In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered Saccharomyces cerevisiae capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type S. cerevisiae fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular β-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting S. cerevisiae strains performed similarly under the anoxic SSF conditions, the hydrolytic S. cerevisiae performed slightly better than the phosphorolytic S. cerevisiae under the microaerobic SSF conditions. Nonetheless, the phosphorolytic S. cerevisiae expressing the mutant CDT-1 showed better ethanol production than the glucose-fermenting S. cerevisiae with an extracellular β-glucosidase, regardless of SSF conditions. These results clearly prove that introduction of the intracellular cellobiose metabolic pathway into yeast can be effective on cellulosic ethanol production in SSF. They also demonstrate that enhancement of cellobiose transport activity in engineered yeast is the most important factor affecting the efficiency of SSF of cellulose.

The present study reports the isolation and analysis of two novel GH1 β-glucosidases from the alkalophilic fungus Stachybotrys microspora, using PCR and Nested-PCR. Three major gene fragments were obtained by PCR: the first two are very similar and constitute a novel gene, which was named Smbgl1A, and the third PCR fragment is part of a different gene, named Smbgl1B. The truncated gene sequences were completely filled using the recent partial whole genome sequencing data of S. microspora (data not yet published). Moreover, we investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects. In fact, RT-PCR analysis showed that while Smbgl1A was expressed when the fungus was grown in the presence of cellulose but not when grown with glucose, Smbgl1B was equally expressed under both conditions. The putative catalytic residues and the conserved glycone binding sites were identified. Zymogram analysis showed the intracellular production of β-glucosidases in S. microspora. The predicted secondary structure exhibited a classical (β/α)8 barrel fold, showing that both SmBGL1A and SmBGL1B belong to the GH1 family. Phylogenetic studies showed that SmBGL1A and SmBGL1B belong to the same branch as β-glucosidases from Stachybotrys chlorohalonata and Stachybotrys chartarum. However, SmBGL1A and SmBGL1B form two distinct clades.

The nucleotide sequences of two mitochondrial genes (cytochrome c oxidase subunit I, COI, and cytochrome b, cyt b) and four nuclear genes (growth hormone gene GH1, ribosomal protein S7 gene RP1, recombination activating gene RAG1, and rhodopsin gene RH) from the Far Eastern redfins of the genus Tribolodon were examined to clarify the status of the southern form of T. hakonensis. Nucleotide sequence analysis of the mitochondrial genes showed differences of 2.6% between individuals of T. hakonensis inhabiting the north and south of the range. Analysis of the nuclear genes showed that Tribolodon sp. (southern form of T. hakonensis) has a mosaic of nuclear genes received from the Pacific redfin T. brandtii and big-scaled redfin T. hakonensis. It is suggested that the new species could have formed as a result of homoploid hybridization between the true T. hakonensis (by original description) and T. brandtii, which probably made it possible for this species to occupy a new ecological niche.

The marine bacteria Saccharophagus degradans (also known as Microbulbifer degradans), are rod-shaped and gram-negative motile γ-proteobacteria, capable of both degrading a variety of complex polysaccharides and fermenting monosaccharides into ethanol. In order to obtain insights into structure-function relationships of the enzymes, involved in these biochemical processes, we characterized a S. degradans β-glycosidase from glycoside hydrolase family 1 (SdBgl1B). SdBgl1B has the optimum pH of 6.0 and a melting temperature T m of approximately 50 °C. The enzyme has high specificity toward short D-glucose saccharides with β-linkages with the following preferences β-1,3>> β-1,4 ≫ β-1,6. The enzyme kinetic parameters, obtained using artificial substrates p-β-NPGlu and p-β-NPFuc and also the disaccharides cellobiose, gentiobiose and laminaribiose, revealed SdBgl1B preference for p-β-NPGlu and laminaribiose, which indicates its affinity for glucose and also preference for β-1,3 linkages. To better understand structural basis of the enzyme activity its 3D model was built and analysed. The 3D model fits well into the experimentally retrieved low-resolution SAXS-based envelope of the enzyme, confirming monomeric state of SdBgl1B in solution.

