Characterization and molecular cloning of thermostable alpha-amylase from Streptomyces sp.To1

Summary A new thermophilic Streptomyces sp. TO1, isolated from Tunisian soil, produced a thermostable alpha-amylase and pullulanase. The gene encoding for the alpha-amylase activity was cloned into the multicopy cloning plasmid pLM1 using S. lividans ZX1 as host strain. The ZX1 / pLM1 strain has the same activity than the initial TO1 strain and about 25 fold higher activity than the ZX1 strain. This alpha-amylase has an optimum of pH and temperature at 6 and 70 °C respectively.     

Ansaetherone,a New Radical Scavenger from Streptomyces sp.

We identified a new radical scavenger, ansaetherone (C₂₆H₃₃NO₇), from a culture of the Streptomyces sp. USF-4727 strain. In our previous study, it was shown that this strain produced four lipoxygenase inhibitors, tetrapetalones A, B, C and D. The chemical structure of ansaetherone was elucidated by the spectroscopic method, indicating that this compound was constructed with an aglycon and a sugar moiety. This chemical structure suggested that ansaetherone was related to the tetrapetalones. This finding provided information regarding tetrapetalone biosynthesis. Ansaetherone showed radical scavenging activity with an ED₅₀ value of 300 μM in our assay.     

Identification of extracellular N-acylhomoserine lactone acylase from a Streptomyces sp. and its application to quorum quenching

N-acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.     

Cloning of a thermostable alpha-amylase gene from Thermomonospora curvata and its expression in Streptomyces lividans

The gene from Thermomonospora curvata CCM 3312 coding for thermostable alpha-amylase (tam) has been cloned in Streptomyces lividans TK 24 and localized to a 2.6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tam gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.     

USF-19A,a New Lipoxygenase Inhibitor from Streptomyces sp.

USF-19A, a soybean Jipoxygenase (SBL) and human 5-lipoxygenase (5-LO) inhibitor, was isolated from Streptomyces sp. USF-19 strain. Its chemical structure was determined by spectroscopic evidence to be a new member of the antimycin antibiotic family. The IC₅₀ value of USF-19A against 5-LO was 28.0 μM.     

Streptomyces sp. LK3 mediated synthesis of silver nanoparticles and its biomedical application

In the present study, the marine actinobacteria mediated biosynthesis of silver nanoparticles (AgNps) was achieved using Streptomyces sp LK3. The synthesized AgNps showed the characteristic absorption spectra in UV–vis at 420 nm, which confirmed the presence of nanoparticles. XRD analysis showed intense peaks at 2θ values of 27.51°, 31.87°, 45.57°, 56.56°, 66.26°, and 75.25° corresponding to (210), (113), (124), (240), (226), and (300) Bragg’s reflection based on the fcc structure of AgNps. The FTIR spectra exhibited prominent peaks at 3,417 cm^?1 (OH stretching due to alcoholic group) and 1,578 cm^?1 (C=C ring stretching). TEM micrograph showed that the synthesized AgNps were spherical in shape with an average size of 5 nm. Surface morphology and topographical structure of the synthesized AgNps were dignified by AFM. The synthesized AgNps showed significant acaricidal activity against Rhipicephalus microplus and Haemaphysalis bispinosa with LC₅₀ values of 16.10 and 16.45 mg/L, respectively. Our results clearly indicate that AgNps could provide a safer alternative to conventional acaricidal agents in the form of a topical antiparasitic formulation. The present study aimed to develop a novel, cost-effective, eco-friendly actinobacteria mediated synthesis of AgNps and its antiparasitic activity.     

Characterization of a xylanolytic complex from Streptomyces sp.

Streptomyces sp. DSM 41796 produced four major extracellular xylanases with Mr of 145, 120, 60 and 45 kDa. Those of 145 and 60 kDa formed a heterodimer. All xylanases, except that of 120 kDa, were induced by xylose, d-arabinose or sucrose, while commercial xylans induced the 60 kDa xylanase in a major proportion than others, and sugar-cane bagasse pith or lemon peel induced predominantly the 45 kDa xylanase.     

Streptomyces multispiralis nov. sp. and a New Antibiotic,Neohumidin

A streptomycete isolated in our laboratories was found to produce a new antibiotic effective for the control of sheath blight of the rice plant. As a result of taxonomical study it was proved to belong to a new species and therefore it was named Streptomyces multispiralis nov. sp. From its fermented broth, the antibiotic was isolated and crystallized from benzene. After the comparison of its physicochemical and biological properties with those of known antibiotics, it was considered to be a new antibiotic and named neohumidin. It inhibited the growth of certain phytopathogenic fungi, saprophytic fungi, yeasts and gram-positive bacteria.     

