Isolation and characterization of a chitinase-secreting mutant of mortierella pusilla with altered interaction with the mycoparasite piptocephalis virginiana

Mortierella pusilla is a susceptible host and supports good growth of the mycoparasite, Piptocephalis virginiana. Uninucleate spores of M. pusilla were sUbjected to N-methyl-N'-nitro-nitrosoguanidine (MNNG). To attain a high mutation frequency , a 1o-minute exposure to 10 mg/ml MNNG was used and lead to the survival of about 10 % of the spores. The exposed spores then were plated on chitin or milk plates. Approximately 30,000 colonies were examined after mutagenesis on the screening media. A strain, MUT23 , with abnormal slow growth morphology was found to delay parasitism by £. virginiana. The particular morphology was not due to auxotrophy, because this strain displayed normal hyphae when glucose was used as the sole carbon source. One interesting phenomenon was that MUT23 showed an extensive clearing zone around the colony on colloidal chitin agar after 20-25 d. On the same conditions, wild type strain did not show this phenotype. In addition, the MUT23 strain produced the same normal hypha as the wild type strain when it was grown on colloidal chitin agar. The MUT23 was also able to produce more spores on colloidal chitin agar than on malt-yeast extract and minimal media. The parasite germ tubes formed appressoria at the point of contact on the cell surface of wild type and MUT23 grown for 6 days cell surface but not on the cel surface of MUT23 grown for 2 days. Thus, interaction between MUT23 strain and the mycoparasite was dependent on MUT23 age. The effect of MUT23 filtrate on germination of the parasite was tested. Lysis of germinated spores of the parasite were observed in concentrated MUT23 filtered solution. MUT23 was compared to the wild type strain for their chitinase production in sUbmerged culture. The chitinase isozymes of both wild type and MUT23 were shown by immunoblotting. Eight distinct chitinase molecules were detected. MUT23 showed markedly higher chitinase activity than the wild type cultured in chitin-containing medium. Maximum chitinase activities of MUT23 were 13.5 fold higher at 20 day of the culture then that of wild type.

Impact of process parameters on chitinase production by an alkalophilic marine Beau6eria bassiana in solid state fermentation

A chitinolytic fungus, Beau6eria bassiana was isolated from marine sediment and significant process parameters influencing chitinase production in solid state fermentation using wheat bran were optimised. The organism was strongly alkalophilic and produced maximum chitinase at pH 9·20. The NaCl and colloidal chitin requirements varied with the type of moistening medium used. Vegetative (mycelial) inoculum was more suitable than conidial inoculum for obtaining maximal enzyme yield. The addition of phosphate and yeast extract resulted in enhancement of chitinase yield. After optimisation, the maximum enzyme yield was 246·6 units g 1 initial dry substrate (U gIDS 1). This is the first report of the production of chitinase from a marine fungus.

Culture medium for isolating chitinolytic bacteria from seawater and plankton

Chitin degradation is a key step in the cycling of nutrients in marine ecosystems and chitinolytic bacteria are the primary agents of this process. Chitinases, produced by bacteria, have potential applications in agriculture, medicine and in a wide range of biotechnological processes. We utilized a simple, rapid and cost-effective method of colloidal chitin preparation and a culture medium, in which colloidal chitin is the sole carbon source for the purpose of counting and isolating chitinolytic bacteria from seawater and plankton. This culture medium could be useful to isolate bacteria with the ability to produce extracellular chitinases for biotechnological applications.

Characterization of a Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) - derived chitinase and its potential for pest control

The larval endoparasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) has a toolbox of biological weapons to secure for host colonization and the successful parasitization of its host Heliothis virescens (F.) (Lepidoptera: Noctuidae). The cDNA of a putative chitinase has been previously isolated and initially characterized from teratocytes of this parasitoid among the plethora of molecules available in the venom and calyx fluids injected by females, oral and/or anal secretions released by the parasitoid larvae and/or produced by the expression of genes of the symbiotic associated polydnavirus. This putative chitinase has been initially associated with the host cuticle digestion to allow for parasitoid egression and with the asepsis of the host environment, acting as an antimicrobial. As chitinases are commonly expressed in plants against plant pathogens, the chitinase derived from the teratocytes of T. nigriceps is a potential tool for the development of insect pest control methods based on the disruption of the perithrophic membrane of herbivores. Therefore, we aimed to characterize the activity of the putative chitinase from teratocytes of T. nigriceps (Tnchi) produced using the Escherichia coli expression system and its potential to control H. virescens larvae when expressed into transgenic tobacco plants. The purified E. coli-produced Tnchi protein showed no chitinolitic activity, but was active in binding with colloidal and crystalline chitins in water and with colloidal chitin in buffered solution (pH = 6.74). Transgenic tobacco plants showed no enhanced chitinolitic activity relative to control plants, but survival of three-day old larvae of H. virescens was severely affected when directly fed on transgenic tobacco leaves expressing the recombinant Tnchi protein. Some properties of the Tnchi protein and the potential use of Tnchi-transgenic plants to control plant pests are discussed. (c) 2012 Elsevier Inc. All rights reserved.

Immobilization Of Papain On Chitin And Chitosan And Recycling Of Soluble Enzyme For Deflocculation Of Saccharomyces Cerevisiae From Bioethanol Distilleries.

Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1-10% w·v(-1)), polyethyleneimine (0.5% v·v(-1)), and tripolyphosphate (1-10% w·v(-1)) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L(-1). Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.

Chitosan Nanoparticles Loaded The Herbicide Paraquat: The Influence Of The Aquatic Humic Substances On The Colloidal Stability And Toxicity.

Polymeric nanoparticles have been developed for several applications, among them as carrier system of pesticides. However, few studies have investigated the fate of these materials in the environment in relation to colloidal stability and toxicity. In nature, humic substances are the main agents responsible for complexation with metals and organic compounds, as well as responsible for the dynamics of these nanoparticles in aquatic and terrestrial environments. In this context, the evaluation of the influence of aquatic humic substances (AHS) on the colloidal stability and toxicity of polymeric nanoparticles of chitosan/tripolyphosphate with or without paraquat was performed. In this study, the nanoparticles were prepared by the ionic gelation method and characterized by size distribution measurements (DLS and NTA), zeta potential, infrared and fluorescence spectroscopy. Allium cepa genotoxicity studies and ecotoxicity assays with the alga Pseudokirchneriella subcapitata were used to investigate the effect of aquatic humic substances (AHS) on the toxicity of this delivery system. No changes were observed in the physical-chemical stability of the nanoparticles due to the presence of AHS using DLS and NTA techniques. However some evidence of interaction between the nanoparticles and AHS was observed by infrared and fluorescence spectroscopies. The ecotoxicity and genotoxicity assays showed that humic substances can decrease the toxic effects of nanoparticles containing paraquat. These results are interesting because they are important for understanding the interaction of these nanostructured carrier systems with species present in aquatic ecosystems such as humic substances, and in this way, opening new perspectives for studies on the dynamics of these carrier systems in the ecosystem.

Nanostructured thin films obtained by electrodeposition over a colloidal crystal template: applications in electrochemical devices

Colloidal particles have been used to template the electrosynthesis of several materials, such as semiconductors, metals and alloys. The method allows good control over the thickness of the resulting material by choosing the appropriate charge applied to the system, and it is able to produce high density deposited materials without shrinkage. These materials are a true model of the template structure and, due to the high surface areas obtained, are very promising for use in electrochemical applications. In the present work, the assembly of monodisperse polystyrene templates was conduced over gold, platinum and glassy carbon substrates in order to show the electrodeposition of an oxide, a conducting polymer and a hybrid inorganic-organic material with applications in the supercapacitor and sensor fields. The performances of the resulting nanostructured films have been compared with the analogue bulk material and the results achieved are depicted in this paper.

Mechanism of point-defect diffusion in a two-dimensional colloidal crystal

The dynamics and mechanism of migration of a vacancy point defect in a two-dimensional (2D) colloidal crystal are studied using numerical simulations. We find that the migration of a vacancy is always realized by topology switching between its different configurations. From the temperature dependence of the topology switch frequencies, we obtain the activation energies for possible topology transitions associated with the vacancy diffusion in the 2D crystal. (C) 2011 American Institute of Physics. [doi:10.1063/1.3615287]

Colloidal Processing of Glass-Ceramics for Laminated Object Manufacturing

Green tapes of Li(2)O-ZrO(2)-SiO(2)-Al(2)O(3) (LZSA) parent glass were produced by aqueous tape casting as the starting material for the laminated object manufacturing (LOM) process. The rheological behavior of the powder suspensions in aqueous media, as well as the mechanical properties of the cast tapes, was evaluated. According to xi potential measurements, the LZSA glass powder particles showed acid surface characteristics and an IEP of around 4 when in aqueous media. The critical volume fraction of solids was about 72 wt% (27 vol%), which hindered the processability of more concentrated slurries. The glass particles also showed an anisometric profile, which contributed to an increase in the interactions between particles during flow. Therefore, the suspensions could not be processed at high solids loadings. Aqueous-based glass suspensions were also characterized by shear thickening after the addition of dispersants. Three slurry compositions were formulated, suitable green tapes were cast, and tapes were successfully laminated by LOM to a gear wheel geometry. A higher tensile strength of the green tapes corresponded to a higher tensile strength of the laminates. Thermal treatment was then applied to the laminates: pyrolysis at 525 degrees C, sintering at 700 degrees C for 1 h, and crystallization at 850 degrees C for 30 min. A 20% volumetric shrinkage was observed, but no surface flaws or inhomogeneous areas were detected. The sintered part maintained the curved edges and internal profile after heat treatment.

THE ROLE OF COLLOIDAL SILICON DIOXIDE IN THE ENHANCEMENT OF THE DRYING OF HERBAL PREPARATIONS IN SUSPENDED STATE

Recently, some research groups have been developing studies aiming to apply spouted beds of inert particles for production of dried herbal extracts. However, mainly due to their complex composition, several problems arise during the spouted bed drying of herbal extracts such as bed instability, product accumulation, particle agglomeration, and bed collapse. The addition of drying carriers, like colloidal silicon dioxide, to the extractive solution can minimize these unwanted effects. The aim of this work was to study the influence of the addition of colloidal silicon dioxide on enhancement of the performance of the drying of hydroalcoholic extract of Bauhinia forficata Link on a spouted bed of inert particles. The physical properties of the herbal extract and of its mixture with colloidal silicon dioxide at several concentrations (20% to 80% related to solids content) were quantified by determination of the surface tension, rheological properties, density, pH, and contact angles with the inert surfaces. Drying performance was evaluated through determination of the elutriation ratio, product recovery ratio, and product accumulation. The product was characterized through determination of the thermal degradation of bioactive compounds and product moisture content. The results indicated that the rheological properties of the extracts and their preparations, the contact angle with inert material, and the work of adhesion play important roles in the spouted bed drying of herbal extracts. Higher concentration of the drying carrier significantly improved the spouted bed drying performance.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa. which is purified by affinity, with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels or TNF alpha. and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification or paracoccin. This procedure provided higher yields than those achieved by means of the technique based oil the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70000 and pl approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright (C) 2009 John Wiley & Sons. Ltd.

Novel colloidal materials for high-throughput screening applications in drug discovery and genomics

Tracking the reaction history is the means of choice to identify bioactive compounds in large combinatorial libraries. The authors describe two approaches to synthesis on silica beads: a) addition of a reporter dye tag during each synthesis step (see Figure), which attaches itself to the bead by colloidal forces, and b) encapsulating arrays of fluorescent dyes into the beads to encode them uniquely, for recognition with a flow cytometer after each reaction step.

Optical barcoding of colloidal suspensions: applications in genomics, proteomics and drug discovery

The enormous amount of information generated through sequencing of the human genome has increased demands for more economical and flexible alternatives in genomics, proteomics and drug discovery. Many companies and institutions have recognised the potential of increasing the size and complexity of chemical libraries by producing large chemical libraries on colloidal support beads. Since colloid-based compounds in a suspension are randomly located, an encoding system such as optical barcoding is required to permit rapid elucidation of the compound structures. We describe in this article innovative methods for optical barcoding of colloids for use as support beads in both combinatorial and non-combinatorial libraries. We focus in particular on the difficult problem of barcoding extremely large libraries, which if solved, will transform the manner in which genomics, proteomics and drug discovery research is currently performed.

Synthesis of optically complex core-shell colloidal suspensions: Pathways to multiplexed biological screening

The ability to generate enormous random libraries of DNA probes via split-and-mix synthesis on solid supports is an important biotechnological application of colloids that has not been fully utilized to date. To discriminate between colloid-based DNA probes each colloidal particle must be 'encoded' so it is distinguishable from all other particles. To this end, we have used novel particle synthesis strategies to produce large numbers of optically encoded particle suitable for DNA library synthesis. Multifluorescent particles with unique and reproducible optical signatures (i.e., fluorescence and light-scattering attributes) suitable for high-throughput flow cytometry have been produced. In the spectroscopic study presented here, we investigated the optical characteristics of multi-fluorescent particles that were synthesized by coating silica 'core' particles with up to six different fluorescent dye shells alternated with non-fluorescent silica 'spacer' shells. It was observed that the diameter of the particles increased by up to 20% as a result of the addition of twelve concentric shells and that there was a significant reduction in fluorescence emission intensities from inner shells as an increasing number of shells were deposited.

Determination of chitin content in fungal cell wall: an alternative flow cytometric method

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted Glycosyl Phosphatidyl Inositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi.