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Streptomyces lunalinharesii strain 235 produces an antimicrobial substance that is active against sulfate reducing bacteria, the major bacterial group responsible for biofilm formation and biocorrosion in petroleum reservoirs. The use of this antimicrobial substance for sulfate reducing bacteria control is therefore a promising alternative to chemical biocides. In this study the antimicrobial substance did not interfere with the biofilm stability, but the sulfate reducing bacteria biofilm formation was six-fold smaller in carbon steel coupons treated with the antimicrobial substance when compared to the untreated control. A reduction in the most probable number counts of planktonic cells of sulfate reducing bacteria was observed after treatments with the sub-minimal inhibitory concentration, minimal inhibitory concentration, and supra-minimal inhibitory concentration of the antimicrobial substance. Additionally, when the treated coupons were analyzed by scanning electron microscopy, the biofilm formation was found to be substantially reduced when the supra-minimal inhibitory concentration of the antimicrobial substance was used. The coupons used for the biofilm formation had a small weight loss after antimicrobial substance treatment, but corrosion damage was not observed by scanning electron microscopy. The absence of the dsrA gene fragment in the scraped cell suspension after treatment with the supra-minimal inhibitory concentration of the antimicrobial substance suggests that Desulfovibrio alaskensis was not able to adhere to the coupons. This is the first report on an antimicrobial substance produced by Streptomyces active against sulfate reducing bacteria biofilm formation. The application of antimicrobial substance as a potential biocide for sulfate reducing bacteria growth control could be of great interest to the petroleum industry., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Four actinomycete strains previously isolated from Brazilian soils were tested for their antimicrobial activity against Bacillus pumilus LF-4 and Desulfovibrio alaskensis NCIMB 13491, bacteria that are well known to be involved in biofilm formation and biocorrosion. Strain 235, belonging to the species Streptomyces lunalinharesii, inhibited the growth of both bacteria. The antimicrobial activity was seen over a wide range of pH, and after treatment with several chemicals and heat but not with proteinase K and trypsin. The antimicrobial substances present in the concentrated supernatant from growth media were partially characterized by SDS-PAGE and extracellular polypeptides were seen. Bands in the size range of 12 to 14.4 kDa caused antimicrobial activity. Transmission electron microscopy of D. alaskensis cells treated with the concentrated supernatant containing the antimicrobial substances revealed the formation of prominent bubbles, the spherical double-layered structures on the cell membrane, and the periplasmic space completely filled with electron-dense material. This is the first report on the production of antimicrobial substances by actinomycetes against bacteria involved in biocorrosion processes, and these findings may be of great relevance as an alternative source of biocides to those currently employed in the petroleum industry.

Streptomyces misionensis strain PESB-25 was screened and selected for its ability to secrete cellulases. Cells were grown in a liquid medium containing sugarcane bagasse (SCB) as carbon source and corn steep liquor (CSL) as nitrogen source, whose concentrations were optimized using response surface methodology (RSM). A peak of endoglucanase accumulation (1.01 U · mL(-1)) was observed in a medium with SCB 1.0% (w/v) and CSL 1.2% (w/v) within three days of cultivation. S. misionensis PESB-25 endoglucanase activity was thermoacidophilic with optimum pH and temperature range of 3.0 to 3.6 and 62° to 70 °C, respectively. In these conditions, values of 1.54 U mL(-1) of endoglucanase activity were observed. Moreover, Mn(2+) was demonstrated to have a hyperactivating effect on the enzyme. In the presence of MnSO4 (8 mM), the enzyme activity increased threefold, up to 4.34 U · mL(-1). Mn(2+) also improved endoglucanase stability as the catalyst retained almost full activity upon incubation at 50 °C for 4 h, while in the absence of Mn(2+), enzyme activity decreased by 50% in this same period. Three protein bands with endoglucanase activity and apparent molecular masses of 12, 48.5 and 119.5 kDa were detected by zymogram.

An actinomycete strain, isolated from a soil sample under a sugar cane plantation in Brazil and identified as Streptomyces viridobrunneus SCPE-09, was selected as a promising cellulolytic strain, and tested for its ability to produce cellulases from agro-industrial residues. Sugar cane bagasse or wheat bran was tested as carbon source, and corn steep liquor tested as nitrogen source. Different concentrations of carbon and nitrogen were tested using factorial design to identify optimal cellulose production. The results showed that media containing wheat bran 2.0% (w/v) and corn steep liquid 0.19% (w/v) lead to the highest production, 2.0 U mL(-1) of CMCase, obtained on the fifth day of fermentation. The pH and temperature profile showed optimal activity at pH 4.9 and 50°C. As for thermostability, endoglucanases were most tolerant at 50°C, retaining more than 80% of maximal activity even after 2 h of incubation. Zymogram analyses using supernatant from growth under optimized conditions revealed the presence of two CMCase bands with apparent molecular masses of 37 and 119 kDa. The combination of pH tolerance and CMCase production from agro-industrial residues by S. viridobrunneus SCPE-09 offers promise for future bioethanol biotechnologies.

