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Abstract: The α-amylase is one of the most promising commercial enzymes with tremendous applications in various industries. Microbial α-amylase shares almost 25-30% of the enzyme market due to its catalytic function in several industries, including sugar, detergent, paper, textile, pharmaceutical industries, etc. The α-amylase hydrolyzes glycosidic linkages of structural components of starch, resulting in maltose, glucose, and high fructose syrups. Starch, the second most abundant organic substance on the Earth, is a readily available, low-cost renewable substrate mainly used in biorefinery and food industries. Amylases are ubiquitous in nature due to their involvement in carbohydrate metabolism. The α-amylases of microbial origin have technical advantages as compared to animal and plant origin. Considering physicochemical properties, bacterial α-amylases are most diverse. However, for industrial purposes, these properties of the biocatalyst, either individually or in a combination, are required to modify through genetic and protein engineering according to the targeted process. The review presents an overview of the current findings of microbial sourced α- amylases, commercial applications, market trends in relevant industries, and achieved improvements in thermostability, catalytic function, pH tolerance, substrate, and product specificities through recombinant DNA technology and protein engineering.

Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 A resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+ , Ca+ , and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

With the progress of biotechnological research and improvements made in bioprocessing with pure cultures, microbial consortia have gained recognition for accomplishing biological processes with improved effectiveness. Microbes are indispensable tool in developing bioprocesses for the production of bioenergy and biochemicals while utilizing renewable resources due to technical, economic and environmental advantages. They communicate with specific cohorts in close proximity to promote metabolic cooperation. Use of positive microbial associations has been recognized widely, especially in food industries and bioremediation of toxic compounds and waste materials. Role of microbial associations in developing sustainable energy sources and substitutes for conventional fuels is highly promising with many commercial prospects. Detoxification of chemical contaminants sourced from domestic, agricultural and industrial wastes has also been achieved through microbial catalysis in pure and co-culture systems. Methanotrophs, the sole biological sink of greenhouse gas methane, catalyze the methane monooxygenasemediated oxidation of methane to methanol, a high energy density liquid and key platform chemical to produce commodity chemical compounds and their derivatives. Constructed microbial consortia have positive effects, such as improved biomass, biocatalytic potential, stability etc. In a methanotroph-heterotroph consortium, non-methanotrophs provide key nutrient factors and alleviate the toxicity from the culture. Non-methanotrophic organisms biologically stimulate the growth and activity of methanotrophs via production of growth stimulators. However, methanotrophs in association with co-cultured microorganisms are in need of further exploration and thorough investigation to study their interaction mode and application with improved effectiveness.

In this study, we investigated the potential of Paenibacillus kribbensis CU01 in producing fusaricidin, a strong antifungal substance, via optimization of metal ions and carbon and nitrogen source, and continuous fermentation. In the cultivation of a 2-l batch, maximal production of fusaricidins (581 mg l−1) was achieved in a modified M9 medium containing metal ions, 10 g l−1 glucose, and 1 g l−1 ammonium chloride. Most of glucose was consumed at a rate of 0.74 g l−1 h−1 within 24 h and fusaricidin production began 15 h after batch cultivation. Continuous fermentation was performed using a 7-l fermenter with 2-l working volume of modified M9 medium containing 10 g l−1 glucose, 1 × 10−3 M FeSO4, and 1 × 10−6 M MnCl2. After 24 h of the start of cultivation, fresh M9 medium was continuously supplied at a flow rate of 2.5 ml min−1, and simultaneously, the same amount of cell culture broth was removed. In a continuous system, the highest fusaricidin concentration (579 mg l−1) was obtained using a dilution rate of 0.075 h−1 with an average productivity of 10.4 mg l−1 h−1 for 24 to 72 h of incubation. Based on these results, it was found that fusaricidin production using P. kribbens CU01 strain increased by at least 28 times the values reported in previous studies.

Methylomonas koyamae LM6 is a potential methanotrophic bacterium of interest for methane bioconversion. Here, we report the complete genome sequence of M. koyamae LM6, which contains 4,337 predicted open reading frames on one chromosome (4,894,002 bp) and one plasmid (186,658 bp), with genes involved in methane oxidation.

Several methods including microwave, Frenchpress, autoclave, bead-beating, ultrasonication, and osmotic shock were compared to identify the most effective microalgal cell disruption method. Botryococcus braunii LB572 was cultured in 5 L flasks containing JM medium mixed with oceanic sediment extract for 13 days. Among the methods tested, enhanced lipid extraction was achieved through microwave treatment (2450MHz, 1250W at 150°C for 20 min). Oleic (C18:1), linolenic (C18:3), and palmitic acids (C16:0) were found to be the major fatty acids among the C14-C24 acids from extracted lipid. In addition, the optimal conditions of transesterification were as follows: 70 mL of methanol, 6 mL of sulfuric acid, 8 mL of chloroform, and boiling at 100°C for 30 min; 85.4% of C14-C24 FAME and 78.5% of C16-C18 FAME were esterified from transesterifiable lipids.

