Antibacterial and Antiviral Properties of Pinus densiflora Essential Oil.

The Korean mountains are home to the Korean red pine (Pinus densiflora). Pine needle oil has been used as a food additive and a traditional herbal medicine; however, any health-related properties of its trunk oil remain unknown. Herein, we assessed antibacterial and antiviral properties of essential oil extracted from the trunk of P. densiflora. Th extracted oil was hydrodistilled using a Clevenger apparatus and analyzed using gas chromatography-mass spectrometry. The antimicrobial activity of the oil was tested using the microbroth dilution technique against 10 bacterial species (6 g-positive and 4 g-negative) and fungi. The extract exerted strong antimicrobial activity against Vibrio parahaemolyticus, Bacillus cereus, Listeria monocytogenes, Propionibacterium acnes, and Malassezia furfur (minimum inhibitory concentration = 10 mL/L). Additionally, it exhibited dose-dependent activity against influenza virus A and feline coronavirus. Furthermore, among 20 identified constituents accounting for 98.7% of the oil contents, the major components included 3-cyclohexene-1-methanol (10.12%), 2-(4-methylcyclohexyl)-2-propanol (9.09%), fenchone (8.14%), O-isopropyltoluene (6.35%), and isothymol methyl ether (6.14%). The P. densiflora trunk essential oil showed antibacterial and antiviral activities that depended on its chemical composition and the microbial strains tested herein. The essential oil can be used as an antimicrobial agent and disinfectant.

Characteristics of rhizosphere and endogenous bacterial community of Ulleung-sanmaneul, an endemic plant in Korea: application for alleviating salt stress.

Microbes influence plant growth and fitness. However, the structure and function of microbiomes associated with rare and endemic plants remain underexplored. To investigate the bacterial community structure of Ulleung-sanmaneul (U-SMN), an endemic plant in Korea, samples were collected from natural and cultivated habitats, and their 16S rDNA was sequenced. The root bacterial community structure differed from those of bulk soil and rhizosphere in both habitats. Endogenous bacteria in cultivated plants were less diverse than wild plants, but Luteibacter rhizovicinus, Pseudomonas fulva, and Sphingomonas pruni were shared. Co-inoculation of Pseudoxanthomonas sp. JBCE485 and Variovorax paradoxus JBCE486 promoted growth and induced salt stress resistance in Arabidopsis and chive. Changes in growth promotion and phenotypes of plants by co-inoculation were mediated by increased auxin production. Each strain colonized the roots without niche competition. The results indicated that host selectivity was influential than environmental factors in formulating endophytic bacterial composition, and domestication simplified the bacterial community diversity. Our results will contribute to the growth and maintenance of endemic U-SMN plants.

Molecular Properties of ß-Carotene Oxygenases and Their Potential in Industrial Production of Vitamin A and Its Derivatives.

ß-Carotene 15,15'-oxygenase (BCO1) and ß-carotene 9',10'-oxygenase (BCO2) are potential producers of vitamin A derivatives, since they can catalyze the oxidative cleavage of dietary provitamin A carotenoids to retinoids and derivative such as apocarotenal. Retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it, and are essential nutrients for humans and highly valuable in the food and cosmetics industries. ß-carotene oxygenases (BCOs) from various organisms have been overexpressed in heterogeneous bacteria, such as Escherichia coli, and their biochemical properties have been studied. For the industrial production of retinal, there is a need for increased production of a retinal producer and biosynthesis of retinal using biocatalyst systems improved by enzyme engineering. The current review aims to discuss BCOs from animal, plants, and bacteria, and to elaborate on the recent progress in our understanding of their functions, biochemical properties, substrate specificity, and enzyme activities with respect to the production of retinoids in whole-cell conditions. Moreover, we specifically propose ways to integrate BCOs into retinal biosynthetic bacterial systems to improve the performance of retinal production.

Biogenesis and Lipase-Mediated Mobilization of Lipid Droplets in Plants.

