Evaluation of inorganic matrixes as supports for immobilization of microbial lipase

EVALUATION OF INORGANIC MATRIXES AS SUPPORTS FOR IMMOBILIZATION OF MICROBIAL LIPASE

H.F.Castro^*, M.L.C.P.Silva and G.L.J.P Silva

Department of Chemical Engineering, School of Chemical Engineering of Lorena,

PO Box 116, 12600-000, Telephone + 55-12-552-6473, Lorena, SP, Brazil

E-mail: [e-mail]

(Received: October 10, 1999 ; Accepted: April 18, 2000)

Abstract - Candida rugosa was immobilized by physical adsorption on several inorganic supports using hexane as coupling medium. The enzymatic activities of the different derivatives were determined by both hydrolysis of olive oil and esterification of n-butanol with butyric acid. The results were compared to previous data obtained by using a controlled porous silica matrix. The goal was to contribute in searching inexpensive supports for optimum lipase performance. All supports examined exhibited good properties for binding the enzyme lipase. Zirconium phosphate was the best support, giving the highest percentage of protein fixation (86%) and the highest retention of lipase activity after immobilization (34%). The operational stability performance for niobium oxide derivative was improved by previously activated the support with silane and glutaraldehyde. Thermal stabilities were also examined by thermal gravimetric analysis (TG).

Keywords: Inorganic matrixes, lipase, immobilization, esterification, operational stability, thermal stability.

INTRODUCTION

Lipases (EC 3.11.3) are versatile catalysts with potential applications in a number of industrial processes. Important uses include their addition to detergents, the manufacture of food ingredients, pitch control in the pulp industry, and biocatalyst of stereoselective transformations (de Castro and Anderson, 1995; Ghandi, 1997; Jarger and Reetz, 1998).

In view of the current high costs of lipases, the possibility of regenerating and reusing the enzyme would be an attractive feature of biocatalysis. Immobilization may protect enzyme to some extent from solvent denaturation. It helps in maintaining homogeneity of enzymes in the reaction media since it avoids aggregation of enzyme particles. In addition to the ease of handling, immobilized enzymes are well suited for the use in continuous packed-bed reactors (de Castro and Anderson, 1995; Balcão et al., 1996).

Support materials with high mechanical strength are desirable particularly in stirred systems. The presence of solvents may necessitate support with high chemical resistance. Various microorganisms grow readily on many substrates catalyzed by lipases which call for microbially resistant supports. The supports should allow the effective utilization of the enzyme by having the enzyme molecule accessible to the substrates. Since the loss of activity is a certainty over time, the support material should be easy to be regenerated with active enzymes (Yahya et al., 1998).

Lipase has been immobilized by several methods, namely adsorption, crosslinking, adsorption followed by crosslinking, covalent attachment and physical entrapment (Balcão et al., 1996). However, the insoluble carriers used are almost always polymeric resins, natural polymeric derivatives, organogels and fibers with limited capacity to reuse the immobilization matrix and therefore creating disposal problems of organic materials. Inorganic carriers are more expensive than their organic commercial counterparts but have the advantage of being reusable which in some circumstances can decrease the immobilization support costs (Fonseca et al., 1993).

Among inorganic supports, silica based carriers and its derivatives with well defined pore size are popular matrixes for immobilization of several enzymes (Trevisan and Mei, 1992; Ison et al., 1994; Zanin et al., 1996; Baron et al., 1996). This support has also been used successfully for the immobilization of microbial lipase (Soares et al., 1999), however its high cost (US$ 67/gram, Sigma) may impose some economical limitations if scale-up is envisaged.

The search for an inexpensive support has motivated our group to undertake this work dealing with the selection of potential inorganic matrices which can be recognized by lipases, at molecular level, as solid surfaces. We investigated the feasibility of using metal phosphates and oxides (zirconium, thorium, tungsten, and niobium) which have important ion exchange properties and showed to be effective as an adsorbent for cations from dilute aqueous or non-aqueous solutions and color removal from industrial wastes (Clearfield, 1982; Clearfield and Thakur, 1980; Thakur and Clearfield, 1981; Watanabe et al., 1974). In this preliminary study, amorphous zirconium phosphate and niobium oxide (amorphous and crystalline) synthesized according to the methodology previously established in our Institution (Medeiros et al., 1996; Serafim et al., 1996) were evaluated as supports for immobilizing Candida rugosa lipase.

