Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris

Jaana H. Toikkanen (Corresponding Author), Marja-Leena Niku-Paavola, Michael Bailey, Juha Immanen, Eija Rintala, Paula Elomaa, Yrjö Helariutta, Teemu H. Teeri, Richard Fagerström

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications.
Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51% identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence.
XET production in shake flask cultivations was better at 22 °C than at 30 °C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET).
Both XET enzymes were produced in 16 l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium.
Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60–70 mg l−1 BXET was produced in the TLFB in complex medium.
Original languageEnglish
Pages (from-to)161-170
JournalJournal of Biotechnology
Volume130
Issue number2
DOIs
Publication statusPublished - 2007
MoE publication typeA1 Journal article-refereed

Fingerprint

Betula
Pichia
xyloglucan - xyloglucosyltransferase
Temperature
Mating Factor
Proteins
Enzymes
Batch Cell Culture Techniques
Plant Cells
Cell growth
Molecular mass
Bioreactors
Transferases
Protein Sorting Signals
Yeast
Cell Wall
Methanol
Amino acids
Yeasts
Amino Acids

Keywords

  • Xyloglucan endotransglycosylase (XET)
  • Pichia pastoris
  • Heterologous expression
  • Bioreactor cultivation
  • Gerbera hybrida
  • Betula pendula

Cite this

Toikkanen, Jaana H. ; Niku-Paavola, Marja-Leena ; Bailey, Michael ; Immanen, Juha ; Rintala, Eija ; Elomaa, Paula ; Helariutta, Yrjö ; Teeri, Teemu H. ; Fagerström, Richard. / Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris. In: Journal of Biotechnology. 2007 ; Vol. 130, No. 2. pp. 161-170.
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abstract = "The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications. Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51{\%} identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence. XET production in shake flask cultivations was better at 22 °C than at 30 °C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET). Both XET enzymes were produced in 16 l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium. Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60–70 mg l−1 BXET was produced in the TLFB in complex medium.",
keywords = "Xyloglucan endotransglycosylase (XET), Pichia pastoris, Heterologous expression, Bioreactor cultivation, Gerbera hybrida, Betula pendula",
author = "Toikkanen, {Jaana H.} and Marja-Leena Niku-Paavola and Michael Bailey and Juha Immanen and Eija Rintala and Paula Elomaa and Yrj{\"o} Helariutta and Teeri, {Teemu H.} and Richard Fagerstr{\"o}m",
year = "2007",
doi = "10.1016/j.jbiotec.2007.03.004",
language = "English",
volume = "130",
pages = "161--170",
journal = "Journal of Biotechnology",
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Toikkanen, JH, Niku-Paavola, M-L, Bailey, M, Immanen, J, Rintala, E, Elomaa, P, Helariutta, Y, Teeri, TH & Fagerström, R 2007, 'Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris', Journal of Biotechnology, vol. 130, no. 2, pp. 161-170. https://doi.org/10.1016/j.jbiotec.2007.03.004

Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris. / Toikkanen, Jaana H. (Corresponding Author); Niku-Paavola, Marja-Leena; Bailey, Michael; Immanen, Juha; Rintala, Eija; Elomaa, Paula; Helariutta, Yrjö; Teeri, Teemu H.; Fagerström, Richard.

In: Journal of Biotechnology, Vol. 130, No. 2, 2007, p. 161-170.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris

AU - Toikkanen, Jaana H.

AU - Niku-Paavola, Marja-Leena

AU - Bailey, Michael

AU - Immanen, Juha

AU - Rintala, Eija

AU - Elomaa, Paula

AU - Helariutta, Yrjö

AU - Teeri, Teemu H.

AU - Fagerström, Richard

PY - 2007

Y1 - 2007

N2 - The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications. Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51% identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence. XET production in shake flask cultivations was better at 22 °C than at 30 °C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET). Both XET enzymes were produced in 16 l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium. Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60–70 mg l−1 BXET was produced in the TLFB in complex medium.

AB - The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications. Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51% identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence. XET production in shake flask cultivations was better at 22 °C than at 30 °C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET). Both XET enzymes were produced in 16 l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium. Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60–70 mg l−1 BXET was produced in the TLFB in complex medium.

KW - Xyloglucan endotransglycosylase (XET)

KW - Pichia pastoris

KW - Heterologous expression

KW - Bioreactor cultivation

KW - Gerbera hybrida

KW - Betula pendula

U2 - 10.1016/j.jbiotec.2007.03.004

DO - 10.1016/j.jbiotec.2007.03.004

M3 - Article

VL - 130

SP - 161

EP - 170

JO - Journal of Biotechnology

JF - Journal of Biotechnology

SN - 0168-1656

IS - 2

ER -