Characterization of a Bifunctional Cytidine 5′-Monophosphate N-acetylneuraminic acid Synthetase Cloned from Streptococcus Agalactiae

Recombinant CMP-sialic acid synthetase, cloned from Streptococcus agalactiae serotype V strain 2603 V/R, is bifunctional having both CMP-sialic acid synthetase and acetylhydrolase (acylesterase) activities. The enzyme is active over a wide pH range with an optimal CMP-sialic acid synthetase activity at pH 9.0 and an optimal acetylhydrolase activity at pH 8.0. A metal cofactor (either Mg²⁺ or Mn²⁺) is required for the CMP-sialic acid synthetase activity but is not for acetylhydrolase activity. Both catalytic functions, however, are impaired by high concentrations of Mn²⁺. Received 10 August 2005; Revisions requested 30 August 2005; Revisions received 1 November 2005; Accepted 2 November 2005     

Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose after batch culture, thereby giving hsBLyS at 3.7 g l⁻¹ and a productivity of 0.11 g l⁻¹ h⁻¹. Revisions requested 1 September 2005 and 11 November 2005; Revisions received 7 November 2005 and 4 January 2006     

Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810

An alkaline cellulase from Bacillus sp. HSH-810 was purified 8.7-fold with a 30% yield and a specific activity of 71 U mg^–1 protein. It was optimally active at pH 10 and 50 °C and was stable from pH 6 to 10 with more than 60% activity remaining after heating at 60 °C for 60 min. The molecular mass of cellulase was 80 kDa. It was inhibited by 50% by Fe³⁺ (1 mM) and Mn²⁺ (0.1 mM) but was relatively insensitive to Hg²⁺ and Pb²⁺ at 1 mM.Revisions requested: 8 October 2004/1 December 2004; Revisions received 29 November 2004/5 January 2005     

Purification and Properties of a β-1,3-glucanase from Chaetomium sp. that is Involved in Mycoparasitism

A β-1,3-glucanase was detected, using laminarin as substrate, in the culture broth of Chaetomium sp. Major activity was associated with a 70 kDa protein band visualized on a polyacrylamide gel. β-1,3-Glucanase was purified by a one-step, native gel purification procedure. Optimal activity was observed at pH 6.0 and 30 °C (over 30 min). It could degrade cell walls of plant pathogens including Rhizoctonia solani, Gibberella zeae, Fusarium sp., Colletotrichum gloeosporioides and Phoma sp. The N-terminal amino acid residues of the purified β-1,3-glucanase are PYQLQTP, which do not exhibit homology to other fungal β-1,3-glucanases suggesting it may be a novel enzyme. Received 20 July 2005; Revisions requested 2 August 2005 and 27 September 2005; Revisions received 16 September 2005 and 3 November 2005; Accepted 6 November 2005     

Textile dye decolorization using cyanobacteria

Cyanobacterial cultures isolated from sites polluted by industrial textile effluents were screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80% after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B (55%). Chlorophyll a synthesis in all cultures was strongly inhibited by the dyes. Visible spectroscopy and TLC confirmed that color removal was due to degradation of the dyes.Revisions requested 10 November 2004/30 November 2004; Revisions received 16 November 2004/ 7 January 2005     

One-step Concentration and Partial Purification of Aspergillus kawachii Non-acidic Polygalacturonases by Adsorption to Glass Fiber Microfilters

The non-acidic polygalacturonases produced by Aspergillus kawachii in a glucose/tryptone medium were adsorbed to a glass fiber microfilter that was used to clarify the fermentation broth. Maximum adsorption occurred at pH 3 under low ionic strength conditions. The adsorbed activity could be readily released with a buffer solution at pH 5. Based upon these observations, a separation process was developed which enabled the broth to be clarified and, at the same time, the non-acidic polygalacturonases to be concentrated 20-fold and purified 100-fold in a unique filtration step. The practical advantage of recovering polygalacturonases by a filtration process lies in the simplicity and efficiency of the operation involved. Received 27 July 2005; Revisions requested 24 August 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Relationship between Poly- β-hydroxybutyrate Production and δ-endotoxin for Bacillus thuringiensis var. kurstaki

A linear relationship between total solid concentration (TSC), δ-endotoxin production [Cry = 0.2795(TSC)−0.2472, R² = 0.8644] and poly-β-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R² = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between δ-endotoxin and PHB accumulation [Cry = 2.1573(PHB)−1.1248, R² = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l⁻¹ was required before the onset of δ-endotoxin production. Revisions requested 28 September 2005 and 4 November 2005; Revisions received 28 October 2005 and 1 February 2006     

Influence of Agitation on the Removal of Nonylphenol by the White-rot Fungi Trametes versicolor and Bjerkandera sp. BOL 13

Bjerkandera sp. BOL 13 removed 95% of nonylphenol (at 9.7 mg nonylphenol l⁻¹ day⁻¹) from aqueous medium after 5 days of incubation in agitated cultures. This removal rate decreased 2.5-fold in static cultures. By comparison, Trametes versicolor removed nonylphenol at 2.8 mg l⁻¹ day⁻¹ under conditions of static incubation, probably due to the action of laccase, but no growth was recorded in the agitated bottles. Received 4 August 2005; Revisions requested 18 August 2005 and 27 September 2005; Revisions received 22 September 2005 and 3 November 2005; Accepted 7 November 2005     

Cloning and Sequence Analysis of the Gene Encoding (6-4)photolyase from Dunaliella salina

DNA photolyase can repair UV-induced DNA damage in a light-dependent manner. A cDNA of (6-4)photolyase from Dunaliella salina (GenBank accession number: AY845324) was cloned, sequenced and its amino acid sequence was deduced. The derived amino acid sequence showed high homology with other (6-4)photolyases and a predicted 3D model was constructed by homology modeling. Revisions requested 20 May 2005 and 18 August 2005; Revisions received 2 August 2005 and 28 November 2005     