Aims: To evaluate cellulase production by Streptomyces malaysiensis in submerged fermentation using brewer's spent grain (BSG) and wheat bran (WB) as carbon source, and corn steep liquor (CSL) as nitrogen source, as compared to yeast extract (YE), and partial characterization of the crude enzyme., Methods and Results: Maximum cellulase production by Streptomyces malaysiensis (720 U l(-1)) occurred within 4 days incubation when using a growth medium containing BSG 0.5% (w/v) and CSL1.2% (w/v). CMCases activity showed to be stable over an acidic pH range (2.0-7.0) and in temperatures of 40-60 degrees C. Zymogram indicated three bands of CMCase activity, with different molecular masses., Conclusion: S. malaysiensis was able to grow and produce good levels of CMCases using solely brewer's spent grain and corn steep liquor as low-cost substrates, making this strain and these low cost by-product worthy for further investigation, and potentially feasible for biotechnological applications in different areas., Significance and Impact of the Study: To our knowledge, this is the first study reporting the use of the low-cost by-products brewer's spent grain and corn steep liquor, as sole substrates for microbial enzyme production.

A novel chitinolytic actinomycete isolated from a Brazilian cerrado soil, designated strain RCQ1071(T), was assigned to the genus Streptomyces on the basis of chemical and morphological characteristics. The almost-complete nucleotide sequence of the 16S rRNA gene of strain RCQ1071(T) was determined and also placed this strain in the genus Streptomyces. Phylogenetic analyses of 16S rRNA gene sequences showed that strain RCQ1071(T) formed a long branch in a group related to Streptomyces albulus, sharing approximately 98 % sequence similarity. Levels of DNA-DNA relatedness between strain RCQ1071(T) and members of this group, namely S. albulus DSM 40492(T), Streptomyces noursei DSM 40635(T) and Streptomyces yunnanensis DSM 41793(T), were 38.3, 27.8 and 46 %, respectively, strongly indicating that strain RCQ1071(T) was not a member of any of these species. The relatively long branch length within a stable clade together with the phenotypic data strongly supported that strain RCQ1071(T) represented a novel species. Based on the combination of physiological, phylogenetic and genomic data, strain RCQ1071(T) is suggested to represent a novel species of the genus Streptomyces, for which the name Streptomyces lunalinharesii sp. nov. is proposed. The type strain is RCQ1071(T) (=ATCC BAA-1231(T) =CIP 108852(T) =DSM 41876(T)).

Chitin from Streptomyces lunalinharesii spores, detected on its outermost surface layer, was isolated and characterized by chemical and spectroscopic methods, transmission electron microscopy and flow cytometry analysis. Gold-chitinase- and gold-lectin (Lycopersicum esculentum agglutinin, LEA)-conjugated labels were used in microscopy experiments, whereas a fluorescence-lectin (LEA) conjugate was used in flow cytometry analysis. Chitin isolation consisted of several steps of hot alkali and nitrous acid treatment, and the final material was obtained in the colloidal form. The infrared and the 13C CP/MAS NMR spectra of Streptomyces sp. colloidal chitin and colloidal chitin obtained from commercial crab shell chitin were very similar. Incubation of the spores with gold-labeled lectin, or gold-labeled recombinant chitinase, showed the presence of gold particles around the spore surface, indicating the specific binding of the lectin or the recombinant chitinase with the chitin present on the outermost surface. Flow cytometry analysis, using the fluorescence-lectin conjugate, confirmed these results. According to scanning electron microscopy, S. lunalinharesii presented spore surface ornamentation belonging to the spiny group. This is the first detailed characterization of chitin on the spore's outermost layer from a Streptomyces species.

Protease production by Streptomyces sp. 594 was obtained after submerged fermentation (SF) and solid-state fermentation (SSF) using feather meal (FM) and corn steep liquor (CSL) as sole sources of carbon and nitrogen. Enzyme productions were 13.4 U ml(-1) in SF and 21.5 U g(-1) in SSF; these values were approximately 86% and 39% higher, respectively, than those obtained previously when yeast extract was used in place of CSL. The proteases, which belong to the serine and metalloproteinase classes, were active at high temperatures (55 degrees C to 90 degrees C) and over a wide range of pH values (5.0 to 10.0). Thus, these thermophilic proteases have shown interesting properties for industrial purposes. As far as we are concerned, this is the first contribution toward the microbial production of thermophilic proteases by a streptomycete using a low-cost medium composed of industrial poultry (FM) and corn processing by-products (CSL).

Aims: Protease production by Streptomyces sp. 594 in submerged (SF) and solid-state fermentation (SSF) using feather meal, an industrial poultry residue, and partial characterization of the crude enzyme., Methods and Results: Streptomyces sp. 594 produced proteases in SF (7.2 +/- 0.2 U ml(-1)) and SSF (15.5 +/- 0.41 U g(-1)), with pH increase in both media. Considering protease activity, values obtained in the liquid extract after SSF (6.3 +/- 0.17 U ml(-1)) were lower than those from SF. The proteases, which belong to serine and metalloproteinase classes, were active over a wide range of pH (5.0-10.0) and high temperatures (55-80 degrees C). Strain 594 was also able to degrade feather in agar and liquid media. Keratinase activity (80 U l(-1)) also confirmed the keratin degrading capacity of this streptomycete., Conclusions: Proteases produced using residues from poultry industry have shown interesting properties for industrial purposes., Significance and Impact of the Study: As far as we are concerned, this is the first contribution towards the production of thermophilic protease by a streptomycete in SSF using a keratinous waste.