This study demonstrates the effects of growth stimulators in oceanic sediment on biomass and lipid production of two oleaginous microalgae, Botryococcus braunii LB572 and Phaeodactylum tricornutum B2089. At the optimal mixing ratio of culture medium and oceanic sediment extract of 6:4 (v/v), specific growth rates of B. braunii LB572 and P. tricornutum B2089 increased 13.0- and 11.3-fold, respectively, compared to a sediment-free medium. The maximum biomass and lipid productions of B. braunii LB572 were 5.54 and 3.09 g L−1, and those of P. tricornutum B2089 were 6.41 and 3.61 g L−1, respectively, indicating that biomass and lipid production in both microalgae increased at least 6- and 8-fold, respectively. Thus, their cultivation time was reduced by at least 6 days. A positive effect of nitrate in the sediment on biomass and lipid production was not found. Fe3+ and Ca2+ promoted biomass and lipid production as their concentrations increased. However, metal ion concentration is not as critical to biomass and lipid production as humic acid, a chelating substance to enhance bioavailability of metal ions to the microalgae. The optimal humic acid concentration for maximal biomass and lipid production was 80 mg L−1, which is the concentration contained in the culture medium mixed with sediment extract at a ratio of 6:4 (v/v). Thus, low-cost oceanic sediment can supply sufficient growth stimulators, especially humic acid, for mass production of biomass and lipid in both microalgae.

MxaJ is a component of type II methanol dehydrogenase (MDH) that mediates electron transfer during methanol oxidation in methanotrophic bacteria. However, little is known about how MxaJ structurally cooperates with MDH and Cytochrome cL . Here, we report for the first time the crystal structure of MxaJ. MxaJ consists of eight α-helices and six β-strands, and resembles the "bi-lobate" folding architecture found in periplasmic binding proteins. Distinctive features of MxaJ include prominent loops and a β-strand around the hinge region supporting the ligand-binding cavity, which might provide a more favorable framework for interacting with proteins rather than small molecules. Proteins 2017; 85:1379-1386. © 2017 Wiley Periodicals, Inc.

We examined the effects of the algicide thiazolidinedione (TD49) and yellow clay on Chattonella marina and assessed their ecological risk for the entire planktonic community. Mesocosm (1000 L) exposure experiments were employed to investigate time-course responses over 9 days. The growth of C. marina was controlled at ≥0.4 μM TD49 but not inhibited in yellow clay treatments. Although the algicidal activity of the 0.4-μM TD49 + 0.4 kg t−1 yellow clay treatment for C. marina was high (72.5 % at 24 h), target alga regrowth occurred. In all treatments, inorganic nutrients such as nitrate + nitrite and phosphate decreased following commencement of the experiment but were >1 μM (limitation concentration) at days 5 and 6, even though consumption pattern of those nutrients was influenced by the TD49 concentration. Depletion of silicate in initial stages played an important role in controlling the shift from diatoms including Chaetoceros and Skeletonema spp. to cryptophytes. Zooplankton were not affected by even the highest the yellow clay treatments and TD concentration of

In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP–purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

Cyclic lipopeptides were produced by Bacillus amyloliquefaciens subsp. plantarum strain BC32-1 that was isolated from yellow loess soil in the Jeonnam province of South Korea. Several lipopeptides were isolated from the bacteria using organic solvent extraction and reverse-phase high-performance liquid chromatography (RP-HPLC). Purified iturin-, surfactin-, and fengycin-type lipopeptides were identified using liquid chromatographymass spectrometry (LC-MS) analysis. Among the lipopeptides, C17-fengycin B showed strong antifungal activity against the phytopathogenic fungus, Fusarium oxysporum f. sp. radicis-lycopersici, and then the fengycin was further characterized by UV, Fourier transform-infrared spectroscopy (FT-IR), and LC-MS/MS analyses. C17-fengycin B was highly produced at quantities of up to 15 µg/mL at 37°C, whereas little amount of the fengycin was produced at 25°C. Purified C17-fengycin B inhibited mycelial growth of F. oxysporum with a minimal inhibitory concentration of 50 µg/mL. This study suggests that C17-fengycin B is a major antifungal component produced by the BC32-1 strain that could be used as an environmentally friendly agent to control the phytopathogenic F. oxysporum.

Natural antibiotics produced by microorganisms have generated considerable interest in various industries. Here we report the potential of antifungal lipopeptides produced by Bacillus amyloliquefaciens subsp. plantarum BC32-1 isolated from ultisols. The BC32-1 strain strongly inhibited mycelial growth of plant pathogenic fungi. Several lipopeptides were purified by organic solvent extraction and reversed-phase high-performance liquid chromatography (RP-HPLC). The purified lipopeptides contained iturin, fengycin, and surfactin-type lipopeptides, as confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Since fengycin-type lipopeptides showed strong antifungal activity, we characterized the chemical structures of fengycin A and B using LC-MS/MS and 2D NMR spectroscopy. This study will provide the structural basis for the development of new antifungal agents for the control of pathogenic fungal diseases.

