RESUMO
Based on sequence alignments and homology modeling, Gly 312 and Lys 436 of the maltogenic amylase from Bacillus sp. US149 (MAUS149) were selected as targets for site-directed mutagenesis to improve the thermostability of the enzyme. Variants of MAUS149 with amino acid substitutions G312A, K436R and G312A-K436R had substrate specificities, kinetic parameters and pH optima similar to those of the wild-type enzyme; however, the enzymes with substitutions K436R and G312A-K436R, had an optimal temperature of 45 °C instead of the 40 °C for the wild-type enzyme. The half-life time at 55 °C increased from 15 to 25 min for the double mutant. Molecular modeling suggests that the increase in thermostability was due to new hydrophobic interactions and the formation of a salt bridge and hydrogen bond in the G312A and K436R variants, respectively. The double mutant could be a potential candidate for application in the bread industry.
Assuntos
Substituição de Aminoácidos/genética , Aminoácidos/genética , Bacillus/enzimologia , Glicosídeo Hidrolases/genética , Mutagênese Sítio-Dirigida/métodos , Temperatura , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Hidrólise , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Mutação/genética , Alinhamento de SequênciaRESUMO
The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the beta-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37 degrees C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19 degrees C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed V(max) and K(m) values of 253 +/- 36 mumol of beta-cyclodextrin/mg/min and 0.36 +/- 0.18 g/L, respectively.
Assuntos
Bacillus/classificação , Bacillus/enzimologia , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Engenharia de Proteínas/métodos , Bacillus/genética , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/fisiologia , Glucosiltransferases/genética , Glucosiltransferases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade da EspécieRESUMO
Pullulanase type I of Geobacillus thermoleovorans US105 strain (PUL US105) was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the open reading frame was connected downstream of the lipase A signal peptide of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction. In addition, pul US105 was fused to the alpha-amylase signal sequence of the Bacillus stearothermophilus US100 strain. The monitoring of the pullulanase activity and Western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. The PUL US105 was purified to homogeneity from the periplasmic fraction, using heat treatment, size exclusion, and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2.
