Mesoporous Silica Materials Labeled for Optical Oxygen Sensing and Their Application to Development of a Silica-Supported Oxidoreductase Biocatalyst

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Abstract

Porous silica materials make great supports for heterogeneous catalysis with immobilized enzymes; however, direct functionalization of their surface through stable attachment of enzymes, reporter molecules, or both is a difficult problem. Overcoming that is necessary for practical implementation. Here, we integrate the development of luminophor-doped oxygen-sensing silica materials with a modular strategy of enzyme immobilization to demonstrate generally applicable design of an oxygen-dependent biocatalyst on a porous silica support. Zbasic2, a highly positively charged silica-binding module of about 7 kDa size, was fused to d-amino acid oxidase, and the resulting chimeric protein was tethered noncovalently via Zbasic2 in defined orientation and in a highly selective manner on silica. The enzyme supports used differed in overall shape and size as well as in internal pore structure. A confocal laser scanning microscopy (CLSM) analysis that employed the oxidases flavin cofactor as the fluorescent reporter group showed a homogeneous internal protein distribution in all supports used. Ru-based organometallic luminophor was adsorbed tightly onto the silica supports, thus enabling internal optical sensing of the O2 available to the enzymatic reaction. Optimization of the surface labeling regarding homogeneous luminophor distribution was guided, and its efficacy was verified by CLSM. Mesostructured silica surpassed controlled pore glass by ≥10-fold in terms of immobilized enzyme effectiveness at high loading of oxidase activity. The effect was shown from detailed comparison of the time-resolved O2 concentration profiles in solution and inside porous support to result exclusively from variable degrees of diffusion-caused limitation in the internal O2 availability. Enzyme immobilized on mesostructured silica approached perfection of a heterogeneous biocatalyst in being almost as effective as the free enzyme (assayed in oxidative deamination of d-methionine), thus emphasizing the large benefit of targeted mass transfer intensification, through proper choice of support parameters, in the development of immobilizates of O2-dependent oxidoreductases on porous silica material.

LanguageEnglish
Pages5984-5993
Number of pages10
JournalACS Catalysis
Volume5
Issue number10
DOIs
StatusPublished - 2 Oct 2015

Fingerprint

Biocatalysts
Silicon Dioxide
Oxidoreductases
Silica
Oxygen
Enzymes
Immobilized Enzymes
Catalyst supports
Microscopic examination
Enzyme immobilization
Proteins
Scanning
Lasers
Organometallics
Pore structure
Methionine
Labeling
Catalysis
Amino acids
Mass transfer

Keywords

  • biocatalysis
  • enzyme immobilization
  • fusion protein
  • intraparticle oxygen gradient
  • optical sensing
  • oxygen-dependent oxidations
  • silica binding module
  • silica materials

ASJC Scopus subject areas

  • Catalysis

Cite this

@article{269e68b7ccd04cd88a0a56b447b4c3f4,
title = "Mesoporous Silica Materials Labeled for Optical Oxygen Sensing and Their Application to Development of a Silica-Supported Oxidoreductase Biocatalyst",
abstract = "Porous silica materials make great supports for heterogeneous catalysis with immobilized enzymes; however, direct functionalization of their surface through stable attachment of enzymes, reporter molecules, or both is a difficult problem. Overcoming that is necessary for practical implementation. Here, we integrate the development of luminophor-doped oxygen-sensing silica materials with a modular strategy of enzyme immobilization to demonstrate generally applicable design of an oxygen-dependent biocatalyst on a porous silica support. Zbasic2, a highly positively charged silica-binding module of about 7 kDa size, was fused to d-amino acid oxidase, and the resulting chimeric protein was tethered noncovalently via Zbasic2 in defined orientation and in a highly selective manner on silica. The enzyme supports used differed in overall shape and size as well as in internal pore structure. A confocal laser scanning microscopy (CLSM) analysis that employed the oxidases flavin cofactor as the fluorescent reporter group showed a homogeneous internal protein distribution in all supports used. Ru-based organometallic luminophor was adsorbed tightly onto the silica supports, thus enabling internal optical sensing of the O2 available to the enzymatic reaction. Optimization of the surface labeling regarding homogeneous luminophor distribution was guided, and its efficacy was verified by CLSM. Mesostructured silica surpassed controlled pore glass by ≥10-fold in terms of immobilized enzyme effectiveness at high loading of oxidase activity. The effect was shown from detailed comparison of the time-resolved O2 concentration profiles in solution and inside porous support to result exclusively from variable degrees of diffusion-caused limitation in the internal O2 availability. Enzyme immobilized on mesostructured silica approached perfection of a heterogeneous biocatalyst in being almost as effective as the free enzyme (assayed in oxidative deamination of d-methionine), thus emphasizing the large benefit of targeted mass transfer intensification, through proper choice of support parameters, in the development of immobilizates of O2-dependent oxidoreductases on porous silica material.",
keywords = "biocatalysis, enzyme immobilization, fusion protein, intraparticle oxygen gradient, optical sensing, oxygen-dependent oxidations, silica binding module, silica materials",
author = "Bolivar, {Juan M.} and Sabine Schelch and Torsten Mayr and Bernd Nidetzky",
year = "2015",
month = "10",
day = "2",
doi = "10.1021/acscatal.5b01601",
language = "English",
volume = "5",
pages = "5984--5993",
journal = "ACS Catalysis",
issn = "2155-5435",
publisher = "American Chemical Society",
number = "10",

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TY - JOUR

T1 - Mesoporous Silica Materials Labeled for Optical Oxygen Sensing and Their Application to Development of a Silica-Supported Oxidoreductase Biocatalyst

AU - Bolivar,Juan M.

AU - Schelch,Sabine

AU - Mayr,Torsten

AU - Nidetzky,Bernd

PY - 2015/10/2

Y1 - 2015/10/2

N2 - Porous silica materials make great supports for heterogeneous catalysis with immobilized enzymes; however, direct functionalization of their surface through stable attachment of enzymes, reporter molecules, or both is a difficult problem. Overcoming that is necessary for practical implementation. Here, we integrate the development of luminophor-doped oxygen-sensing silica materials with a modular strategy of enzyme immobilization to demonstrate generally applicable design of an oxygen-dependent biocatalyst on a porous silica support. Zbasic2, a highly positively charged silica-binding module of about 7 kDa size, was fused to d-amino acid oxidase, and the resulting chimeric protein was tethered noncovalently via Zbasic2 in defined orientation and in a highly selective manner on silica. The enzyme supports used differed in overall shape and size as well as in internal pore structure. A confocal laser scanning microscopy (CLSM) analysis that employed the oxidases flavin cofactor as the fluorescent reporter group showed a homogeneous internal protein distribution in all supports used. Ru-based organometallic luminophor was adsorbed tightly onto the silica supports, thus enabling internal optical sensing of the O2 available to the enzymatic reaction. Optimization of the surface labeling regarding homogeneous luminophor distribution was guided, and its efficacy was verified by CLSM. Mesostructured silica surpassed controlled pore glass by ≥10-fold in terms of immobilized enzyme effectiveness at high loading of oxidase activity. The effect was shown from detailed comparison of the time-resolved O2 concentration profiles in solution and inside porous support to result exclusively from variable degrees of diffusion-caused limitation in the internal O2 availability. Enzyme immobilized on mesostructured silica approached perfection of a heterogeneous biocatalyst in being almost as effective as the free enzyme (assayed in oxidative deamination of d-methionine), thus emphasizing the large benefit of targeted mass transfer intensification, through proper choice of support parameters, in the development of immobilizates of O2-dependent oxidoreductases on porous silica material.

AB - Porous silica materials make great supports for heterogeneous catalysis with immobilized enzymes; however, direct functionalization of their surface through stable attachment of enzymes, reporter molecules, or both is a difficult problem. Overcoming that is necessary for practical implementation. Here, we integrate the development of luminophor-doped oxygen-sensing silica materials with a modular strategy of enzyme immobilization to demonstrate generally applicable design of an oxygen-dependent biocatalyst on a porous silica support. Zbasic2, a highly positively charged silica-binding module of about 7 kDa size, was fused to d-amino acid oxidase, and the resulting chimeric protein was tethered noncovalently via Zbasic2 in defined orientation and in a highly selective manner on silica. The enzyme supports used differed in overall shape and size as well as in internal pore structure. A confocal laser scanning microscopy (CLSM) analysis that employed the oxidases flavin cofactor as the fluorescent reporter group showed a homogeneous internal protein distribution in all supports used. Ru-based organometallic luminophor was adsorbed tightly onto the silica supports, thus enabling internal optical sensing of the O2 available to the enzymatic reaction. Optimization of the surface labeling regarding homogeneous luminophor distribution was guided, and its efficacy was verified by CLSM. Mesostructured silica surpassed controlled pore glass by ≥10-fold in terms of immobilized enzyme effectiveness at high loading of oxidase activity. The effect was shown from detailed comparison of the time-resolved O2 concentration profiles in solution and inside porous support to result exclusively from variable degrees of diffusion-caused limitation in the internal O2 availability. Enzyme immobilized on mesostructured silica approached perfection of a heterogeneous biocatalyst in being almost as effective as the free enzyme (assayed in oxidative deamination of d-methionine), thus emphasizing the large benefit of targeted mass transfer intensification, through proper choice of support parameters, in the development of immobilizates of O2-dependent oxidoreductases on porous silica material.

KW - biocatalysis

KW - enzyme immobilization

KW - fusion protein

KW - intraparticle oxygen gradient

KW - optical sensing

KW - oxygen-dependent oxidations

KW - silica binding module

KW - silica materials

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U2 - 10.1021/acscatal.5b01601

DO - 10.1021/acscatal.5b01601

M3 - Article

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T2 - ACS Catalysis

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SN - 2155-5435

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