Bifidobacterium longum l-Arabinose Isomerase—Overexpression in Lactococcus lactis, Purification, and Characterization

N. Salonen, Antti Nyyssölä, K. Salonen, O. Turunen

Research output: Contribution to journalArticleScientificpeer-review

18 Citations (Scopus)

Abstract

Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum l-AI was characterized using d-galactose and l-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0–6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K m value was 120 mM for l-arabinose and 590 mM for d-galactose. The V max was 42 U mg−1 with l-arabinose and 7.7 U mg−1 with d-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg2+, Mn2+). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum l-AI as the catalyst at 35 °C, equilibrium yields of 36 % d-tagatose and 11 % l-ribulose with 1.67 M d-galactose and l-arabinose, respectively, as the substrates were reached.
Original languageEnglish
Pages (from-to)392-405
JournalApplied Biochemistry and Biotechnology
Volume168
Issue number2
DOIs
Publication statusPublished - 2012
MoE publication typeA1 Journal article-refereed

Fingerprint

Lactococcus lactis
Arabinose
Purification
Enzymes
Metal ions
Galactose
Isomerases
Substrates
Metals
Ions
Ethylenediaminetetraacetic acid
Catalyst activity
Phosphates
Catalysts
Bifidobacterium longum
Edetic Acid
Catalytic Domain

Keywords

  • L-Arabinose isomerase
  • Bifidobacterium longum
  • D-Tagarose
  • protein production
  • Lactococcus lactis

Cite this

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title = "Bifidobacterium longum l-Arabinose Isomerase—Overexpression in Lactococcus lactis, Purification, and Characterization",
abstract = "Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum l-AI was characterized using d-galactose and l-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0–6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K m value was 120 mM for l-arabinose and 590 mM for d-galactose. The V max was 42 U mg−1 with l-arabinose and 7.7 U mg−1 with d-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg2+, Mn2+). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum l-AI as the catalyst at 35 °C, equilibrium yields of 36 {\%} d-tagatose and 11 {\%} l-ribulose with 1.67 M d-galactose and l-arabinose, respectively, as the substrates were reached.",
keywords = "L-Arabinose isomerase, Bifidobacterium longum, D-Tagarose, protein production, Lactococcus lactis",
author = "N. Salonen and Antti Nyyss{\"o}l{\"a} and K. Salonen and O. Turunen",
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Bifidobacterium longum l-Arabinose Isomerase—Overexpression in Lactococcus lactis, Purification, and Characterization. / Salonen, N.; Nyyssölä, Antti; Salonen, K.; Turunen, O.

In: Applied Biochemistry and Biotechnology, Vol. 168, No. 2, 2012, p. 392-405.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Bifidobacterium longum l-Arabinose Isomerase—Overexpression in Lactococcus lactis, Purification, and Characterization

AU - Salonen, N.

AU - Nyyssölä, Antti

AU - Salonen, K.

AU - Turunen, O.

PY - 2012

Y1 - 2012

N2 - Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum l-AI was characterized using d-galactose and l-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0–6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K m value was 120 mM for l-arabinose and 590 mM for d-galactose. The V max was 42 U mg−1 with l-arabinose and 7.7 U mg−1 with d-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg2+, Mn2+). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum l-AI as the catalyst at 35 °C, equilibrium yields of 36 % d-tagatose and 11 % l-ribulose with 1.67 M d-galactose and l-arabinose, respectively, as the substrates were reached.

AB - Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum l-AI was characterized using d-galactose and l-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0–6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K m value was 120 mM for l-arabinose and 590 mM for d-galactose. The V max was 42 U mg−1 with l-arabinose and 7.7 U mg−1 with d-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg2+, Mn2+). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum l-AI as the catalyst at 35 °C, equilibrium yields of 36 % d-tagatose and 11 % l-ribulose with 1.67 M d-galactose and l-arabinose, respectively, as the substrates were reached.

KW - L-Arabinose isomerase

KW - Bifidobacterium longum

KW - D-Tagarose

KW - protein production

KW - Lactococcus lactis

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DO - 10.1007/s12010-012-9783-8

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EP - 405

JO - Applied Biochemistry and Biotechnology

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SN - 0273-2289

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