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

Huei Fen Lo, Ya Hui Chen, Nai-Wan Hsiao, Hsiang Ling Chen, Hui Yu Hu, Wen Hwei Hsu, Long Liu Lin

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 languageEnglish
Pages (from-to)411-416
Number of pages6
JournalWorld Journal of Microbiology and Biotechnology
Volume21
Issue number4
DOIs
Publication statusPublished - 2005 Jun 1

Fingerprint

Bacilli
aspartic acid
Amylases
histidine
Histidine
amylases
Aspartic Acid
Bacillus
Stabilization
His-His-His-His-His-His
Enzymes
enzymes
Mutagenesis
site-directed mutagenesis
chelates
Molecular mass
Threonine
Site-Directed Mutagenesis
Chromatography
Nickel

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Plant Science
  • Biochemistry
  • Food Science
  • Microbiology
  • Biotechnology

Cite this

Lo, Huei Fen ; Chen, Ya Hui ; Hsiao, Nai-Wan ; Chen, Hsiang Ling ; Hu, Hui Yu ; Hsu, Wen Hwei ; Lin, Long Liu. / Stabilization of a truncated Bacillus sp. strain TS-23 α-amylase by replacing histidine-436 with aspartate. In: World Journal of Microbiology and Biotechnology. 2005 ; Vol. 21, No. 4. pp. 411-416.
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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 journalArticle

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AU - Chen, Ya Hui

AU - Hsiao, Nai-Wan

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AU - Hu, Hui Yu

AU - Hsu, Wen Hwei

AU - Lin, Long Liu

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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.

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