Cytosolic lipid droplets (LDs) derived from the endoplasmic reticulum (ER) mainly contain neutral lipids, such as triacylglycerols (TAGs) and sterol esters, which are considered energy reserves. The metabolic pathways associated with LDs in eukaryotic species are involved in diverse cellular functions. TAG synthesis in plants is mediated by the sequential involvement of two subcellular organelles, i.e., plastids - plant-specific organelles, which serve as the site of lipid synthesis, and the ER. TAGs and sterol esters synthesized in the ER are sequestered to form LDs through the cooperative action of several proteins, such as SEIPINs, LD-associated proteins, LDAP-interacting proteins, and plant-specific proteins such as oleosins. The integrity and stability of LDs are highly dependent on oleosins, especially in the seeds, and oleosin degradation is critical for efficient mobilization of the TAGs of plant LDs. As the TAGs mobilize in LDs during germination and post-germinative growth, a plant-specific lipase-sugar-dependent 1 (SDP1)-plays a major role, through the inter-organellar communication between the ER and peroxisomes. In this review, we briefly recapitulate the different processes involved in the biogenesis and degradation of plant LDs, followed by a discussion of future perspectives in this field.

Antioxidant, Whitening, Antiwrinkle, and Anti-Inflammatory Effect of Ajuga spectabilis Nakai Extract.

Since ancient times, plants have been a good source of natural antioxidants. Plants remove active oxygen through antioxidants and contain various active ingredients. These active ingredients of plants are used to alleviate skin aging and chronic diseases. Ajuga spectabilis Nakai (AS) is a perennial plant, is endemic to Korea, and has the characteristics of alpine plants. The aim of this study was to assure the possibility of using AS as a functional natural and cosmetic material. For this, we carried out biologically activated material characteristic evaluations about antioxidant, wrinkle reduction, and anti-inflammatory effects using AS extract. To carry out this experiment, we extracted AS extract from AS water extract (AS-W) and AS 70% ethanol extract (AS-E). AS-E showed the highest DPPH activity and tyrosinase inhibitory activity. After, the measurement of metalloprotease (MMP)-1 inhibition effect showed the AS-W and AS-E activation at the concentration of 100 µg/mL. In addition, at the same concentration, from the result of the measurement of the biosynthesis quantity of pro-collagen type-1 we knew that its excellent effect appeared in AS-E (CCD-986sk). The inhibition of NO production in AS-W and AS-E was confirmed in LPS-induced mouse macrophage RAW264.7 cells. On cell viability, it was judged that AS-E had no toxicity because it showed a high cell viability at a high concentration, and it was used for the anti-inflammatory activity. Inhibition of NO production worked only in AS-E; inflammatory cytokine TNF-α and IL-6 were suppressed in a concentration-dependent manner in AS-E. AS is believed to be used as a natural cosmetic material because it has been proven to have antioxidant, whitening, wrinkle-improving, and anti-inflammatory effects. Therefore, the results indicate that AS extract can play an important role as a functional natural material and a cosmetic material for whitening, wrinkle reduction, and anti-inflammatory effect.

Improved production of deglucosylated platycodin D from saponins from balloon flower leaf by a food-grade enzyme using high hydrostatic pressure.

Platycosides, saponins contained in balloon flower, which have been used as food health supplements for respiratory diseases, have diverse pharmacological effects. Platycosides exhibit better pharmacological activity by hydrolyzing their own sugars. However, to date, there have been no studies on the production of deglucosylated platycodin D suitable for food applications. In this study, Pluszyme 2000P, which was derived from Aspergillus niger, a food-grade microorganism, was used to completely convert platycoside E into deglucosylated platycodin D. For an efficient and economical production of deglucosylated platycodin D, the productivity was improved approximately 2.4 times by application of high hydrostatic pressure and the discarded balloon flower leaf was used as a substrate. As a result, deglucosylated platycodin D was produced with the highest concentration (3.49 mg/mL) and productivity (581.7 mg/L/h) reported so far. Our results contribute to functional saponin production and the related food industries.