MATERIALS AND METHODS

Materials

Commercial Candida rugosa lipase (Type VII) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St Louis, MO, United States). The lipase was a crude preparation with a nominal specific activity of 835 units.mg^-1 solid and 10% protein based on the Lowry method protein assay (Lowry et al., 1954). Low acidity olive oil (Carbonel) was purchased at a local market. Substrates for esterification reactions were dehydrated, with 0.32 cm molecular sieves (aluminum sodium silicate, type 13 X-BHD Chemicals, Toronto, Canada), previously activated in an oven at 350°C for 6 hours. Solvents were standard laboratory grade. Alcohol, organic acid and other reagents were purchased either from Aldrich Chemical Co. (Milwaukee, WI, USA) or Sigma Chemical Co. (St Louis, MO, USA).

Supports

Crystalline hydrous niobium oxide (CNO), amorphous hydrous niobium oxide (ANO) and amorphous zirconium phosphate (ZrP) were prepared following methodology previously established (Medeiros et al., 1996, Serafim et al., 1996). Details of the characterization procedures for the textural and ion exchange properties of the solids have been published elsewhere (Medeiros et al., 1996, Serafim et al., 1996).

Crystalline hydrous niobium oxide was obtained by precipitation from the corresponding niobium metallic in an acidic mixture solution (HF/HNO₃) followed by addition of ammonium hydroxide. Amorphous hydrous niobium oxide was prepared by alkaline fusion with K₂CO₃ at 1000^oC for 6 hours, followed by addition of hot water and nitric acid 1.0 M. Amorphous zirconium phosphate (ZrP) was obtained by precipitation from the corresponding ZrOCl₂.H₂O in an H₃PO₄ aqueous solution followed by addition of ammonium hydroxide. All supports were dried at 100^oC and the main particle size was between 75 and 180 mm. The surface areas, S_BET in m².g^-1, determined by nitrogen adsorption from BET method were 18, 110 and 234 m².g^-1 for CNO, ANO and ZrP, respectively.

Immobilization Procedure

Lipase was immobilized by physical adsorption on the selected supports according to the procedure previously described for Celite (de Castro et al., 1999). Alternatively, lipase was also covalently bound on crystalline hydrous niobium oxide (CNO) previously treated with g-aminopropyltriethoxysilane (g-APTS), followed by reaction of the pretreated beads with glutaraldehyde solution, according to the procedure described for controlled pore silica (Soares et al., 1999). For each gram of dry support, 0.25 grams of lipase was used. The enzyme was dissolved in 10 ml of 100 mM of sodium phosphate buffer (pH 7.0) and mixed with the support under low stirring during 2 hours at room temperature. After this, 10 ml of hexane was added to the mixture enzyme-support and coupling took place overnight at 4°C. The derivative was filtered (Whatman filter paper 41) and thoroughly rinsed with hexane. Analyses of the hydrolytic activities carried out on initial and spent lipase solutions and immobilized preparations were also used to determine the coupling yield (h%) according to the following expression:

(1)

Protein Assay

Protein was determined according to Lowry et al.(1954) using bovine serum albumin (BSA) as a standard. The amount of bound protein was determined indirectly from the difference between the amount of protein introduced into the coupling reaction mixture and the amount of protein in the filtrate and in the washing solutions.

Moisture Content

The moisture content of the immobilized preparations was determined by Karl Fisher titration (Mettler DL18).

Hydrolysis Assay

Hydrolytic activities of free and immobilized lipase were assayed by the olive oil emulsion method according to the modification proposed by Soares et al. (1999). The substrate was prepared by mixing 50 ml of olive oil with 50 ml of gum arabic solution (7% w/v). The reaction mixture containing 5 ml of the emulsion, 2 ml of 100 mM sodium phosphate buffer (pH 7.00) and either free (1 ml, 5 mg.ml^-1) or immobilized (250 mg) lipase was incubated for 10 min at 37°C. The reaction was stopped by the addition of 10 ml of acetone-ethanol solution (1:1). The liberated fatty acids were titrated with 25 mM potassium hydroxide solution in the presence of phenolphthalein as an indicator. One unit (U) of enzyme activity was defined as the amount of enzyme which liberated 1 mmol of free fatty acid per min under the assay conditions.

