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

Institute of Molecular Biology, National Chung Hsing University, 臺中市, Taiwan, Taiwan
World Journal of Microbiology and Biotechnology (Impact Factor: 1.78). 05/2005; 21(4):411-416. DOI: 10.1007/s11274-004-1764-9


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 54 kDa. 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 K
value, more than 66% increase in the value of catalytic efficiency (k
) was observed in H436D, H436K, and H436Y. At 70 °C, H436D exhibited an increased half-life with respect to the wild-type enzyme.

40 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His6-tagged EcDCP (His6-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05mM IPTG induction at 26°C for 12h. The recombinant enzyme was purified to homogeneity by Ni2+-NTA resin and had a molecular mass of approximately 75kDa. The temperature and pH optima for His6-EcDCP were 37°C and 7.0, respectively. In the presence of 200mM NaCl, His6-EcDCP was stimulated by 1.5 fold. The K M and k cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83mM and 168.3s−1, respectively. His6-EcDCP activity was dramatically inhibited by 10mM EDTA, 0.25mM 1.10-phenanthroline, and 2.5mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His6-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.
    Full-text · Article · Feb 2008 · World Journal of Microbiology and Biotechnology
  • [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.
    No preview · Article · May 2008 · PROCESS BIOCHEMISTRY
  • [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.
    No preview · Article · May 2008 · World Journal of Microbiology and Biotechnology
Show more