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This study focused on the applying endo-xylanases to reduce the use of bleaching agent, coupled with the production of xylooligosaccharides (XOs), in mulberry paper making. Paper mulberry pulp (PMP) was consecutively prepared from paper mulberry bark by traditional NaOH-treatment, and two types of thermostable endo-xylanase from Streptomyces thermovulgaris TISTR1948 (wild-type and recombinant endo-xylanases) were employed in the biobleaching of PMP. This process was optimized to achieve maximum XOs yields and the highest PMP quality. The optimal condition was an enzyme dosage of 125 U/g PMP at 12 h of reaction time, both in a 500 mL laboratory bottle and a 150 L reactor. The mixture obtained from the reactor was separated as liquid of XOs derived from PMP (PMP-XOs) and solid biobleached PMP. The PMP-XOs from wild-type endo-xylanase were composed of 31.6% xylopentaose (X5), 30.9% xylohexaose and higher-degree XOs (X ≥ 6), and 11.7% xylobiose (X2), whereas 76.6% of X5 and 8.6% of X2 were the main products from recombinant endo-xylanase. The PMP-XOs derived from both endo-xylanase types exhibited high antioxidant activities, reducing power, phenolic contents, and prebiotic efficacy. In addition, the application of both endo-xylanases enhanced the brightness of PMP by 5.1% and 3.5%, and reduced the kappa number by 9.1% and 3.6%, respectively. Biobleached PMP was subsequently subjected to the NaOCl bleaching step to produce the mulberry paper. This approach could reduce NaOCl consumption by 20–25%, making it an environmentally friendly alternative. The production of valuable prebiotics, such as PMP-XOs, further enhances the economic viability of this approach. more...

SummaryRecent research has accumulating evidence to support that gut microbiota can be a newly discovered organ/tissue for humans. Although the functions of gut microbiota have been linked to almost all aspects of human physiological, most findings so far are still either belonged to correlational research or at preliminary stage of causal study due to the complexity of gut microbiota as well as lack of proper tools and methods. Thus, development of new approaches is essential and necessary. Lipid droplet is a cellular organelle governing cell lipid homeostasis that is directly related to human metabolic syndromes. Previously we and other groups found lipid droplets in several bacterial strains. We wonder if gut bacteria have lipid droplets. If so, these bacteria may play key roles to regulate gut lipid content, remove unnecessary lipids and produce useful lipid molecules for host. Here we identified lipid droplet-like structures in freshly isolated bacteria from mouse small and large intestines, as well as mouse and human feces. We also purified and analyzed lipid droplets from a cloned human gut bacterium Streptomyces thermovulgaris. Together, we demonstrate the existence of lipid droplets in gut microbiota and established an approach to study them. more...

Production of xylooligosaccharides (XOs) from corncob using the thermostable endo-xylanase from Streptomyces thermovulgaris TISTR1948 was investigated using KOH pretreatment, followed by enzymatic hydrolysis. The optimal reaction time for production of XOs was 12 h, after which xylobiose comprised a majority of products, and a low xylose content was observed. The optimal conditions for production of XOs were studied using a central composite design. At an enzyme concentration of 129.43 U/g of substrate, 53.80°C, and pH 6.17, the yield of XOs reached 162.97 mg/g of substrate or 752.15 mg/g of hemicellulose in KOH-pretreated corncob. The prebiotic properties of XOs derived from corncob were also investigated using in vitro fermentation of those XOs with the known probiotic strains Lactobacillus casei TISTR1463, L. lactis TISTR1464, and L. plantarum TISTR1465. XOs derived from corncob were comparable to commercial XOs for an ability to enhance the growth of the specified probiotic lactobacilli. more...

A thermostable xylanase gene ( stx I) obtained from Streptomyces thermonitrificans NTU-88 on domain analysis revealed an N-terminal catalytic domain featuring homology to a known xylanase within the glycoside hydrolase family 11. Recombinant STXI retained more than 60% of its activity following its incubation for at 60 °C for 24 h. These characteristics were close to thermophile and mesophile Streptomyces strains. The main hydrolysis products of xylan degraded by STXI included large xylooligosaccharide fragments. These results indicated that STXI was a typical endoxylanase. As regards the phylogenetic relationships of GH11, STXI and the other xylanase deriving from Streptomyces were included in a subgroup of the aerobic bacterial group. This result implied that the evolutionary relationships between the various xylanases deriving from Streptomyces strains were convergent. more...

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. more...

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. more...

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. more...

A thermostable D-xylose–glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by γ-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE–Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12™ columns. The N-terminal amino acid sequence and amino acid analysis shows 73–92% homology with xylose–glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12™ column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60–85°C at pH 7.0, using D-glucose, and up to 50–60°C at pH 7.6, using D-xylose as substrates. The activation energy (Ea= 46 kJ·mol–1) and the critical temperature (Tc= 60°C) were determined by fluorescence spectroscopy. Tcis in close coincidence with the melting temperature of denaturation (Tm= 59°C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 kJ·mol–1. The specific activity (kmvalues) for D-xylose-glucose isomerase at 70°C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, recpectively.Key words: D-xylose-glucose isomerase, protein sequencing, protein stability, protein denaturation. more...

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. more...

