Extracellular α-amylase from Streptomyces rimosus

Summary A purification procedure for an extracellular -amylase from Streptomyces rimosus, oxytetracycline-producing strain, is described. The enzyme obtained was shown to be an acidic (pI 4.75) monomer with a relative molecular mass (M_r) of 43 000, containing three cysteines involved in the catalytic activity of the enzyme. Its amino-terminal part has 57–67% homology with amylases from other Streptomyces species. S. rimosus -amylase is sensitive to higher temperatures, and partially stabilized by Ca²⁺ ions. It hydrolyses starch (optimum at pH 5.0–6.0) in an endohydrolase manner giving rise to maltotriose, maltotetraose and higher oligosaccharides. Starch granules, except those from rice, were not significantly affected by the isolated -amylase.     

Cloning and expression of the α-amylase gene and oxytetracycline production in Streptomyces rimosus

An 8.4 kb Sau3AI DNA fragment containing the Streptomyces rimosus TM-55 -amylase gene (amy) was ligated to a vector pIJ702, named pCYL01, and cloned into amylase deficient mutant S. lividans M2 (amy ^–). Subcloning study showed that the amy gene was localized in 3.3 kbKpnI-PstI fragment. The molecular weight of the purified -amylases of S. lividans M2/pCYL01 and S. rimosus TM-55 were estimated to be 65.7 kDa. Different sizes of recombinant plasmids carrying the amy gene had been retransferred into the parental strain of S. rimosus TM-55. Among these S. rimosus transformants, TM-55/pCYL01, TM-55/pCYL12 and TM-55/pCYL36 showed amylase activity 1.36- to 2.05-fold at the seventh day (1.61 to 2.42 units vs 1.18 units), and oxytetracycline (OTC) production 2.00- to 2.50-fold at the ninth day (approximate 140 to 170 g ml^–1 vs 72 g ml^–1), higher than that of S. rimosus TM-55 alone, respectively. These results showed that industrial microorganisms could be improved by genetic and metabolic engineering.     

Zur Wirkung von α-Strahlen auf Plasma und Zellorganellen vonAllium cepa-Epidermen

Ohne ZusammenfassungAusgeführt im Rahmen von Untersuchungen, die von der Atomkommission des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung unterstützt werden. Der Verfasser dankt für die Gewährung der wertvollen finanziellen Hilfe.     

Streptomyces erythraeus trypsin inactivates α1-antitrypsin

Streptomyces erythraeus trypsin (SET) is a serine protease that is secreted extracellularly by S. erythraeus. We investigated the inhibitory effect of α(1)-antitrypsin on the catalytic activity of SET. Intriguingly, we found that SET is not inhibited by α(1)-antitrypsin. Our investigations into the molecular mechanism underlying this observation revealed that SET hydrolyzes the Met-Ser bond in the reaction center loop of α(1)-antitrypsin. However, SET somehow avoids entrapment by α(1)-antitrypsin. We also confirmed that α(1)-antitrypsin loses its inhibitory activity after incubation with SET. Thus, our study demonstrates that SET is not only resistant to α(1)-antitrypsin but also inactivates α(1)-antitrypsin.     

An α-l-Arabinofuranosidase from Streptomyces purpurascens IFO 3389

By screening 46 strains of Actinomycetes for their ability to hydrolyze arabinan, 16 strains were found to have α-l-arabinofuranosidase activity, and Streptomyces purpurascens IFO 3389 was selected as the most promising of the sixteen. An α-l-arabinofuranosidase [EC 3.2.1.55] has been highly purified from the culture fluid of this organism grown on beet arabinan as the carbon source. The molecular weight of the native enzyme was determined to be 495, 000 by gel filtration and that of the subunit to be 62,000 by SDS polyacrylamide gel electrophoresis. The pI value was 3.9. The purified enzyme was active on p-nitrophenyl α-l-arabinofuranoside and arabino-oligomers, and inactive on arabinan, arabinoxylan and arabinogalactan. The optimum pH was 6.5. The enzyme was inhibited by Hg²⁺, Ag⁺ and l-arabino-γ-lactone. The values of Km and V_max for p-nitrophenyl α-l-arabinofuranoside were determined to be 8.2 × 10^?5 m and 89.3 μmol per min per mg of protein, respectively.     