Activity and action pattern of Bacillus licheniformis alpha-amylase in aqueous ethanol

A purified B. licheniformis alpha-amylase in a mixture of ethanol-aqueous buffer (1:1, v/v) retains half the activity shown in water alone. In ethanol-aqueous buffer (7:3, v/v) about 20% of the activity is retained. The pattern of oligosaccharides produced from amylose changed with ethanol concentration; in aqueous buffer the products are: DP 1 and 2, 33.7%; DP 3, 28.5%; DP 4, 4.4% and DP 5, 33.4%. Whereas in ethanol-aqueous buffer (7:3, v/v) the products are DP 1 and 2, 66.8%; DP 3, 17.3%; DP 4, 4.1% and DP 5, 11.8%. These results suggest that a change in substrate affinity at the active centre subsites is induced in the ethanol-aqueous buffer medium.     

Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp

A thermostable and cellulase-free xylanase has been produced from Streptomyces sp. QG-11-3 in solid substrate fermentation using wheat bran and eucalyptus kraft pulp as the prime solid substrates. The maximum xylanase yield obtained using these two substrates were 2360 U/g and 1200 U/g dry solid substrate at substrate:moisture ratios of 1:3 and 1:2.5, respectively. In immobilized cell system using polyurethane foam (PUF) and three nonwoven fabrics, namely, polyester, silk, and cotton, the xylanase yields were enhanced by 2.5-fold (203 U/ml), 1.91-fold (155 U/ml), 1.54-fold (125 U/ml), and 1.47-fold (119 U/ml), respectively, compared to the xylanase yield in liquid-batch fermentation (81 U/ml). In the biobleaching experiments, the xylanase dose of 3.5 U/g moisture free pulp exhibited the optimum bleach boosting of eucalyptus kraft pulp at pH 8.5 and 50 degrees C after 2 h of treatment. When xylanase treated pulp was subsequently treated with 4.5% chlorine, it resulted in reduction of kappa number by 25%, enhanced the brightness (%ISO) by 20% and improved the pulp properties such as tensile strength and burst factor by up to 63% and 8%, respectively.     

Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25

Directed evolution coupled with a high-throughput robotic screen was employed to broaden the industrial use of the maltogenic alpha-amylase Novamyl from Bacillus sp. TS-25. Wild-type Novamyl is currently used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. However, the enzyme is rapidly inactivated during the baking process of bread made with low pH recipes and Novamyl thus has very limited beneficial effect for this particular application. In an effort to improve the performance of Novamyl for low pH bread applications such as sourdough and rye, two error-prone PCR libraries were generated, expressed in Bacillus subtilis and screened for variants with improved thermal stability and activity under low pH conditions. Variants exhibiting improved performance were iteratively recombined using DNA shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10 degrees C increase in thermal stability at pH 4.5, one of which demonstrated substantial anti-staling properties in low pH breads.     

Purification and primary structure of proteinous alpha-amylase inhibitor from Streptomyces chartreusis.

A new polypeptide inhibitor, AI-409, that inhibits human salivary alpha-amylase, was purified from a fermentation broth of Streptomyces chartreusis strain No. 409. This protein consists of a single-chain polypeptide of 78 amino acid residues, and includes two disulfide bridges. The primary structure of AI-409 and the locations of the disulfide bridges were identified by enzymatic digestion and the automatic Edman technique. Enzymatic digestion was done with trypsin, carboxypeptidase Y, and chymotrypsin. One of the disulfide bridges was between Cys(10) and Cys(26), and the other between Cys(44) and Cys(71).     

Biosynthesis of a alpha-amylase and protease by Streptomyces olivaceus 142. III. Some aspects of alpha-amylase induction.

The induction of alpha-amylase in Streptomyces olivaceus 142 depends on the phase of growth of culture and the nature of the carbon sources upon which the cells were grown prior to exposure to inducer. The most susceptible to induction are cells from the initial hours of growth and glycerol -- grown cells. Reduction in the susceptibility of cells to alpha-amylase induction is probably a result of catabolic repression.     

SstI: a restriction endonuclease from Streptomyces sp. stanford.

A strain of Streptomyces has been isolated which is a convenient source of a new restriction endonuclease. The enzyme has been prepared from extracts of these cells and its cleavage sites localized on phage lambda DNA. The enzyme, termed SstI, produces cohesive ends and should be useful for molecular cloning experiments.     

