Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96?hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral–alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.     

Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

In Synechocystis sp. PCC 6803, the loop domain (aa 1–70) of the phycobilisome core-membrane linker, L_CM, was found to interact with the glycosyl transferase homolog, Sll1466. Growth of a Sll1466 knock-out mutant was slightly faster in low light, but strongly inhibited in high light; the phenotype is discussed in relation to the regulation of light energy transfer to photosystem II. At the molecular level, the mutant shows the following changes compared to the wild type: (1) a smaller size and higher mobility of phycobilisomes on the thylakoid membrane, and (2) a changed lipid composition of the thylakoid membrane, especially decreased amounts of digalactosyl diacylglycerol. These results indicate a profound regulatory role for Sll1466 in regulating photosynthetic energy transfer.     

Quantitative trait loci and candidate genes associated with starch pasting viscosity characteristics in cassava (Manihot esculenta Crantz)

Starch pasting viscosity is an important quality trait in cassava (Manihot esculenta Crantz) cultivars. The aim here was to identify loci and candidate genes associated with the starch pasting viscosity. Quantitative trait loci (QTL) mapping for seven pasting viscosity parameters was carried out using 100 lines of an F₁ mapping population from a cross between two cassava cultivars Huay Bong 60 and Hanatee. Starch samples were obtained from roots of cassava grown in 2008 and 2009 at Rayong, and in 2009 at Lop Buri province, Thailand. The traits showed continuous distribution among the F₁ progeny with transgressive variation. Fifteen QTL were identified from mean trait data, with Logarithm of Odds (LOD) values from 2.77–13.01 and phenotype variations explained (PVE) from10.0–48.4%. In addition, 48 QTL were identified in separate environments. The LOD values ranged from 2.55–8.68 and explained 6.6–43.7% of phenotype variation. The loci were located on 19 linkage groups. The most important QTL for pasting temperature (PT) (qPT.1LG1) from mean trait values showed largest effect with highest LOD value (13.01) and PVE (48.4%). The QTL co‐localised with PT and pasting time (PTi) loci that were identified in separate environments. Candidate genes were identified within the QTL peak regions. However, the major genes of interest, encoding the family of glycosyl or glucosyl transferases and hydrolases, were located at the periphery of QTL peaks. The loci identified could be effectively applied in breeding programmes to improve cassava starch quality. Alleles of candidate genes should be further studied in order to better understand their effects on starch quality traits.     

Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.     

原人参二醇型皂苷水解酶及制备人参皂苷Compound K的研究进展

人参皂苷Compound K (CK)是一种具有抗癌抗炎等药理活性的化合物。目前在天然人参中暂未鉴定出,工业上主要通过原人参二醇型皂苷的去糖基化进行制备。相对于传统的物理、化学的去糖基化法,利用原人参二醇型皂苷水解酶制备CK具有特异性强、绿色环保和高效稳定的优点。本文根据水解酶作用的糖基连接碳原子的差异将原人参二醇型皂苷水解酶分成了3类,发现大多数能制备CK的水解酶为Ⅲ型原人参二醇型皂苷水解酶。此外,对水解酶在制备CK中的应用进行了总结评估,旨在为人参皂苷CK的大规模制备及其在食品和药品行业中的开发提供参考。     

Catalytic properties of β-cyclodextrin glucanotransferase from alkalophilic<Emphasis Type="Italic">Bacillus</Emphasis> sp. BL-12 and intermolecular transglycosylation of stevioside

The catalytic properties of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilicBacillus sp. BL-12 specific for the intermolecular transglycosylation of stevioside were investigated. The molecular mass of purified β-CGTase by ultra-filtration and β-cyclodextrin polymer affinity chromatography was estimated to be 90 kDa, which is high compared to other known bacterial CGTases. The optimal pH and temperature were 9.0 and 50°C, respectively, and thermal stability at 40°C was elevated 10-fold in the presence of 1% maltodextrin. The kinetic parameters of the new β-CGTase from alkalophilicBacillus sp. BL-12 indicate that it is more suitable for transglycosylation than the cyclization reaction. Maltodextrin was the most suitable glycosyl donor for transglycosylation of stevioside. The transglycosylation of stevioside was carried out using 60 units of CGTase per gram of maltodextrin, 20 g/L stevioside as the glycosyl acceptor, and 50 g/L maltodextrin as the gycosyl donor at 40°C for 6 h, and a conversion yield of stevioside as high as 76% was obtained.     

Carbohydrate de-N-acetylases acting on structural polysaccharides and glycoconjugates

Deacetylation of N-acetylhexosamine residues in structural polysaccharides and glycoconjugates is catalyzed by different families of carbohydrate esterases that, despite different structural folds, share a common metal-assisted acid/base mechanism with the metal cation coordinated with a conserved Asp-His-His triad. These enzymes serve diverse biological functions in the modification of cell-surface polysaccharides in bacteria and fungi as well as in the metabolism of hexosamines in the biosynthesis of cellular glycoconjugates. Focusing on carbohydrate de-N-acetylases, this article summarizes the background of the different families from a structural and functional viewpoint and covers advances in the characterization of novel enzymes over the last 2–3 years. Current research is addressed to the identification of new deacetylases and unravel their biological functions as they are candidate targets for the design of antimicrobials against pathogenic bacteria and fungi. Likewise, some families are also used as biocatalysts for the production of defined glycostructures with diverse applications.     

Microbial and viral chitinases: Attractive biopesticides for integrated pest management

The negative impact of the massive use of synthetic pesticides on the environment and on human health has stimulated the search for environment-friendly practices for controlling plant diseases and pests. Among them, biocontrol, which relies on using beneficial organisms or their products (bioactive molecules and/or hydrolytic enzymes), holds the greatest promise and is considered a pillar of integrated pest management. Chitinases are particularly attractive to this purpose since they have fungicidal, insecticidal, and nematicidal activities. Here, current knowledge on the biopesticidal action of microbial and viral chitinases is reviewed, together with a critical analysis of their future development as biopesticides.     

Immobilized Cyclodextrin Glycosyl Transferase for the Continuous Production of Cyclodextrins

Cyclodextrin glycosyl transferase (E.C: 2.4.1.19) from Bacillus, macerans and from a Bacillus sp. isolate was immobilized by two methods, viz. to epoxy-activated Sepharose and to alkylamine silica treated with glutaraldehyde. Because of the ready availability, low cost ($0.01/g), good surface area (30 M²/g) and ease of operation of a continuous cylindrical reactor, the high silica fabric was chosen. The immobilized enzyme had a pH optimum shifted to the alkaline side (from 6.5 to 7.5) and had a reduced temperature optimum (from 60°C to 50-55°C). Reuse efficiency showed 65% reduction in the overall activity of the immobilized enzyme after 10 cycles of 48 h each. Continuous operation at 55°C of a cylindrical reactor of 141 ml capacity, using the immobilized enzyme (80 g of high - silica fabric containing 114 mg of purified enzyme) gave a maximum productivity of 10.2 g of cyclodextrins L^-1 h,^-1, at a dilution rate of 0.32 h^-1 and a substrate concentration of 20 g L^-1. The half life of the biocatalyst was found to be 22 days, which could be further improved by using a lower operating temperature. Over the useful life time of the immobilized biocatalyst (22 days), the total Cyclodextrin produced was of the order of 88 Kg.