We report here the cross-reaction of RNA polymerase II antiserum with histones H1(0) and H5 and the complementary cross-reactions of antisera to the globular domain of histone H1(0) (GH1(0)) and histone H5 (GH5) with RNA polymerase II. Immunoblotting of RNA polymerase II antiserum with fragments of histone H1(0) localized the cross-reaction at the junction of the globular and C-terminal domains of histone H1(0). The structural homology implied by these cross-reactions is interesting in light of reports that suggest H1(0) may play a role in differentiation and development.

A Gram-reaction-negative bacterial strain, designated <em>GH1</em>-19^T, was isolated from a tidal mudflat sample collected in Gangwha Island, Republic of Korea. Cells of the novel micro-organism were strictly aerobic, non-sporulating, motile and rod-shaped. Growth occurred at 10-40 °C (optimum, 30 °C), pH 6-9 (pH 8) and in the presence of 1-9 % NaCl (3 %). Comparative analysis of complete or nearly complete 16S rRNA gene sequences exhibited that strain <em>GH1</em>-19^T formed a distinct cluster between <i>Marimonas arenosa</i> CAU 1311^T (97.42 % sequence similarity) and <i>Aquicoccus porphyridii</i> L1 8-17^T (97.35 %). Similarity levels of 16S rRNA gene sequences between the novel strain and other members of the family <i>Rhodobacteraceae</i> were below 96.6 %. The isoprenoid quinone was Q-10. The major fatty acids were C_{18 : 1}ω<i>7</i>c, C_{16 : 0}, summed feature 3 (C_{16 : 0}<i>ω</i>7<i>c</i> and/or C_{16 : 0}<i>ω</i>6<i>c</i>) and C_{12 : 0} 3-OH. The polar lipids consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid, an unidentified phospholipid and an unidentified lipid. The G+C content of the DNA was 63.2 mol% (draft genome). DNA-DNA relatedness value between the novel strain and the type strain of <i>Marimonas arenosa</i> was 12.7±9.0 %. On the basis of data from phenotypic, chemotaxonomic and DNA-DNA hybridization studies together with phylogenetic analyses, strain <em>GH1</em>-19^T (=KCTC 62376^T=DSM 106292^T) represents a novel species of the genus <i>Marimonas</i>, for which the name <i>Marimonas</i><i>lutisalis</i> sp. nov. is proposed, with the emended description of the genus <i>Marimonas</i>.

A marine proteobacterium, designated strain GH1-16^T, was isolated from a sample of tidal mudflat collected at the seashore of Gangwha Island, Republic of Korea and the taxonomic status was examined by a polyphasic approach. The isolate was Gram-reaction-negative, strictly aerobic, catalase- and oxidase-positive, non-motile, short-rod-shaped and produced yellow-coloured colonies. An absolute requirement for Na⁺ was observed. The major respiratory quinone was ubiquinone-10. The major polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid. The dominant cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C17 : 1ω6c. The DNA G+C content was 60.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GH1-16^T was closely related to members of the genus Altererythrobacter. The closest relative was Altererythrobacter marensis MSW-14^T (97.3 % sequence similarity) followed by Altererythrobacter aquaemixtae JSSK-8^T (96.8 %) and Altererythrobacter epoxidivorans JCS350^T (96.7 %). The DNA relatedness of strain GH1-16^T against its closest relative was 21.8-25.0 %. On the basis of data obtained by a polyphasic taxonomic approach, strain GH1-16^T (=KCTC 52845^T=NBRC 113275^T) is considered to represent a novel species of the genus Altererythrobacter, for which the name Altererythrobacter lutipelagi sp. nov. is proposed.

Members within the family Rhodbacteraceae are morphologically and genetically highly diverse, and originate mostly from coastal marine environments. In this study, a novel species of this family, designated M0103^T, was isolated from the surface of a sea snail Littorina scabra. Strain M0103^T is Gram-stain-negative, halophilic, non-motile and non-Bacteriochlorophyll a-producing bacterium. Several phenotypic characteristics of the isolate were similar to other species within this family, such as the sole respiratory quinone Q-10 and major fatty acid components C_{18 : 1} ω18 : 0 and C_{16 : 0}. Strain M0103^T contains a diphosphatidylglycerol, a phosphatidylglycerol, a phosphatidylcholine, a phosphatidy ethanolamine, a phosphatidylinositol, five unidentified phospholipids and four unidentified polar lipids. Based on the 16S rRNA gene sequence analysis, this isolate showed the closest phylogenetic relationship with 'Palleronia pontilimi' GH1-23^T (95.1 %). Values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) of genome sequences were of 70.1-76.4 % and 18.3-20.9 % between the isolate and 24 closely related type strains. Analysis the 4.0 Mb genome of strain M0103^T revealed several putative genes associated with cellular stress resistance, which may play protective roles for the isolate in the adaptation to a marine environment. Phylogenetic, phenotypic and chemotaxonomic analyses suggested that strain M0103^T represents a novel genus and novel species of the family Rhodobacteraceae, for which the name Mesobaculum littorinae gen. nov., sp. nov. is proposed. The type strain is M0103^T (=MCCC 1K03619^T=KCTC 62358^T).