Streptomyces daliensis sp. nov. from soil

A novel actinomycete strain YIM 31724^T was isolated from a soil sample collected from Dali, Yunnan Province, People’s Republic of China. The strain is characterized by white to yellow white aerial mycelia, spiral spore chains and smooth spore surface. The cell wall of strain YIM 31724^T contained LL-diaminopimelic acid (A₂pm) and traces of meso-A₂pm. Whole-cell hydrolysates contained mainly glucose and small amounts of galactose and xylose. The menaquinones were MK-9(H₆) (31%) and MK-9(H₈) (69%). Phosphatidylethanolamine was the diagnostic phospholipid. The DNA G+C content of strain YIM 31724^T was 67.2 mol%. Phylogenetic analysis indicated that the strain belongs to the genus Streptomyces, with highest similarity to Streptomyces rimosus subsp. rimosus JCM 4667^T (rRNA gene sequence similarity value of 98.9%) and Streptomyces erumpens DSM 40941^T (rRNA gene sequence similarity value of 98.7%). Based on its phenotypic and genotypic characteristics, including low DNA–DNA hybridization results, strain is proposed as the type strain of a novel species, Streptomyces daliensis sp. nov.     

Aureolic acids from a marine-derived Streptomyces sp. WBF16

A marine-derived actinomycete (Streptomyces sp. WBF16) exhibiting antitumor activities was investigated. The strain was identified using morphological, biochemical and genetic techniques. 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces coelicolor A3 (2). Furthermore, a new aureolic acid (Chromomycin B, 1), along with Chromomycin A₂ (2) and Chromomycin A₃ (3) were isolated from its secondary metabolites. Their structures were determined by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. Compounds 1–3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1 and HUVEC.     

Evaluation of the possible proteomic application of trypsin from Streptomyces griseus

Trypsin (EC 3.4.21.4) is the protease of choice for proteome analysis using mass spectrometry of peptides in sample digests. In this work, trypsin from Streptomyces griseus (SGT) was purified to homogeneity from pronase. The enzyme was evaluated in in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analyses of the digests. We recognized a remarkable cleavage performance of SGT. The number of produced and matching tryptic peptides was higher than in the case of commonly used bovine trypsin (BT) and allowed us to obtain higher identification scores in database searches. Interestingly, SGT was found to also generate nonspecific peptides whose sequencing by MALDI-TOF/TOF tandem mass spectrometry (MS/MS) revealed a partial F-X, Y-X, and W-X cleavage specificity. To suppress autolysis, either arginine or arginine plus lysine residues in SGT were modified by chemical reagents. In consequence, the autolytic pattern of SGT was reduced significantly, but specific activity dropped dramatically. As demonstrated by relative quantification of peptides at different times, SGT is more stable at 37 °C than is its bovine counterpart. We conclude that SGT represents a convenient alternative for proteomic applications involving protein digestion. Moreover, parallel digestions of sample aliquots by SGT and BT provide the possibility of combining partially different results (unique matching peptides) to improve protein identification.     

Biosynthesis of alpha-amylase and protease by Streptomyces olivaceus 142. I. Regulation of alpha-amylase activity.

Streptomyces olivaceus 142 produces amylase in the logarithmic phase of growth of the culture. The synthesis of the enzyme is induced by maltose and starch. In the case of maltose the synthesis is induced by a contaminating compound, probably being a higher than maltose polymer of glucose. The synthesis of amylase is negatively controlled by catabolic repression. The level of the activity of the enzyme depends not only on the biosynthesis but also on it proteolytic degradation.     

Molecular cloning and expression in Streptomyces lividans of a proteinous alpha-amylase inhibitor (HaimII) gene from Streptomyces griseosporeus

The gene encoding a proteinous alpha-amylase inhibitor (HaimII) of Streptomyces griseosporeus YM-25 has been cloned in Escherichia coli K12 using a deoxyinosine-containing synthetic oligonucleotide as the probe. A 1.6 kilobases BamHI fragment was confirmed to hybridize with the probe and subcloned in an E. coli-S. lividans shuttle vector. The plasmid clone was transferred into S. lividans by transformation. An appreciable amount of alpha-amylase inhibitor activity was found in the culture medium of S. lividans harboring the plasmid. As the specificity was indistinguishable from that of HaimII produced by the original S. griseosporeus strain, we concluded that the HaimII protein was synthesized in S. lividans and excreted into the medium.