To the Editor: Invasive infections with Streptomyces spp. are rare; in reference to two cases reported in Emerging Infectious Diseases (1,2), we describe here the first documented case of bacteremia with Streptomyces thermovulgaris. An 81-year-old woman was admitted to the emergency room of Diakonessenhuis, Utrecht, the Netherlands, with severe abdominal pain in the right lower quadrant and feculent vomitus. The patient had a history of Crohn's disease, for which she had undergone resection of the ileum and cecum, and was receiving high-dose corticosteroid therapy (prednisone 25 mg daily). The patient also had steroid-induced osteoporosis and an internal pacemaker. On admission, the patient had a temperature of 35.6°C, a leukocyte count of 8.4 x 109/L with 30% bandforms, and a C-reactive protein level of 18 mg/L. Moreover, the patient had severe metabolic acidosis and was hemodynamically unstable, suggesting septic shock subsequent to a presumed bowel perforation. Blood samples were drawn and cultured, and empiric treatment was initiated with ceftriaxone and metronidazole. Exploratory laparotomy showed colonic inflammation. The patient was transferred to the intensive care unit postoperatively. On day 9 of hospitalization, the blood culture taken on day 1 before antimicrobial drug was started, turned positive in the automatic blood culture system. Gram staining showed gram-positive nocardioform rods, and subculture on tryptic soy agar with 7% sheep blood yielded polymorphous colonies that sunk into the agar and showed filamentous growth. Despite intensive antimicrobial and supportive therapy, sepsis progressed into multiple organ failure with severe neuropathy. On day 25 of hospitalization, the patient died. Autopsy demonstrated no possible infective focus except severe inflammation of the colon and distal ileum. Permission for cerebral section was not granted. The blood isolate showed strictly aerobic growth at 37°C and 50°C with β-hemolysis and was positive for catalase, caseinase, gelatinase, and nitrate reduction; the isolate was negative for oxidase, urease, and esculin hydrolysis and was nonmotile at 37°C. The isolate was sent to the National Institute of Public Health, Bilthoven, the Netherlands, for identification. Biochemical analysis, fatty acid analysis, and 16S rRNA typing identified the strain as S. thermovulgaris. The strain was susceptible to ceftriaxone (MIC 0.32 µg/mL) by Etest (BA Biodisk, Solna, Sweden). Further susceptibility testing performed by agar diffusion showed that the organism was also susceptible to amoxicillin, vancomycin, a combination of trimethoprim and sulfamethoxazole, and erythromycin. Bacteremia produced by a strain of S. thermovulgaris, as we report here, is the first documented case of isolation of this microorganism from human material. The streptomycetes are classified as a separate genus within the aerobic actinomycetes and are most well known for the many antimicrobial substances isolated from the approximately 600 different species (3). Streptomycetes, aerobic, spore-forming, gram-positive bacteria with filamentous growth, are ubiquitous in soil and can cause mycetomas (4). Streptomycetes are widely distributed in terrestrial and aquatic habitats with soil, fodder, and compost as their primary reservoirs. The amount of actinomycetes (the taxonomic group to which the streptomycetes belong) in soil is estimated to be 107–108 microorganisms per gram (5), and in total biomass equal to that of all other bacteria together and slightly less than that of fungi. S. thermovulgaris belongs to the thermophilic streptomycetes, which do not grow at temperatures more...

Bed performance of an immobilized biocatalyst composed of Streptomyces cells immobilized on a protein carrier susceptible to compaction, is studied in a fixed bed bioreactor with agitator at low or zero rotation. Adherence to the plug flow mixing regime and no significant external mass transfer were registered in the range of liquid velocities observed. more...

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. more...

A filamentous actinomycete, designated strain D65(T), was isolated from a root of a wild tree, Alnus nepalensis D. Don (Nepalese Alder), collected in Xishuangbanna, China. It produced the bioactive agents daidzein and N-acetyltyramine and had morphological and chemical properties characteristic of streptomycetes. Pink to brownish red diffusible pigments were produced on several ISP media. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain D65(T) formed a distinct phyletic line that was most closely, albeit loosely, associated with Streptomyces hebeiensis YIM 001(T), Streptomyces aurantiogriseus NRRL B-5416(T), Streptomyces griseoviridis NBRC 12874(T), Streptomyces niveoruber NBRC 15428(T) and Streptomyces thermovulgaris NBRC 13473. A number of phenotypic properties allowed differentiation of the strain from related Streptomyces species. Therefore strain D65(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces alni sp. nov. is proposed. The type strain is D65(T) (=CGMCC 4.3510(T)=NRRL B-24611(T)). more...

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. more...

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. more...

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. more...

SUMMARY: Fifty thermophilic streptomycetes from diverse habitats were compared through 135 unit characters with 201 representative mesophilic streptomycetes from an earlier numerical phenetic study. Data were examined using the simple matching (S SM), Jaccard (S J) and pattern (D P) coefficients, and clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) technique. In all three analyses, the thermophilic streptomycetes formed an aggregate taxon composed of three major (seven to nineteen strains), five minor (two to three strains) and two single-member clusters. Cluster composition was not affected by the statistics used. The numerical phenetic data showed that thermophilic streptomycetes form several distinct centres of variation, four of which correspond to previously described species; a further taxon was also considered to merit species status. It is proposed that Streptomyces thermolineatus sp. nov. be recognized and the name Streptomyces macrosporus Krassilnikov et al. 1968 be revived. Emended descriptions are given for Streptomyces megasporus (Krassilnikov et al., 1968) Agre 1983, Streptomyces thermoviolaceus Henssen 1957 and Streptomyces thermovulgaris Henssen 1957. more...