RESUMO
We report kinetic data of penicillin hydrolysis catalyzed by beta-lactamase entrapped in reverse micelles formed with cetyl trimethylammonium bromide (CTAB), n-octane, hexanol and aqueous buffer. The K(cat) of this diffusion-limited reaction can be improved in aqueous buffer by a factor of 1.1-1.2 just by increasing the phosphate buffer concentration from 50 to 100 mM. In reverse micelles, increasing the buffer concentration has little effect on K(cat) when the size of the empty micelle is below the size of the protein. However, in larger micelles, the effect is enhanced and the K(cat) improves several fold, changing the form of the curve of K(cat) versus Wo from bell-shaped to almost hyperbolic. The results indicate that micellar exchange and internal diffusion may limit the reaction in reverse micelles and provide further evidence that the form of the curve depends on other factors besides the relationship between the size of the enzyme and that of the empty reverse micelle.
Assuntos
Compostos de Cetrimônio/metabolismo , Escherichia coli/enzimologia , Penicilinas/metabolismo , beta-Lactamases/metabolismo , Catálise , Cetrimônio , Difusão , Micelas , Fosfatos/metabolismo , Compostos de Potássio/metabolismoRESUMO
The ability of alpha-amylases from different sources to carry out reactions of alcoholysis was studied using methanol as substrate. It was found that while the enzymes from Aspergillus niger and Aspergillus oryzae, two well-studied saccharifying amylases, are capable of alcoholysis reactions, the classical bacterial liquefying alpha-amylases from Bacillus licheniformis and Bacillus stearothermophilus are not. The effect of starch and methanol concentration, temperature and pH on the synthesis of glucosides with alpha-amylase from A. niger was studied. Although methanol may inactivate alpha-amylase, a 90% substrate relative conversion can be obtained in 20% methanol at a high starch concentration (15% w/v) due to a stabilizing effect of starch on the enzyme. As the products of alcoholysis are a series of methyl-oligosaccharides, from methyl-glucoside to methyl-hexomaltoside, alcoholysis was indirectly quantified by high performance liquid chromatography analysis of the total methyl-glucoside produced after the addition of glucoamylase to the alpha-amylase reaction products. More alcoholysis was obtained from intact soluble starch than with maltodextrins or pre-hydrolyzed starch. The biotechnological implications of using starch as substrate for the production of alkyl-glucosides is analyzed in the context of these results.
Assuntos
Aspergillus/enzimologia , Bacillus/enzimologia , Metanol/metabolismo , Amido/metabolismo , alfa-Amilases/metabolismo , Aspergillus niger/enzimologia , Aspergillus oryzae/enzimologia , Geobacillus stearothermophilus/enzimologia , Cinética , Especificidade por SubstratoRESUMO
By mutating Ala-289 by Phe or Tyr in the Bacillus stearothermophilus alpha-amylase, we induced this enzyme to perform alcoholytic reactions, a function not present in the wild-type enzyme. This residue was selected from homology analysis with neopullulanase, where the residue has been implicated in the control of transglycosylation [Kuriki et al. (1996) J. Biol. Chem. 271, 17321-173291. We made some inferences about the importance of electrostatic and geometrical modifications in the active site environment of the amylase to explain the behavior of the modified enzyme.
Assuntos
Substituição de Aminoácidos , Geobacillus stearothermophilus/enzimologia , Glicosiltransferases/metabolismo , alfa-Amilases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Glicosídeo Hidrolases/metabolismo , Glicosilação , Glicosiltransferases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Análise de Sequência , alfa-Amilases/genéticaRESUMO
The hydrolytic enzymes, alpha-amylases, and the cyclodextrin glycosyltransferases (CGTases) are key enzymes in the depolymerization of starch. These two groups of enzymes are evolutionarily related. We propose that the transferase activity is likely to have evolved from an ancestral hydrolase. Sequence analysis provides support for this hypothesis. Consequently, we have conducted an experimental study to test the possible adaptive value for evolving a CGTase. We found that when an alpha-amylase and a CGTase are combined more glucose is generated from starch than would be expected from the independent action of either of these enzymes. Thus, we propose that the biological role of CGTases is to work in concert with alpha-amylases for the efficient saccharification of starch. This observation can be useful in industrial processes aimed at producing syrups with high contents of glucose or maltose.
Assuntos
Bactérias/enzimologia , Evolução Molecular , Glucosiltransferases/genética , Filogenia , Plantas/enzimologia , alfa-Amilases/genética , Bactérias/classificação , Bactérias/genética , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Plantas/classificação , Plantas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Software , alfa-Amilases/química , alfa-Amilases/metabolismoRESUMO
This article discusses the techniques of site-specific mutagenesis and protein engineering and their application in the study of enzyme active sites and the mechanism of enzyme action. Particular emphasis is given to beta-lactamase.
Assuntos
Enzimas/química , Engenharia de Proteínas/métodos , Ativação Enzimática , Mutagênese Sítio-Dirigida , Especificidade por SubstratoRESUMO
This article discusses the techniques of site-specific mutagenesis and protein engineering and their application in the study of enzyme active sites and the mechanism of enzyme action. Particular emphasis is given to beta-lactamase
Assuntos
Enzimas/química , Engenharia de Proteínas/métodos , Ativação Enzimática , Mutagênese Sítio-Dirigida , Especificidade por SubstratoRESUMO
A number of considerations are made on the efficiency of mutagenesis techniques used in protein engineering, particularly those that include a random component. The expected outcome of different protocols is analyzed using computer programs. Special emphasis is made on the effect that the degeneracy of the genetic code has on the bias of the representation of amino acid replacements. The consequences of using alternative methods is analyzed in terms of the likelihood of obtaining underrepresented amino acid substitutions in mutant libraries. A consideration is also made of the outcome of combinatorial mutagenesis experiments with regard to the size of the amino acid window and the multiplicity of replacements that could be sampled with different methods. Optimal mutagenesis rates for specific conditions could be derived from the presented data and the computer program made available with this paper.