Keywords: Mesobaculum; Mesobaculum littorinae; cellular stress resistance.

A novel Gram-reaction-negative bacterium, designated strain GH1-34^T, was isolated from a sample of tidal mudflat collected at the seashore of Gangwha Island, Republic of Korea. Cells of the bacterium were strictly aerobic, catalase- and oxidase-positive, motile by means of a polar flagellum and rod shaped. It was found to grow at 0-5 % (w/v) NaCl, 20-45 °C and pH 6-10. The major isoprenoid quinone was Q-10. The polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid and an unidentified glycolipid. The predominant fatty acids were C18 : 1ω7c and C19 : 0cyclo ω8c. The G+C content of the DNA was 70.9 mol%. Comparative 16S rRNA gene sequence analysis revealed that strain GH1-34^T formed a tight cluster with the type strain of Lutibaculum baratangense with 98.3 % sequence similarity; levels of the 16S rRNA gene sequence similarity between the novel strain and other representatives of the order 'Rhizobiales' were <95.0 %. DNA-DNA relatedness between the organism and L. baratangense KCTC 22669^T was 33 %, Based on the results of phenotypic analysis and DNA-DNA hybridization experiments, strain GH1-34^T (=KCTC 52847^T=NBRC 113277^T) represents a novel species of the genus Lutibaculum, for which the name Lutibaculum pontilimi sp. nov. is proposed.

A novel marine bacterium, designated strain GH1-24^T, was isolated from a tidal mudflat sample collected at Gangwha Island, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequence comparison exhibited that the novel isolate was most closely related to Sneathiella and formed a tight cluster with Sneathiella glossodoripedis JCM 23214^T with 98.1 % sequence similarity. The 16S rRNA gene sequence similarities to other Sneathiella species were low: Sneathiella chinensis LMG 23452^T (96.1 %) and Sneathiellachungangensis CAU 1294^T (95.8 %). Cells of the organism were Gram-reaction-negative, strictly aerobic, catalase- and oxidase-positive, motile rods and showed growth at 0.5-6 % NaCl, 20-42 °C and pH 6.0-8.0. The G+C content of the DNA was 54.9 mol%. The major isoprenoid quinone was Q-10. The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C19 : 0cyclo ω8c. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid. The results of phenotypic and phylogenetic analyses and DNA-DNA relatedness studies suggest that the isolate can be a member of a novel species of the genus Sneathiella, for which the name Sneathiellalimimaris sp. nov. is proposed. The type strain is GH1-24^T (=KCTC 52846^T=NBRC 113276^T).

Stevia rebaudiana Bertoni is an important economic crop that is well known for its secondary metabolites, steviol glycosides (SGs), found in leaves. Because the enzymes of deglycosylation (glycoside hydrolases) play important roles in SGs biosynthetic processes, our study is focused on the functions of β-glucosidases in SGs catabolism in stevia. We cloned and characterized 19 stevia GH1 genes based on transcriptomic sequences. The 19 genes were divided into five putative subfamilies in Arabidopsis. Conserved motifs in the SrGH1 proteins were analysed using the online motif-based sequence analysis tool, MEME. Most of the identified proteins contain the conserved 'TFNEP' motif (contains the catalytic acid/base) and 'ITENG' motif (contains the catalytic nucleophile). Furthermore, the steviol glycoside content and expression of these 19 genes were characterized under constant darkness. The dark treatment lowered the steviol glycoside content significantly, while SrBGLU16 responded to darkness and was markedly upregulated. This study is the first transcriptome-wide analysis of the GH1 family in Stevia rebaudiana. The sequences of 19 SrGH1 members and their expression when grown in darkness were characterized. Among the 19 genes, SrBGLU16 was markedly upregulated by darkness. Thus, we identified SrBGLU16 for further investigation as a possible steviol glycoside beta-glucosidase.

