DNA sequencing with chain terminating inhibitors

Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the β-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.

The biological degradation of cellulose

β-Glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the s...

Microbial beta-glucosidases: cloning, properties, and applications.

Exposure of food proteins to certain processing conditions induces two major chemical changes:  racemization of all l-amino acids to d-isomers and concurrent formation of cross-linked amino acids such as lysinoalanine. Racemization of l-amino acids residues to their d-isomers in food and other proteins is pH-, time-, and temperature-dependent. Although racemization rates of the 18 different l-amino acid residues in a protein vary, the relative rates in different proteins are similar. The diet contains both processing-induced and naturally formed d-amino acids. The latter include those found in microorganisms, plants, and marine invertebrates. Racemization impairs digestibility and nutritional quality. The nutritional utilization of different d-amino acids varies widely in animals and humans. In addition, some d-amino acids may be both beneficial and deleterious. Thus, although d-phenylalanine in an all-amino-acid diet is utilized as a nutritional source of l-phenylalanine, high concentrations of d-tyrosin...

Chemistry, nutrition, and microbiology of D-amino acids.

The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 A resolution, and a comparison with related enzymes.

This review describes the most recent developments in the biotechnological applications of penicillin acylases. This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid and the synthesis of semisynthetic β-lactam antibiotics. In addition, penicillin acylases can also be employed in other useful biotransformations, such as peptide synthesis and the resolution of racemic mixtures of chiral compounds. Particular emphasis is placed on advances in detection of new enzyme specificities towards other natural penicillins, enzyme immobilization, and optimization of enzyme-catalyzed hydrolysis and synthesis in the presence of organic solvents.

Biotechnological applications of penicillin acylases: state-of-the-art.

Endoglucanase III (EG III) was purified to homogeneity from the culture medium of Trichoderma reesei QM 9414. It has a molecular mass of 48 kDa, and an isoelectric point of 5.1. Maximal activity was observed between pH4 and 5. Celloligosaccharides and their chromophoric derivatives were used as substrates, and the reaction products were analysed by quantitative h.p.l.c. Nucleophilic competition experiments (between methanol and water) allowed unequivocal assessment of cleavage sites. EG III preferentially released cellobiose (or the corresponding glycoside) from the reducing end of the higher cellodextrins. A putative binding model containing five subsites is proposed. The pH-dependence of 4′-methylumbelliferyl beta-cellotrioside hydrolysis indicates the presence of a protonated group with a pK 5.5 in the reaction mechanism, and the possible involvement of a carboxy group is corroborated by a temperature study (delta Hion = -15.9 J/mol). This, together with independent evidence from affinity-labelling experiments [Tomme, Macarron and Claeyssens (1991) Cellulose 991, New Orleans, Abstr. 32] and n.m.r. studies [Gebbler, Gilkes, Claeyssens, Wilson, Beguin, Wakarchuk, Kilburn, Miller, Warren and Withers (1992) J. Biol. Chem. 267, 12559-12561], favours the assumption of a lysozyme-type (retention of configuration, two essential carboxy groups) mechanism for this family A cellulase.

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Mode of action of endoglucanase III from Trichoderma reesei.

In recent years, glycosiltransferases have arisen as standard biocatalysts for the enzymatic synthesis of a wide variety of natural and non-natural nucleosides. Such enzymatic synthesis of nucleoside analogs catalyzed by nucleoside phosphorylases and 2′-deoxyribosyltransferases (NDTs) has demonstrated to be an efficient alternative to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection–deprotection steps. This minireview exhaustively covers literature reports on this topic with the final aim of presenting NDTs as an efficient option to nucleoside phosphorylases for the synthesis of natural and non-natural nucleosides. Detailed comments about structure and catalytic mechanism of described NDTs, as well as their possible biological role, substrate specificity, and advances in detection of new enzyme specificities towards different non-natural nucleoside synthesis are included. In addition, optimization of enzymatic transglycosylation reactions and their application in the synthesis of natural and non-natural nucleosides have been described. Finally, immobilization of NDTs is shown as a practical procedure which leads to the preparation of very interesting biocatalysts applicable to industrial nucleoside synthesis.

New insights on nucleoside 2′-deoxyribosyltransferases: a versatile Biocatalyst for one-pot one-step synthesis of nucleoside analogs

At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.

Enhanced production of penicillin V acylase from Streptomyces lavendulae.

Penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae showed both enhanced activity and stability in mixed water/glycerol and water/glycols solvents. The catalytic activity was increased up to a critical concentration of these cosolvents, but further addition of the latter led to a gradual protein deactivation. The highest stabilizing effect was achieved in the presence of glycerol. Thermal stability was increased proportionally to the concentration of glycerol and glycols in the reaction mixture only if the amount added is below the threshold concentration. Reaction conditions that allow simultaneously enhanced activity and stability in the hydrolysis of penicillin V catalyzed by penicillin V acylase from S. lavendulae could be established.

I. de la Mata

Papers

Biotechnological applications of penicillin acylases: state-of-the-art.

Mode of action of endoglucanase III from Trichoderma reesei.

New insights on nucleoside 2′-deoxyribosyltransferases: a versatile Biocatalyst for one-pot one-step synthesis of nucleoside analogs

Enhanced production of penicillin V acylase from Streptomyces lavendulae.

Activation and stabilization of penicillin V acylase from streptomyces lavendulae in the presence of glycerol and glycols.