A novel hexavalent chromium (CrVI)-removing Bacillus sp. was isolated from leather industry wastewatercontaminated soil. This potential isolate was subjected to Cr(VI) removal under free and immobilized states in a stirred batch reactor (SBR). Two biokinetic parameters, Vmax and Km, and the effective diffusivity (De) for various bead sizes were calculated from Lineweaver-Burk and Eadie-Hoftsee plots, respectively. With respect to bead size, De decreased significantly from a maximum of 3.024 × 10−6 cm2/sec in the 0.20 cm bead to 2.948 × 10−6, 1.775 × 10−7, and 1.144 × 10−7 cm2/sec in the 0.40-, 0.60- and 0.80 cm beads, respectively. Additionally, steady and unsteady state modeling of diffusional mass transfer into the immobilized beads was conducted to determine the mass transfer rate as a function of time and the beads’ radial profile. Furthermore, the space-time yield (STY) was modeled according to the residence time of the reactor. The reactor’s STY was reasonable and could be further boosted by increasing the fractional biocatalyst.

The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MPT (MDH Mas ), was determined at 1.7 A resolution. The active form of MDH Mas (or MDHI Mas ) is a heterotetrameric α2β2, where each β-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two β-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a β-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg2+ ion, instead of the Ca2+ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the β-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHI Mas integrity in the sea water environment to provide a firm basis for complex formation with MxaJ Mas or Cyt cL. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.

Harmful algal blooms (HABs) can lead to substantial socio-economic losses and extensive damage to aquatic ecosystems, drinking water sources and human health. Common algicidal techniques, including ozonation, ultrasonic treatment and dispersion of algae-killing chemicals, are unsatisfactory both economically and ecologically. This study therefore presents a novel alternative strategy for the efficient control of deleterious algae via the use of host-specific virus-like particles (VLPs) combined with chemically synthesized algicidal compounds. The capsid protein of HcRNAV34, a single-stranded RNA virus that infects the toxic dinoflagellate, Heterocapsa circularisquama, was expressed in and purified from Escherichia coli and then self-assembled into VLPs in vitro. Next, the algicidal compound, thiazolidinedione 49 (TD49), was encapsidated into HcRNAV34 VLPs for specific delivery to H. circularisquama. Consequently, HcRNAV34 VLPs demonstrated the same host selectivity as naturally occurring HcRNAV34 virions, while TD49-encapsidated VLPs showed a more potent target-specific algicidal effect than TD49 alone. These results indicate that target-specific VLPs for the delivery of cytotoxic compounds to nuisance algae might provide a safe, environmentally friendly approach for the management of HABs in aquatic ecosystems.

Azo dyes are recalcitrant and xenobiotic nature makes these compounds a challenging task for continuous biodegradation up to satisfactorily levels in large-scale. In the present report, the biodegradation efficiency of alginate immobilized indigenous Aeromonas sp. MNK1 on Methyl Orange (MO) in a packed bed reactor was explored. The experimental results were used to determine the external mass transfer model. Complete MO degradation and COD removal were observed at 0.20 cm bead size and 120 ml/h flow rate at 300 mg/l of initial dye concentration. The degradation of MO decreased with increasing bead sizes and flow rates, which may be attributed to the decrease in surface of the beads and higher flux of MO, respectively. The experimental rate constants (k ps) for various beads sizes and flow rates were calculated and compared with theoretically obtained rate constants using external film diffusion models. From the experimental data, the external mass transfer effect was correlated with a model J D = K Re (-(1 - n)). The model was tested with K value (5.7) and the Colburn factor correlation model for 0.20, 0.40 and 0.60 bead sizes were J D = 5.7 Re (-0.15), J D = 5.7 Re (-0.36) and J D = 5.7 Re (-0.48), respectively. Based on the results, the Colburn factor correlation models were found to predict the experimental data accurately. The proposed model was constructive to design and direct industrial applications in packed bed reactors within acceptable limits.

In order to evaluate the potential to control the fish-killing dinoflagellate Cochlodinium polykrikoides, we compared the algicidal effects of the thiazolidinedione derivative TD49 with those of yellow clay in 10-L microcosms. The responses of higher trophic level marine organisms and microbial loop communities to the algicide were also evaluated. In the yellow clay treatments, the concentration of C. polykrikoides was slightly reduced at day 1 of the experiment but remained higher than that of the control, suggesting that the reduction ratio of C. polykrikoides was

