Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: Cloning, expression, and enzyme characterization

Sang Kee Kang, Kwang Keun Cho, Jong Kun Ahn, Jin Duck Bok, Seung Ha Kang, Jung Hee Woo, Hong Gu Lee, Seung Kwon You, Yun Jaie Choi

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Three thermostable lactose-hydrolases, namely, two β-glycosidases (bglA and bglB) and one β-galactosidase (bgaA) genes were cloned from the genomic library of Thermus sp. IB-21. The bglA, bglB, and bgaA consisted of 1311 bp (436 amino acid residues), 1296 bp (431aa), and 1938 bp (645 aa) of nucleotides with predicted molecular masses of 49,066, 48,679, and 72,714 Da, respectively. These enzymes were overexpressed in Escherichia coli BL21(DE3) using pET21b(+) vector system. The recombinant enzymes were purified to homogeneity by a heat precipitation (70°C, 40 min) and a Ni 2+-affinity chromatography. The molecular masses of the purified enzymes estimated by SDS-PAGE agreed with their predicted values. All the purified enzymes showed their optimal pH at around 5.0-6.0. In contrast, the temperature profiles for activity and thermostability patterns were different for each enzyme. BglB β-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70°C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70°C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.

Original languageEnglish
Pages (from-to)337-346
Number of pages10
JournalJournal of Biotechnology
Volume117
Issue number1
DOIs
Publication statusPublished - 2005 Apr 6

Keywords

  • Lactose intolerance
  • Lactose-hydrolase
  • Low-lactose milk
  • Thermostability
  • Thermus sp.
  • β-Galactosidase
  • β-Glycosidase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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