Biomolecular Catalysis - ACS Publications - American Chemical Society

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Chapter 12

Nanoporous Sol-Gel Supports Enzymatic Hydrolysis of Chlorophyll in Organic Media 1

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Yunyu Yi , Selim Kermasha , and Ronald Neufeld *

Downloaded by CORNELL UNIV on June 8, 2017 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch012

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Department of Chemical Engineering, Queen's University, Kingston, Ontario K7L 3N6, Canada Department of Food Science and Agricultural Chemistry, McGill University, 21111 Lakeshore Road, Ste-Anne de Bellevue, Quebec H9X 3V9, Canada 2

Entrapment of membrane proteins is a challenging task compared to that involving soluble proteins. Chlorophyllase, a membrane protein, was entrapped in nanoporous tetramethoxysilane (TMOS)-derived sol-gel, demonstrating higher protein mass and activity yield than that in polysaccharide gel and hybrid gel. Both sol-gel formulation process and chemistry may affect apparent activity of sol-gel entrapped chlorophyllase, thus need to be taken into account to improve entrapped chlorophyllase activity. Even though the external transfer of chlorophyll to the sol-gel matrix can be minimized by increasing mixing rate up to 150 rpm, internal diffusion of chlorophyll in nanoporous sol-gel was largely restricted, confirmed by the measured diffusion coefficient of 10- m /s. The apparent activity was also affected by the availability of hydrophobic chlorophyll substrate within the hydrophilic gel, which was controlled by partitioning of chlorophyll between a water/organic solvent medium and the gel phase. 14

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© 2008 American Chemical Society

Kim et al.; Biomolecular Catalysis ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Introduction Chlorophyllase (EC 3.1.1.14) is a membrane glycoprotein, present in the photosynthetic membranes of higher plants and algae (1). In vitro, chlorophyllase catalyzes the hydrolysis of hydrophobic chlorophyll (2) suggesting application in the enzymatic removal of chlorophyll, coextracted as a contaminant into Canola oil. Hydrolysis of chlorophyll in contaminated oil by chlorophyllase enzyme has not as yet been demonstrated due to the fact that chlorophyllase is not produced commercially, and both enzyme and substrate are highly water immiscible (3). Thus the enzymatic hydrolysis of chlorophyll, a water-insoluble substrate, was carried out in non-aqueous media to solubilize chlorophyll and increase access of chlorophyll to the enzyme. Thus far, a wide variety of nonaqueous media have been developed for chlorophyllase-catalyzed reactions aimed at optimizing chlorophyllase biocatalysis, including water/miscible organic solvent (4), aqueous/hexane biphasic organic solvent (5), biphasic system (6), and ternary micellar systems containing polysorbates (7) and nonionic surfactant (8). The biocatalysis of chlorophyllase was also studied in ternary micellar systems but with pheophytin as substrate (9,10). Successful immobilization would allow the reuse of enzyme, which is costly and difficult to produce. Meanwhile, immobilized enzymes demonstrate enhanced stability against harsh environments, such as that of Canola oil and organic solvent, compared to the free enzyme counterpart. Hence, chlorophyllase was entrappd in polysaccharide gel, hybrid gel and tetramethoxysilane (TMOS)based sol-gel in earlier investigations (11,12). Chlorophyllase demonstrated higher mass and activity yield upon entrapment in TMOS-based sol-gel. This paper is then focused on optimizing catalytic performance of nanoporous sol-gel entrapped chlorophyllase in a water/acetone reaction medium. Sol-gel derived silica was reported as entrapment matrix for a wide-range of enzymes (13, 14), whereas only a few reports describe the encapsulation of membrane proteins in sol-gel (12, 15-20). As the field is relatively new, the solgel immobilization process is not well understood (21), and some results are difficult to understand. For example, encapsulation of the same class of enzyme, but derived from alternate sources can result in different activity yields, even though the same sol-gel process has been followed (22). These differences may be due to sol-gel reactions being highly complex, and thus there is little effective control over the structure of the gel at a molecular level (21). Improving entrapped enzyme activity is always one of the objectives while developing enzyme preparation for industrial application. Apparent activity of entrapped enzyme depends on the intrinsic activity of enzyme in the specific microenvironment of entrapment matrix, such as pH, ionic strength, hydrophobic or electrostatic interaction between enzyme molecule and matrix. Also substrate diffusion within a nanoporous matrix to reach the enzyme is largely hindered. For example, translational diffusion of small molecules, such as acetone, in pores

Kim et al.; Biomolecular Catalysis ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Downloaded by CORNELL UNIV on June 8, 2017 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch012

201 with average diameter of 3 nm is reduced by a factor of 5 (23). Any factor that leads to small pore size of sol-gel leads to greater restriction. Apparent activity of entrapped enzyme would also depend on the accessibility of enzyme. For solgel entrapped enzyme, not all the enzyme entrapped is readily available to substrate (13,22), as some protein may be deeply entrapped in enclosed pores as a more cross-linked gel network forms during aging. For entrapping chlorophyllase in a nanoporous TMOS-derived sol-gel, the factors that may affect activities are (a) gelation time, defined as the time between the addition of protein to pre-gel sol and the loss of sol fluidity (24); (b) acidic pH when using HC1 as catalyst for hydrolysis of sol-gel precursor (25); (c) alcohol produced as a result of hydrolysis of sol-gel precursor (25); (d) gel morphology and thus the accessibility of substrate (26); and (e) drying stress (15). Sol-gel chemistry during formulation of the pre-gel sol would affect the activity of entrapped chlorophyllase through the first four factors. Sol-gel process involving aging and drying conditions would affect the activity of entrapped chlorophyllase through the last two factors. Hence, this paper outlines the sol-gel process and chemical parameters that need to be considered to improve entrapped chlorophyllase activity in a water/acetone organic solvent system (15,26). Some other factors, including diffusion/partition coefficient, of substrate within or to the nanoporous sol-gel support, may decrease the apparent activity of chlorophyllase entrapped in nanoporous sol-gel, and thus is also presented (26). This main objective then was to establish the ground work for industrial chlorophyll removal from Canola oil by an enzymatic approach.

Optimization of Sol-gel Process on Catalytic Performance of Entrapped Chlorophyllase As shown in Figure 1, the entrapment process starts with a pre-gel sol consisting of partially hydrolysed precursor containing the active encapsulant, in this case chlorophyllase. Sol transforms to a gel over a few minutes or hours (t , gelation time), depending on composition. Fresh gel is aged at 4°C to develop a more chemically condensed gel network. Gel is then dried to remove water/alcohol from the porous matrix. Both aging and drying conditions likely affect chlorophyllase activity through altering enzyme hydration, or through changes in gel morphology affecting accessibility of the substrate chlorophyll to the enzyme chlorophyllase. g

Aging Conditions Stronger gel structures develop with longer aging times, reducing gel shrinkage during drying, thus preserving the conformation of entrapped enzyme

Kim et al.; Biomolecular Catalysis ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Downloaded by CORNELL UNIV on June 8, 2017 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch012

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Figure I. Schematic illustration of the process of entrapping chlorophyllase in TMOS-derived nanoporous sol-gel

to a larger extent [27]. However, longer aging time may lead to a small pore size, thus decreasing the apparent activity of sol-gel entrapped enzyme. An example would be the apparent activity of lipase decreased upon aging for 25 days at 4°C (28). Aging time ranging from 1 day to 1 week was studied with chlorophyllase. An increase in apparent activity was found with aging time (15), indicating that aging for up to 1 week did not lead to a significant decrease of matrix pore size. Various researchers adopted aging times for entrapped enzymes, ranging from hours to weeks or months, however for practical reasons, a 24 h period was adopted toward an optimal formulation, even though stronger gel networks are preferable. Aging temperature is another factor that may affect the gel structure, as well as entrapped enzyme activity. Aging at 4°C is preferable for maintaining enzyme activity, and thus is most commonly used.

Drying Conditions The natural environment for enzyme is water, as an essential water layer (bound water) surrounds the enzyme molecule, maintaining activity or correct conformation (29). Drying or dehydration thus may denature enzyme if the essential water layer is removed (30). During drying, free water is likely be removed during early stages of drying, thus trie enzyme is likely to retain activity to a large degree as long as the bound water remains associated. However, the removal of bound water during later stages of drying may cause detrimental conformational changes to proteins. For example, antibody denatured during 100 h of drying (31) and lipase completely lost its activity when air-dried to less than 50% of initial water, which was not restored by water replacement (32). Hence,

Kim et al.; Biomolecular Catalysis ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Downloaded by CORNELL UNIV on June 8, 2017 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch012

203 extensive drying is generally detrimental to proteins. In the case of chlorophyllase, air-drying for 6 h would still lead to a highly active chlorophyllase preparation, whereas negligible activity was observed when drying time exceeded 12 h, as seen in Figure 2(15). The decrease of entrapped chlorophyllase activity could also be due to drying stress as a result of high capillary forces developed during evaporation of solvents from gel nanopores. The drying approach that eliminates the capillary force is likely to reduce the drying stress imposed. This hypothesis was supported by a higher specific activity observed with freeze-dried gel than that of air-dried, vacuum-dried and solvent-dried gel (15) and is consistent with that reported by Kamakami and Yoshida (32) with sol-gel entrapped lipase. Even though freeze-drying is preferable to obtain a more active chlorophyllase preparation, air-drying for 3 h is more convenient for practical reasons, and thus used toward optimization.

Sol-gel Chemistry on Catalytical Performance of E n t r a p p e d Chlorophyllase Sol-gel chemistry or formulation determines the gelation time (24), and microenvironment of the sol-gel matrix. It may also affect pore morphology of sol-gel through altering the relative hydrolysis and condensation rate (33). Hence, sol-gel chemistry can affect the catalytical performance of sol-gel entrapped chlorophyllase either directly through altering intrinsic activity of chlorophyllase, or indirectly through altering the gel morphology and thus accessibility of chlorophyllase.

Water/silane Molar Ratio Water/silane molar ratio in the starting sol can affect the relative hydrolysis and condensation rate, impacting on pore morphology of sol-gel, and thus the accessibility of chlorophyllase to substrate. There is always an optimum water/silane molar ratio for a specific enzyme. An optimum chlorophyllase activity was observed at an R-value of 30, with lower activities obtained at both lower and higher R (15). Chen and Lin (34) and Reetz et al. (22) reported similar findings with sol-gel entrapped lipase. At low R value, reduced lipase activity was explained by enzyme aggregation and alcohol denaturation in the pre-gel sol. Sol with R-values over 24 were thought to form sol-gel through alternate gelation behavior, resulting in increasing amounts of residual enzyme in the aqueous supernatant, lowering entrapped lipase activity. In the present study, a change in gelation behavior was not observed. In contrast, over 94% encapsulation yields were observed for all levels of R examined. An examination

Kim et al.; Biomolecular Catalysis ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Downloaded by CORNELL UNIV on June 8, 2017 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch012

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