Zur Kenntnis des Fettsäureabbaus durch Schimmelpilze

Ohne Zusammenfassung     

Zur Kenntnis des Fettsäureabbaus durch Schimmelpilze

Ohne Zusammenfassung     

Zur Spezifität des Calciumnachweises durch Tetracyclin

Zusammenfassung Tetracycline sind zum histochemischen Nachweis von Calcium ungeeignet. Weder die Vitalfärbung noch die Färbung von Gewebsschnitten liefern brauchbare Ergebnisse. Es besteht keine Relation zwischen Calciumgehalt und Intensität der Tetracyclinfärbung. Bei der Vitalfärbung reagieren Tetracycline wahrscheinlich bereits mit freiem Calcium des Blutes unter Ausbildung eines Tetracyclin-Calcium-Komplexes. Erst im Calciumkomplex ist Tetracyclin zur Reaktion mit Gewebsstukturen befähigt. Die Bedeutung der Vitalfärbung liegt in der Sichtbarmachung potentieller Bindungsorte für Calcium im Gewebe. — Die völlig unspezifischen Ergebnisse der Schnittfärbung können durch die mit der histologischen Gewebspräparation verbundenen Eiweißdenaturierung erklärt werden.

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On the specificity of tetracycline staining for calcium

Summary Tetracyclines are not suitable for the histochemical demonstration of calcium sites in tissue. Both, staining of tissue sections and vital staining, give insufficient results. Calcium content and intensity of tetracycline staining are not correlated. In all probability in vital staining a tetracycline-calcium complex is formed with free calcium of the blood, before any reaction with tissue structures can take place. Potential bindings sites for calcium are demonstrated. The staining of tissue sections on the other hand is fully nonspecific; this is explained by the protein denaturation that occurs during the histological preparation of the tissue.

     

Stereo- and Regioselective Hydroxylation of α-Ionone by Streptomyces Strains

A total of 215 Streptomyces strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15 Streptomyces strains, S. fradiae Tü 27, S. arenae Tü 495, S. griseus ATCC 13273, S. violaceoniger Tü 38, and S. antibioticus Tü 4 and Tü 46 converted α-ionone to 3-hydroxy-α-ionone with significantly higher hydroxylation activity compared to that of β-ionone. Hydroxylation of racemic α-ionone [(6R)-(−)/(6S)-(+)] resulted in the exclusive formation of only the two enantiomers (3R,6R)- and (3S,6S)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the Streptomyces strains tested proceeds with both high regio- and stereoselectivity.Ionones and their derivatives are important intermediates in the metabolism of terpenoids, e.g., in carotenoid biosynthesis, and have been isolated from many sources (1a, 11). Compounds with a trimethylcyclohexane building block constitute essential aroma elements in many plant oils and thus have attracted the attention of the flavor and fragrance industry (3). Further, ionone derivatives, e.g., 3-hydroxy-β-ionone, could prove valuable intermediates for the chemoenzymatic synthesis of carotenoids, e.g., for astaxanthin and zeaxanthin (5).Microbial transformation of α- and/or β-ionone to a number of hydroxy and oxo derivatives has been reported for several fungal strains (2, 4, 8, 9, 18), mainly of the genus Aspergillus, but not for bacterial strains. 3-Hydroxy-α-ionone was observed, among other metabolites, when Cunninghamella blakesleeana ATCC 8688 (2) or Aspergillus niger JTS 191 (18) was used.Many species of the order Actinomycetes are known to catalyze a broad spectrum of xenobiotic transformations. Several cytochrome P-450-dependent monooxygenases from Streptomyces strains, which catalyze the hydroxylation of a wide range of substrates, have been investigated on the molecular level (12) and thus provide an interesting potential as biocatalysts for specific hydroxylation reactions by recombinant techniques.As a first step in this direction, we now report the screening of 215 Streptomyces strains for their capacity to hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives in a regio- and stereoselective manner. The structure and stereochemistry of the main biotransformation product were characterized unequivocally by nuclear magnetic resonance (NMR) spectroscopy.     

