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A Novel Reversible pH-Triggered Release Immobilized Enzyme System

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Abstract

A novel immobilized enzyme system supported by poly(acrylic acid/N,N’-methylene-bisacryl-amide) hydrogel microspheres was prepared. This system exhibited characteristics of reversible pH-triggered release. The morphology, size, and chemical structure were examined through optical microscopy, particle size analyzer, and Fourier transform infrared spectrometer. Immobilization and release features were further investigated under different conditions, including pH, time, and microsphere quantity. Results showed the microspheres were regularly spherical with 3.8 ~ 6.6 μm diameter. Loading efficiencies of bovine serum albumin immobilized by gel entrapment and adsorption methods were 93.9% and 56.2%, respectively. The pH-triggered protein release of the system occurred when medium pH was above 6.0, while it was hardly detected when medium pH was below 6.0. Release efficiencies of entrapped and adsorbed protein were 6.38% and 95.0%, respectively. Hence, adsorption method was used to immobilize trypsin. Loading efficiency of 77.2% was achieved at pH 4.0 in 1 h. Release efficiency of 91.6% was obtained under optimum pH catalysis condition set at 8.0 and trypsin was free in solutions with retention activity of 63.3%. And 51.5% of released trypsin could be reloaded in 10 min. The results indicate this kind of immobilized enzyme system offers a promising alternative for enzyme recovery in biotechnology.

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Abbreviations

AAc:

acrylic acid

MBA:

N,N′-methylene-bisacrylamide

BSA:

bovine serum albumin

TEMED:

N,N,N′,N′-tetramethylethylenediamine

BAEE:

N-Benzoyl-L-arginine ethylester hydrochloride

p(AAc/MBA):

poly(acrylic acid/N,N′-methylene-bisacryl-amide)

D pH :

main particle size of p(AAc/MBA) microspheres at certain pH

ηL :

loading efficiency

ηR :

release efficiency

ηU :

recovery efficiency

Ci :

initial BSA concentration introduced

Cf :

final BSA concentration in supernatants after immobilization

Cr :

BSA concentration in supernatants after release

Cu :

BSA concentration in supernatants after recovery

Vl :

loading solution volume

Vr :

release solution volume

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Acknowledgment

This study was supported by grants from the National Natural Science Foundations of China (Nos. 30772658 and 30570494).

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  1. Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, 710049, China

    Lili Gai & Daocheng Wu

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  1. Lili Gai
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  2. Daocheng Wu
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Correspondence to Daocheng Wu.

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Gai, L., Wu, D. A Novel Reversible pH-Triggered Release Immobilized Enzyme System. Appl Biochem Biotechnol 158, 747–760 (2009). https://doi.org/10.1007/s12010-008-8373-2

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