Thiazolidinedione (TD) derivatives have been found to have an algicidal effect on harmful algal bloom microalgae. In this study, 75 TD derivatives were synthesized and analyzed for algicidal activity. Among these synthetic TDs, 18 TD derivatives showed specific algicidal activity on two strains belonging to the classes Raphidophyceae (Chattonella marina and Heterosigma akashiwo) and Dinophyceae (Cochlodinium polykrikoides). Two strains belonging to Bacillariophyceae (Navicula pelliculosa and Phaeodactylum EPV), one strain belonging to Dinophyceae (Amphidinium sp.), and a Eustigmatophycean microalga (Nannochloropsis oculata) showed less sensitivity to the TD derivatives than the other two phyla. The most reactive TD derivative, compound 2 (TD118), was selected and tested for morphological and physiological changes. TD118 effectively damaged the cell membrane of C. marina, H. akashiwo and C. polykrikoides. The O2 evolution and photosystem II efficiency (F v /F m ) of C. marina, H. akashiwo and C. polykrikoides were also severely reduced by TD118 treatment. Amphidinium sp., N. pelliculosa, Phaeodactylum EPV and N. oculata showed less reduction of O2 evolution and the F v /F m by TD118. These results imply that the species-specific TD structure relationship may be due to structural and/or physiological differences among microalgal species.

A series of naphthoquinone-benzothiazole conjugates were synthesized as algicides, and their efficacies against harmful algal blooming species, such as Chattonella marina, Heterosigma akashiwo and Cochlodinium polykrikoides, were examined. The introduction of substituted benzothiazole at the C2 position of 1,4-naphthoquinone (compounds 1–9) resulted in higher algicidal activity against C. polykrikoides than the C6 conjugates (compounds 10–20). On the other hand, of the C6 conjugates, compounds 11 and 12 exhibited better algicidal activity against H. akashiwo, C. marina, and C. polykrikoides than the C2 conjugates. Further structure-activity analysis indicated that a replacement of the methoxy groups with hydroxyl groups (compounds 21–26) decreased the algicidal activity significantly. Among the various synthetic naphthoquinonebezothiazole conjugates tested, compound 12 was found to affect the most significant decrease in the level of C. polykrikoides growth, with an IC50 of 0.19 μM. Compound 11 was found to be the most potent inhibitor against H. akashiwo and C. polykrikoides, with IC50 values of 0.32 and 0.12 μM, respectively. Overall, these results highlight a possible method for controlling and inhibiting red tide forming algae using NQ derivatives.

The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding l -alanine dehydrogenase in Mycobacterium smegmatis . The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of l -alanine. The purified AldR protein exists as a homodimer in the absence of l -alanine, while it adopts the quaternary structure of a homohexamer in the presence of l -alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by l -alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N 2 -ATC-N 2 -TC and one putative AldR binding site with the sequence GA-N 2 -GTT-N 2 -TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of l -alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

Culture experiments involving 23 algae strains were conducted to evaluate the algicidal effects of a newly developed algicidal thiazolidinedione (TD) derivative (TD49) on non-harmful and harmful algal bloom (HAB) species. We also assessed the effect of various concentrations of TD49 on various growth phases (lag, logarithmic, and stationary) of the harmful algae Heterocapsa circularisquama (Dinophyceae) and Heterosigma akashiwo (Raphidophyceae; hereafter, Heterosigma) and the non-harmful diatoms Skeletonema costatum and Chaetoceros didymus. The inhibitory ratios (%) for H. circularisquama and Heterosigma at 2.0 μM TD49 were significantly higher than those at other concentra- tions, and the inhibitory ratio varied depending on growth phase and species as follows: logarithmic≥stationary>lag phaseforH.circularisquamaandlogarithmic≥lag>stationary phase for Heterosigma. Although the inhibitory ratios for C. didymus were similar to those for the two harmful algae (H. circularisquama and Heterosigma), inhibitory effects on S. costatum were not apparent at >2.0 μM in any growth phase. The algicidal activity of TD49 on the harmful and non- harmful algae was as follows: unarmored HAB species>ar- mored HAB species>diatom species>cryptophyte species. TD49 was algicidal to most HABs but had a little inhibitory effect on some non-harmful algae, implying that TD49 has selective algicidal activity. Our results indicate that TD49 is potentially of use in the control of HAB species within semi- enclosed bays.

The DevSR two-component system in Mycobacterium smegmatis consists of the DevS histidine kinase and the DevR response regulator. It is a regulatory system that is involved in the adaptation of mycobacteria to hypoxic and NO stresses. Using the yeast two-hybrid assay and pull-down assay, it was demonstrated that the phosphoaccepting Asp (Asp54) of DevR is important for protein-protein interactions between DevR and DevS. The negative charge of Asp54 of DevR was shown to play an important role in protein-protein interactions between DevR and DevS. When the Lys104 residue, which is involved in transmission of conformational changes induced by phosphorylation of the response regulator, was replaced with Ala, the mutant form of DevR was not phosphorylated by DevS and functionally inactive in vivo. However, the K104A mutation in DevR only slightly affected protein-protein interactions between DevR and DevS.