Keywords: Glycosyl hydrolase family 1; Hydrolysis; Stevia rebaudiana bertoni; Steviol glycosides; β-glucosidase.

The minor flavonoid baohuoside I from Herba epimedii has better bioactivities than its precursor compounds icariin and other major epimedium flavonoids. In this study, a novel β-glucosidase gene (Igag_0940) was cloned and expressed to improve the conversion efficiency in the process of baohuoside I production. For the first time, the recombinant IagBgl1 was purified and then identified uniquely as a trimer in GH 1 family protein from Archaea. The maximum activity of recombinant IagBgl1 was exhibited at 95 °C, pH 6.5, and it retained more than 70% after incubation at 90 °C for 4 h. IagBgl1 had a high catalytic activity towards icariin with a K_cat/K_m ratio of 488.19 mM^-1·s^-1. Under optimized conditions (65 °C, pH 6.5, 0.8 U/mL enzyme, and 90 min), 10 g/L icariin was transformed into 7.564 g/L baohuoside I with a molar conversion of 99.48%. Meanwhile, 2.434 g/L baohuoside I was obtained from 10 g/L total epimedium flavonoids by a two-step conversion system built with IagBgl1 and two other thermostable enzymes. This is the first report of enzymatic conversion for producing baohuoside I by thermostable enzymes.

Keywords: Baohuoside I; Ignisphaera aggregans; The total epimedium flavonoids; Thermostability; β-glucosidase.

This work was performed to study Castilla Rose (Purshia plicata) as a potential source of polyphenols obtained by solid-state fermentation (SSF)-assisted extraction using the microorganism Aspergillus niger GH1 and to evaluate the antioxidant activity of the extracted compounds. First, water absorption capacity (WAC) of the plant material, radial growth of the microorganism, determination of best fermentation conditions, and maximum accumulation time of polyphenols were tested. Then, a larger-scale fermentation, polyphenols isolation by column liquid chromatography (Amberlite XAD-16) and recovered compounds identification by HPLC-MS were made. Finally, the antioxidant activity of the recovered compounds was tested by ABTS, DPPH, and lipid oxidation inhibition assays. The best fermentation conditions were temperature 25 °C and inoculum 2 × 10⁶ spores/g, while the maximum extraction time of polyphenols was 24 h (173.95 mg/g). The HPLC/MS analysis allowed the identification of 25 different polyphenolic compounds, and the antioxidant activity of the obtained polyphenols was demonstrated, showing ABTS assay the most effective with inhibition of 94.34%.

Keywords: Aspergillus niger GH1; Castilla Rose; antioxidant activity; polyphenols; solid-state fermentation.

Polyacylated anthocyanins with multiple glycosyl and aromatic acyl groups tend to make flowers display bright and stable blue colours. However, there are few studies on the isolation and functional characterization of genes involved in the polyacylated anthocyanin biosynthesis mechanism, which limits the molecular breeding of truly blue flowers. Senecio cruentus is an important potted ornamental plant, and its blue flowers contain 3',7-polyacylated delphinidin-type anthocyanins that are not reported in any other plants, suggesting that it harbours abundant gene resources for the molecular breeding of blue flowers. In this study, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of blue, carmine and white colours of cineraria cultivars "Venezia" (named VeB, VeC, and VeW, respectively), we found that 3',7-polyacylated anthocyanin, cinerarin, was the main pigment component that determined the blue colour of ray florets of cineraria. Based on the transcriptome sequencing and differential gene expression (DEG) analysis combined with RT- and qRT-PCR, we found two genes encoding uridine diphosphate glycosyltransferase, named ScUGT1 and ScUGT4; two genes encoding acyl-glucoside-dependent glucosyltransferases which belong to glycoside hydrolase family 1 (GH1), named ScAGGT11 and ScAGGT12; one gene encoding serine carboxypeptidase-like acyltransferase ScSCPL2; and two MYB transcriptional factor genes ScMYB2 and ScMYB4, that were specifically highly expressed in the ray florets of VeB, which indicated that these genes may be involved in cinerarin biosynthesis. The function of ScSCPL2 was analysed by virus-induced gene silencing (VIGS) in cineraria leaves combined with HPLC-MS/MS. ScSCPL2 mainly participated in the 3' and 7-position acylation of cinerarin. These results will provide new insight into the molecular basis of the polyacylated anthocyanin biosynthesis mechanism in higher plants and are of great significance for blue flower molecular breeding of ornamental plants.