Biotransformation of racemic diisophorone by <Emphasis Type="Italic">Cephalosporium aphidicola</Emphasis> and <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Racemic diisophorone (500 mg) was converted by Cephalosporium aphidicola and Neurospora crassa over 10 days at 25 °C to 8β-hydroxydiisophorone in yields of 10% (52 mg) and 20% (103 mg), respectively. The structure was established by IR, specific rotation, mass spectral, 1D and 2D-NMR studies.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 8 April 2005 and 10 May 2005     

Purification and Characterization of a Cold-active Iron Superoxide Dismutase from a Psychrophilic Bacterium, Marinomonas sp. NJ522

Marinomonas sp. NJ522, isolated from Antarctic sea ice, produces a cold-active iron superoxide dismutase (SOD; EC 1.15.1.1). The purified SOD was dimeric and had an approx. Mr of 48 kDa. Highest activity was detected from pH 8 to 10 and at 40 °C (assayed over 10 min). Activity at 0 °C was nearly 35% of the maximum activity. Received 25 August 2005; Revisions requested 30 August 2005 and 26 September 2005; Revisions received 12 September 2005 and 25 October 2005; Accepted 1 November 2005     

Redirecting Carbon Flux in Torulopsis glabrata from Pyruvate to α-Ketoglutaric Acid by Changing Metabolic Co-factors

The nutrition conditions needed to redirect the carbon flux in Torulopsis glabrata, a pyruvate hyper-production yeast, from pyruvate to α-ketoglutaric acid (KG) were investigated in a stirred fermentor. A minor amount of KG (1.3 gl⁻¹) was produced when NaOH was used to control the pH, while 12 g KG l⁻¹ was produced when CaCO₃ was used instead. When thiamine and biotin were included in the medium, 13 g KG l⁻¹ and 68 g pyruvate l⁻¹ were produced after 48 h when glucose was nearly consumed (approximately 5 gl⁻¹). With fermentation continuing for a further 16 h, the concentration of pyruvate decreased to 31 gl⁻¹, and KG increased to 30 gl⁻¹. KG thus accumulated at the expense of pyruvate consumption. Received 2 June 2005; Revisions requested 30 June 2005 and 1 September 2005; Revisions received 1 September 2005 and 28 October 2005; Accepted 28 October 2005     

Production of active pigment epithelium-derived factor in<Emphasis Type="Italic">E. coli</Emphasis>

Human pigment epithelium-derived factor (PEDF), a neurotrophic factor, is the most potent natural inhibitor of angiogenesis. To produce the active PEDF, the gene coding for the human PEDF protein was expressed in E. coli. The rPEDF protein was expressed at 457 mg l^–1 as a soluble protein. The yield of purified GST fusion protein was 14 mg ll^–1. Purified rPEDF inhibited tube formation in endothelial cells.Revisions requested 30 November 2004; Revisions received 25 January 2005     

Extraction of High Quality of RNA and Construction of a Suppression Subtractive Hybridization (SSH) Library from Chestnut Rose (Rosa roxburghii Tratt)

Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and β-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 μg RNA g⁻¹ fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes. Qiang Xu and Xiaopeng Wen - Contribute to this work equally Revisions requested 3 November 2005; Revisions received 18 January 2006     

Isolation and Characterization of a Novel Thraustochytrid-like Microorganism that Efficiently Produces Docosahexaenoic Acid

A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 °C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n−3) at 43% of the total fatty acids. It had a growth rate of 0.38 h⁻¹. The DHA production rate of 2.8 ± 0.7 g l⁻¹ day⁻¹ is the highest value reported for any microorganism. Received 7 October 2005; Revisions requested 7 October 2005; Revisions received 15 November 2005; Accepted 15 November 2005     

Characterization of an Anti-Thioredoxin Monoclonal Antibody

An anti-E. coli thioredoxin monoclonal antibody, IMM-3C6, which showed high specificity to thioredoxin as assessed by indirect ELISA, was generated using hybridoma technology. The affinity constant of IMM-3C6 to thioredoxin was 0.40×10⁹ m⁻¹ and its sensitivity to thioredoxin fusion protein in dot blotting was 50 ng. In sandwich ELISA, it detected thioredoxin fusion protein between 16 and 150 ng/ml. By using IMM-3C6 as the ligand, thioredoxin fusion protein was successfully purified by affinity chromatography. IMM-3C6 was confirmed to be a useful tool for immunoassay and purification of thioredoxin fusion proteins. These authors contributed equally to the work. Received 21 September 2005; Revisions requested 7 October 2005; Revisions received 10 November 2005; Accepted 11 November 2005     

Colloid Lithography-Induced Polydimethylsiloxane Microstructures and their Application to Cell Patterning

Colloidal lithography was used to make a novel array (2-D) of micro-rings, dots, and interconnected-honeycomb structures. These geometries are controlled using the curing temperature-dependent rheological properties of the siloxane elastomer precursor. Serratia marcescens was patterned on the interconnected honeycomb microstructure demonstrating a potential application for microbioanalytical devices, microfluidics, and bio-micro-electromechanical systems. Received 26 August 2005; Revisions requested 23 September 2005; Revisions received 10 November 2005; Accepted 11 November 2005     

Expression and Secretion of Bifidobacterium adolescentis Amylase by Bifidobacterium Longum

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli–Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium. Both authors contributed equally Received 17 June 2005; Revisions requested 13 July 2005 and 26 September 2005; Revisions received 12 September 2005 and 8 November 2005; Accepted 11 November 2005