Sergei Braun

Biochemically active sol-gel glasses: The trapping of enzymes ☆

Abstract We describe the preparation and properties of a biochemically active sol-gel glass, obtained by trapping the enzyme alkaline phosphatase (ALP) in a polymerizing tetramethoxysilane. The immobilized purified ALP from bovine intestinal mucosa had a 30% activity yield and an improved stability to thermal deactivation compared to a solution. The composite bioactive glass was preserved in water at room temperature for two months without loosing activity. A non-Michaelis-Menten kinetics was observed. The concept of preparing bioactive materials by the sol-gel method seems to be general. Thus, other enzymes (chitinase, aspartase, β-glucosidase) were successfully trapped.

Mycelial morphology and metabolite production

Abstract Mycelial microorganisms are exploited extensively in the commercial production of a wide range of secondary metabolites. They can be cultured as free mycelia, as aggregated forms (pellets/flocs), or as artificially bound/entrapped cells, though problems are associated with the culture of each morphological type. Since the morphological type can strongly influence metabolite production, the methodology for inducing pellet formation, and the type of pellets produced are an important consideration for effective metabolite production.

Biocatalysis by sol-gel entrapped enzymes

Abstract Attachment of enzymes to insoluble matrix is an essential step in the development of biocatalysts. Transparent xerogels containing various enzymes were obtained by mixing a solution of an enzyme with tetra-methoxy orthosilicate (TMOS) at room temperature followed by gelation and drying. Effective immobilization was usually obtained at initial pH values > 7, where there is a change in the gelation mechanism from predominant hydrolysis/condensation to predominant direct polymerization of silicate precursors. The properties of sol-gel matrix, namely, transparency, large hydrophilic surface and good chemical and thermal stability, make it an ideal material for both biocatalysts and optical sensor devices. An example of a simple optical glucose sensor is demonstrated.

Photoprotection of Bacillus thuringiensis kurstaki from ultraviolet irradiation

Irradiation of Bacillus thuringiensis var. kurstaki HD1 at 300-350 nm for up to 12 hr using a photochemical reactor results in a rapid loss of its toxicity to larvae of Heliothis armigera. Photoprotection of the toxic component was obtained by adsorption of cationic chromophores such as acriflavin (AF), methyl green, and rhodamine B to B. thuringiensis. AF gave the best photoprotection and a level of 0.42 mmol/g dye absorbed per gram of B. thuringiensis was highly toxic even after 12 hr of ultraviolet (uv) irradiation as compared to the control (77.5 and 5% of insect mortality, respectively). Ultraviolet and Fourier-transform infrared spectroscopic studies indicate molecular interactions between B. thuringiensis and AF. The nature of these interactions and energy or charge transfer as possible mechanisms of photoprotection are discussed. It is speculated that tryptophan residues are essential for the toxic effect of B. thuringiensis. It is suggested that photoprotection is attained as energy is transferred from the excited tryptophan moieties to the chromophore molecules.

Conversion of Fumaric Acid to l-Malic by Sol-Gel Immobilized Saccharomyces cerevisiae in a Supported Liquid Membrane Bioreactor

Conversion of fumaric acid (FA) to l‐malic acid (LMA) was carried out in a bioreactor divided by two supported liquid membranes (SLMs) into three compartments: Feed, Reaction, and Product. The Feed/Reaction SLM, made of tri‐ n‐octylphosphine oxide (vol 10%) in ethyl acetate, was selective toward the substrate, fumaric acid ( SFA/LMA = 10). The Reaction/Product SLM, made of di(2‐ethylhexyl) phosphate (vol 10%) in dichloromethane, was selective toward the product, l‐malic acid ( SLMA/FA = 680). Immobilized yeast engineered to overproduce the enzyme fumarase [E.C. 4.2.1.2] was placed in the Reaction compartment and served as the catalyst. The yeast was immobilized in small glasslike beads of alginate‐silicate sol‐gel matrix. The construction of the bioreactor ensured unidirectional flow of the substrate from the Feed to the Reaction and of the product from the Reaction to the Product compartments, with the inorganic counterion traveling in the opposite direction. The conversion of almost 100%, above the equilibrium value of ca. 84% and higher than that for the industrial process, 70%, was achieved. In contrast to the existing industrial biocatalytic process resulting in l‐malic acid salts, direct production of the free acid is described.

The Crystal Structures of the Psychrophilic Subtilisin S41 and the Mesophilic Subtilisin Sph Reveal the Same Calcium-Loaded State.

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold‐adapted serine protease produced by Antarctic bacilli, at 1.4 Å resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 Å resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 Å. The final models for S41 and Sph include a calcium‐loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium‐binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium‐binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main‐chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures. Proteins 2009.

Biochemical Aspects of Sol-Gel Science and Technology

To our Readers S. Braun, D. Avnir. Entrapment of Parathion Hydrolase from Pseudomonas spp. in Sol-Gel Glass C. Dosoretz, et al. Immobilization of Urease on Composite Fibre by Using a Gel Formation of Cellulose Acetate and Titanium Iso-Propoxide H. Hatayama, et al. Biogels of Cytochrome c: Cytochrome c Peroxidase Complex Studied by Electron Paramagnetic Resonance Spectroscopy C.T. Lin, et al. Bioactive Ca10(PO4)6(OH)2-TiO2 Composite Coating Prepared by Sol-Gel Process P. Li, et al. Characterization of Hydrophobic Sol-Gel Materials Containing Entrapped Lipases M.T. Reetz, et al. Immunoassays in Sol-Gel Matrices J. Livage, et al. Iodide Fluorescence Quenching of Sol-Gel Immobilized BSA C.L. Wambolt, S.S. Saavedra. Mechanisms of Hydroxyapatite Formation on Porous Gel-Silica Substrates M.M. Pereira, L.L. Hench. In vitro Protein Interactions with a Bioactive Gel-Glass K.D. Lobel, L.L. Hench. Atrazine Degradation by Pseudomonas Strain ADP Entrapped in Sol-Gel Glass M. Rietti-Shati, et al. Biological Activity of Functionalized SiO2 Thin Films Prepared by Sol-Gel Method R. Collino, et al. Immobilization of Plant Cell in Hybrid Sol-Gel Materials R. Campostrini, et al. Phycobiliproteins Encapsulated in Sol-Gel Glass Zhongping Chen, et al. Encapsulation of the Ferritin Protein in Sol-Gel Derived Silica Glasses E.H. Lan, et al. Enzymatic Activity of Oxalate Oxidase and Kinetic Measurements by Optical Methods in Transparent Sol-Gel Monoliths S.A. Yamanaka, et al. Sol-Gel Derived Ceramic-Carbon Enzyme Electrodes: Glucose-Oxidase as a Test Case S. Sampath, et al. Use of Sol-Gel Entrapment Techniques for the Resolution of Itaconic Acid Biosynthetic Pathway in Aspergillus terreus E. Bresser, S. Braun. Sol-Gel Entrapment of Monoclonal Anti-Atrazine Antibodies A. Turniansky, et al.

Bacillus thuringiensis potency bioassays against Heliothis armigera, Earias insulana, and Spodoptera littoralis larvae based on standardized diets

Bacillus thuringiensis screening programs based on the official potency bioassay using third-instar larvae and on a neonate bioassay were developed for Heliothis armigera, Earias insulana, and Spondoptera littoralis. In these bioassays, the diets were standardized to be suitable, with minor modifications, for feeding of the three lepidopterans. The bioassay protocol was based on determination of the LC50 of the microbial standard HD-1-S-80 in the insects susceptible to B. thuringiensis var. kurstaki strains. This was followed by preliminary screening of B. thuringiensis strains at the LC50 of the B. thuringiensis standard. The B. thuringiensis strains causing 100% mortality at this LC50 in the larvae were selected for potency determinations. The neonate bioassay was suitable for accurate determinations of potencies also in S. littoralis--a representative of insects weakly susceptible to the HD-1 standard. The role of the official and the neonate bioassays in developing microbial control programs is discussed.

Conversion of citric acid to itaconic acid in a novel liquid membrane bioreactor

A novel liquid membrane reactor was designed to carry out the conversion of citric acid to itaconic (methylene succinic) acid by the fungus Aspergillus terreus. This bioreactor contained two supported liquid membranes (SLMs) that divided it into three compartments: (1) the feed chamber containing citric acid; (2) the reaction chamber containing the enzyme A terreus cells; and (3) the product collection chamber containing the counter-ion. The specificity of SLMs allows the unidirectional flow, of the substrate from the feed to the reaction chamber, and of the product from the reaction to the product chamber in exchange for a counter-ion, thus maintaining low concentrations of both the product and the substrate, and limiting their inhibitory effects on the conversion process. The yield of itaconic acid in the membrane bioreactor was about two orders of magnitude higher than in the batch process. © 2000 Society of Chemical Industry

Turbidostat culture of Saccharomyces cerevisiae W303‐1A under selective pressure elicited by ethanol selects for mutations in SSD1 and UTH1

We investigated the genetic causes of ethanol tolerance by characterizing mutations selected in Saccharomyces cerevisiae W303-1A under the selective pressure of ethanol. W303-1A was subjected to three rounds of turbidostat, in a medium supplemented with increasing amounts of ethanol. By the end of selection, the growth rate of the culture has increased from 0.029 to 0.32 h(-1) . Unlike the progenitor strain, all yeast cells isolated from this population were able to form colonies on medium supplemented with 7% ethanol within 6 days, our definition of ethanol tolerance. Several clones selected from all three stages of selection were able to form dense colonies within 2 days on solid medium supplemented with 9% ethanol. We sequenced the whole genomes of six clones and identified mutations responsible for ethanol tolerance. Thirteen additional clones were tested for the presence of similar mutations. In 15 of 19 tolerant clones, the stop codon in ssd1-d was replaced with an amino acid-encoding codon. Three other clones contained one of two mutations in UTH1, and one clone did not contain mutations in either SSD1 or UTH1. We showed that the mutations in SSD1 and UTH1 increased tolerance of the cell wall to zymolyase and conclude that stability of the cell wall is a major factor in increased tolerance to ethanol.