Production of D-Allose From D-Allulose Using Commercial Immobilized Glucose Isomerase.

Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can potentially provide a continuous production system for industrial applications of D-allose.

Synergistic nanoarchitecture of mesoporous carbon and carbon nanotubes for lithium-oxygen batteries.

A rechargeable lithium-oxygen battery (LOB) operates via the electrochemical formation and decomposition of solid-state Li2O2 on the cathode. The rational design of the cathode nanoarchitectures is thus required to realize high-energy-density and long-cycling LOBs. Here, we propose a cathode nanoarchitecture for LOBs, which is composed of mesoporous carbon (MPC) integrated with carbon nanotubes (CNTs). The proposed design has the advantages of the two components. MPC provides sufficient active sites for the electrochemical reactions and free space for Li2O2 storage, while CNT forests serve as conductive pathways for electron and offer additional reaction sites. Results show that the synergistic architecture of MPC and CNTs leads to improvements in the capacity (~ 18,400 mAh g- 1), rate capability, and cyclability (~ 200 cycles) of the CNT-integrated MPC cathode in comparison with MPC.

Phosphate sugar isomerases and their potential for rare sugar bioconversion.

Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-ribose-5-phosphate isomerase and D-mannose-6-phosphate isomerase to produce D-allose and L-ribose, respectively.

Bakkenolides and Caffeoylquinic Acids from the Aerial Portion of Petasites japonicus and Their Bacterial Neuraminidase Inhibition Ability.

Petasites japonicus have been used since a long time in folk medicine to treat diseases including plague, pestilential fever, allergy, and inflammation in East Asia and European countries. Bioactive compounds that may prevent and treat infectious diseases are identified based on their ability to inhibit bacterial neuraminidase (NA). We aimed to isolate and identify bioactive compounds from leaves and stems of P. japonicas (PJA) and elucidate their mechanisms of NA inhibition. Key bioactive compounds of PJA responsible for NA inhibition were isolated using column chromatography, their chemical structures revealed using 1 H NMR, 13 C NMR, DEPT, and HMBC, and identified to be bakkenolide B (1), bakkenolide D (2), 1,5-di-O-caffeoylquinic acid (3), and 5-O-caffeoylquinic acid (4). Of these, 3 exhibited the most potent NA inhibitory activity (IC50 = 2.3 ± 0.4 µM). Enzyme kinetic studies revealed that 3 and 4 were competitive inhibitors, whereas 2 exhibited non-competitive inhibition. Furthermore, a molecular docking simulation revealed the binding affinity of these compounds to NA and their mechanism of inhibition. Negative-binding energies indicated high proximity of these compounds to the active site and allosteric sites of NA. Therefore, PJA has the potential to be further developed as an antibacterial agent for use against diseases associated with NA.

Conversion of Glycosylated Platycoside E to Deapiose-Xylosylated Platycodin D by Cytolase PCL5.

Platycosides, the saponins abundant in Platycodi radix (the root of Platycodon grandiflorum), have diverse pharmacological activities and have been used as food supplements. Since deglycosylated saponins exhibit higher biological activity than glycosylated saponins, efforts are on to enzymatically convert glycosylated platycosides to deglycosylated platycosides; however, the lack of diversity and specificities of these enzymes has limited the kinds of platycosides that can be deglycosylated. In the present study, we examined the enzymatic conversion of platycosides and showed that Cytolase PCL5 completely converted platycoside E and polygalacin D3 into deapiose-xylosylated platycodin D and deapiose-xylosylated polygalacin D, respectively, which were identified by LC-MS analysis. The platycoside substrates were hydrolyzed through the following novel hydrolytic pathways: platycoside E → platycodin D3 → platycodin D → deapiosylated platycodin D → deapiose-xylosylated platycodin D; and polygalacin D3 → polygalacin D → deapiosylated polygalacin D → deapiose-xylosylated polygalacin D. Our results show that cytolast PCL5 may have a potential role in the development of biologically active platycosides that may be used for their diverse pharmacological activities.

