Streptomyces Thermovulgaris Research Articles

Cloning, Expression, and Characterization of a Metalloprotease from Thermophilic Bacterium Streptomyces thermovulgaris.

Proteases hydrolyze proteins and reduce them to smaller peptides or amino acids. Besides many biological processes, proteases play a crucial in different industrial applications. A 792 bp protease gene (nprB) from the thermophilic bacterium Streptomyces thermovulgaris was cloned and expressed in E. coli BL21 using pET 50b (+). Optimal recombinant protease expression was observed at 1 mM IPTG, 37 °C for 4 h. The resulting protease was observed in soluble form. The molecular mass estimated by SDS-PAGE and Western blot analysis of the protease (NprB) fused with His and Nus tag is ~70 KDa. The protease protein was purified by Ammonium sulfate precipitation and immobilized metal ion affinity chromatography. The optimum pH and temperature for protease activity using casein as substrate were 7.2 and 70 °C, respectively. The mature protease was active and retained 80% of its activity in a broad spectrum of pH 6-8 after 4 h of incubation. Also, the half-life of the protease at 70 °C was 4 h. EDTA (5 mM) completely inhibited the enzyme, proving the isolated protease was a metalloprotease. NprB activity was enhanced in the presence of Zn2+, Mn2+, Fe2+ and Ca2+, while Hg2+ and Ni2+ decreased its activity. Exposure to organic solvents did not affect the protease activity. The recombinant protease was stable in the presence of 10% organic solvents and surfactants. Further characterization showed that zinc-metalloprotease is promising for the detergent, laundry, leather, and pharmaceutical industries.

Optimization of Cellulase-Free Xylanase Production by Thermophilic Streptomyces thermovulgaris TISTR1948 through Plackett-Burman and Response Surface Methodological Approaches

Cellulase-free xylanase production by thermophilic Streptomyces thermovulgaris TISTR1948 was cultivated in a basal medium with rice straw as sole source of carbon and as an inducible substrate. Variable medium components were selected in accordance with the Plackett-Burman experimental design. The optimization conditions of physical factors (pH and temperature levels) were then combined in further studies through the response surface methodology approach. Only two significant components, rice straw and yeast extract, were chosen for the optimization studies. A second-order quadratic model was constructed by central composite design (CCD). The model revealed that both pH and temperature levels were significant, and were dependent on xylanase production. Under these experimental designs, the xylanase yield increased from 51.11 to 274.49 U/mL (3,400 to 10,000 U/g of rice straw) or about 537% higher than an unoptimized basal medium. The optimum conditions to achieve maximum yield of xylanase were 27.45 g/L of rice straw and 5.42 g/L of yeast extract under relatively neutral conditions of pH 7.11, 50.03 °C, and a incubation period.

Molecular basis for thermal properties of Streptomyces thermovulgaris fumarase C hinge at hydrophilic amino acids R163, E170 and S347

Industrially, the use of high temperatures (40-60 degrees C) in the L: -malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50 degrees C and highly stable even after 24 h of incubation at 40 degrees C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30 degrees C and was rapidly denatured at 50 degrees C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10 degrees C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C.

Effect of inoculation in composting processes: Modifications in lignocellulosic fraction

Three microbial isolates, identified as Bacillus shackletonni, Streptomyces thermovulgaris and Ureibacillus thermosphaericus were tested as inoculants in composting processes in relation to their capacity to improve lignocellulose degradation. Different wastes from agricultural activities were used as raw material for the heaps: pepper plant waste (PPW) as the main component and olive-oil mill waste (OMW), almond shell (AS), pruning waste (PW) and rice straw (RS) as additives. Cellulose was more extensively degraded than hemicellulose and lignin, although the use of inoculants ( B. shackletonni and S. thermovulgaris) improved the action of the autochthonous microbiota just in the AS heaps. A higher efficiency was observed for lignin, since lower concentrations of this polymer were detected in the inoculated heaps in relation to control heaps. U. thermosphaericus was the most efficient microorganism since inoculation with this strain decreased the final lignin content in a range between 17.23% and 24.34%. S. thermovulgaris and B. shackletonni led to a higher reduction of the lignin levels in the OMW and PW heaps (14.25% and 19.07% less lignin than control heaps) and OMW (13%), respectively. The composting process can therefore be improved by means of inoculation if the microorganisms used for this purpose are appropriate for the characteristics of the raw material.

