Elsevier

Polymer

Volume 50, Issue 24, 16 November 2009, Pages 5693-5700
Polymer

Immobilization of carbonic anhydrase by embedding and covalent coupling into nanocomposite hydrogel containing hydrotalcite

https://doi.org/10.1016/j.polymer.2009.09.067Get rights and content

Abstract

Poly(acrylic acid-co-acrylamide)/hydrotalcite (PAA-AAm/HT) nanocomposite hydrogels activated by N-hydroxysuccinimide (NHS) in the presence of N, N'-dicyclohexylcarbodiimide (DCC) were used to immobilize carbonic anhydrase (CA) by embedding and covalent coupling. Cryo Scanning Electron Microscope (CryoSEM) proved the presence of free water in the porous network structures of the swollen hydrogels. Fluorescence microscopy indicated the existence of the immobilized enzyme in the hydrogels. Compared with un-activated hydrogels, activated hydrogels could improve the amount of the immobilization of enzyme, and maximum enzyme loading is about 4.6 mg/g of support for the activated hydrogels. The porous embedding and multi-point covalent linkage between enzyme and hydrogels strengthened the secondary structure stability of enzyme and thus enhanced enzyme stability in the presence of organic solvent and at high temperature. The immobilized enzyme in the activated hydrogel with enhanced structural stability offers great potential as a method to stabilize enzyme for various applications.

Introduction

Carbonic anhydrase (CA) is a zinc metalloenzyme that efficiently catalyzes the reversible hydration of carbon dioxide to bicarbonate and a proton with a maximum turnover rate of 106 mol [CO2] mol−1 [CA] sec−1. CA is the most efficient catalyst for CO2 hydration and dehydration, particularly for application at low pCO2. Great efforts have been focused on the exploration of CA catalysis in, such as, CO2 adsorption in a space shuttle or a submarine, CO2 hydration in an oxygenator, CO2 reduction in the atmosphere, and the enrichment of natural gas [1], [2], [3], [4].

The specificity of enzyme promises great improvements in various applications such as chemical conversions, biosensor, and bioremediation. However, the short catalytic lifetime of enzyme presently limits their usefulness. Immobilization of enzyme has been taken to improve catalytic stability of enzymes and can expand the applications of the natural catalysts [5], [6]. There are many materials, including polymers and inorganic supports, to be used to immobilize enzyme, and good activity retention and enhanced thermostability are often observed [7], [8], [9], [10], [11], [12], [13], [14].

Many attempts have been made to immobilize CA, and encapsulation of CA can provide an efficient route to enhance enzyme stability. For example, coating CA with a Michael-adduct-based membrane could retain only 7% of the original activity [15]. The uniform CA nanogel with enhanced structural stability against denaturation and aggregation expands the applications of CA catalysis, particularly those carried out at high temperatures. However, enzyme stability in the presence of organic solvent was not investigated [16]. Another source of considerable is hydrogels. Porous superabsorbent hydrogels display advantageous for using in pharmaceutical field, e.g. as scaffold for cell growth because they present adequate sites for attachment and growth of enough cells to survive in vitro [17], [18]. Thereby, there has been an increasing interest on the synthesis of hydrogels for enzyme immobilization. Several researchers reported that hydrogel carriers such as poly(acrylamide-co-acrylic acid), and Ca-alginate provide a protective microenvironment for enzymes and yield higher stabilities [19], [20]. Simultaneity, in our previous work, poly(acrylic acid-co-acrylamide)/hydrotalcite (PAA-AAm/HT) nanocomposite hydrogels have been used to immobilize CA and could retain over 85% of the enzyme activity [21]. The reason may lie on the formation of a microenvironment almost all composed of free water inside the network porous of the hydrogel. However, free water in the network porous of the hydrogel has not been proved. Furthermore, the amount of the immobilization of enzyme is lower. In order to enhance the amount of the immobilization of enzyme, PAA-AAm/HT nanocomposite hydrogels could be activated by N-hydroxysuccinimide and then used to immobilize CA enzyme. The porous embedding and multi-point covalent linkage between CA molecules and the hydrogels will improve the amount of the immobilization of enzyme, but also strengthen the secondary structure stability and thus enhanced enzyme stability in the presence of organic solvent or at high temperature. Therefore, the immobilized CA in the activated nanocomposite hydrogel with enhanced structural stability offer great potential as a method to stabilize enzyme for various applications.

Herein, PAA-AAm/HT nanocomposite hydrogels were firstly activated by N-hydroxysuccinimide (NHS) in the presence of N, N'-dicyclohexylcarbodiimide (DCC). And then, activated hydrogels were used to immobilize carbonic anhydrase (CA). The CO2 hydration activities of free enzymes and immobilized enzymes were evaluated in detail. The amounts of the enzyme immobilized in the hydrogels before and after activation were also compared. The method has shown some interesting and valuable advantages, such as, high amount of the immobilization and improved stability in the presence of organic solvent and at high temperature.

Section snippets

Materials

Acrylic acid (AA, C.P.) and acrylamide (AAm, A.R.) were purified by rotary evaporation at 74 °C/25 mmHg and recrystallization in acetone to remove polymerization inhibitor, respectively. N, N'-methylenebisacrylamide (NMBA, A.R.), kalium persulfate (KPS, A.R.), trishydroxymethylaminomethane (Tris, B.R.), and N, N'-dicyclohexylcarbodiimide (DCC, C.P.) were bought from Sinopharm Chemical Reagent Co., Ltd, China. Sodium methyl allyl sulfonate (SMAS, 99%) was purchased from Aldrich. N

Morphology and water (salt) absorbency of PAA-AAm/HT nanocomposite hydrogel

Morphology of dried nanocomposite hydrogel is shown in Fig. 2. PAA-AAm/HT nanocomposite hydrogels obtained by inverse suspension polymerization disperse uniformly in the form of regular spherical particles and sizes are around 100 nm. In addition, TEM technique confirmed the exfoliated structure of PAA-AAm/HT nanocomposite hydrogel. The microstructure of the nanocomposite hydrogels is clearly accompanied by the TEM pattern (see Fig. 3). Exfoliated layers of HT were prevalent, and some primary

Conclusions

Embedded and covalently immobilized enzymes by using activated PAA-AAm/HT nanocomposite hydrogels were investigated in this study. Among the advantages of this approach are: (i) easy immobilization procedure, (ii) high immobilization capacity; (iii) good thermal stability for various biotechnological applications such as a part of enzyme reactor. Experimental results show that three dimensional structure of enzyme did not affect from immobilization procedure. Thermal stability and organic

Acknowledgement

This work was supported financially by the National Natural Science Foundation of China (No. 20776123) and the National Basic Research Program of China (No. 2007CB707805). The authors thank Hanmin Chen (Equipment & Technology Service Platform, College of Life Sciences, Zhejiang University) for CryoSEM analyses.

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