Keywords: Senecio cruentus; acyltransferase; blue flower; glucosyltransferase; polyacylated anthocyanins.

Nine plant-associated bacterial strains designated as L1I52^T, NRK F1, NRK F15, NRK F16, NRK F41, NRK F42, NRK F47, NRK F49, and NRK F50 originating from the roots and rhizosphere region of a coastal saline tolerant pokkali rice were taxonomically characterized in this study. Genomic fingerprinting using Enterobacterial Repetitive Intergenic Consensus (ERIC) primers discriminated the nine strains based on the DNA fingerprint patterns indicating that they were not clonal in origin. Phylogenetic analysis using 16S rRNA and other five housekeeping genes (gyrB, glyA, atpA, dnaK and murG) revealed that the novel strains constituted a single novel species within the genus Flavobacterium. In all tree construction methods, the novel strains formed a distinct phylogenetic branch, with Flavobacterium daejeonense GH1-10^T, F. sufflavum BBQ-12^T, and F. glycines Gm-149^T as their nearest phylogenetic neighbours. However, average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) comparison between the draft genomes of L1I52^T (representative isolate) and it's nearest phylogenetic neighbours were well below the proposed threshold values (<95 % and <70 %) used for species discrimination. Thus, based on the phenotypic, genotypic and chemotaxonomic data obtained in this study, we describe a novel Flavobacterium species for which we propose the name Flavobacterium pokkalii sp.nov., with strain L1I52^T (=MTCC 12454^T=KCTC 42429^T) as the type strain. In addition, L1I52^T is a potential plant growth promoting rhizobacteria as they can promote pokkali rice growth and we identified several plant associated gene features in the genome of L1I52^T that are potentially involved in plant microbe interactions.

Keywords: Brackish rice; Flavobacterium; PGPR; Pokkali; Rhizobacteria.

In bacteria, mono- and disaccharides are phosphorylated during the uptake processes through the vastly spread transport system phosphoenolpyruvate-dependent phosphotransferase. As an initial step in the phosphorylated disaccharide metabolism pathway, 6-phospho-β-glucosidases and 6-phospho-β-galactosidases play a crucial role by releasing phosphorylated and nonphosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. Here, an X-ray structure of a glycoside hydrolase family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglH, was determined at 2.2 Å resolution, and its substrate specificity was investigated. The sequence of BlBglH was compared to the sequences of 58 other GH1 enzymes using sequence alignments, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BlBglH was found to have sequence features characteristic of the 6-phospho-β-glucosidase activity enzymes. Motif and structural observations highlighted the importance of loop L8 in 6-phospho-β-glucosidase activity enzymes. To further affirm enzyme specificity, molecular docking and molecular dynamics simulations were performed using the crystallographic structure of BlBglH. Docking was carried out with a 6-phospho-β-glucosidase enzyme activity positive and negative control ligand, followed by 400 ns of MD simulations. The positive and negative control ligands were PNP6Pglc and PNP6Pgal, respectively. PNP6Pglc maintained favorable interactions within the active site until the end of the MD simulation, while PNP6Pgal exhibited instability. The favorable binding of substrate stabilized the loops that surround the active site. Binding free energy calculations showed that the PNP6Pglc complex had a substantially lower binding energy compared to the PNP6Pgal complex. Altogether, the findings of this study suggest that BlBglH possesses 6-phospho-β-glucosidase enzymatic activity and revealed sequence and structural differences between bacterial GH1 enzymes of various activities.

