) and Mg⁺⁺ were required for the inactivating reaction. It was found that this enzyme inactivated only DHSM but not other aminoglycosidic antibiotics such as kanamycin, aminodeoxykanamycin, neomycin, paromomycin, lividomycin and gentamicin.     

The purification and spectral properties of polyphenol oxidase I fromNicotiana tabacum

Polyphenol oxidase plays a key role in plant defense systems. We report the first-time purification of polyphenol oxidase (PPO 1.14.18.1) from fresh leaves of tobacco (Nicotiana tabacum) using acetone powder, ammonium sulfate precipitation, and column chromatography with DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-75. PPO I was purified approximately 71-fold (3200 U/mg). The MALDI-TOF-MS spectrum showed that the enzyme was purified to a pure protein with a molecular weight of 35700 Da. The optimum pH of PPO I was 7, the optimum temperature was 40°C, and the Km value was 6.8 mM using catechol as the substrate at pH 6.5 and with 0.05 M H₃PO₄−NaOH buffer. The maximum emission peak of PPO I was 339 nm with 16 nm of blue-shifted compared with 355 nm of free tryptophan. The UV/VIS spectra and the absence of an EPR signal are indicative of type-3 coppers, but not type-1 or type-2 coppers. PPO I and mushroom PPO have the same active center for a pair of coupled antiferromagnetic copper ions.     

Studies on continuous enzyme reactions. IV. Preparation of a DEAE-sephadex–aminoacylase column and continuous optical resolution of acyl-DL-amino acids

Conditions for the preparation of an aminoacylase column using DEAE-Sephadex as a carrier were investigated. The aminoacylase column having the highest activity was obtained when 7500 μmoles/hr. of partially purified aminoacylase was charged into a column packed with 9 ml. of DEAE-Sephadex A-25 (bead type, hydroxy form). By employing a DEAE-Sephadex–aminoacylase column, conditions for continuous optical resolution of acyl-DL -amino acids were investigated. When a solution of 0.2M acetyl-DL -methionine (pH 7.0, containing 5 × 10^?4M Co²⁺) or 0.2M acetyl-DL -phenylalanine (pH 6.0, containing 5 × 10^?4M Co²⁺) was passed through the aminoacylase column at the flow rate of SV = 2.5 or 2.0, respectively, at 50°C., the highest rate of hydrolysis of both substrates was attained. From the column effluents, enzymatically hydrolyzed L -methionine and L -phenylalanine were isolated in a good yield.     

Purification and some properties of endopolygalacturonase from Rhizopus sp. LKN

An endopolygalacturonase of Rhizopus sp. strain LKN, one of several isolates from tempe starter (ragi), was purified 235-fold by CM-Sephadex C-50, DEAE-Sephadex A-50 ion exchange chromatographies and Sephadex G-75 gel filtration. The purified enzyme was homogeneous by SDS-PAGE with a M _r of 38.5 kDa. Its K _m value for pectic acid was 2 mg/ml. It was stable at pH 4.5 to 11 and up to 50°C, with optimum activity at pH 4.5 to 4.75 and 55 to 60°C. Some ionic compounds enhanced the enzyme activity, whereas tannic acid at 0.5 mm caused about 90% inhibition.The authors are with the Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan.