In the present work, we isolated and identified Aspergillus sydowii NYKA 510 as the most potent laccase producer. Its medium constituents were optimized to produce the highest possible amount of laccase, which was after 7 days at 31℃ and pH 5.2. Banana peel and peptone excelled in inducing laccase production at concentrations of 15.1 and 2.60 g/l, respectively. Addition of copper sulfate elevated enzyme yield to 145%. The fungus was employed in a microbial fuel cell (MFC). The best performance was obtained at 2000 Ω achieving 0.76 V, 380 mAm^-2, 160 mWm^-2, and 0.4 W. A project to design a self-sufficient lighting unit was implemented by employing a system of 2 sets of 4 MFCs each, connected in series, for electricity generation. A scanning electron microscopy image of A. sydowii NYKA 510 was utilized in algorithmic form generation equations for the design. The mixed patterning and patterned customized mass approach were developed by the authors and chosen for application in the design.

The innate immune response serves as a first-line-of-defense mechanism for a host against viral infection. Viruses must therefore subvert this anti-viral response in order to establish an efficient life cycle. In line with this fact, Kaposi's sarcoma-associated herpesvirus (KSHV) encodes numerous genes that function as immunomodulatory proteins to antagonize the host immune system. One such mechanism through which KSHV evades the host immunity is by encoding a viral homolog of cellular interferon (IFN) regulatory factors (IRFs), known as vIRFs. Herein, we summarize recent advances in the study of the immunomodulatory strategies of KSHV vIRFs and their effects on KSHV-associated pathogenesis.

β-Glucosidases and β-xylosidases are two categories of enzymes that could cleave out non-reducing, terminal β-D-glucosyl and β-D-xylosyl residues with release of D-glucose and D-xylose, respectively. In this paper, two functional β-glucosidase Dth3 and β-xylosidase Xln-DT from Dictyoglomus thermophilum were heterologously expressed in E.coli BL21 (DE3). Dth3 and Xln-DT were relatively stable at 75℃ and were tolerant or even stimulated by glucose and xylose. Dth3 was highly tolerant to glucose with a K_i value of approximately 3 M. Meanwhile, it was not affected by xylose in high concentration. The activity of Xln-DT was stimulated 2.13-fold by 1 M glucose and 1.29-fold by 0.3 M xylose, respectively. Furthermore, the βglucosidase Dth3 and β-xylosidase Xln-DT showed excellent selectivity to cleave the outer C-6 and C-3 sugar moieties of ASI, which established an effective and green method to produce the more pharmacologically active CAG, an exclusive telomerase activator. We measured temperature, pH and dosage of enzyme using a single-factor experiment in ASI biotransformation. After optimization, the optimal reaction conditions were as follows: 75℃, pH 5.5, 1 U of Dth3 and 0.2 U of Xln-DT, respectively. Under the optimized conditions, 1 g/l ASI was transformed into 0.63 g/l CAG with a corresponding molar conversion of 94.5% within 3 h. This is the first report to use the purified thermostable and sugar-tolerant enzymes from Dictyoglomus thermophilum to hydrolyze ASI synergistically, which provides a specific, environment-friendly and cost-effective way to produce CAG.

The purpose of this study was to determine the probiotic properties of Lactobacillus brevis KCCM 12203P isolated from the Korean traditional food kimchi and to evaluate the antioxidative activity and immune-stimulating potential of its heat-killed cells to improve their bio-functional activities. Lactobacillus rhamnosus GG, which is a representative commercial probiotic, was used as a comparative sample. Regarding probiotic properties, L. brevis KCCM 12203P was resistant to 0.3% pepsin with a pH of 2.5 for 3 h and 0.3% oxgall solution for 24 h, having approximately a 99% survival rate. It also showed strong adhesion activity (6.84%) onto HT-29 cells and did not produce β-glucuronidase but produced high quantities of leucine arylamidase, valine arylamidase, β-galactosidase, and N-acetyl-β-glucosaminidase. For antioxidant activity, it appeared that viable cells had higher radical scavenging activity in the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, while in the 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, heat-killed cells had higher antioxidant activity. Additionally, L. brevis KCCM 12203P showed higher lipid oxidation inhibition ability than L. rhamnosus GG; however, there was no significant difference (p < 0.05) between heat-killed cells and control cells. Furthermore, heat-killed L. brevis KCCM 12203P activated RAW 264.7 macrophage cells without cytotoxicity at a concentration lower than 10⁸ CFU/ml and promoted higher gene expression levels of inducible nitric oxide synthase, interleukin-1β, and interleukin-6 than L. rhamnosus GG. These results suggest that novel L. brevis KCCM 12203P could be used as a probiotic or applied to functional food processing and pharmaceutical fields for immunocompromised people.

Resistant starch (RS) is metabolized by gut microbiota and involved in the production of short-chain fatty acids, which are related to a variety of physiological and health effects. Therefore, the availability of RS as a prebiotic is a topic of interest, and research on gut bacteria that can decompose RS is also important. The objectives in this study were 1) to isolate a human gut bacterium having strong degradation activity on non-gelatinized RS, 2) to characterize its RS-degrading characteristics, and 3) to investigate its probiotic effects, including a growth stimulation effect on other gut bacteria and an immunomodulatory effect. Bifidobacterium adolescentis P2P3 showing very strong RS granule utilization activity was isolated. It can attach to RS granules and form them into clusters. It also utilizes high-amylose corn starch granules up to 63.3%, and efficiently decomposes other various types of commercial RS without gelatinization. In a coculture experiment, Bacteroides thetaiotaomicron ATCC 29148, isolated from human feces, was able to grow using carbon sources generated from RS granules by B. adolescentis P2P3. In addition, B. adolescentis P2P3 demonstrated the ability to stimulate secretion of Th1 type cytokines from mouse macrophages in vitro that was not shown in other B. adolescentis. These results suggested that B. adolescentis P2P3 is a useful probiotic candidate, having immunomodulatory activity as well as the ability to feed other gut bacteria using RS as a prebiotic.

Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine. Here, we isolated an endolysin LysSAP8, as one of the peptidoglycan hydrolases, derived from the bacteriophage SAP8 infecting S. aureus. This endolysin was tagged with a 6×His at the C-terminal of the target protein and purified using affinity chromatography. LysSAP8 demonstrated lytic activity against a broad spectrum of bacteria, which included a majority of the staphylococcal strains tested in this study as well as the methicillin-resistant S. aureus (MRSA); however, no such activity was observed against other gram-positive or gram-negative bacteria. Additionally, LysSAP8 could maintain bactericidal activity until 0.1 nM working concentration and after heat treatment at 37℃ for 30 min. The ability of LysSAP8 to lyse cells under varying conditions of temperature (4-43℃), pH (3-9), and NaCl concentrations (0-1,000 mM), and divalent metal ions (Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Hg²⁺, and Zn²⁺) was examined. At the optimized condition, LysSAP8 could disrupt approximately 3.46 log CFU/ml of the planktonic cells in their exponential phase of growth within 30 min. In this study, we have suggested that LysSAP8 could be a potent alternative as a biocontrol agent that can be used to combat MRSA.

Carotenoids are organic pigments with antioxidant properties and are widespread in nature. Here, we isolated five microbes, each forming yellow-colored colonies and harboring C₃₀ carotenoid biosynthetic genes (crtM and crtN). Thereafter, Lactobacillus plantarum subsp. plantarum KCCP11226, which showed the highest carotenoid production, was finally selected and the produced pigment was identified as C₃₀ carotenoid 4,4'-diaponeurosporene. This strain exhibited the highest survival rate under oxidative stress and its carotenoid production was also enhanced after exposure to 7 mM H₂O₂. Moreover, it showed the highest ability to scavenge DPPH free radical. Our results suggested that L. plantarum subsp. plantarum KCCP11226, which produces 4,4'-diaponeurosporene as a natural antioxidant, may be a functional probiotic.

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.

Isomaltooligosaccharides (IMOs) have good prebiotic effects, and long IMOs (LIMOs) with a degree of polymerization (DP) of 7 or above show improved effects. However, they are not yet commercially available, and require costly enzymes and processes for production. The N-terminal region of the thermostable Thermoanaerobacter thermocopriae cycloisomaltooligosaccharide glucanotransferase (TtCITase) shows cyclic isomaltooligosaccharide (CI)-producing activity owing to a catalytic domain of glycoside hydrolase (GH) family 66 and carbohydrate-binding module (CBM) 35. In the present study, we elucidated the activity of the C-terminal region of TtCITase (TtCITase-C; Met740-Phe1,559), including a CBM35-like region and the GH family 15 domain. The domain was successfully cloned, expressed, and purified as a single protein with a molecular mass of 115 kDa. TtCITase-C exhibited optimal activity at 40℃ and pH 5.5, and retained 100% activity at pH 5.5 after 18-h incubation. TtCITase-C synthesized α-1,6 glucosyl products with over seven degrees of polymerization (DP) by an α-1,6 glucosyl transfer reaction from maltopentaose, isomaltopentaose, or commercialized maltodextrins as substrates. These results indicate that TtCITase-C could be used for the production of α-1,6 glucosyl oligosaccharides with over DP7 (LIMOs) in a more cost-effective manner, without requiring cyclodextran.

The gut microbiome influences the health and well-being of dogs. However, little is known about the impact of breed on the fecal microbiome composition in dogs. Therefore, we aimed to investigate the differences in the fecal microbiome in three breeds of dog fed and housed under the same conditions, namely eight Maltese (8.0 ± 0.1 years), eight Miniature Schnauzer (8.0 ± 0.0 years), and nine Poodle dogs (8.0 ± 0.0 years). Fresh fecal samples were collected from the dogs and used to extract metagenomic DNA. The composition of the fecal microbiome was evaluated by 16S rRNA gene amplicon sequencing on the MiSeq platform. A total of 840,501 sequences were obtained from the 25 fecal samples and classified as Firmicutes (32.3-97.3% of the total sequences), Bacteroidetes (0.1-62.6%), Actinobacteria (0.2-14.7%), Fusobacteria (0.0-5.7%), and Proteobacteria (0.0-5.1%). The relative abundance of Firmicutes was significantly lower in the Maltese dog breed than that in the other two breeds, while that of Fusobacteria was significantly higher in the Maltese than in the Miniature Schnauzer breed. At the genus level, the relative abundance of Streptococcus, Fusobacterium, Turicibacter, Succinivibrio, and Anaerobiospirillum differed significantly among the three dog breeds. These genera had no correlation with age, diet, sex, body weight, vaccination history, or parasite protection history. Within a breed, some of these genera had a correlation with at least one blood chemistry value. This study indicates that the composition of the fecal microbiome in dogs is affected by breed.

Tetraselmis is a green algal genus, some of whose species are important in aquaculture as well as biotechnology. In algal culture, fluorescent lamps, traditional light source for culturing algae, are now being replaced by a cost-effective light-emitting diodes (LEDs). In this study, we investigated the effect of LED light of different wavelengths (white, red, yellow, and blue) on the growth of Tetraselmis suecica and its associated microbial community structures using the next-generation sequencing (NGS). The fastest growth rate of T. suecica was shown in the red light, whereas the slowest was in yellow. The highest OTUs (3426) were identified on day 0, whereas the lowest ones (308) were found on day 15 under red light. The top 100 OTUs associated with day 0 and day 5 cultures of T. suecica under the red and yellow LED were compared. Only 26 OTUs were commonly identified among four samples. The highest numbers of unique OTUs were identified at day 0, indicating the high degree of initial microbial diversity of the T. suecica inoculum. The red light-unique OTUs occupied 34.98%, whereas the yellow-specific OTUs accounted for only 2.2%. This result suggested a higher degree of interaction in T. suecica culture under the red light, where stronger photosynthesis occurs. Apparently, the microbial community associated with T. suecica related to the oxygen produced by algal photosynthesis. This result may expand our knowledge about the algae-bacteria consortia, which would be useful for various biotechnological applications including wastewater treatment, bioremediation, and sustainable aquaculture.

A Gram-staining-negative, aerobic, light brown pigment bacterium, designated strain CE80^T was isolated from marine sponge Callyspongia elegans in Jeju Island, Republic of Korea. Strain CE80^T grew optimally at 25℃, in the range of pH 5.0-11.0 (optimum 7.0-8.0), and with 1.0-5.0% NaCl (optimum 1-3% (w/v)). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain CE80^T belonged to the genus Labrenzia and was closely related to L. suaedae YC6927^T (98.3%), L. alexandrii DFL-11^T (96.6%), L. aggregata IAM 12614^T (96.6%) L. marina mano18^T (96.5%) and L. alba CECT 5094^T (96.2%). The major fatty acids of strain CE80^T were C_18:1 ω7c, and summed feature. The polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamin, one unidentified aminolipid, one phospholipid and four unidentified lipids. The DNA G+C content of strain CE80^T was 55.9 mol%. The major respiratory quinone was Q-10. DNA-DNA relatedness between strain CE80^T and L. suaedae YC6927^T was 56.1±2.8%. On the basis of physiological and biochemical characterization and phylogenetic and chemotaxonomic analysis, strain CE80^T represents a novel species of the Labrenzia, for which the name Labrenzia callyspongiae sp. nov., is proposed. The type strain is CE80^T (=KCTC 42849^T =JCM 31309^T).

Recently, outbreaks of food-borne diseases linked to fresh produce have been an emerging public health concern worldwide. Previous research has shown that when human pathogens co-exist with plant pathogens, they have improved growth and survival rates. In this study, we have assessed whether Escherichia coli O157:H7 benefits from the existence of a phytopathogenic bacterium and the underlying mechanisms were further investigated. When Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and E. coli O157:H7 were co-inoculated by either dipping or infiltration methods, the populations of E. coli O157:H7 increased; however, no effect was observed when type three secretion system (T3SS) mutants were used instead, suggesting that E. coli O157:H7 benefits from the presence of Pst DC3000. In addition, this study confirmed that the E. coli O157:H7 populations increased when they occupied the tomato leaf intercellular space; this colonization of the interior of the leaves was possible due to the suppression of the PAMP-triggered immunity (PTI) by Pst DC3000, in particular with the AvrPto effector. In conclusion, our data support a plausible model that E. coli O157:H7 benefits from the presence of Pst DC3000 via AvrPto suppression of the PTI resistance.

The alkaliphilic, calcium carbonate precipitating Bacillus sp. strain AK13 can be utilized in concrete for self-repairing. A statistical experimental design was used to develop an economical medium for its mass cultivation and sporulation. Two types of screening experiment were first conducted to identify substrates that promote the growth of the AK13 strain: the first followed a one-factor-at-a-time factorial design and the second a two-level full factorial design. Based on these screening experiments, barley malt powder and mixed grain powder were identified as the substrates that most effectively promoted the growth of the AK13 strain from a range of 21 agricultural products and by-products. A quadratic statistical model was then constructed using a central composite design and the concentration of the two substrates was optimized. The estimated growth and sporulation of Bacillus sp. strain AK13 in the proposed medium were 3.08 ± 0.38 × 10⁸ and 1.25 ± 0.12 × 10⁸ CFU/ml, respectively, which meant that the proposed low-cost medium was approximately 45 times more effective than the commercial medium in terms of the number of cultivatable bacteria per unit price. The spores were then powdered via a spray-drying process to produce a spore powder with a spore count of 2.0 ± 0.7 × 10⁹ CFU/g. The AK13 spore powder was mixed with cement paste, yeast extract, calcium lactate, and water. The yeast extract and calcium lactate generated the highest CFU/ml for AK13 at a 0.4:0.4 ratio compared to 0.4:0.25 (the original ratio of the B4 medium) and 0.4:0.8. Twenty-eight days after the spores were mixed into the mortar, the number of vegetative cells and spores of the AK13 strain had reached 10⁶ CFU/g within the mortar. Cracks in the mortar under 0.29 mm were healed in 14 days. Calcium carbonate precipitation was observed on the crack surface. The mortar containing the spore powder was thus concluded to be effective in terms of healing micro-cracks.

Thermophilic anaerobic digestion (TAD) is characterized by higher biogas production rates as a result of assumedly faster microbial metabolic conversion rates compared to mesophilic AD. It was hypothesized that the thermophilic microbiome with its lower diversity than the mesophilic one is more susceptible to disturbances introduced by alterations in the operating factors, as an example, the supply of nitrogen-rich feedstock such as poultry manure (PM). Laboratory scaled TAD experiments using cattle slurry and increasing amounts of PM were carried out to investigate the (in-) stability of the process performance caused by the accumulation of ammonium and ammonia with special emphasis on the microbial community structure and its dynamic variation. The results revealed that the moderate PM addition, i.e., 25% (vol/vol based on volatile substances) PM, resulted in a reorganization of the microbial community structure which was still working sufficiently. With 50% PM application, the microbial community was further stepwise re-organized and was able to compensate for the high cytotoxic ammonia contents only for a short time resulting in consequent process disturbance and final process failure. This study demonstrated the ability of the acclimated thermophilic microbial community to tolerate a certain amount of nitrogen-rich substrate.

The isolation of respiratory viruses, especially from clinical specimens, often shows poor efficiency with classical cell culture methods. The lack of suitable methods to generate virus particles inhibits the development of diagnostic assays, treatments, and vaccines. We compared three inoculation methods, classical cell culture, the addition of a JAK2 inhibitor AZD1480, and centrifugation-enhanced inoculation (CEI), to replicate human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). In addition, a combined method using AZD1480 treatment and CEI was used on throat swabs to verify that this method could increase virus isolation efficiency from human clinical specimens. Both CEI and AZD1480 treatment increased HRSV and HMPV genome replication. Also, the combined method using CEI and AZD1480 treatment enhanced virus proliferation synergistically. The combined method is particularly suited for the isolation of interferon-sensitive or slowly growing viruses from human clinical specimens.

Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to the genus Betacoronavirus and causes severe morbidity and mortality in humans especially when infected patients have underlying diseases such as chronic obstructive pulmonary disease (COPD). Previously, we demonstrated that MERS-CoV-encoded ORF8b strongly inhibits MDA5- and RIG-I-mediated induction of the interferon beta (IFN-β) promoter activities. Here, we report that ORF8b seemed to regulate MDA5 or RIG-I differentially as protein levels of MDA5 were significantly down-regulated while those of RIG-I were largely unperturbed. In addition, ORF8b seemed to efficiently suppress phosphorylation of IRF3 at the residues of 386 and 396 in cells transfected with RIG-I while total endogenous levels of IRF3 remained largely unchanged. Furthermore, ORF8b was able to inhibit all forms of RIG-I; full-length, RIG-I-1-734, and RIG-I-1-228, the last of which contains only the CARD domains. Taken together, it is tempting to postulate that ORF8b may interfere with the CARD-CARD interactions between RIG-I and MAVS. Further detailed analysis is required to delineate the mechanisms of how ORF8b inhibits the MDA5/RIG-I receptor signaling pathway.

Several animal species including pigs are directly involved in the zoonotic transmission of the hepatitis E virus (HEV) to humans. This study was conducted to detect HEV in bovine and porcine raw livers by nested reverse transcription-polymerase chain reaction. Zoonotic HEV strains were identified in 1.0 and 3.0% of the tested bovine and porcine livers, respectively. HEV-4 was detected in the bovine livers, but both HEV-3 and HEV-4 were identified in the porcine livers. These results indicate that zoonotic transmission of HEV may occur via consumption of raw or undercooked livers of pigs and cattle.