Article

Stabilization of a truncated Bacillus sp. strain TS-23 α-amylase by replacing histidine-436 with aspartate

World Journal of Microbiology and Biotechnology (Impact Factor: 1.35). 05/2005; 21(4):411-416. DOI: 10.1007/s11274-004-1764-9

ABSTRACT Histidine-436 of a truncated Bacillus sp. strain TS-23 α-amylase (His6-tagged ΔNC) has been known to be responsible for thermostability of the enzyme. To understand further the structural role of this residue, site-directed mutagenesis was conducted to replace His-436 of His6-tagged ΔNC with aspartate, lysine, tyrosine or threonine. Starch-plate assay showed that all Escherichia coli M15 transformants conferring the mutated amylase genes retained the amylolytic activity. The over-expressed proteins have been purified to near homogeneity by nickel-chelate chromatography and the molecular mass of the purified enzymes was approximately 54kDa. The specific activity for H436T was decreased by more than 56%, while H436D, H436K, and H436Y showed a higher activity to that of the wild-type enzyme. Although the mutations did not lead to a significant change in the Km value, more than 66% increase in the value of catalytic efficiency (kcat/Km) was observed in H436D, H436K, and H436Y. At 70C, H436D exhibited an increased half-life with respect to the wild-type enzyme.

0 Followers
 · 
163 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: One deletion mutant was constructed from the structural gene of a truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) by site-directed mutagenesis. BACΔNC and BACΔNC/ΔR210-S211 were overexpressed in recombinant Escherichia coli M15 cells and purified to nearly homologous by nickel-chelate chromatography. BACΔNC and BACΔNC/ΔR210-S211 were very similar with respect to specific activity, kinetic parameters, pH–activity profile, and temperature–activity curve. An increased half-life at 70 °C was observed for BACΔNC/ΔR210-S211, suggesting that Arg210-Ser211 deletion leads to a conformational change of the enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for the wild-type enzyme and BACΔNC/ΔR210-S211, but they showed a different sensitivity towards temperature-induced denaturation. These results indicated that the rigidity of the enzyme has been altered by Arg210-Ser211 deletion.
    PROCESS BIOCHEMISTRY 05/2008; DOI:10.1016/j.procbio.2008.01.020 · 2.52 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The functional and structural significance of glutamic acid 219 of a N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was explored by the approach of site-directed saturation mutagenesis. The expressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular mass was determined to be approximately 54 kDa by SDS/PAGE. Except E219F, E219P, and E219W, all other mutant enzymes exhibited a lower shift in their optimum temperatures with respect to the wild-type enzyme. A decreased thermostability was also found in all of the mutant enzymes when compared with the wild-type form of BACΔNC. Except E219F, E219P, and E219W mutant enzymes, greater than 2-fold decrease in k cat and a similar substrate affinity relative to the wild-type BACΔNC were observed for the rest mutant enzymes. Based on these observations, it is suggested that Glu-219 apparently plays an important role in the thermostability of BACΔNC.
    World Journal of Microbiology and Biotechnology 01/2008; 24(5):619-626. DOI:10.1007/s11274-007-9518-0 · 1.35 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: BACDeltaNC/Delta RS is a thermostable variant derived from the truncated alpha-amylase (BAC Delta NC) of alkaliphilic Bacillus sp. strain TS-23. With the aim of enhancing its resistance towards chemical oxidation, Met231 of BAC Delta NC/Delta RS was replaced by leucine to create BAC Delta NC/Delta RS/M231L. The functional significance of the 31 C-terminal residues of BAC Delta NC/Delta RS/M231L was also explored by site-directed mutagenesis of the 483 th codon in the gene to stop codon (TAA), thereon the engineered enzyme was named BAC Delta NC/Delta RS/M231L/Delta C31. BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 were very similar to BAC Delta NC in terms of specific activity, kinetic parameters, pH-activity profile, and the hydrolysis of raw starch; however, the engineered enzymes showed an increased half-life at 70 degrees C. The intrinsic fluorescence and circular dichroism spectra were nearly identical for wild-type and engineered enzymes, but they exhibited a different sensitivity towards GdnHCl-induced denaturation. This implicates that the rigidity of the enzyme has been changed as the consequence of mutations. Performance of the engineered enzymes was evaluated in the presence of commonly used detergent compounds and some detergents from the local markets. A high compatibility and performance of both BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 may be desirable for their practical uses in the detergent industry.
    Journal of Bioscience and Bioengineering 06/2010; 109(6):531-8. DOI:10.1016/j.jbiosc.2009.11.012 · 1.79 Impact Factor