TY - JOUR
T1 - Stabilization of a truncated Bacillus sp. strain TS-23 α-amylase by replacing histidine-436 with aspartate
AU - Lo, Huei Fen
AU - Chen, Ya Hui
AU - Hsiao, Nai Wan
AU - Chen, Hsiang Ling
AU - Hu, Hui Yu
AU - Hsu, Wen Hwei
AU - Lin, Long Liu
PY - 2005/6/1
Y1 - 2005/6/1
N2 - 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 His 6-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 m value, more than 66% increase in the value of catalytic efficiency (k cat /K m ) was observed in H436D, H436K, and H436Y. At 70°C, H436D exhibited an increased half-life with respect to the wild-type enzyme.
AB - 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 His 6-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 m value, more than 66% increase in the value of catalytic efficiency (k cat /K m ) was observed in H436D, H436K, and H436Y. At 70°C, H436D exhibited an increased half-life with respect to the wild-type enzyme.
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U2 - 10.1007/s11274-004-1764-9
DO - 10.1007/s11274-004-1764-9
M3 - Article
AN - SCOPUS:22944476426
VL - 21
SP - 411
EP - 416
JO - Mircen Journal of Applied Microbiology and Biotechnology
JF - Mircen Journal of Applied Microbiology and Biotechnology
SN - 0265-0762
IS - 4
ER -