Streptomyces malaysiensis AMT-3, isolated from a Brazilian cerrado soil, showed proteolytic activities detected by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum proteinase production was obtained when using 2.5% wheat bran and 0.1% yeast extract in the culture medium, after 5 days incubation at 30 degrees C. The enzymatic complex degraded gelatin optimally at pH 7.0, and under these conditions eight proteolytic bands (four serine-proteinases and four metaloproteinases), ranging from 20 to 212 kDa, were detected on the culture supernatant filtrates. In addition, a 35-kDa proteinase was thermostable at 60 degrees C for 120 min. These results point out to the applicability of gelatin zymograms in the characterization of crude enzymatic complexes. According to our results, this enzymatic complex could be used for biotechnological applications.

Streptomyces are important microorganisms because of their capacity to produce numerous bioactive molecules. In the present work protease production, by Streptomyces sp. 594 isolated from a Brazilian Cerrado soil, was maximized by optimizing a low-cost culture medium composition (casitone and sugarcane molasses) using statistical experimental design. The final protease activity (56 U/mL) was 2.8-fold and 58-fold higher than that obtained in the beginning of this study, and in a previous work, using an actinomycete selection medium, respectively. Protease production, not growth associated, appeared to be modulated by an inducer system, whereby the C/N ratio seemed to play a significant role.

Aims: To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells., Methods and Results: Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested., Conclusions: These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis., Significance and Impact of the Study: Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.

Aims: The chitinolytic activity of an actinomycete, isolated from a tropical acidic ferrasol (FAO) under cerrado (savanna) vegetation, is reported., Methods and Results: Selection of the strain was based on spot inoculation on solid colloidal chitin medium. The use of chemotaxonomic, morphological and physiological procedures placed it in the Streptomyces genus, but identification to species level could not be achieved. A protein with endochitinase activity was isolated and purified from the supernatant fluid by concentration, precipitation, hydrophobic interaction, gel filtration and adsorption procedures. The molecular size of the purified chitinase was estimated by gel filtration to be 70 kDa, and its pI was 6.1. The enzyme had temperature and pH optima of 40 degrees C and 8.0, respectively, and showed thermal (30-70 degrees C) and pH (4-9) stabilities. Antifungal activity of the selected strain was observed following in vitro experiments using growing cells, crude extract or the purified endochitinase, and by detecting growth inhibition of the tested phytopathogenic fungi., Conclusion: Strain Streptomyces RC 1071 could not be placed into any known species, suggesting a new taxon. The purified endochitinase presented similar molecular weight, optimum temperature and pH activity, and stability of other endochitinolytic enzymes reported in the literature. In all three in vitro experiments performed, inhibition of growth of the phytopathogenic fungi used as test organisms was observed., Significance and Impact of the Study: Some of the endochitinase characteristics such as thermal stability, as well as pH tolerance, are very interesting for biotechnological purposes. In addition, due to its antifungal activity, Streptomyces RC 1071 seems promising for use in biological control.

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their supernatants after growth in a microcrystalline cellulose medium, using carboxymethylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates. Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

The crude enzyme extracts from five actinomycetes selected from a cerrado soil presented very good endochitinolytic activity when compared to a commercial chitinase. Exochitinase and chitobiase activities were also detected. They were identified as Streptomyces, but could not be characterized to species level, probably corresponding to new ones. The crude extracts, obtained from growth on fungal mycelium plus chitin of three of the strains, have shown a very pronounced activity against phytopathogenic fungi. In tests using growing cells, all five strains were active. These data suggest that these strains are potential biocontrol agents.

Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI) medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4% and 10.4% yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate, was 9-10 and the optimum temperature was 37 degrees C. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth.

Streptomyces cyaneus, a micro-organism isolated from Brazilian cerrado soil, produces an extracellular proteinase (SCP), which was purified 22-fold to homogeneity from culture supernatant fluid, using a single aprotinin-agarose affinity chromatography step. It is produced at a level corresponding to approximately 15% of total protein, but its physiological function has yet to be determined. The molecular mass of this S. cyaneus proteinase was estimated to be 120 kDa by gel filtration high performance liquid chromatography, and it migrates by SDS-PAGE as a single band of 30 kDa. It was optimally active at 25 degrees C and pH 9.0, and was fully inhibited by the serine-proteinase inhibitors PMSF and TPCK. A Km value of 1. 86 x 10-5 mmol l-1, and Vmax of 2.0 x 10-2 mmol l-1 (Abs247 nm microg-1 min-1), were calculated for alpha-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate.