RESUMEN
Penicillin G acylase is the key enzyme used in the industrial production of ß-lactam antibiotics. This enzyme hydrolyzes penicillin G and related ß-lactam antibiotics releasing 6-aminopenicillanic acid, which is an intermediate in the production of semisynthetic penicillins. To improve the enzymatic activity of Escherichia coli penicillin acylase, sequential rounds of error-prone polymerase chain reaction were applied to the E. coli pac gene. After the second round of evolution, the best mutant M2234 with enhanced activity was selected and analyzed. DNA sequence analyses of M2234 revealed that one amino acid residue (K297I), located far from the center of the catalytic pocket, was changed. This mutant (M2234) has a specific activity 4.0 times higher than the parent enzyme and also displayed higher stability at pH 10.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Mutagénesis , Penicilina Amidasa/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Sustitución de Aminoácidos , Estabilidad de Enzimas , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Concentración de Iones de Hidrógeno , Mutación Missense , Penicilina Amidasa/química , Penicilina Amidasa/genética , Mutación Puntual , Ingeniería de Proteínas/métodos , Análisis de Secuencia de ADNRESUMEN
Five hundred ninety-seven bacterial isolates from Turkish hot spring water sources were screened for their ability to produce extracellular α-amylase. Among them, a high enzyme-producing Bacillus subtilis isolate, A28, was selected, and its α-amylase gene was cloned and expressed in Escherichia coli by a ligase-independent method. α-Amylase from the recombinant strain was purified to homogeneity by Q-Sepharose anion exchange and Sephacryl S-100 gel filtration chromatographies. The final yield of the enzyme was about 22.5 % of the initial activity, with a 16.4-fold increase in specific activity compared with the culture lysate. The optimum temperature and pH of the enzyme were 70 °C and 6.0, respectively. The enzyme was highly active at acidic-neutral pH range of 4.5-7.0. The amy28 α-amylase retained 100 % of its activity after incubation at 50 °C for 90 min. Co(+2), Cu(2+), Fe(2+), Fe(3+), Ni(+2), and Zn(+2) caused significant inhibition in enzyme activity, which was not affected by Na(+), Mg(2+), Li(+), and Ba(2+). The activity was inhibited about 70 % upon treatment of the enzyme with 10 mM ethylenediaminetetraacetic acid. However, Ca(2+) ions known as high temperature stabilizer for other amylases did not stimulate the activity of the enzyme. Due to pH stability and thermostability of the recombinant amylase, this enzyme may be suitable in starch processing, brewing, and food industries.
Asunto(s)
Amilasas/genética , Amilasas/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Clonación Molecular/métodos , Compuestos de Amonio/farmacología , Amilasas/química , Amilasas/aislamiento & purificación , Quelantes/farmacología , Estabilidad de Enzimas , Escherichia coli/genética , Genoma Bacteriano/genética , Concentración de Iones de Hidrógeno , Metales/farmacología , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tensoactivos/farmacología , TemperaturaRESUMEN
To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. pumilus isolates. Using a high-throughput activity assay, the mutant with highest activity was selected. This chimeric mutant (L3-3), carrying two crossover positions and three point mutations, has a specific activity 6.4 and 8.2 times higher than the two parent enzymes. The mutant also is more tolerant to various detergents and organic solvents, and has a 9 times longer half-life at 50 °C. Homology modeling of mutant L3-3, based on the highly homologous B. subtilis lipase A, shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity. Increased specific activity may result from the location of mutations close to the active site. Together, our results show that it is possible to evolve, by DNA shuffling, B. pumilus lipase variants with improved applicability as biocatalysts, even if the two parent enzymes are highly similar.
Asunto(s)
Bacillus/enzimología , Evolución Molecular Dirigida/métodos , Lipasa/metabolismo , Bacillus/genética , Barajamiento de ADN/métodos , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Calor , Lipasa/química , Lipasa/genética , Modelos Moleculares , Mutación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de ProteínaRESUMEN
Penicillin G acylase (PA) is an important enzyme used in the industrial production of b-lactam antibiotics. In this study, the effects of mutations in the translation initiation region of the Escherichia coli pac gene, encoding periplasmic PA, were examined. Several mutations led to increased amounts of PA activity, including those that lengthened the spacer region between the ribosome binding site and the ATG start codon, and those with altered codons on positions +2 and +4 relative to the start codon. These results indicated that the wild-type sequence of the pac gene does not provide maximum expression levels and that the strategies applied in this study can be used to improve production of PA in E. coli. Unexpectedly, our study also suggested that translocation of PA was, in contrast to earlier reports, shown not to require the Twin-arginine translocation pathway for transport into the periplasm.