A Thermotolerant Xylan-Degrading Enzyme Is Produced by Streptomyces malaysiensis AMT-3 Using by-Products From the Food Industry

Abstract This study evaluated the production of endoxylanases by Streptomyces malaysiensis AMT-3 in submerged fermentation using by-products of the food industry at 28ºC. In shake-flasks experiments, the highest endoxylanase activity of 45.8 U.mL-1 was observed within 6 days in a medium containing (w/v) 2.5% wheat bran and 1.2% corn steep liquor. The same culture conditions were used to evaluate the enzyme production in a 2 L stirred tank reactor under different agitation (300, 450 and 600 rev.min-1) and aeration (30 and 60 L.h-1) conditions. The use of 450 rev.min-1 coupled to an aeration of 90 L.h-1 resulted on 81.3 U.mL-1 endoxylanase activity within 5 days. The effect of temperature and pH on endoxylanase activity and stability showed the highest activity at 60 ºC and pH 6.0. Zymography showed the presence of three xylanolytic bands with molecular masses of 690, 180 and 142 kDa. The results showed that the thermotolerant actinobacterial endoxylanase can be produced in high titers using by-product of the food industry.

Streptomyces odonnellii sp. nov., a proteolytic streptomycete isolated from soil under cerrado (savanna) vegetation cover.

A novel streptomycete, strain 594T, isolated from Brazilian soil collected under cerrado (savanna) vegetation cover is described. Strain 594T produced thermophilic chitinolytic proteases in assays containing feather meal and corn steep liquor as sole sources of carbon and nitrogen. The strain produced white to grey aerial mycelium and spiral chains of spiny-surfaced spores on the aerial mycelium and did not produce diffusible pigments. The ll-isomer of diaminopimelic acid was present in the cell wall and menaquinones were predominantly MK-9(H6) (52 %) and MK-9(H8) (30 %) with 6 % MK-9(H4) and slightly less than 1 % MK-9(H2). Polar lipids present were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unknown phospholipid. The major fatty acids were anteiso-C15 : 0, anteiso-C16 : 0, anteiso-C14 : 0 and anteiso-C17 : 0. The G+C content of the genomic DNA was 70.4 mol%. Phylogenetic analysis of the nearly complete 16S rRNA gene sequence indicated that it differed from described Streptomyces species. Multilocus sequence analysis (MLSA) using five housekeeping genes (atpD, gyrB, rpoB, recA and trpB) comparing Streptomyces type strains showed that the MLSA distance of strain 594T to the most closely related species was greater than the 0.007 threshold. The in silico DNA-DNA relatedness between the genome sequence of strain 594T and that of the phylogenetically nearest species was well below the species level recommendation. There was thus multiple evidence justifying the description of this strain as representing a novel species, for which the name Streptomyces odonnellii sp. nov. is proposed. The type strain is 594T (=IMPPG 594T=DSM 41949T=NRRL B-24891T).

Streptomyces lunalinharesii 235 prevents the formation of a sulfate-reducing bacterial biofilm

ABSTRACT 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.

Streptomyces lunalinharesii 235 prevents the formation of a sulfate-reducing bacterial biofilm.

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.

Streptomyces misionensis PESB-25 produces a thermoacidophilic endoglucanase using sugarcane bagasse and corn steep liquor as the sole organic substrates.

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.

Production and partial characterization of cellulases and Xylanases from Trichoderma atroviride 676 using lignocellulosic residual biomass.

Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL(-1) for xylanase, 1.9 U mL(-1) for endoglucanase, 0.25 U mL(-1) for FPase, and 0.17 U mL(-1) for ß-glucosidase) after 3-4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50-60 °C and pH 4.0-5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.

Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

Keratinase Production by Three Bacillus spp. Using Feather Meal and Whole Feather as Substrate in a Submerged Fermentation.

Three Bacillus species (B. subtilis LFB-FIOCRUZ 1270, B. subtilis LFB-FIOCRUZ 1273, and B. licheniformis LFB-FIOCRUZ 1274), isolated from the poultry industry, were evaluated for keratinase production using feathers or feather meal as the sole carbon and nitrogen sources in a submerged fermentation. The three Bacillus spp. produced extracellular keratinases and peptidases after 7 days. Feather meal was the best substrate for keratinase and peptidase production in B. subtilis 1273, with 412 U/mL and 463 U/ml. The three strains were able to degrade feather meal (62-75%) and feather (40-95%) producing 3.9-4.4 mg/ml of soluble protein in feather meal medium and 1.9-3.3 mg/ml when feather medium was used. The three strains produced serine peptidases with keratinase and gelatinase activity. B. subtilis 1273 was the strain which exhibited the highest enzymatic activity.

Culturable fungal diversity of shrimp Litopenaeus vannamei boone from breeding farms in Brazil

Litopenaeus vannamei, which is the most common shrimp species cultivated in the northeast of Brazil, is very susceptible to microbial diseases, and this consequently affects productivity. There are reports of bacteria, viruses and protozoa in these shrimp, but not fungi. This study aims to isolate and identify fungi present in shrimp Litopenaeus vannamei, and in their nursery waters, at two breeding farms in Brazil. The pathogenic potential of the isolates was assessed through the qualitative detection of proteases and aflatoxin B production. The 146 isolated fungi comprised 46 species. Aspergillus, Penicillium and Furarium were the three most relevant genera and Aspergillus flavus was the predominant species with a total of 33 isolates. Most of the isolated species are known as potentially pathogenic to humans and other animals. Eighteen isolates of A. flavus and two of A. parasiticus were able to produce aflatoxin B and 33 out of the 46 species produced protease, indicating that these fungi may also become pathogenic to shrimp and their consumers.

Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate.

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.

Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3.

Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

Streptomyces lunalinharesii sp. nov., a chitinolytic streptomycete isolated from cerrado soil in Brazil.

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)).