Cloning and characterization of α-L-rhamnosidase from Chloroflexus aurantiacus and its application in the production of isoquercitrin from rutin.

OBJECTIVE: This study was conducted to characterize recombinant α-L-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin. RESULTS: The α-L-rhamnosidase from C. aurantiacus was cloned and expressed in Escherichia coli BL21 and purified as a soluble enzyme. α-L-rhamnosidase purified from C. aurantiacus has a molecular mass of approximately 105 kDa and is predicted to exist as a homodimer with a native enzyme of 200 kDa. The purified enzyme exhibited the highest specific activity for rutin among the reported isoquercitrin producing α-L-rhamnosidases and was applied in the production of isoquercitrin from rutin. Under the optimised conditions of pH 6.0, 50 °C, 0.6 U mL-1 α-L-rhamnosidase, and 30 mM rutin, α-L-rhamnosidase from C. aurantiacus produced 30 mM isoquercitrin after 2 h with a 100% conversion yield and productivity of 15 mM h-1. CONCLUSIONS: We achieved a high productivity of isoquercitrin from rutin. Moreover, these results suggest that α-L-rhamnosidase from C. aurantiacus is an effective isoquercitrin producer.

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs.

Avian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance.

Effect of high hydrostatic pressure treatment on isoquercetin production from rutin by commercial α-L-rhamnosidase.

OBJECTIVES: To optimize conversion of rutin to isoquercetin by commercial α-L-rhamnosidase using high hydrostatic pressure (HHP). RESULTS: The de-rhamnosylation activity of α-L-rhamnosidase for isoquercetin production was maximal at pH 6.0 and 50 °C using HHP (150 MPa). The enzyme showed high specificity for rutin. The specific activity for rutin at HHP was 1.5-fold higher than that at atmospheric pressure. The enzyme completely hydrolysed 20 mM rutin in tartary buckwheat extract after 2 h at HHP, with a productivity of 10 mM h(-1). The productivity and conversion were 2.2- and 1.5-fold higher at HHP than at atmospheric pressure, respectively. CONCLUSIONS: This is the first report concerning the enzymatic hydrolysis of isoquercetin in tartary buckwheat at HHP.

Characterization of a recombinant mannobiose 2-epimerase from Spirochaeta thermophila that is suggested to be a cellobiose 2-epimerase.

A purified recombinant enzyme from Spirochaeta thermophila, that is suggested to be a cellobiose 2-epimerase, was a 47 kDa monomer with a specific activity of 29.2 U min(-1) for mannobiose. The epimerization activity of the recombinant enzyme for mannobiose was maximal at pH 7.0 and 60 °C with a half-life of 124 h. The enzyme exhibited a higher epimerization activity for mannose or the mannose moiety at the reducing end of ß- and α-1,4-glycosyl-mannose than for glucose or the glucose moiety of ß- and α-1,4-glycosyl-glucose. The enzyme was identified as a mannobiose 2-epimerase by evaluating its substrate specificity with not only glucose-containing sugars but also mannose-containing sugars. The activities of the reported cellobiose 2-epimerases from Caldicellulosiruptor saccharolyticus, Dictyoglomus turgidum and Ruminococcus marinus for mannobiose were higher than those for cellobiose, strongly suggesting that these enzymes are not cellobiose 2-epimerases but are mannobiose 2-epimerases.

ß-glucosidase from Penicillium aculeatum hydrolyzes exo-, 3-O-, and 6-O-ß-glucosides but not 20-O-ß-glucoside and other glycosides of ginsenosides.

