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 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.
| Original language | English |
|---|---|
| Pages (from-to) | 411-416 |
| Number of pages | 6 |
| Journal | World Journal of Microbiology and Biotechnology |
| Volume | 21 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2005 Jun 1 |
Fingerprint
All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology
- Plant Science
- Biochemistry
- Food Science
- Microbiology
- Biotechnology
Cite this
}
Stabilization of a truncated Bacillus sp. strain TS-23 α-amylase by replacing histidine-436 with aspartate. / Lo, Huei Fen; Chen, Ya Hui; Hsiao, Nai-Wan; Chen, Hsiang Ling; Hu, Hui Yu; Hsu, Wen Hwei; Lin, Long Liu.
In: World Journal of Microbiology and Biotechnology, Vol. 21, No. 4, 01.06.2005, p. 411-416.Research output: Contribution to journal › Article
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.
UR - http://www.scopus.com/inward/record.url?scp=22944476426&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=22944476426&partnerID=8YFLogxK
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 -