Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides

L.N. Corici, A.E. Frissen, D.J. van Zoelen, I.F. Eggen, F. Peter, C.M. Davidescu, C.G. Boeriu

Research output: Contribution to journalArticleAcademicpeer-review

16 Citations (Scopus)

Abstract

Alcalase 2.4L FG, a commercial preparation of Subtilisin A, was physically entrapped in glass sol–gel matrices using alkoxysilanes of different types mixed with tetramethoxysilane (TMOS). The materials were used for catalyzing C-terminal amidation of Z-Ala-Phe-OMe in a mixture of tert-butanol/DMF. From the screening of silane monomers in the sol–gel coating process, it was concluded that dimethyldimethoxysilane (DMDMOS) gave the best performance, and Alcalase immobilized therein exhibited the highest activity in the ammonolysis of Z-Ala-Phe-OMe. The percentage of protein immobilization was in the range of 68–98%, and total amidation activity of the immobilized Alcalase was up to 1.76 µmol/h/mg gel. We investigated the immobilization efficiency for a protein mass range of 2.8–9.7 mg per mmol total silanes, to determine the immobilization capacity of the biosilica support. The optimum enzyme loading capacity in the silica matrix was 115 mg/g dry silica xerogel (11.5%, w/w). The amount of the DMDMOS silicate was optimized by adjusting the molar ratio of silane mixture (DMDMOS and TMOS at 1:1). Biocatalyst sol–gel particles prepared at optimum immobilization conditions retained 100% of the original activity even after 14 cycles of repeated use. Reproducibility of the immobilization technique was also investigated by evaluating the catalytic efficiency of the obtained preparations. The thermal stability of the protease at 70 °C increased threefold upon entrapment in sol–gel materials, and twofold under storage for 50 days at ambient temperature.
Original languageEnglish
Pages (from-to)90-97
JournalJournal of Molecular Catalysis. B, Enzymatic
Volume73
Issue number1-4
DOIs
Publication statusPublished - 2011

Fingerprint

Subtilisins
Bacilli
Silanes
Amides
Immobilization
Peptides
Silica
Silicon Dioxide
Proteins
Xerogels
Biocatalysts
tert-Butyl Alcohol
Butenes
Catalyst supports
Silicates
Screening
Thermodynamic stability
Enzymes
Gels
Monomers

Keywords

  • organic-solvents
  • serine proteases
  • enzyme
  • stabilization
  • encapsulation
  • entrapment
  • stability
  • lipases
  • biocatalysts
  • activation

Cite this

@article{c3a8a277e1fa426c9197269ad36f41fc,
title = "Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides",
abstract = "Alcalase 2.4L FG, a commercial preparation of Subtilisin A, was physically entrapped in glass sol–gel matrices using alkoxysilanes of different types mixed with tetramethoxysilane (TMOS). The materials were used for catalyzing C-terminal amidation of Z-Ala-Phe-OMe in a mixture of tert-butanol/DMF. From the screening of silane monomers in the sol–gel coating process, it was concluded that dimethyldimethoxysilane (DMDMOS) gave the best performance, and Alcalase immobilized therein exhibited the highest activity in the ammonolysis of Z-Ala-Phe-OMe. The percentage of protein immobilization was in the range of 68–98{\%}, and total amidation activity of the immobilized Alcalase was up to 1.76 µmol/h/mg gel. We investigated the immobilization efficiency for a protein mass range of 2.8–9.7 mg per mmol total silanes, to determine the immobilization capacity of the biosilica support. The optimum enzyme loading capacity in the silica matrix was 115 mg/g dry silica xerogel (11.5{\%}, w/w). The amount of the DMDMOS silicate was optimized by adjusting the molar ratio of silane mixture (DMDMOS and TMOS at 1:1). Biocatalyst sol–gel particles prepared at optimum immobilization conditions retained 100{\%} of the original activity even after 14 cycles of repeated use. Reproducibility of the immobilization technique was also investigated by evaluating the catalytic efficiency of the obtained preparations. The thermal stability of the protease at 70 °C increased threefold upon entrapment in sol–gel materials, and twofold under storage for 50 days at ambient temperature.",
keywords = "organic-solvents, serine proteases, enzyme, stabilization, encapsulation, entrapment, stability, lipases, biocatalysts, activation",
author = "L.N. Corici and A.E. Frissen and {van Zoelen}, D.J. and I.F. Eggen and F. Peter and C.M. Davidescu and C.G. Boeriu",
year = "2011",
doi = "10.1016/j.molcatb.2011.08.004",
language = "English",
volume = "73",
pages = "90--97",
journal = "Journal of Molecular Catalysis. B, Enzymatic",
issn = "1381-1177",
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number = "1-4",

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Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides. / Corici, L.N.; Frissen, A.E.; van Zoelen, D.J.; Eggen, I.F.; Peter, F.; Davidescu, C.M.; Boeriu, C.G.

In: Journal of Molecular Catalysis. B, Enzymatic, Vol. 73, No. 1-4, 2011, p. 90-97.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides

AU - Corici, L.N.

AU - Frissen, A.E.

AU - van Zoelen, D.J.

AU - Eggen, I.F.

AU - Peter, F.

AU - Davidescu, C.M.

AU - Boeriu, C.G.

PY - 2011

Y1 - 2011

N2 - Alcalase 2.4L FG, a commercial preparation of Subtilisin A, was physically entrapped in glass sol–gel matrices using alkoxysilanes of different types mixed with tetramethoxysilane (TMOS). The materials were used for catalyzing C-terminal amidation of Z-Ala-Phe-OMe in a mixture of tert-butanol/DMF. From the screening of silane monomers in the sol–gel coating process, it was concluded that dimethyldimethoxysilane (DMDMOS) gave the best performance, and Alcalase immobilized therein exhibited the highest activity in the ammonolysis of Z-Ala-Phe-OMe. The percentage of protein immobilization was in the range of 68–98%, and total amidation activity of the immobilized Alcalase was up to 1.76 µmol/h/mg gel. We investigated the immobilization efficiency for a protein mass range of 2.8–9.7 mg per mmol total silanes, to determine the immobilization capacity of the biosilica support. The optimum enzyme loading capacity in the silica matrix was 115 mg/g dry silica xerogel (11.5%, w/w). The amount of the DMDMOS silicate was optimized by adjusting the molar ratio of silane mixture (DMDMOS and TMOS at 1:1). Biocatalyst sol–gel particles prepared at optimum immobilization conditions retained 100% of the original activity even after 14 cycles of repeated use. Reproducibility of the immobilization technique was also investigated by evaluating the catalytic efficiency of the obtained preparations. The thermal stability of the protease at 70 °C increased threefold upon entrapment in sol–gel materials, and twofold under storage for 50 days at ambient temperature.

AB - Alcalase 2.4L FG, a commercial preparation of Subtilisin A, was physically entrapped in glass sol–gel matrices using alkoxysilanes of different types mixed with tetramethoxysilane (TMOS). The materials were used for catalyzing C-terminal amidation of Z-Ala-Phe-OMe in a mixture of tert-butanol/DMF. From the screening of silane monomers in the sol–gel coating process, it was concluded that dimethyldimethoxysilane (DMDMOS) gave the best performance, and Alcalase immobilized therein exhibited the highest activity in the ammonolysis of Z-Ala-Phe-OMe. The percentage of protein immobilization was in the range of 68–98%, and total amidation activity of the immobilized Alcalase was up to 1.76 µmol/h/mg gel. We investigated the immobilization efficiency for a protein mass range of 2.8–9.7 mg per mmol total silanes, to determine the immobilization capacity of the biosilica support. The optimum enzyme loading capacity in the silica matrix was 115 mg/g dry silica xerogel (11.5%, w/w). The amount of the DMDMOS silicate was optimized by adjusting the molar ratio of silane mixture (DMDMOS and TMOS at 1:1). Biocatalyst sol–gel particles prepared at optimum immobilization conditions retained 100% of the original activity even after 14 cycles of repeated use. Reproducibility of the immobilization technique was also investigated by evaluating the catalytic efficiency of the obtained preparations. The thermal stability of the protease at 70 °C increased threefold upon entrapment in sol–gel materials, and twofold under storage for 50 days at ambient temperature.

KW - organic-solvents

KW - serine proteases

KW - enzyme

KW - stabilization

KW - encapsulation

KW - entrapment

KW - stability

KW - lipases

KW - biocatalysts

KW - activation

U2 - 10.1016/j.molcatb.2011.08.004

DO - 10.1016/j.molcatb.2011.08.004

M3 - Article

VL - 73

SP - 90

EP - 97

JO - Journal of Molecular Catalysis. B, Enzymatic

JF - Journal of Molecular Catalysis. B, Enzymatic

SN - 1381-1177

IS - 1-4

ER -