MxaJ is a component of type II methanol dehydrogenase (MDH) that mediates electron transfer during methanol oxidation in methanotrophic bacteria. However, little is known about how MxaJ structurally cooperates with MDH and Cytochrome c

To assess the algicidal effects of the thiazolidinedione derivative TD49 on the unarmored dinoflagellate Heterocapsa circularisquama and to evaluate the response of the planktonic community and the environment to this chemical, we undertook mesocosm (1,300 L) and small-scale experiments. The reduction ratio for H. circularisquama in each experiment was dependent on the concentration of TD49. At a TD49 concentration >0.4 μM, the abundance of H. circularisquama decreased by 99 % in the small-scale experiment and by 84 % in the mesocosm during the initial 2 days. At 0.2 μM TD49, the abundance of H. circularisquama decreased by up to 85 % in the small-scale experiment, whereas the abundance in the mesocosm increased, implying the absence of an algicidal effect. The decrease in planktonic organisms, including H. circularisquama, following TD49 treatment was correlated with abrupt declines in culture pH and dissolved oxygen concentration. Following addition of TD49, there was a significant increase in the abundance of diatoms and cryptophyta species, and after 8 days, the dominant species in the TD49 treatments shifted to small pennate diatoms including Cylindrotheca and Entomoneis species. The growth of some species among the zooplankton community was promoted at low TD49 concentrations (≤0.4 μM), whereas high concentrations (≥1.0 μM) had a negative effect. This study demonstrates that TD49 is an effective agent for the control for H. circularisquama blooms and that large-scale mesocosms play a crucial role in assessing the application of algicides such as TD49 in natural environments.

Dinoflagellates are considered one of the most abundant and diverse groups of marine microplankton and viruses are recognized as one of the significant factors affecting the plankton dynamics. Here, we report basic characteristics of a new dinoflagellate-infecting virus, Heterocapsa pygmaea DNA virus (HpygDNAV) which infects a toxic dinoflagellate, H. pygmaea. HpygDNAV is a polyhedral large virus (ca. 160?170 nm in diameter) propagating in its host`s cytoplasm. Because of the virion size, appearance in thin sections, and propagation characteristics, HpygDNAV is assumed to harbor a large double-stranded DNA genome; i.e., HpygDNAV is most likely a nucleocytoplasmic large DNA virus (NCLDV) belonging to the family Phycodnaviridae. Its infectivity is strain-specific, rather than species-specific, as is the case for other algal viruses. The burst size and latent period are estimated to be roughly 100?250 infectious units cell?1 and < 96 h, respectively.

The algicidal effects of the thiazolidinedione derivative TD49 on Heterosigma akashiwo and Chattonella marina (Raphidophyceae) were assessed, and the response of the planktonic community and environment to the algicide was evaluated in a microcosm, quantifying 12 L. The abundance of over 80 % of H. akashiwo and C. marina declined in a day significantly in microcosms to which TD49 was added (final concentration 2 μM), and this was correlated with an abrupt decline in the culture pH. The number of protists (i.e., ciliates) other than H. akashiwo and C. marina gradually increased with time in the TD49 treatments, implying that the decline in numbers of H. akashiwo and C. marina cells resulting from TD49 treatment was a major factor in the growth of the other organisms. However, TD49 may be toxic to aquatic zooplankton communities, even though it is a highly selective algicide for harmful algae bloom species. The study indicates that TD49 is an effective agent for the control for H. akashiwo and C. marina blooms in enclosed and eutrophic water bodies.

In this study, the ability of Chlorella vulgaris and Dunaliella salina to use biomass resources for anaerobic digestive biogas production was examined. The differences in cell wall structure pretreatments affecting the yield of soluble products showed that D. salina is a better candidate for biogas production than C. vulgaris. There was no significant difference between pretreated and non-pretreated D. salina in terms of methane production yield by inocula obtained from anaerobic digestion systems. Therefore, D. salina is a suitable algal biomass for biogas production due to its high biomass productivity, simple pretreatment needs, and easy conversion to biogas.

To evaluate the algicidal impact of a newly developed algicide thiazolidinedione derivative,TD49, on dinophyceae Akashiwo sanguinea in aquatic ecosystems, tentative culture experiments for thetarget species were conducted in small (SS), middle (MS), and large scale (LS) culture vessels. When TD49was introduced at the final concentration of 2 µM in SS and MS, as well as 1 µM in LS, the abundance of A.sanguinea decreased significantly in all the treatments. On the other hand, total phytoplankton abundance,except A. sanguinea in the TD49 treatments, gradually increased with culture time, which implies that a celldestruction of A. sanguinea by TD49 is a major cause of the population growth by other phytoplanktonspecies. Also, A. sanguinea was easily destroyed, which was likely to be a source of extracellularsubstances. In particular, a pH decrease was significant in the treatments than in the control, which indicatesthat the water in the treatments has been acidified, due to an increase in the heterotrophic metabolisms ofbacteria and degradation of A. sanguinea cells. Our results indicate that the TD49 substance is the potentialagents for the control of A. sanguinea in the enclosed and eutrophic water bodies.Key words :Akashiwo sanguinea, algicidal effect, thiazolidinedione, ecosystem*Corresponding author. E-mail : yokim@kordi.re.kr