A novel ß-glucosidase from Penicillium aculeatum was purified as a single 110.5-kDa band on SDS-PAGE with a specific activity of 75.4 U mg⁻¹ by salt precipitation and Hi-Trap Q HP and Resource Q ion exchange chromatographies. The purified enzyme was identified as a member of the glycoside hydrolase 3 family based on its amino acid sequence. The hydrolysis activity for p-nitrophenyl-ß-D-glucopyranoside was optimal at pH 4.5 and 70 °C with a half-life of 55 h. The enzyme hydrolyzed exo-, 3-O-, and 6-O-ß-glucosides but not 20-O-ß-glucoside and other glycosides of ginsenosides. Because of the novel specificity, this enzyme had the transformation pathways for ginsenosides: Rb1 → Rd → F2 → compound K, Rb2 → compound O → compound Y, Rc → compound Mc1 → compound Mc, Rg3 → Rh2 → aglycone protopanaxadiol (APPD), Rg1 → F1, and Rf → Rh1 → aglycone protopanaxatriol (APPT). Under the optimum conditions, the enzyme converted 0.5 mM Rb2, Rc, Rd, Rg3, Rg1, and Rf to 0.49 mM compound Y, 0.49 mM compound Mc, 0.47 mM compound K, 0.23 mM APPD, 0.49 mM F1, and 0.44 mM APPT after 6 h, respectively.

Characterization of a recombinant L-rhamnose isomerase from Dictyoglomus turgidum and its application for L-rhamnulose production.

A putative recombinant enzyme from Dictyoglomus turgidum was characterized and immobilized on Duolite A568 beads. The native enzyme was a 46 kDa tetramer. Its activity was highest for L-rhamnose, indicating that it is an L-rhamnose isomerase. The maximum activities of both the free and immobilized enzymes for L-rhamnose isomerization were at pH 8.0 and 75 °C in the presence of Mn(2+). Under these conditions, the half-lives of the free and immobilized enzymes were 28 and 112 h, respectively. In a packed-bed bioreactor, the immobilized enzyme produced an average of 130 g L-rhamnulose l(-1) from 300 g L-rhamnose l(-1) after 240 h at pH 8.0, 70 °C, and 0.6 h(-1), with a productivity of 78 g l(-1) h(-1) and a conversion yield of 43 %. To the best of our knowledge, this is the first report describing the enzymatic production of L-rhamnulose.

Development of novel sugar isomerases by optimization of active sites in phosphosugar isomerases for monosaccharides.

Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases.

Borate enhances the production of lactulose from lactose by cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus.

Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus was used in the presence of borate to increase the production of lactulose from lactose. Maximum production of lactulose occurred using a 1:1M ratio of borate-lactose. Under this condition, the enzyme produced 614 g l(-1) lactulose from 700 g l(-1) lactose after incubation at pH 7.5 and 80 °C for 3h, with a conversion yield of 88% and a productivity of 205 g l(-1) h(-1). The yield and productivity of lactulose production obtained in the present study are among the highest achieved through chemical or biological synthesis.

Characterization of a recombinant cellobiose 2-epimerase from Dictyoglomus turgidum that epimerizes and isomerizes ß-1,4- and α-1,4-gluco-oligosaccharides.

A recombinant putative N-acyl-D-glucosamine 2-epimerase from Dictyoglomus turgidum was identified as a cellobiose 2-epimerase by evaluating its substrate specificity. The purified enzyme was a 46 kDa monomer with a specific activity of 16.8 µmol min(-1) mg(-1) for cellobiose. The epimerization activity was maximal at pH 7.0 and 70 °C with a half-life of 55 h. The isomerization of the glucose at the reducing end of ß-1,4- and α-1,4-linked gluco-oligosaccharides to a fructose moiety by the enzyme took place after the epimerization of the glucose to a mannose moiety. The enzyme converted cellobiose to 12.8 % 4-O-ß-D-glucopyranosyl-D-mannose and 54.6 % 4-O-ß-D-glucopyranosyl-D-fructose as an equilibrium and converted lactose to 12.8 % epilactose and 54.3 % lactulose.