Monolignol glucosides are storage forms of monolignols, which are polymerized to lignin to strengthen plant cell walls. The conversion of monolignol glucosides to monolignols is catalyzed by monolignol β-glucosidases. Rice Os4BGlu18 β-glucosidase catalyzes hydrolysis of the monolignol glucosides, coniferin, syringin, and p-coumaryl alcohol glucoside more efficiently than other natural substrates. To understand more clearly the basis for substrate specificity of a monolignol β-glucosidase, the structure of Os4BGlu18 was determined by X-ray crystallography. Crystals of Os4BGlu18 and its complex with δ-gluconolactone diffracted to 1.7 and 2.1 Å resolution, respectively. Two protein molecules were found in the asymmetric unit of the P212121 space group of their isomorphous crystals. The Os4BGlu18 structure exhibited the typical (β/α)8 TIM barrel of glycoside hydrolase family 1 (GH1), but the four variable loops and two disulfide bonds appeared significantly different from other known structures of GH1 β-glucosidases. Molecular docking studies of the Os4BGlu18 structure with monolignol substrate ligands placed the glycone in a similar position to the δ-gluconolactone in the complex structure and revealed the interactions between protein and ligands. Molecular docking, multiple sequence alignment, and homology modeling identified amino acid residues at the aglycone-binding site involved in substrate specificity for monolignol β-glucosides. Thus, the structural basis of substrate recognition and hydrolysis by monolignol β-glucosidases was elucidated.

A novel Gram-stain-negative, motile by gliding, and aerobic bacterial strain, designated SA31T, was isolated from reclaimed saline land soil near a lake in Taean-gun, South Korea. Cells of the isolate formed ivory-coloured colonies. Growth occurred at 10-35 °C (optimum 25-30 °C), pH 6.0-9.0 (optimum pH 7.0-7.5), and 0-2.0 % (w/v) NaCl (optimum 0 %). Based on similarities of 16S rRNA gene sequences, strain SA31T was mostly affiliated with the genus Flavobacterium, exhibiting the highest sequence similarities with Flavobacterium palustre S44T (96.0 %), Flavobacterium glycines Gm-149T (95.9 %), Flavobacterium defluvii EMB117T (95.7 %) and Flavobacterium daejeonense GH1-10T (95.6 %). Phylogenetic analysis based on 16S rRNA gene sequences also indicated that strain SA31T was clustered with Flavobacterium daejeonense GH1-10T and Flavobacterium glycines Gm-149T. The predominant fatty acids (>7 % of total) of strain SA31T were iso-C15 : 0, summed feature 3 (C16 : 1ω7с and/or C16 : 1ω6с), iso-C17 : 0 3-OH and iso-C15 : 0 3-OH. The major polar lipids of the isolate comprised phosphatidylethanolamine, one unknown aminolipid, one unknown glycolipid, one unknown aminophospholipid and three unknown lipids. The major respiratory quinone was MK-6. The genomic DNA G+C content of strain SA31T was 33.5 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, strain SA31T represents a novel species the genus Flavobacterium, for which the name Flavobacterium eburneum sp. nov. is proposed. The type strain is SA31T (=KACC 18743T=JCM 31221T).

Tannase is an enzyme that catalyses the hydrolysis of ester bonds present in tannins. Most of the scientific reports about this biocatalysis focus on aspects related to tannase production and its recovery; on the other hand, reports assessing the molecular aspects of the tannase gene or protein are scarce. In the present study, a tannase gene fragment from several Aspergillus strains isolated from the Mexican semidesert was sequenced and compared with tannase amino acid sequences reported in NCBI database using bioinformatics tools. The genetic relationship among the different tannase sequences was also determined. A conserved region of 7 amino acids was found with the conserved motif GXSXG common to esterases, in which the active-site serine residue is located. In addition, in Aspergillus niger strains GH1 and PSH, we found an extra codon in the tannase sequences encoding glycine. The tannase gene belonging to semidesert fungal strains followed a neutral evolution path with the formation of 10 haplotypes, of which A. niger GH1 and PSH haplotypes are the oldest.

Flourensia cernua foliage was used in a solid-state fungal bioprocess to identify factors that could affect β-glucosidase production such as growth medium components and partial identification of molecules from the plant material. Under an exploratory experimental design, each variable had their distinctive result on conditions, which affects and could further improve β-glucosidase production. Under the experimental design, 1482 U/L of β-glucosidase were detected, which marks an improvement in production compared to levels obtained in a control treatment with an activity of 1092 U/L. It was shown that inoculum, water content and pH were the factors with the greater effect on β-glucosidase production. Polyphenolic content and cellulosic fiber in the form of raw fiber were measured to assess compound degradation of the plant material. Although fiber content was apparently unaffected, polyphenolic content decreased; β-glucosidase was produced by A. niger GH1. This behavior could be associated with fiber level and polyphenolic content because molecules of this type can be hydrolyzed by β-glucosidase. According to our results, F. cernua biomass can be used as a carbon source for β-glucosidase production in a short culture time.