Esterification Assay

Reaction systems consisted of heptane (20 ml), n-butanol (250 mM), butyric acid (300 mM) and immobilized lipase (1.0 gram, dry weight). The mixture was incubated at 37⁰C for 24 hours with continuous shaking at 150 rpm. The product formed was determined by gas chromatography using a 6 ft 5% DEGS on Chromosorb WHP, 80/10 mesh column (Hewlett Packard, Palo Alto, CA, USA) and hexanol as internal standard. Activity was expressed as mmol of butyl butyrate formed per minute per gram of dry support.

Operational Stability

The operational stability of the immobilized enzyme was assayed by using 1.0 gram of immobilized lipase in successive batches performed under the same conditions as described for esterification activity assay. Twenty-four hours after starting each batch, the immobilized lipase was removed from the reaction medium and rinsed with hexane, in order to extract any substrate or product eventually retained in the matrix. One hour later (length of time required for the solvent to evaporate) the immobilized derivative was introduced in a fresh medium

Thermal Analysis

Thermal gravimetric analysis (TG-DTG) was performed in a Shimadzu thermogravimetric instrument, TGA-50 model. Samples weighting 10 mg were examined at heating rates of 10^oC .min^-1 in a dry nitrogen flux from 30 to 600^oC.

RESULTS AND DISCUSSION

Immobilization of Lipase onto Inorganic Materials by Adsorption

Three materials (see material and methods for abbreviations) were tested as support matrixes for the immobilization of Candida rugosa lipase by adsorption. In this method, the enzyme was bound to the support without activation using hexane as coupling medium. The results obtained are shown in Table 1.

The amount of protein adsorbed on each support was in the range of 16 to 21 mg/ gram support (dry weight) which gave 64 to 86% protein recovered. After filtration, all derivatives had similar moisture content (25%). The best result with respect to both protein (86%) and catalytic activity (34%) recovered after immobilization was obtained with zirconium phosphate (ZrP). Further information on the catalytic properties of the derivatives was obtained by performing esterification assays (last row in Table 1). These results indicated that ZrP had the most favourable configuration for immobilization of Candida rugosa lipase leading to preparations with high activities to perform both hydrolysis and esterification reactions.

Recycle Potential of Adsorbed Lipase Preparations

The operational stability of the adsorbed lipase derivatives was determined during successive batch esterification reactions at 37°C (Table 2). Thus after the first assay (about 24 hours), each derivative was recovered, washed with hexane and reused with fresh substrate mixture at the same conditions as the first experiment. After an initial loss of activity which is probably due to inactivation lost or loss of adsorbed or only weakly stabilized lipase, the ZrP lipase derivative gave stable performance activity over 5 reaction cycles (half life of 136 hours). However, both niobium oxide (crystalline and amorphous) supports showed poor operational stability resulting in high activity lost (over 75%) after five recycles.

Physical adsorption method is relatively easy and simple for immobilization and leads to minimal structural change of the enzyme during the immobilization process. However, bonding forcing such as hydrogen bonds, Van der Walls forces, and/or hydrophobic interactions are not strong and cause desorption of enzymes from the support during repeated reactions. To overcome this, it is recommended to use a porous support material so that suitable amount of enzyme can be spread on a surface area without conformational changes. Therefore, the increased stability of immobilized lipase on ZrP might result from the improved enzyme retention in this support which has the highest surface area (234 m².g^-1 ). From these results, zirconium phosphate may be selected as the most favorable support matrix for immobilizing Candida rugosa lipase.

Immobilization by Covalent Binding

The direct coupling of lipase on niobium oxide crystalline (CNO) structure as first proposed gave the lowest operational stability (activity dropped 85% after five recycles). To overcome this limitation another experiment was performed in which lipase was immobilized according to the procedure described for controlled porous silica (Soares et al., 1999). In this method the lipase was covalently bound on CNO previously activated with g-aminopropyltriethoxysilane (g-APTS), followed by transformation of the aminoalkyl groups attached to the niobium oxide surfaces into Schiff’s bases by treatment with glutaraldehyde.