Anaerobic digestion is an alternative method to digest food wastes and to produce methane that can be used as a renewable energy source. We investigated bacterial and archaeal community structures in a three-stage methane production process using food wastes with concomitant wastewater treatment. The three-stage methane process is composed of semianaerobic hydrolysis/acidogenic, anaerobic acidogenic, and strictly anaerobic methane production steps in which food wastes are converted methane and carbon dioxide. The microbial diversity was determined by the nucleotide sequences of 16S rRNA gene library and quantitative real-time PCR. The major eubacterial population of the three-stage methane process was belonging to VFA-oxidizing bacteria. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (Methanoculleus). Family Picrophilaceae (Order Thermoplasmatales) was also observed as a minor population. The predominance of hydrogenotrophic methanogen suggests that the main degradation pathway of this process is different from the classical methane production systems that have the pathway based on acetogenesis. The domination of hydrogenotrophic methanogen (Methanoculleus) may be caused by mesophilic digestion, neutral pH, high concentration of ammonia, short HRT, and interaction with VFA-oxidizing bacteria (Tepidanaerobacter etc.).

Heterocapsa circularisquama RNA virus (HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates. The ability of HcRNAV coat protein (HcRNAV CP) to self-assemble into virus-like particles (VLPs) in vitro suggested that heterologous expression was possible, and that the VLPs might be ideal nanocontainers for the targeted delivery of genes and chemicals. In this paper, we report the expression of a codon-optimized HcRNAV 109 CP gene in Pichia pastoris and the production of self-assembled HcRNAV VLPs using large-scale fermentation. The HcRNAV 109 CP gene was synthesized according to the codon preference of P. pastoris and cloned into a pPICZA vector. The recombinant plasmid pPICZA-CPsyns was transformed into P. pastoris by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS polyacrylamide gel electrophoresis. After large-scale fermentation, the yield of HcRNAV CPsyns reached approximately 2.5 g L−1 within 4 d. The HcRNAV VLPs were purified using PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and were subsequently analyzed using UV spectrophotometry and transmission electron microscopy. Fluorescence dye-labeled myoglobin was loaded into the cages of the HcRNAV VLPs and the encapsulation was confirmed by fluorescence spectroscopy. The results point to the possible utilization in pharmacology or nanotechnology of HcRNAV VLPs produced by P. pastoris fermentation.

In this study, we analyzed a bacterial community closely associated with Cochlodinium polykrikoides that caused harmful algal blooming in the sea. Filtration using a plankton mesh and percoll gradient centrifugation were performed to eliminate free-living bacteria. Attached bacteria were analyzed by culture-dependent and culture-independent methods. Five culturable bacterial strains were isolated and identified from the C. polykrikoides mixed bacterial community. The isolates belonged to α-Proteobacteria (Nautella sp., Sagittula sp., and Thalassobius sp.) and γ-Proteobacteria (Alteromonas sp. and Pseudoalteromonas sp.). All of the 5 isolates showed algicidal activity against C. polykrikoides and produced extracellular compounds responsible for algicidal properties after entering the stationary phase. The algicidal compounds produced by the 5 isolates were heat-stable and had molecular masses of less than 10,000 Da. Furthermore, the algicidal compounds were relatively specific for C. polykrikoides in terms of their algicidal activities. Culture-independent analysis of the bacterial community in association with C. polykrikoides was performed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). On the basis of the PCR-DGGE profile, Sagittula sp. was identified as a dominant species in the bacterial community of C. polykrikoides.

Previous studies have shown that functionalized gold nanoparticles (AuNPs) can be used as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNA to living systems. Here, we report the ability of functionalized AuNP to deliver RNA aptamers into the nuclei of human cells. An in vitro-synthesized RNA aptamer specific to the β-catenin protein was delivered into the HepG2 human cell line more efficiently via functionalized AuNP than liposome-based delivery, and resulted in nearly complete inhibition of β-catenin binding to the p50 subunit of NF-κB in the nucleus. This inhibition led to repression of NF-κB p50-dependent transcription of CRP. Also, the β-catenin aptamer in the nucleus led to down-regulation of β-catenin-mediated transcriptional activity through the TCF complex and resulted in decrease in the levels of cyclin D, and c-myc mRNA by ∼47% and ∼57%, respectively. In addition, we used functionalized AuNP to deliver another RNA aptamer targeted to the p50 subunit of NF-κB into the A549 human cell line, and this was sufficient to induce apoptosis of the cells. Our findings demonstrate that AuNP GDS can be used to deliver small, highly structured RNA aptamers into the nucleus of human cells where they modulate the activity of transactivators by interacting with target proteins.