Synergy of two thermophiles enables decomposition of poly-ɛ-caprolactone under composting conditions

The ultimate degradation (i.e. complete mineralization) of biodegradable polymers proceeds through hydrolysis to the production of degradation intermediates (primary degradation) that are then taken into the microbial cell and further degraded to CO2 and water. We first isolated thermophilic actinomycete (Streptomyces thermonitrificans PDS-1), which has the activity of ultimate degradability, from compost in which poly-epsilon-caprolactone (PCL) degraded vigorously. We next tried to investigate the detailed mechanisms of degradation of the PCL in compost by developing a new experimental method in which isolated microorganisms are used to inoculate sterilized compost raw materials containing PCL. It was confirmed that the ultimate degradation of PCL could not be achieved by the action of the strain PDS-1 alone, and that a supplementary microorganism (Bacillus licheniformis HA1) isolated from compost utilizes the degradation intermediates and also increases the activity of the other primary microorganism (PDS-1) by adjusting the pH. We could thus show experimental proof of synergy between two thermophiles in the ultimate degradation of a biodegradable polymer in compost.

Influence of microbial inoculation and co-composting material on the evolution of humic-like substances during composting of horticultural wastes

Four different raw materials (olive-oil mill, pruning waste, rice straw and almond shell waste) used as additives were mixed with pepper plant wastes for composting purposes. Three bacterial strains isolated from prior composting processes and identified as Bacillus shackletonni, Streptomyces thermovulgaris and Ureibacillus thermosphaericus were added to every windrow and their effect on humic-like substances evolution was studied. In all the cases, an increase in the humification indices (humic to fulvic acids ratio, humification ratio and humification index) was obtained. On the other hand, inoculation significantly affected the humification indices, and differences due to both raw materials and microbial inoculants could be observed. When inoculated in almond shell and rice straw heaps respectively, B. shackletonni and U. thermosphaericus induced higher humification levels than those obtained for other raw material/inoculant combinations. Thus, the improvement of composting processes by means of inoculation seems to depend on properties of raw materials and microorganisms applied.

Synthesis and antibacterial activity of substituted 1,2,3,4-tetrahydropyrazino [1,2- a] indoles

A series of substituted 1,2,3,4-tetrahydropyrazino [1,2- a] indole derivatives have been synthesized and tested against the Gram positive and Gram negative strains of bacteria namely Staphylococcus aureus (MTCCB 737), Salmonella typhi (MTCCB 733), Pseudomonas aeruginosa (MTCCB 741), Streptomyces thermonitrificans (MTCCB 1824) and Escherichia coli (MTCCB 1652). All synthesized compounds showed mild to moderate activity. However, compounds 4d– f were found to have potent activity against pathogenic bacteria used in the study. Their MIC ranged from 3.75 to 60 μg/disc. In vitro toxicity tests demonstrated that toxicity of 4d– f was not significantly different than that of gentamycin. However, at higher concentration (1000–4000 μg/ml) difference was highly significant.

Investigations on anti-Aspergillus properties of bacterial products

To investigate the anti-Aspergillus properties of bacterial products. In the present study, 12 bacterial strains were screened for antifungal activity against Aspergilli. The culture supernatant and lysates of Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli (BL21, DH5alpha, HB101, XL Blue), Klebsiella pneumoniae, Streptomyces thermonitrificans, Streptococcus pneumoniae, Enterobacter aerogenes, Staphylococcus aureus and Salmonella typhi were examined for antifungal activity in protein concentration ranging from 1000.0 to 7.8 microg ml-1 using microbroth dilution assay. The lysate of Salm. typhi and E. coli BL21 exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 15.6-31.2 microg ml-1 by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, a concentration of 3.1 microg disc-1 of Salm. typhi lysate showed significant activity against Aspergilli. Escherichia coli BL21 exhibited similar activity at 6.2 microg disc-1. The work on identification of molecule endowed with antimycotic properties is in progress. The products of Salm. typhi and E. coli demonstrated significant activity against Aspergillus species. This is the first time that E. coli has been reported for anti-Aspergillus activity. It could be an important source of biologically active compounds useful for developing better new antifungal drugs/or probiotics.

Extracellular nuclease from a thermophile, Streptomyces thermonitrificans: production, purification and partial characterization of – double strand preferential – deoxyribonuclease activity

A thermophilic strain of Streptomyces thermonitrificans produced high levels of extracellular nuclease (designated as nuclease Stn β) when grown on nutrient broth glucose medium. Maximal nuclease activity (51 U ml −1) was obtained, in 40 h, when the culture was grown on modified NBG medium at 45 °C. The enzyme was purified to homogeneity with an overall recovery of 5.6% and a specific activity of 10,833. The relative molecular mass of the purified enzyme, determined by gel filtration, was 22.4 kDa and it showed an obligate requirement for Mn 2+ for activity. The optimum pH and temperature of nuclease Stn β were 8.0 and 45 °C, respectively. The enzyme was inhibited by Mg 2+, Co 2+, Cu 2+, Zn 2+ and Hg 2+, inorganic phosphate, pyrophosphate, dithiothreitol, β-mercaptoethanol, EDTA and NaCl. Nuclease Stn β was stable to high concentrations of urea and organic solvents but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Stn β is a multifunctional enzyme with substrate specificity in the order of dsDNA > ssDNA ≫ RNA. The end products of dsDNA hydrolysis were predominantly oligonucleotides (85–90%) and small amounts of 5′ mononucleotides (10–15%) suggesting an endo mode of action.

