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Spectinabilin (neoaureothin) is a rare nitrophenyl-substituted polyketide produced by some Streptomyces species. This compound is known to exhibit various biological activities such as anticancer, antibiotic, immunomodulatory, antimalarial and nematicidal effects. Despite being a valuable secondary metabolite for the development of novel drugs, the production yield of spectinabilin is Streptomyces sp. AN091965. One of the mutants, S-N87 showed up to about 10-fold spectinabilin productivity (354.8 ± 7.8 mg/L) compared to the parental strain Streptomyces sp. AN091965 (37.6 ± 5.6 mg/L) in flask culture conditions, representing the highest spectinabilin yield reported thus far. In addition, this strain showed a stable yield of 2.27 g/L even in a scaled-up environment (150 L tank fermentation), which suggests that the selected mutant is a genetically stable and robust strain. Further, the mutant Streptomyces sp. S-N87 that enhanced spectinabilin production stably showed a significant increase in nematicidal activities against pine wilt nematode compared to the parental strain. The present study is the first to develop a Streptomyces mutant that over-produces spectinabilin by traditional mutagenesis. Further studies such as whole-genome analysis and genetic modification are needed to provide a theoretical basis and insights into the polyketide synthase pathway of this talented mutant.

Polyurethane (PU), currently replacing existing synthetic materials worldwide, is a synthetic polymer derived from polyols, isocyanates, and a chain extender added by condensation reactions. PU wastes which are difficult to recycle, are commonly discarded in landfills and flow into ecosystems, thereby causing serious environmental problems. In recent years, insect-associated microbes have become a promising, eco-friendly strategy as an alternative to plastic recycling. This study aimed to evaluate the potential of Serratia sp. HY-72 strain isolated from the intestine of the Asian mantis (Hierodula patellifera) for PU degradation. The 65 kDa family I.3 lipase which degrades PU was identified and characterized, with a specific activity of 2,883 U mg−1. The bacterial filtrates and the recombinant lipase degraded Impranil (a colloidal polyester-PU dispersion, 100 g l−1) by 85.24 and 78.35% after 72 h incubation, respectively. Fourier transform infrared spectroscopy analysis revealed changes in Impranil functional groups, with decreased C=O functional group and aliphatic chain signals, and increased N-H bending with C-N stretching and C-O stretching. The current study also revealed that the HY-72 strain biodegraded the commercial PU foams (polyester- and polyether- PU) with 23.95 and 10.95% weight loss after 2 weeks, respectively with changes in surface morphology and structure such as cracks, roughness, and surface roughening. Altogether, this is one of the few studies reporting biodegradation of PU by the insect-associated microbe. These findings suggest that the insect-associated microbe could be a promising resource for biodegradation and recycling of plastic waste.

Polyurethane (PU), currently replacing existing synthetic materials worldwide, is a synthetic polymer derived from polyols, isocyanates, and a chain extender added by condensation reactions. PU wastes which are difficult to recycle, are commonly discarded in landfills and flow into ecosystems, thereby causing serious environmental problems. In recent years, insect-associated microbes have become a promising, eco-friendly strategy as an alternative to plastic recycling. This study aimed to evaluate the potential of

The GluM gene (1491-bp) coding for a β-glucosidase comprising a single catalytic glycoside hydrolase family 1 domain from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium funkei HY-13, was cloned and over-expressed in Escherichia coli BL21. The recombinant histidine-tagged enzyme (rGluM: 56 kDa) displayed the highest cleavage activity toward p-nitrophenyl (pNP)-β- d -glucopyranoside at pH 5.0 and 40 °C. The β-glucosidase activity of rGluM was enhanced over 1.8-fold of its original activity in the presence of 1 mM Ca2+, Ni2+, Mn2+, and Co2+ ions, respectively, while it was highly sensitive to 5 mM N-bromosuccinimide and 1 mM Hg2+. The susceptibility of some pNP-sugar derivatives and d -cellobiose to rGluM was evaluated to be in the order of pNP-β- d -glucopyranoside > pNP-β- d -galactopyranoside > d -cellobiose > pNP-β- d -cellobioside > pNP-β- d -mannopyranoside. The kcat/Km values of rGluM toward pNP-β- d -glucopyranoside, pNP-β- d -galactopyranoside, and d -cellobiose were 302.28, 179.73, and 6.40 mM-1 s-1, respectively. At a concentration below 1.0 M, d -galactose was a potent activator of rGluM with β-glucosidase activity enhanced by approximately 160% in a dose-dependent manner. Moreover, the d -glucose ( d -xylose (≤ 700 mM) stimulation of rGluM suggests that it can be exploited as a potential biocatalyst to generate d -glucose molecules in d -cellobiose degradation.

Background Nematodes are parasitic animals that cause over 100 billion US dollars loss in agricultural business. The whole-genomes of two Streptomyces strains, Streptomyces spectabilis KCTC9218T and Streptomyces sp. AN091965, were sequenced. Both strains produce spectinabilin, an antinematode drug. Its secondary metabolism was examined to aid the development of an efficient nematicidal drug-producing host strain. Results The whole-genome sequences of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 were analyzed using PacBio and Illumina sequencing platforms, and assembled using hybrid methodology. The total contig lengths for KCTC9218T and AN091965 were 9.97 Mb and 9.84 Mb, respectively. A total of 8,374 and 8,054 protein-coding genes, as well as 39 and 45 secondary metabolite biosynthetic gene clusters were identified in KCTC9218T and AN091965, respectively. 18.4 ± 6.45 mg/L and 213.89 ± 21.30 mg/L of spectinabilin were produced by S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Pine wilt disease caused by nematode was successfully prevented by lower concentration of spectinabilin injection than that of abamectin recommended by its manufacturer. Production of multiple antinematode drugs, including spectinabilin, streptorubin B, and undecylprodigiosin was observed in both strains using high-resolution liquid chromatography mass spectrometry (LC–MS) analysis. Conclusions Whole-genome sequencing of spectinabilin-producing strains, coupled with bioinformatics and mass spectrometry analyses, revealed the production of multiple nematicidal drugs in the KCTC9218T and AN091965 strains. Especially, Streptomyces sp. AN091965 showed high production level of spectinabilin, and this study provides crucial information for the development of potential nematicidal drug producers.

The supplementation of pig diets with exogenous enzymes is widely used with the expectation that it will improve the efficiency of nutrient utilization, thereby, improving growth performance. This study aims to evaluate the effects of a 0.1% (v/v) multi-enzyme (a mixture of arazyme (2,500,000 Unit/kg), xylanase (200,000 Unit/kg) and mannanase (200,000 Unit/kg)) supplementation derived from invertebrate symbiotic bacteria on pig performance. Here, 256 growing pigs were assigned to control and treatment groups, respectively. The treatment group exhibited a significantly reduced average slaughter age; the final body weight and average daily gain increased compared with that of the control group. In the treatment group, the longissimus muscle showed a remarkable decrease in cooking loss, shear force, and color values with increased essential and non-essential amino acid concentrations. Furthermore, the concentrations of mono- and polyunsaturated fatty acids in the treatment group increased. Feed additive supplementation increased the family of Ruminococcaceae and genera Lactobacillus, Limosilactobacillus, Turicibacter, and Oscillibacter, which play a positive role in the host physiology and health. Predicted metabolic pathway analysis confirmed that operational taxonomic units and predicted amino acid biosynthesis pathways were strongly associated. The results suggest that applying exogenous enzymes derived from invertebrate symbiotic bacteria enhances animal performance.

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

Endophytic bacteria, a rich source of bioactive secondary metabolites, are ideal candidates for environmentally benign agents. In this study, an endophytic strain, Streptomyces sp. AE170020, was isolated and selected for the purification of nematicidal substances based on its high nematicidal activity. Two highly active components, aureothin and alloaureothin, were identified, and their chemical structures were determined using spectroscopic analysis. Both compounds suppressed the growth, reproduction, and behavior of Bursaphelenchus xylophilus. In in vivo experiments, the extracts of strain Streptomyces sp. AE170020 effectively suppressed the development of pine wilt disease in four-year-old plants of Pinus densiflora. The potency of secondary metabolites isolated from endophytic strains suggests applications in controlling Bursaphelenchus xylophilus and opens an avenue for further research on exploring bioactive substances against the pine wood nematode.

Endo-β-1,3-glucanase plays an essential role in the deconstruction of β-1,3-d-glucan polysaccharides through hydrolysis. The gene (1650-bp) encoding a novel, bi-modular glycoside hydrolase family 64 (GH64) endo-β-1,3-glucanase (GluY) with a ricin-type β-trefoil lectin domain (RICIN)-like domain from Cellulosimicrobium funkei HY-13 was identified and biocatalytically characterized. The recombinant enzyme (rGluY: 57.5 kDa) displayed the highest degradation activity for laminarin at pH 4.5 and 40 °C, while the polysaccharide was maximally decomposed by its C-terminal truncated mutant enzyme (rGluYΔRICIN: 42.0 kDa) at pH 5.5 and 45 °C. The specific activity (26.0 U/mg) of rGluY for laminarin was 2.6-fold higher than that (9.8 U/mg) of rGluYΔRICIN for the same polysaccharide. Moreover, deleting the C-terminal RICIN domain in the intact enzyme caused a significant decrease (&gt, 60%) of its ability to degrade β-1,3-d-glucans such as pachyman and curdlan. Biocatalytic degradation of β-1,3-d-glucans by inverting rGluY yielded predominantly d-laminaripentaose. rGluY exhibited stronger growth inhibition against Candida albicans in a dose-dependent manner than rGluYΔRICIN. The degree of growth inhibition of C. albicans by rGluY (approximately 1.8 μM) was approximately 80% of the fungal growth. The superior anti-fungal activity of rGluY suggests that it can potentially be exploited as a supplementary agent in the food and pharmaceutical industries.

The gene (1488-bp) encoding a novel GH10 endo-β-1,4-xylanase (XylM) consisting of an N-terminal catalytic GH10 domain and a C-terminal ricin-type β-trefoil lectin domain-like (RICIN) domain was identified from Luteimicrobium xylanilyticum HY-24. The GH10 domain of XylM was 72% identical to that of Micromonospora lupini endo-β-1,4-xylanase and the RICIN domain was 67% identical to that of Actinospica robiniae hypothetical protein. The recombinant enzyme (rXylM: 49kDa) exhibited maximum activity toward beechwood xylan at 65°C and pH 6.0, while the optimum temperature and pH of its C-terminal truncated mutant (rXylM△RICIN: 35kDa) were 45°C and 5.0, respectively. After pre-incubation of 1h at 60°C, rXylM retained over 80% of its initial activity, but the thermostability of rXylM△RICIN was sharply decreased at temperatures exceeding 40°C. The specific activity (254.1Umg-1) of rXylM toward oat spelts xylan was 3.4-fold higher than that (74.8Umg-1) of rXylM△RICIN when the same substrate was used. rXylM displayed superior binding capacities to lignin and insoluble polysaccharides compared to rXylM△RICIN. Enzymatic hydrolysis of β-1,4-d-xylooligosaccharides (X3-X6) and birchwood xylan yielded X3 as the major product. The results suggest that the RICIN domain in XylM might play an important role in substrate-binding and biocatalysis.

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (&gt, 1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase.

To evaluate the biocatalytic characteristics of a new endo-β-1,4-d-mannan-degrading enzyme (ManP) from Paenibacillus sp. strain HY-8, a gut bacterium of the longicorn beetle Moechotypa diphysis. Purified ManP (32 kDa) with an N-terminal amino acid sequence of APSFAVGADFSYVPG displayed the greatest degree of biocatalytic activity toward locust bean gum (LBG) at 55 °C and pH 7.0. The enzyme degraded LBG, guar gum, ivory nut mannan, and mannooligosaccharides (M2–M5), but did not exhibit any hydrolytic activity against structurally unrelated substrates. The biocatalytic activity of ManP against LBG and guar gum was 695 and 450 U mg−1, respectively. Especially, enzymatic hydrolysis of mannobiose yielded a mixture of mannose (16.6 %) and mannobiose (83.4 %), although the degree of mannobiose degradation by ManP with was relatively limited. The present results suggest that ManP is an endo-β-1,4-mannanase and is distinct from various other characterized endo-β-1,4-mannanases.

The gene (1608-bp) encoding a GH6 endo-β-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0-10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley β-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including β-1,3-D-glucan, β-1,4-D-galactomannan, and β-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley β-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.

The nutritional requirements for antimicrobial activity of Streptomyces rimosus AG-P1441 were optimized using statistically-based experimental designs at a flask level. Based on a one-factor-at-a-time (OFAT) approach, glucose, corn starch and soybean meal were identified as the carbon and nitrogen sources having a significant effect on antimicrobial productivity. As a result of investigating the effect of glucose concentration, the highest antimicrobial activity was observed at 3% concentration. Response surface methodology (RSM) was then applied to optimize the growth medium components (corn starch, soybean meal, MgCl2 and glutamate). Antimicrobial productivity increased sharply when the medium consisted of 3% glucose, 3.5% corn starch, 2.5% soybean meal, 1.2 mM MgCl2 and 5.9 mM glutamate. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in antimicrobial activity, evaluated by the paper disc assay, revealed a 29 mm inhibition zone diameter against Phytophthora capsici.

The nutritional requirements for antimicrobial activity of Streptomyces rimosus AG-P1441 were optimized using statistically-based experimental designs at a flask level. Based on a one-factor-at-a-time (OFAT) approach, glucose, corn starch and soybean meal were identified as the carbon and nitrogen sources having a significant effect on antimicrobial productivity. As a result of investigating the effect of glucose concentration, the highest antimicrobial activity was observed at 3% concentration. Response surface methodology (RSM) was then applied to optimize the growth medium components (corn starch, soybean meal, MgCl2 and glutamate). Antimicrobial productivity increased sharply when the medium consisted of 3% glucose, 3.5% corn starch, 2.5% soybean meal, 1.2 mM MgCl2 and 5.9 mM glutamate. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in antimicrobial activity, evaluated by the paper disc assay, revealed a 29 mm inhibition zone diameter against Phytophthora capsici.

The XylP gene, which encodes endoxylanase in Bacillus sp. HY-20, was subcloned, and two expression plasmids, pG-xylP and pGMF-xylP were constructed. These plasmids, which contain different signal sequences, XylP s.s and MFα opt s.s, respectively, for the secretory expression of endoxylanase, were transformed into Saccharomyces cerevisiae SEY2102 and FY833, respectively. The recombinant endoxylanases were successfully expressed, with a total activity range of 23.7-70.1 unit/ml according to the expression system and host strain. The endoxylanase activity in SEY2102/pGMF-xylP reached a maximum of 88.1 unit/ml in baffled flask culture. Most of the recombinant endoxylanase was efficiently secreted in the extracellular fraction, and the MFαopt s.s was more efficient for secreting endoxylanase in yeast than the XylP s.s. Therefore, the expression system developed in this study produces large extracellular amounts of endoxylanase using S. cerevisiae as the host strain, and it could be used in bioethanol production and industrial applications.

In the present study, the inhibitory effect of arazyme on allergic inflammation was investigated by evaluating the alteration of cytokine production and expression of skin barrier proteins in immune and HaCaT human keratinocyte cells. THP‑1 human monocytic and EoL‑1 human eosinophilic cells were treated with Dermatophagoides pteronissinus extract (DpE). Monocyte chemotactic protein‑1 (MCP‑1)/CCL2, interleukin (IL)‑6 and IL‑8 increased following DpE treatment and arazyme significantly blocked the increase of MCP‑1, IL‑6 and IL‑8 expression in cell types. Secretion of MCP‑1, IL‑6 and IL‑8 induced by lipopolysaccharide in THP‑1 cells was also inhibited by arazyme treatment. Arazyme inhibited the secretion of IL‑6 and IL‑8 due to phorbol 12‑myristate 13‑acetate and calcium ionophores in human mast cells. Arazyme blocked the secretion of thymus and activation‑regulated chemokine (TARC)/CCL17, MCP‑1, IL‑6 and IL‑8 due to tumor necrosis factor‑α (TNF‑α) and interferon‑γ (IFN‑γ) in HaCaT cells. TNF‑α and IFN‑γ suppressed the expression of skin barrier proteins, including filaggrin, involucrin and loricrin. By contrast, arazyme increased the expression of filaggrin, involucrin and loricrin. These results may contribute to the development of a therapeutic drug for the treatment of allergic diseases, including atopic dermatitis.

The potential of non-ionic polysorbate surfactants as alternative inducers of medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHAs) for the production of diverse bacterial MCL-PHA depolymerases was evaluated. When grown with corn oil as the sole carbon substrate, Pseudomonas alcaligenes LB19 preferentially produced lipolytic enzymes, but its MCL-PHA depolymerase was not induced by the substrate. However, the results of activity staining and sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis clearly revealed that Tween 20 induced simultaneous production of lipolytic enzymes and the MCL-PHA depolymerase with the molecular mass (26.5 kDa) of P. alcaligenes LB19, which has been previously identified. Moreover, the co-production of two functionally distinct hydrolytic enzymes induced by Tween 20 was commonly observed in various Gram-positive and Gram-negative bacteria that were fed the substrate. Thus, it is expected that non-ionic polysorbate surfactants including Tween 20 can be widely exploited as promising universal substrates for the facile and efficient production of diverse MCL-PHA depolymerases.

Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)-induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular signal-regulated kinase 1/2 (ERK1/2) activation under LPS-stressed conditions via protein kinase C δ (PKCδ) activation. Furthermore, PKCδ-induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS-treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition was restored in an ERK1/2-dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition-induced ERK1/2 activation via PKCδ relieved LPS-mediated endotoxemia. This finding suggests that IL-10 hyperexpression resulting from GSK-3 inhibition-induced ERK activation could be a new therapeutic pathway for endotoxemia.

The gene (2304-bp) encoding a novel xylanolytic enzyme (XylK2) with a catalytic domain, which is 70% identical to that of Cellulomonas flavigena DSM 20109 GH6 β -1,4-cellobiohydrolase, was identified from an earthworm ( Eisenia fetida )-symbiotic bacterium, Cellulosimicrobium sp. strain HY-13. The enzyme consisted of an N-terminal catalytic GH6-like domain, a fibronectin type 3 (Fn3) domain, and a C-terminal carbohydrate-binding module 2 (CBM 2). XylK2ΔFn3-CBM 2 displayed high transferase activity (788.3 IU mg −1 ) toward p -nitrophenyl (PNP) cellobioside, but did not degrade xylobiose, glucose-based materials, or other PNP-sugar derivatives. Birchwood xylan was degraded by XylK2ΔFn3-CBM 2 to xylobiose (59.2%) and xylotriose (40.8%). The transglycosylation activity of the enzyme, which enabled the formation of xylobiose (33.6%) and xylotriose (66.4%) from the hydrolysis of xylotriose, indicates that it is not an inverting enzyme but a retaining enzyme. The endo- β -1,4-xylanase activity of XylK2ΔFn3-CBM 2 increased significantly by approximately 2.0-fold in the presence of 50 mM xylobiose.

The signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells. Therefore, blocking the aberrant activity of STAT3 in tumor cells is a validated therapeutic strategy. To discover novel inhibitors of STAT3 activity, we screened against microbial natural products using a dual-luciferase assay. Using the microbial metabolome library, we identified cosmomycin C (CosC), which was isolated from the mycelium extract of Streptomyces sp. KCTC19769, as a STAT3 pathway inhibitor. CosC inhibited STAT3 (Tyr705) phosphorylation and subsequent nuclear translocation in MDA-MB-468 breast cancer cells. CosC-mediated inhibition of STAT3 signaling pathway was confirmed by suppressed expression of STAT3 downstream target proteins including cyclin D1, Bcl-xL, survivin, Mcl-1, and VEGF in CosC-treated MDA-MB-468 cells. Flow cytometry showed that CosC caused accumulation in the G0–G1 phase of the cell cycle and induced apoptosis via PARP cleavage and caspase-3 activation. Based on these findings, CosC may be a potential candidate for modulation of STAT3 pathway.

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50 °C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, β-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-β-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

The virulence against the two-spotted spider mite, Tetranychus urticae Koch, was evaluated with entomopathogenic fungus Paecilomyces lilacinus (Thom) Samson HY-4 which is isolated from coleopteran insect and registered to Korean and US patents. Virulence tests were conducted with conidial suspensions () of P. lilacinus HY-4 against T. urticae adults and positive results were recorded in laboratory conditions. The spraying device was also developed for the efficient and exact evaluation of treatment. The developed spraying device was named as SD-tower sprayer and its efficacy of spraying conidia was evaluated. The accumulated mortality caused by P. lilacinus HY-4 using SD-tower spray was about 73% at 6 days after inoculation. This suggest that the isolate P. lilacinus HY-4 may be considered as promising for a new approach to prevent adult infestations by T. urticae.

A novel GH10 endo-β-1,4-xylanase (XylG) gene from Streptomyces thermocarboxydus HY-15, which was isolated from the gut of Eisenia fetida, was cloned, over-expressed, and characterized. The XylG gene (1182 bp) encoded a polypeptide of 393 amino acids with a deduced molecular mass of 43,962 Da and a calculated pI of 6.74. The primary structure of XylG was 69% similar to that of Thermobifida fusca YX endo-β-1,4-xylanase. It was most active at pH 6.0 and 55 °C. The susceptibilities of xylans to XylG were as follows: oat spelt xylan > birchwood xylan > beechwood xylan. The XylG also showed high activity (474 IU/mg) toward p-nitrophenylcellobioside. Moreover, at pH 6.0 and 50 °C, the Vmax and Km values of the XylG were 127 IU/mg and 2.51 mg/ml, respectively, for oat spelt xylan and 782 IU/mg and 5.26 mM, respectively, for p-nitrophenylcellobioside. A homology model indicated that XylG folded to form a (β/α)8-barrel with two catalytic residues of an acid/base (Glu181) and a nucleophile (Glu289). The formation of a disulfide bond between Cys321 and Cys327 were predicted by homology modeling.

A xylanolytic bacterium, Cellulosimicrobium sp. HY-13, was isolated from the digestive tract of an earthworm, Eisenia fetida . The purified cellulase-free endo-β-1,4-xylanase (XylK) produced by strain HY-13 was found to contain an N-terminal amino acid sequence of APSTLEAAAE and to have a relative molecular mass of 36 kDa. It was most active at pH 6.0 and 55 °C and had V max and K m values toward oat spelt xylan of 4067 IU/mg and 2.78 mg/ml, respectively. XylK primarily degraded xylan to a series of xylooligosaccharides composed of xylobiose to xylotetraose, but it could not further hydrolyze xylobiose to xylose. The results of the present study suggest that the relatively highly active XylK lacking exo-xylanolytic activity is a promising candidate for the efficient production of non-digestible xylooligosaccharides that may have beneficial effects to gastrointestinal health via promotion of the growth of probiotics.

Arazyme is a novel protease produced by the HY-3 strain of Aranicola proteolyticus, which is a Gram-negative aerobic bacterium that has been isolated from the intestine of the spider Nephila clavata. This study focused on the hepatoprotective effect of Arazyme on carbon tetrachloride (CCl4)-induced acute hepatic injury in senescence marker protein 30 (SMP30) knock-out (KO) mice and SMP30 wild-type (WT) mice. WT mice and SMP30 KO mice were divided into eight groups as follows: (i) two negative control groups (G1, G5) which were treated with a single intraperitoneal (i.p.) olive oil injection. (ii) Two positive control groups (G2, G6) which received a single i.p. CCl4 (0.4mL/kg) injection. (iii) Two vitamin C-treated groups (G3, G7) which received a single oral administration of vitamin C (100mg/kg) and were injected with a single i.p. CCl4 (0.4mL/kg). (iv) Two Arazyme-treated groups (G4, G8) which received a single oral administration of Arazyme (500mg/kg) and were injected with a single i.p. CCl4 (0.4mL/kg). Through present study, we could find that Arazyme-treated groups showed decreased degree of liver injury, increased expression of SMP30, decreased expression of phospho-Smad3 (p-Smad3), elevated expression of antioxidant proteins including sorbitol dehydrogenase, dihydropteridine reductase (DHPR), dehydrofolate reductase (DHFR), NADH dehydrogenase, glutathione S-transferase kappa 1 (GSTK1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) compared with non-Arazyme-treated groups. Therefore, it is concluded that Arazyme plays a significant role in protecting injured hepatocytes by increasing the expression of SMP30, inhibiting the transforming growth factor-beta (TGF-beta)/Smad pathway and elevating the expression of antioxidant proteins.

A series of cinnamyl compounds related to 2′-hydroxycinnamaldehyde were synthesized and their antitumor effects against human cancer cells evaluated. Hydroxylamine derivative 6 inhibited the growth of human cancer cells and human colon tumor xenograft in nude mice. Its antitumor effects belong to the induction of apoptosis and arresting cell cycle at G2/M phase, which is confirmed by detection of apoptosis markers and cell cycle analysis.