Using such a method, both hydrolytic and synthetic activities were enhanced (Table 3) and even more important than that, higher operational stability was obtained (Figure 1). Such results were favorable comparable to those described by Soares et al. (1999). This suggested that silica matrix could be also replaced by CNO with great advantage in the process costs.

Thermal Stability

The thermal stability of enzymes is one of the important criteria for long-term and commercial application. The activity of immobilized enzyme is known to be more resistant against heat than that native state. Thermal gravimetric analysis (TG) provides an important tool for thermal stability studies of macromolecules (Airoldi and Oliveira, 1992; Aly, 1996). This technique has enabled us to determine the temperature range at which a heated sample undergoes a major conformational change by means of monitoring the thermal weight loss profile. In the case of free lipase and immobilized lipase derivatives such temperature range can be related to the protein unfolding and thus to the enzyme denaturation. Thermal degradation temperatures were determined for free lipase, all tested supports (crystalline hydrous niobium oxide (CNO); amorphous hydrous niobium oxide (ANO) and amorphous zirconium phosphate (ZrP) and the resulting immobilized lipase derivatives (lipase-CNO, lipase-ANO and lipase-ZrP). Thermal weight loss results are showed in Table 4 and examples of TG curves are given in Figures 2 to ⁴.

Figure 2 shows the TG curve for free lipase which was characterized by two weight loss peaks. In the first one, at temperature range from 30 to 180^oC, characterized by a low weight loss (4.3%) due to the dehydration of the interstitial water containing in the free lipase sample. From 180 to 600^oC, continuous weight loss was observed indicating a complete decomposition of the organic structure of lipase.

The amorphous zirconium phosphate TG curve (Figure 3) shows only one weight loss peak (about 11%), at temperature from 30 to 400^oC. This was also associated to the dehydration of the interstitial water. Over 500^oC, complete condensation of the mono hydrogen phosphate groups occurred as expected for this material under such temperature (Airoldi and Oliveira, 1992). The thermogravimetric curve for ANO support (figure not shown) indicated a large weight lost (10.5%) at temperature lower than 200°C and a small one (4.9%) from 200 to 400^oC. The former weight loss was due to the elimination of the free water and the latter to the water that it held in the support pores (Aly, 1996). The TG curve profile for CNO support showed distinct shape, only one step of 14% weight loss at temperature range of 30-400°C was detected also due to the elimination of interstitial water.

The TG curve for the lipase immobilized on ZrP (^{Figure 4}) indicated that maximum rate of weight loss (22.0%) occurred in the range 30 to 100^oC, probably due to the elimination of both hydrated water and organic structure of lipase. The thermogravimetric curve for the lipase immobilized on ANO (figure not shown) indicated weight loss of 13% in one step at temperature range of 30 to 400^oC. The same behavior was observed for the lipase immobilized on CNO with a weight loss of 15.4% at the same temperature range. The thermogravimetric curve for the lipase immobilized on sylanized CNO showed weight loss in two steps. The first was characterized by a weight loss (13.6%) at 30 to 200^oC due to the elimination of hydration water and the second step was characterized by 5% weight loss at 200 to 400°C corresponding to water loss that is held in the pores.

The results indicated that upon immobilization, the profile curves for all lipase derivatives shifted towards higher temperatures suggesting that a strong interaction between enzyme and all tested supports occurred which enhanced the conformation stability of the native form. Further investigation on the thermal stability of such derivatives is under progress.

CONCLUSIONS

Over the last 15 years significant advances have been made in the use of enzymes in non-conventional media. These advances are ushering in a new age in the application of enzymes to organic syntheses. Our efforts in this area have focused on the development of suitable support materials for biotransformations in non-conventional media. To understand the phenomena which lead to enzyme inactivation in these systems, we have turned to various thermodynamic approaches to characterize the supports and understand fundamentals aspects of enzyme stability. In this work we have demonstrated that cheap metal inorganic supports are biocompatible with lipases rendering immobilized derivatives with similar or better characteristics than those obtained with controlled porous silica.

ACKNOWLEDGMENTS

This work was supported by research grants from the Brazilian Research Council (CNPq) and FAPESP.

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Publication Dates

• Publication in this collection
  16 Mar 2001
• Date of issue
  Dec 2000

History

• Accepted
  18 Apr 2000
• Received
  10 Oct 1999