Zur Frage der Virusübertragung durch Samen,insbesondere desX-,Y- und Blattrollvirus der Kartoffel

Ohne Zusammenfassung     

Streptomyces rimosus M4018 中氧化应激转录抑制因子Rex的克隆表达及与rex 操纵子的体外结合活性

【目的】为了研究龟裂链霉菌Streptomyces rimosus M4018中的rex基因对自身rex operator(ROP)的调控机制。【方法】根据天蓝色链霉菌Streptomyces coelicolor A3(2)中rex基因的同源序列设计引物进行PCR,从S.rimosus M4018中获得其rex基因(Sr-rex)。同时,通过染色体步移的方法,获得其上游的ROP序列。采用体外凝胶迁移的方法,分析了Sr-Rex对ROP的调控作用。【结果】获取的Sr-rex基因核苷酸序列长度为846 bp,预测的编码氨基酸序列与S.coelicolor A3(2)中Rex的同源性为84%,获得GenBank登录号:GQ849479。圆二色光谱显示Sr-Rex的结构以α螺旋和β折叠为主,与软件预测相符。凝胶迁移实验表明,Sr-Rex能与S.rimosus M4108中扩增到的ROP片段特异性结合。同时,以Rex:ROP的最小结合序列为基础,设计了一条22 bp的单链DNA片段,和Sr-Rex的最大结合摩尔浓度比约为5:1。高浓度的NADH抑制两者的结合活性,而NAD+对结合没有影响。【结论】在S.rimosus M4108中,Rex是通过响应胞内NAD(H)水平的方式来调控ROP的表达的。     

Beeren und Farbenwahl durch V?gel

Summary Three chance observations on colour preference in birds feeding on berries are presented.Blackbirds (Turdus merula) in Bavaria fed in a cherry-tree irrespective of the colour of the cherries. They began feeding when the first cherries ripened and turned from greenish-yellow to light red. As the cherries were offered in different colours at the same time, and pell-mell in the same tree, the birds presumably quickly learned by trial and error to eat cherries of different colours.A little later Blackbirds, which must have been partly if not exclusively the same individuals, started to feed on red currants. Red and white currants were offered to them at the same time, and side by side but on different bushes. In this case there was a very clear spontaneous selection of red in preference to white. White berries were only eaten after the red bushes had been cleared up by the birds.Sardinian Warblers (Sylvia melanocephala) in Sardinia, feeding on berries ofRhamnus alaterna, which are coral red and hard when unripe and turn to blackish and soft getting ripe, prefered the black ones. It is presumed that they conditioned themselves to black and soft by trial and error learning. Generalisations as to birds preferring certain colours in berries they feed on can not be drawn from any special case.     

Inhibitory effect of components from Streptomyces species on α-glucosidase and α-amilase of different origin

The search for the effective and safe α-glucosidase and α-amylase inhibitors from Actinomycetaceae being antidiabetic agents is actual problem. Twenty one Streptomyces spp. of soil samples collected from different places of China were screened for the ability to produce this kind of inhibitory activities. Fermentation broth of isolated strains had absorbance between 350–190 nm. The Streptomyces strains PW003, ZG636, and ZG731 were characterized by special absorption at 280, 275, and 400 nm, respectively. Ten of the collected actinomycete strains had the ability to inhibit α-glucosidase or/and α-amylase and the fermentation broth of the same strain had inhibitory activity varied greatly depending on the enzyme source. In the process to screen the leading compounds used as antidiabetic agents, human α-glucosidase and α-amylase were revealed as the best used in trail compared with the same enzymes from other sources. Active α-glucosidase inhibitor was isolated from Streptomyces strain PW638 fermentation broth and identified as acarviostatin I03 by MS and NMR spectrometry. Its IC₅₀ value was 1.25 and 12.23 μg/ml against human intestinal N-terminal maltase-glucoamylase and human pancreatic α-amylase, respectively.