Immobilization of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> α-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study, α-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions of immobilization. The immobilized α-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum temperature also shifted from 50°C to 57°C compared with soluble enzyme. Immobilized α-galactosidase was used in batch, repeated batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the fourth cycle. The performance of immobilized α-galactosidase was tested in a fluidized bed reactor at different flow rates and 86% reduction of RO in soymilk was obtained at 25 ml h⁻¹ flow rate. The study revealed that immobilized α-galactosidase in continuous mode is efficient in reduction of RO present in soymilk.     

Production and partial purification of α-amylase from a novel isolate <Emphasis Type="Italic">Streptomyces gulbargensis</Emphasis>

Extracellular amylase production by a newly isolated alkali-thermotolerant strain Streptomyces gulbargensis DAS 131 was optimized and characterized. The highest amylase production was achieved by growing S. gulbargensis DAS 131 in media with 1% starch. Strain exhibited maximal activity at pH 9.0 and 45 degrees C and relatively stable in alkaline conditions (pH 11). Starch and peptone were found to be the good source of carbon and nitrogen with a yield of 2,216.6 and 2,156.1 U, respectively. Maltose and maltotriose were the main end products of starch hydrolysis, indicating alpha-amylase activity. SDS-PAGE analysis revealed a monomeric form with a molecular weight of 55 kDa.     

Molecular polymorphism of α-galactosidase <Emphasis Type="Italic">MEL</Emphasis> genes of <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts

Molecular genetic analysis of melibiose-fermenting Saccharomyces strains isolated from fermentative processes and natural sources in different world regions was conducted to deduce the evolutionary diversity of Saccharomyces yeasts and find new α-galactosidase MEL genes. The species S. bayanus, S. mikatae, and S. paradoxus were shown to have a single copy of MEL and not accumulate polymeric genes, unlike some S. cerevisiae populations. The polymeric genes MELp1 and MELp2 were identified in S. paradoxus for the first time. Genes identical by 98.7% are located on the chromosomes X and VI, respectively. Phylogenetic analysis indicates that MEL genes of the Saccharomyces yeasts are species-specific.     

The ‘gifted’ actinomycete <Emphasis Type="Italic">Streptomyces leeuwenhoekii</Emphasis>

Streptomyces leeuwenhoekii strains C34^T, C38, C58 and C79 were isolated from a soil sample collected from the Chaxa Lagoon, located in the Salar de Atacama in northern Chile. These streptomycetes produce a variety of new specialised metabolites with antibiotic, anti-cancer and anti-inflammatory activities. Moreover, genome mining performed on two of these strains has revealed the presence of biosynthetic gene clusters with the potential to produce new specialised metabolites. This review focusses on this new clade of Streptomyces strains, summarises the literature and presents new information on strain C34^T.     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> inhibition of α-amylases of stored-product mite <Emphasis Type="Italic">Acarus siro</Emphasis>

The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of -amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mites gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops     

Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Gill cell toxicity of northern boreal scyphomedusae <Emphasis Type="Italic">Cyanea</Emphasis> <Emphasis Type="Italic">capillata</Emphasis> and <Emphasis Type="Italic">Aurelia</Emphasis> <Emphasis Type="Italic">aurita</Emphasis> measured by an in vitro cell assay

Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1, was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml⁻¹ (corresponding to 0.2 μg 10⁴ cells⁻¹), and a total loss of activity was observed above 40.0 μg ml⁻¹ (corresponding to 4.0 μg 10⁴ cells⁻¹). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 10⁴ cells⁻¹, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex venoms and, in some cases, more potent venoms than smaller animals.     

<Emphasis Type="Italic">Chromobacterium violaceum</Emphasis> and its important metabolites — <Emphasis Type="Italic">review</Emphasis>

C. violaceum appeared as important bacterium in different applications and mainly these aspects are related to the production of violacein. This review discusses the last reports on biosynthetic pathways, production, genetic aspects, biological activities, pathological effects, antipathogenic screening through quorum sensing, environmental effects and the products of C. violaceum with industrial interest. An important discussion is on biological applications in medicine and as industrial products such as textile and in cosmetics.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.