Extracellular nuclease from a thermophile, Streptomyces thermonitrificans: purification and characterization of the deoxyribonuclease activity

An extracellular nuclease from Streptomyces thermonitrificans (designated as nuclease Stn alpha) was purified to homogeneity with an overall yield of 2.8%. The Mr of the purified enzyme was 39.6 kDa. The purified enzyme showed an exclusive requirement of Mn2+ for its activity but is not a metalloprotein. The optimum pH for ds- and ssDNA hydrolysis were 7.0 and 7.5 whereas, the optimum temperature was 40 and 45 degrees C, respectively. The enzyme was inhibited by divalent cations, inorganic phosphate and pyrophosphate but not by 3' and 5' mononucleotides. Nuclease Stn alpha is a multifunctional enzyme and its substrate specificity is in the order of dsDNA>ssDNA>>RNA. The end products of both ds- and ssDNA hydrolysis were predominantly oligonucleotides (80-85%) and a small amount of 3' mononucleotides (10-15%) suggesting an endo mode of action.

Extracellular nuclease from a thermophile, Streptomyces thermonitrificans: purification and characterization of the deoxyribonuclease activity

An extracellular nuclease from Streptomyces thermonitrificans (designated as nuclease Stn α) was purified to homogeneity with an overall yield of 2.8%. The M r of the purified enzyme was 39.6 kDa. The purified enzyme showed an exclusive requirement of Mn 2+ for its activity but is not a metalloprotein. The optimum pH for ds- and ssDNA hydrolysis were 7.0 and 7.5 whereas, the optimum temperature was 40 and 45 °C, respectively. The enzyme was inhibited by divalent cations, inorganic phosphate and pyrophosphate but not by 3′ and 5′ mononucleotides. Nuclease Stn α is a multifunctional enzyme and its substrate specificity is in the order of dsDNA > ssDNA ≫ RNA. The end products of both ds- and ssDNA hydrolysis were predominantly oligonucleotides (80–85%) and a small amount of 3′ mononucleotides (10–15%) suggesting an endo mode of action.

Thermostable malate synthase of Streptomyces thermovulgaris.

The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40-60 degrees C) than those of scMS (28-37 degrees C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9-91.4% amino acid identities, with stMS possessing slightly more charged residues (approximately 31%) than its mesophilic counterparts (approximately 28-29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.

Extracellular deoxyribonuclease production by a thermophile, Streptomyces thermonitrificans.

A thermophilic bacterial strain, Streptomyces thermonitrificans, produced high levels of extracellular deoxyribonuclease (DNase) when grown on NBG medium (containing 1% peptone, 0.3% beef extract, 1% glucose and 0.5% NaCl). Maximum DNase activity (140 U x ml(-1)) was obtained, in 24 h, when the culture was grown on modified NBG medium (containing 1.3% beef extract, 1% glucose, 0.5% NaCl and 50 microM Mn2+ at 45 degrees C. The crude enzyme showed higher activity on native DNA than on sonicated and heat denatured DNA. Moreover, addition of Mn2+ in the assay mixture resulted in a significant stimulation (10-15 fold) of the enzyme activity.

Classification of thermophilic streptomycetes, including the description of Streptomyces thermoalcalitolerans sp. nov.

A polyphasic taxonomic study was undertaken to clarify relationships within and between representative thermophilic alkalitolerant streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA-DNA relatedness studies, support the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and S. thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of priority, S. thermonitrificans is a later subjective synonym of S. thermovulgaris. Similarly, 10 out of the 11 representative thermophilic alkalitolerant isolates had a combination of properties consistent with their classification as S. thermovulgaris. The remaining thermophilic alkalitolerant isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic thermophilic isolate, Streptomyces strain NAR85, was identified as S. thermodiastaticus.

Purification and characterization of the protease enzymes of Streptomyces thermovulgaris grown in rapemeal-derived media

When grown in a particulate-free, protein-rich medium derived from rapemeal (termed medium B), Streptomyces thermovulgaris produced multiple protease enzymes. The main protease activity was attributed to two types of serine protease, denoted as SV1 and SV2. A metallo protease component (SV3) and an azocaseinase component (SV4) were also present. Protease SV1 had a molecular weight of 30 kDa and a pI of 5.8. Protease SV2 was characterized by a high thermostability in the presence of calcium ions and had a pI of 8.4. This enzyme had a molecular weight of 60 kDa, but we suggest that this is the dimeric form, with 30 kDa being the monomer unit. The method chosen for initial downstream processing influenced both the yield and type of protease purified. When cell-free supernatant fluid was concentrated using ultrafiltration, rather than acetone precipitation, a higher percentage and a greater range of proteases were recovered. The medium used for the growth of Strep. thermovulgaris also appeared to affect the type of protease produced. A more diverse range of proteases were produced on rapemeal-derived medium when compared to yeast extract medium.

Protease production by Streptomyces thermovulgaris grown on rapemeal‐derived media

A range of actinomycete species was tested for their ability to grow on particulate and particle-free rapeseed meal-derived media. Streptomycetes grew on both types of medium and produced a number of extracellular enzymes. Highest activities of protease were produced by Streptomyces thermovulgaris and reflected the high available protein content of rapemeal. Enzyme production and growth were analysed in fermentor-grown batch cultures of S. thermovulgaris using the particle-free rapemeal broth termed medium B. Growth was biphasic and the majority of the protease was produced during the second slower phase. Analysis of the protease as azocaseinase activity revealed a high degree of thermostability in the presence of calcium such that approximately 20% of the activity remained after incubation at 70 degrees C for 24 h. Gel filtration suggested that S. thermovulgaris synthesized more than one kind of protease and this was confirmed by using specific peptide substrates and inhibitors which revealed the presence of distinct serine and metallo-type enzymes.

Medium optimization for the production of glucose isomerase from thermophilic Streptomyces thermonitrificans

A thermophilic strain of Streptomyces thermonitrificans produced a high activity of intracellular glucose isomerase (12 U/ml) when grown in a medium containing 1% (w/v) xylose, supplemented with 2% (w/v) sorbitol as the second carbon source, at 50°C for 16 h. Addition of Mg(2+) enhanced enzyme production but the organism could grow and produce the enzyme in the absence of Co(2+).

Mushroom worker's lung: Serologic reactions to thermophilic actinomycetes present in the air of compost tunnels

Vast numbers of spores of the thermophilic actinomycetes Excellospora flexuosa, Thermomonospora alba, T. curvata and T. fusca were collected from the air in fermentation tunnels during the spawning of mushroom compost, i.e. over 10(9) CFU m-3 of air. Five different genera of fungi, namely, Aspergillus, Aureobasidium, Cladosporium, Penicillium and Scytalidium, were found at only 10(3) CFU m-3 of air. Agaricus bisporus, used for spawning, was absent. Sera of 10 mushroom growers affected by Mushroom Worker's Lung (MWL) were tested by a qualitative dot-ELISA for antibodies against the spores of these micro-organisms. All 10 were positive for one or more of the actinomycetes E. flexuosa, T. alba, T. curvata and T. fusca. No antibodies were found against Streptomyces thermovulgaris, Thermoactinomyces vulgaris and T. sacchari nor against the fungi Aspergillus fumigatus, Penicillium brevicompactum, P. chrysogenum, Scytalidium thermophilum and Trichoderma viride. Sera of 11 of 14 workers engaged in routine spawning of compost in tunnels reacted positively with 1 or more of the actinomycetes. Their 10log serum titres increased with the duration of employment to an upper limit of 2.5. The sera of 19 non-exposed individuals were negative. Because high numbers of spores of E. flexuosa, T. alba, T. curvata and T. fusca were present in the air that was used for successful inhalation provocation of mushroom workers with MWL and because of the elevated serum titres of these workers, we presume these organisms to contribute in the occurrence of MWL.

D-Glucose Isomerization using an Immobilized Glucose Isomerase preparation from Streptomyces Thermovulgaris Strain 127

The kinetics of D-glucose isomerization to D-fructose by an original whole-cell immobilized biocatalyst preparation based on Streptomyces thermovulgaris (strain 127) have been studied under various process conditions, substrate concentration, particle size. Two versions of different activity were applied. For the biocatalyst of moderate activity the kinetic constants in the MICHAELIS -MENTEN equation for reversible reaction were determined. Effectiveness factors for both preparations were calculated and compared with other reported data.

Respiratory chain composition and activity in some thermotolerant streptomycetes

The cytochrome composition of membrane preparations from 3 thermotolerant species of Streptomyces was determined from difference spectra. All contained b, c and a-type haemoproteins and in addition, 2 species (Streptomyces thermoviolaceus and Streptomyces thermoflavus) possessed cytochrome oxidase d. A CO-reacting b cytochrome similar to cytochrome oxidase o could also be detected in all 3 organisms. Thermotolerance of the respiratory chains showed no correlation with cytochrome composition. In general, Streptomyces thermonitrificans had the most thermostable activities, whilst those of S. thermoviolaceus were the most thermolabile. Growth temperature exerted no qualitative effects on respiratory chain composition but did affect the thermostability of some substrate-supported oxidase activities.