A voltammetric enzyme electrode was developed based on nicotinamide-independent trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), which catalyses the oxidation of trimethylamine (TMA) to dimethylamine and formaldehyde. A quaternized osmium hydrogel polymer, poly(vinylimidazole-[Os(4,4′-dimethyl-2,2′-bipyridine)2Cl]+/2+) with ethylamine (PVI-Os-EA), was prepared as a potential redox mediator in an electrochemical biosensor. TMA was detected using TMADH that was co-immobilized with an osmium hydrogel polymer on electrodeposited gold nanoparticles (Au-NPs) on screen-printed carbon electrodes (SPCEs). The Au-NPs deposited onto SPCEs provided about a three times higher electrochemical response compared to that of a planar gold electrode. As TMA was catalyzed by wired TMADH, the electrical signal was monitored at 0.3 V versus Ag/AgCl by cyclic voltammetry and chronoamperometry. The anode currents increased linearly in proportion to the TMA concentration over the 0 ∼ 2.5 mM range with a detection limit of 1 μM (R = 0.9972).

An outdoor enclosure experiment was carried out near the southern coast of Korea in order to investigate the effect of enclosed environments on planktonic community structure. The experiment was conducted in 6 floating, 220-liter cylindrical enclosures maintained for short periods (17 days). Rapid decreases of inorganic nutrients (N. P, Si), and in the abundance of large diatoms, Coscinodiscus spp., were observed after only one day from the start of the experiment. Decreases in chlorophyll a progressed through the experimental period, although the small diatoms, Chaetoceros spp. and Thalassiosira sp., increased considerably during the initial days. The continuous supply of dissolved organic carbon from crashing stocks of planktonic organisms sustained rapid growth of heterotrophic bacteria. The bacterial increase was followed by increase of heterotrophic nanoflagellates and ciliated protozoa. Noctiluca scintillans flourished as a dominant zooplankter until the end of the experiment, feeding on phytoplankton during the first half of the observations and on the abundant heterotrophic microorganisms during the second half. The results suggested that the enclosed environment provoked a structural shift of the plankton community from autotrophy to heterotrophy and indicated additionally that the trophic status in plankton communities was reflected by an enclosed environment formed by artificial structures in the coastal zone.

A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP(+) remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP(+), and a mutant bFMO(Y207S), which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP(+) and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP(+) revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMO(Y207S) exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole.

Several molecular techniques have been used to study viruses under different environmental conditions and to examine the genetic diversity of natural virus communities. Here, natural marine virus samples were collected from six different southern coastal regions of Korea and subjected to PCR amplification with five different degenerate primers specific for either the DNA polymerase or capsid protein gene of algal viruses. PCR products ranging from 300 to 700 bp were observed on agarose gel analysis, and major PCR bands were purified and cloned. PCR using primers specific for the viral DNA polymerase gene and for the coat protein gene yielded 332 and 366 clones, respectively. Of the clones analyzed, 147 (44%) DNA polymerase gene clones and 326 (89%) coat protein clones showed similarity to known virus genes. Clustering and assembly revealed 23 and 38 unique sequences for the DNA polymerase and coat protein genes, respectively. BLASTX and phylogenetic analyses of these sequences revealed close relationships with various virus groups, including one of the major algal virus groups, Phycodnaviridae . Pairwise nucleotide comparisons among sequences classified into the same group revealed the extent of genetic diversity in both polymerase and coat protein gene sequences. These results indicated the presence of genetic diversity within similar virus groups in the marine system. Additionally, PCR products with the same sequences were recovered from different locations, indicating the presence of the same virus in different geographic locations in the southern coastal region of the Korean Peninsula.

Two types of methanol dehydrogenase (MDH) were obtained from a novel marine methylotrophic bacterium, Methylophaga aminisulfidivorans MP(T), grown on methanol. Type I MDH consisted of two identical dimers of α (65.98 kDa) and β (7.58 kDa) subunits organized to form the α(2)β(2) tetramer. Type II MDH contained an additional MxaJ protein (27.86 kDa) and had more specific activity than type I MDH. The K(m) values of type I and II MDH for methanol under cytochrome c(L) reduction assay system were estimated to be 50.3 and 13.0 μM, respectively, and the isoelectric points of type I and II MDH were determined to be 5.4 and 5.8, respectively. The average molar ratios of α:β, α:MxaJ, and β:MxaJ in type II MDH were approximately 1:0.99, 1:0.41 and 1:0.42, respectively. Based on these results, the original conformation of the MDH of M. aminisulfidivorans MP(T) is most likely the α(2)β(2)-MxaJ complex. During purification, the lysozyme and freeze-thawing cell disruption method significantly increased the amount of type II MDH in the soluble fraction compared with strong physical disruption methods such as sonication and French Press.

A cytosolic superoxide dismutase (SOD) was purified and characterized from a fast-growing Mycobacterium sp. strain JC1 DSM 3803 grown on methanol. The native molecular weight of the purified SOD was estimated to be 48 kDa. SDS-PAGE revealed a subunit of 23 kDa, indicating that the enzyme is a homodimer. The enzyme activity was inhibited by H(2)O(2) and azide. The purified SOD contained 1.12 and 0.56 g-atom of Mn and Fe per mol of enzyme, respectively, suggesting that it may be a Fe/Mn cambialistic SOD. The apo-SOD reconstitution study revealed that Mn salts were more specific than Fe salts in the SOD activity. The gene encoding the SOD was identified from the JC1 cosmid genomic library by PCR screening protocol. The cloned gene, sodA, had an open reading frame (ORF) of 624 nt, encoding a protein with a calculated molecular weight of 22,930 Da and pi of 5.33. The deduced SodA sequence exhibited 97.6% identity with that of Mycobacterium fortuitum Mn-SOD and clustered with other mycobacterial Mn-SODs. A webtool analysis on the basis of SOD sequence and structure homologies predicted the SOD as a tetrameric Mn-SOD, suggesting that the protein is a dimeric Mn-SOD having tetramer-specific sequence and structure characteristics.

Methylophaga aminisulfidivorans MP(T) is a marine methylotrophic bacterium that utilizes C(1) compounds such as methanol as a carbon and energy source. The released electron from oxidation flows through a methanol-oxidizing system (MOX) consisting of a series of electron-transfer proteins encoded by the mox operon. One of the key enzymes in the pathway is methanol dehydrogenase (MDH), which contains the prosthetic group pyrroloquinoline quinone (PQQ) and converts methanol to formaldehyde in the periplasm by transferring two electrons from the oxidation of one methanol molecule to the electron acceptor cytochrome c(L). In order to obtain molecular insights into the oxidation mechanism, a native heterotetrameric α(2)β(2) MDH complex was directly purified from M. aminisulfidivorans MP(T) grown in the presence of methanol and crystallized. The crystal diffracted to 1.7 Å resolution and belonged to the monoclinic space group P2(1) (unit-cell parameters a = 63.9, b = 109.5, c = 95.6 Å, β = 100.5°). The asymmetric unit of the crystal contained one heterotetrameric complex, with a calculated Matthews coefficient of 2.24 Å(3) Da(-1) and a solvent content of 45.0%.

Bio-indigo was produced in 3000-L batch and 5-L continuous fermentation of a recombinant Escherichia coli DH5α harboring a novel gene encoding flavin-containing monooxygenase (FMO). Batch fermentation in a 3000-L fermenter produced 911 ± 22 mg L−1 of indigo from 2 g L−1 tryptophan as a substrate (yield 46.9%) under the following culture conditions as follows: culture temperature 30 °C, pH 7.0, agitation speed 200 rpm, and aeration 3 vvm. Sufficient oxygen (aeration rate and agitation speed) was critical for bio-indigo production. For continuous fermentation in a 5-L fermenter, the volumetric productivity was found to be 11.3 mg L−1 h−1 up to 110 h (final accumulated bio-indigo was 23 g) with a constant dilution rate (D) of 0.084 h−1 (constant feeding rate of 0.167 L h−1 with medium containing 3 g L−1 tryptophan). Recombinant E. coli cells have the ability to withstand the toxicity of high concentration of accumulated indigo in batch fermentation. In continuous fermentation, the recombinant cells exhibited high plasmid stability up to 110 h, after which they lost the plasmid.

The objective of this study was to develop chromatographic methods for the determination of the modification degree and the characterization of poly(ethylene glycol)-modified polyamidoamine dendrimers (PEG–PAMAMs). The PEG–PAMAMs were prepared by reacting PAMAM generation 4 with monomethoxy PEG–nitrophenyl carbonate (mPEG–NPC). The modification degrees of PEG–PAMAMs were determined by quantifying 4-nitrophenol released from mPEG–NPC after PEGylation reaction using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The PEG–PAMAMs, which have poor UV absorbances, were characterized by HPLC with charged aerosol detection. This study demonstrates that the combination of these two detectors is a powerful tool for the preparation and characterization of PEG–PAMAMs.

Using a specialized ribosome system, previous studies have identified G791 in Escherichia coli 16S rRNA as an invariant and essential residue for ribosome function. To investigate the functional role of G791, we searched for multicopy suppressors that partially restored the protein synthesis ability of mutant ribosomes bearing a G to U substitution at position 791 (U791 ribosomes). Analyses of isolated multicopy suppressors showed that overexpression of initiation factor 1 (IF1) enhanced the protein synthesis ability of U791 ribosomes. In contrast, overexpression of initiation factor 2 (IF2) or IF3 did not enhance the protein synthesis ability of wild-type or U791 ribosomes, and overexpression of IF1 did not affect the function of wild-type or mutant ribosomes bearing nucleotide substitutions in other regions of 16S rRNA. Analyses of sucrose gradient profiles of ribosomes showed that overexpression of IF1 marginally enhanced the subunit association of U791 ribosomes and indicated lower binding affinity of U791 ribosomes to IF1. Our findings suggest the involvement of IF1 in the restoration of the P-site function that was impaired by a nucleotide substitution at residue G791.