The growth hormone (GH) releasing effect of thyrotropin-releasing hormone (TRH) and galanin, a 29-amino acid peptide widely distributed in mammalian CNS, alone or in combination was investigated in cultured rat pituitary tumor cells (GH1). TRH stimulated GH secretion in GH1 cells (maximal stimulation at the dose of 0.1 microM). Galanin alone had a significant GH inhibitory effect in GH1 cells at all the doses used. When the two peptides were administered in combination, no significant changes as compared to baseline levels were observed. The results of this study indicate that galanin has potent direct inhibitory effects on baseline and TRH-stimulated GH release from rat tumor cells.

Bromocriptine in concentrations up to 10(-4) M was studied for morphological and endocrinological effects upon the GH3 cell line as well as the GH1 and AtT-20 cell lines. The cells (10(5)/ml) were incubated with RPMI 1640 or in some experiments Dulbecco's Modified Eagle's Medium supplemented with 10% FCS. Bromocriptine was added in concentrations of 10(-4) to 10(-8) mol/L and aliquots of medium were obtained at 2 and 24 hs for the determination of growth hormone and prolactin. Significant reductions in concentrations of growth hormone and prolactin as well as cell number were observed with a concentration of bromocriptine of 10(-4) M at 24 hs. The electron microscopic appearance of GH3 cells treated with 10(-4) mol/L concentrations of bromocriptine for 24 hs demonstrated extensive and marked vacuolization in the cytoplasm which had already appeared 2 hs after treatment with bromocriptine. In bromocriptine-treated (10(-4) mol/L for 24 hs) GH1 cells and AtT-20 cells, the morphologic features were essentially unchanged, compared to the untreated group. Since many previous reports demonstrated a defective dopamine receptor system in GH3 cells, it must be concluded that bromocriptine has an extradopaminergic action which is selectively observed in GH3 cell.

An adenylate cyclase present in a PRL-producing tumor cell line, GH1, which is selectively stimulated by chlorpromazine, has been characterized with respect to several biochemical properties. The parameters studied include divalent metal ion specificity, guanyl nucleotide interaction, and sensitivity to sodium fluoride. The effects of calcium and the calcium chelator, EGTA, were also tested on the chlorpromazine response. The data reported herein establish the optimal conditions for the activation of adenylate cyclase by chlorpromazine in homogenates of GH1 cells. In addition, the results from this study identify a heat-stable protein in these cells which regulates cyclase activity. This protein, which can be released into the supernatant by the pretreatment of GH1 cells with EGTA, is an absolute requirement for chlorpromazine stimulation of adenylate cyclase activity in these cells. The activation of adenylate cyclase by chlorpromazine in homogenates of normal rat pituitaries was demonstrated as well as the presence of a protein factor which regulates this activity.

UNASSIGNED

The screening of rice mutants with improved cellulose to glucose saccharification efficiency (SE) identifies reduced xylan and/or ferulic acid, and a qualitative change of lignin to impact SE. To ensure the availability of sustainable energy, considerable effort is underway to utilize lignocellulosic plant biomass as feedstock for the production of biofuels. However, the high cost of degrading plant cell wall components to fermentable sugars (saccharification) has been problematic. One way to overcome this barrier is to develop plants possessing cell walls that are amenable to saccharification. In this study, we aimed to identify new molecular factors that influence saccharification efficiency (SE) in rice. By screening 22 rice mutants, we identified two lines, 122 and 108, with improved SE. Reduced xylan and ferulic acid within the cell wall of line 122 were probable reasons of improved SE. Line 108 showed reduced levels of thioglycolic-released lignin; however, the amount of Klason lignin was comparable to the wild-type, indicating that structural changes had occurred in the 108 lignin polymer which resulted in improved SE. Positional cloning revealed that the genes responsible for improved SE in 122 and 108 were rice CONSTITUTIVE PHOTOMORPHOGENIC 1 (OsCOP1) and GOLD HULL AND INTERNODE 1 (GH1), respectively, which have not been previously reported to influence SE. The screening of mutants for improved SE is an efficient approach to identify novel genes that affect SE, which is relevant in the development of crops as biofuel sources.