Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2

Barbara G. Klug-Santner, Wolfgang Schnitzhofer, Maria Vrsanska, Jörg Weber, Pramod Agrawal, Vincent Nierstrasz, Georg M. Guebitz

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Abstract

An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3 ± 4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC–MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 °C in Tris/HCl buffer. It showed a half-life at 30 °C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 °C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a β-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 g l−1 and a vmax of 0.72 g l−1 min−1. The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.
Original languageUndefined
Pages (from-to)390-401
JournalJournal of biotechnology
Volume121
Issue number3
DOIs
Publication statusPublished - 2006

Keywords

  • Bacillus pumilus
  • IR-78469
  • Pectate lyase
  • Bioscouring
  • METIS-235843

Cite this

Klug-Santner, B. G., Schnitzhofer, W., Vrsanska, M., Weber, J., Agrawal, P., Nierstrasz, V., & Guebitz, G. M. (2006). Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2. Journal of biotechnology, 121(3), 390-401. https://doi.org/10.1016/j.jbiotec.2005.07.019
Klug-Santner, Barbara G. ; Schnitzhofer, Wolfgang ; Vrsanska, Maria ; Weber, Jörg ; Agrawal, Pramod ; Nierstrasz, Vincent ; Guebitz, Georg M. / Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2. In: Journal of biotechnology. 2006 ; Vol. 121, No. 3. pp. 390-401.
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title = "Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2",
abstract = "An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3 ± 4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC–MS of PL revealed 15{\%} homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 °C in Tris/HCl buffer. It showed a half-life at 30 °C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 °C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50{\%} of esterification. The best substrate was 29.5{\%} methylated pectin. PL cleaved polygalacturonic acid via a β-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 g l−1 and a vmax of 0.72 g l−1 min−1. The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80{\%} of pectin was proven by means of ruthenium red dyeing and HPAEC (65{\%}). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.",
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Klug-Santner, BG, Schnitzhofer, W, Vrsanska, M, Weber, J, Agrawal, P, Nierstrasz, V & Guebitz, GM 2006, 'Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2' Journal of biotechnology, vol. 121, no. 3, pp. 390-401. https://doi.org/10.1016/j.jbiotec.2005.07.019

Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2. / Klug-Santner, Barbara G.; Schnitzhofer, Wolfgang; Vrsanska, Maria; Weber, Jörg; Agrawal, Pramod; Nierstrasz, Vincent; Guebitz, Georg M.

In: Journal of biotechnology, Vol. 121, No. 3, 2006, p. 390-401.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2

AU - Klug-Santner, Barbara G.

AU - Schnitzhofer, Wolfgang

AU - Vrsanska, Maria

AU - Weber, Jörg

AU - Agrawal, Pramod

AU - Nierstrasz, Vincent

AU - Guebitz, Georg M.

PY - 2006

Y1 - 2006

N2 - An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3 ± 4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC–MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 °C in Tris/HCl buffer. It showed a half-life at 30 °C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 °C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a β-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 g l−1 and a vmax of 0.72 g l−1 min−1. The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.

AB - An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3 ± 4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC–MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 °C in Tris/HCl buffer. It showed a half-life at 30 °C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 °C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a β-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 g l−1 and a vmax of 0.72 g l−1 min−1. The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.

KW - Bacillus pumilus

KW - IR-78469

KW - Pectate lyase

KW - Bioscouring

KW - METIS-235843

U2 - 10.1016/j.jbiotec.2005.07.019

DO - 10.1016/j.jbiotec.2005.07.019

M3 - Article

VL - 121

SP - 390

EP - 401

JO - Journal of biotechnology

JF - Journal of biotechnology

SN - 0168-1656

IS - 3

ER -

Klug-Santner BG, Schnitzhofer W, Vrsanska M, Weber J, Agrawal P, Nierstrasz V et al. Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2. Journal of biotechnology. 2006;121(3):390-401. https://doi.org/10.1016/j.jbiotec.2005.07.019