Because the activation of matrix metalloproteinases (MMP) is a key factor in the metastatic process, compounds with the ability to inhibit MMP activity have a potential in the treatment of tumor. From the examination of 2000 plant extracts, obovatal isolated from the extract of the leaves of Magnolia obovata THUNB was a potent inhibitor of MMP-2 enzyme in vitro. In human fibrosarcoma cells (HT1080) activated with MMP-2, obovatal inhibited MMP-2 enzyme activity and expression. In addition, the compound blocked migration and invasion of the cells. This study demonstrates that obovatal exerts its anticancer effects through blocking migration and invasion by inhibition of MMP-2 expression and activity, and also will be a good lead molecule for the development of anti-tumor drug.

Two biflavonoids, ginkgetin (1) and sciadopitysin (2), were isolated from the MeOH extract of the young branches of Taxus cuspidata, which inhibited phosphatase of regenerating liver-3 (PRL-3) with IC50 values of 25.8 and 46.2 microM, respectively. This is the first report on PRL-3 inhibitors, isolated from natural sources.

The screening of natural products that preferentially inhibit growth of H-ras transformed rat2 cells vs. rat2 cells was performed to identify H-ras specific growth inhibitor. A lanostane-type triterpene acid, dehydrotrametenolic acid (3beta-hydroxylanosta-7,9(11),24-trien-21-oic acid), was isolated from the sclerotium of Poria cocos (Polyporaceae). Dehydrotrametenolic acid selectively inhibited the growth of H-ras transformed cells with a GI(50) value of 40 microM. FACS analysis indicated that the compound exerted its anti-proliferation effects through cell cycle arrest at G2/M phase and accumulation of sub-G1 population. Dehydrotrametenolic acid-induced apoptosis was further confirmed with chromosomal DNA fragmentation, caspase-3 activation, and degradation of PARP and Lamin A/C degradation. The compound also regulated the expression of H-ras, Akt and Erk, which are the downstream proteins of H-ras signaling pathways. The results suggest that dehydrotrametenolic acid can be a potential anticancer agent against H-ras transformed tumor.

Actinomycin D was revealed as an inhibitor of Shc/Grb2 interaction in cell lines from our recent study. Shc and Grb2 proteins are important molecules in Ras signaling pathways leading to cellular differentiation and proliferation, which require dramatic morphological changes. It was detected by transmission electron microscopy that actinomycin D induced significant changes in cellular ultrastructures of B104-1-1 cells and confirmed that the changes were due to inhibition of Shc/Grb2 interaction by actinomycin D rather than its inhibitory effect on transcription. Because actinomycin D was dispersed mainly in cytoplasm and Shc peptide (synthetic 13 amino acid tyrosine phosphorylated polypeptide) successfully displaced actinomycin D binding to its cellular targets while the other polypeptide from PDGF receptor could not. We examined the effect of actinomycin D on growth of B104-1-1 tumor xenografted in nude mice. Tumor growth was inhibited in vivo after treatment with this inhibitor. Efficacy was correlated with a reduction in the levels of Shc/Grb2 binding in excised tumors. These results suggest that actinomycin D inhibited Shc/Grb2 interaction in B104-1-1 tumor xenografted in nude mice.

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nM, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.

We investigated the biochemical properties of a 51.5 kDa metalloprotease (arazyme, 3.4.24.40) secreted into the culture medium by Aranicola proteolyticus, a symbiotic bacterium of the spider Nephila clavata. The enzyme was purified to apparent homogeneity by ion exchange chromatography in a Resource Q column (FPLC system). The substrate specificity requirements of purified arazyme were examined using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = ethylenediaminedinitrophenyl). Three series of peptides were assayed to map the S2, S1 and S ′ 1 subsites: Abz-KXRFSKQ-EDDnp, Abz-KLXFSKQ-EDDnp and Abz-KLRXSKQ-EDDnp (X are natural amino acids). The results indicated that S1 subsite has a broad specificity, being Gly the preferred amino acid for this subsite followed by positively charged residues (Arg and His). The S2 and S ′ 1 subsites accommodated better hydrophobic residues with aliphatic or aromatic side chains (Leu, Phe). The pH effect on hydrolysis of Abz-KLFFSKQ-EDDnp indicated that optimal hydrolysis occurred at pH 8.0 or higher. The effect of NaCl on the arazyme activity depends on the substrate, but in general the activity was reduced with this salt. The temperature did not affect the enzyme from 10 to 45 °C, after which activity decreased sharply. Arazyme presented high hydrolytic activity on substance P and peptides related to bradykinin. In addition, arazyme activity was resistant to the treatment by pepsin, trypsin and chymotrypsin. In conclusion, arazyme has a broad hydrolytic profile and works in very aggressive conditions, which justify its potential use in therapeutics and biotechnological applications.

Four cyclopentenediones, farnesyl protein transferase inhibitors, and anti-tumor compounds were isolated from the methanolic extract of the fruits of Lindera erythrocarpa Makino (Lauraceae). The structure of the compounds was determined by spectral data including NMR and mass spectrometry, and cyclopentenediones such as methyllinderone (1), methyllucidone (2), lucidone (3), and linderone (4) were identified by comparing their reported spectral data with that of the literature values. Compounds 1-4 inhibited farnesyl protein transferase with IC50 value of 55.3+/-4.1, 42+/-1.9, 103+/-5.1, and 40+/-3.5 microM, respectively. Isolated compounds also inhibited the growth of various human cancer cell lines in a dose-dependent manner. Especially, Compounds 1 and 2 selectively inhibited the growth of H-ras-transformed rat-2 cell lines in comparison with normal rat-2 cells with a GI50 value of 0.3 and 0.85 microM, respectively. Methyllucidone strongly inhibited the growth of human cancer cells and colon tumor xenografted in nude mice. The anti-tumor effects of the compound were further confirmed with caspase-3 activation and degradation of PARP. The results suggest that methyllucidone can be a potential anti-cancer agent against H-ras-transformed tumor and will also be a good lead molecule for the development of anti-tumor drug.

Diarylheptanoids [curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), bisdimethoxymethylcurcumin (4), and 1,2-dihydrobis(de-O-methyl)curcumin (5)] were isolated from the methanolic extract of Curcuma longa L. and a new cyclic diarylheptanoid (6) and a known Compound 7 were isolated from fruits of Alnus japonica Steud. Diarylheptanoids (1-3) inhibited farnesyl protein transferase (FPTase) with an IC50 of 29-50 microM. The other compounds very mildly inhibited FPTase, therefore, the inhibitory activity on FPTase very much depends on the structure of diarylheptanoids.

Flavonoids are well-known phytochemicals that are produced by various plants in high quantities. The chemopreventive activity of flavonoids is dependent on their structural features. The studies of structure-FPTase inhibitory activity indicated that the number, position and substitution of hydroxyl groups of the A and B rings of flavonoid, and unsaturation of the C2-C3 bond are important factors affecting inhibition on FPTase by flavonoids. A couple of flavonoids inhibited FPTase and also the growth of human tumor cell lines, especially butein, which strongly inhibited the growth of colon cancer cell line (HCT116). However, flavanones and flavanols did not inhibit FPTase nor the growth of tumor cells.

Members of the Artemisia genus are important medicinal plants found throughout the world. Arteminolides A–D ( 1 – 4 ), isolated from the aerial parts of Artemisia, have an inhibitory activity on farnesyl-protein transferase (FPTase; EC 2.5.1.29) in in vitro assay. This study was carried out with the purpose of validating anti-tumor effects of the compounds in human tumor cells and mouse xenograft model. The arteminolides inhibited tumor cell growth in a dose-dependent manner. Furthermore, arteminolide C ( 3 ) blocked in vivo growth of human colon and lung tumor xenograft without the loss of body weight in nude mice.

Cinnamaldehydes have been shown to have inhibitory effects on farnesyl protein transferase (FPTase; EC 2.5.1.29) in vitro, angiogenesis, cell-cell adhesion, and tumor cell growth and to be immunomodulators. However, the mechanisms responsible for these effects remain unknown. To elucidate the molecular mechanism of the cinnamaldehyde derivative CB403 for growth inhibition, CB403 was synthesized from 2'-hydroxycinnamaldehyde. CB403-treated cells were weakly adherent to the culture dishes. In addition, CB403 inhibited tumor growth in these cells in a concentration-dependent manner. FACS analysis using human cancer cells treated with this compound showed cell cycle arrest in mitosis, which was correlated with a marked increase in the amount of cyclin B1. Furthermore, CB403 blocked in vivo growth of human colon and breast tumor xenografts without loss of body weight in nude mice. These results support the hypothesis that the cinnamaldehyde derivative CB403 exerts cytostatic properties by inducing mitotic arrest in cancer cells.