Magnetic alginate microparticles for purification of a-amylases

M. Safafikova^a, I. Roy^b, M.N. Gupta^b, I. Safafik^c

¹ Laboratory of Biochemistry and Microbiology, Institute of Landscape Ecology, Na Sadkach 7, 3 70 05 Ceske Budejovice, Czech Republic

^b Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India

^c Department of General Biology, University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic

Abstract

Spherical magnetic alginate microparticles (25-60 fim in diameter) were prepared using the microemulsion system, with water-saturated 1-pentanol as the organic phase. The limited solubility of 1-pentanol in water enabled simple removal of the organic solvent from the prepared beads with water solution. The prepared alginate microparticles were used as magnetic affinity adsorbents for specific purification of a-amylases. Enzyme activity was eluted by 1.0 M maltose. a-Amylases from Bacillus amy-loliquefaciens and porcine pancreatic acetone powder were purified 9- and 12-fold with 88 and 96% activity recovery, respectively.

Keywords: Alginate; Ferrofluid; Magnetic fluid; Magnetic microparticles; Magnetic nanoparticles; Amylases

1. Introduction

Alginates (polysaccharides consisting of man-nuronic and guluronic acids) are naturally occurring biopolymers and have many applications in various areas of biosciences and biotechnology (e.g. as a matrix for the entrapment and/or delivery of a variety of proteins and cells), and in the food and beverage industry (as a thickening or a gelling agent and a colloidal stabilizer; Smidsrod and Skjåk-Bræk, 1990).

Alginates offer a relatively inert aqueous environment within the matrix and high gel porosity allows high diffusion rates of macromolecules. Alginates also show unexpected affinity towards a-amylases (Roy et al., 2000).

For biotechnology applications, alginate is mainly used in the bead form usually prepared by a syringe with a needle; the diameter and shape of the beads formed is dependent on the size of the needle and the viscosity of the alginate solution (Smidsrod and Skjåk-Bræk, 1990).

Several well known methods (atomization, spraying and water-in-oil emulsifica-tion methods) can also be used to prepare alginate microbeads that are less than 0.2 mm in diameter (Gombotz and Wee, 1998).

Alginate beads with incorporated ferromagnetic materials represent very promising materials for various applications. Magnetically modified beads easily manipulated with an external magnetic field can be used when working with difficult to handle samples, such as biological crude broths, which are often viscous suspensions (Safarik and Safarikova, 1997; Safarikova and Safarik, 2001). Magnetic alginate beads have been already used for affinity separation of various starch-degrading enzymes such as a-amylase, (3-amylase, glucoamylase and pullulanase (Teotia and Gupta, 2001, 2002). Other applications involve immobilization of yeast cells (Brady et al., 1996) and immobilization of affinity ligands (Burns et al., 1985). The goal of our work was to develop a simple procedure for rapid preparation of magnetic alginate microparticles using microemulsion procedure in a water-in-oil emulsion system. The microemulsion was formed using water-saturated 1-pentanol as the oil phase. It is further shown that these microparticles also constitute effective affinity materials for a-amylases. The efficiency of the alginate microparticles was compared with alginate macroparticles described in an earlier work (Teotia and Gupta, 2001). Alginate microparticles were also used for the isolation of porcine pancreatic a-amylase from porcine pancreatic acetone powder containing a number of contaminating hydrolases. Thus, the selectivity of the alginate as a macroaffinity ligand was established.

2. Materials and methods

2.1. Materials

Sodium alginate was from BDH Laboratory Supplies, United Kingdom. 1-Pentanol was from Aldrich, USA while sodium dodecylsulphate (SDS) was from Serva, Germany. Citrate-based magnetic fluid was prepared as described recently (Domingo et al., 2001); the concentration of magnetite was 37 mg/ml (determined using a spectrophotometric procedure; Kiwada et al., 1986). Porcine pancreatic acetone powder (sold as pancreatin) was purchased from Sisco Research Laboratories, Mumbai, India. BAN 240 L (commercial preparation of a-amylase from Bacillus amyloliquefa-ciens) was a product of Novo Nordisk A/S, Denmark and was obtained from Arun & Co., Mumbai, India. All other chemicals used were of analytical grade.

2.2. Preparation of magnetic alginate microparticles

Two milliliters of 2% sodium alginate solution was added to 60 mg of SDS in a test tube. After thorough mixing and SDS dissolution, 600 fxl of citrate stabilized ferrofluid was added and the content was mixed well again. Eight milliliters of water-saturated 1-pentanol was then added and the whole content

was vortexed using a vortex mixer (IKA minishaker, Model MS1, obtained from Fisher Scientific, Czech Republic) at the maximum speed for ca. 5 min. Then the content of the test tube was rapidly poured to another vortexed test tube containing 10 ml of 5% calcium chloride solution and vortexing continued for another 2 min. Then the bead suspension was allowed to stand for 15 min, the beads were separated using an appropriate magnetic separator and washed repeatedly with 5% calcium chloride solution until the 1-pentanol was washed out. Finally, the suspension was filtered through a sieve (mesh size: 100 mm). The beads were stored in 5% calcium chloride solution at 4 °C. The size of magnetic alginate microparticles was measured using optical microscopy.

2.3. Estimation of enzyme activities and amount of protein

Activity of a-amylases was estimated using starch as the substrate (Dekker, 1977). In the case of porcine pancreatic amylase, the assay was carried out at pH 6.9 (Dekker, 1977), whereas in the case of the bacterial enzyme, the assay was done at pH 5.6 [Product sheet on BAN, NOVO Nordisk A/S (1990)]. Bradford assay was used for the protein determination with bovine serum albumin as a standard (Bradford, 1976).

2.4. Binding capacity of magnetic alginate microparticles for a-amylases

Binding capacities of the microparticles were measured by equilibrating 1 ml of different concentrations of the enzyme (bacterial enzyme, 0.5-1 ml; porcine pancreatic enzyme, 0.4-1 ml, appropriately diluted in the respective assay buffers up to a total volume of 1 ml) with 1 ml of the magnetic alginate microparticles (settled volume, after decanting the excess buffer) overnight at 25 °C. After equilibration, aliquots were removed and the amylase activity in the supernatant was measured to calculate bound amylase activity per ml of the alginate microparticles.

2.5. Purification of a-amylases

The magnetic alginate microparticles (16 ml, settled volume) were equilibrated with 20 ml each of porcine pancreatic and bacterial a-amylase preparations at

25 °C for 1 h. After equilibration, the microparticles were washed with the respective assay buffers till the enzyme activity in the washings dropped to zero. The microparticles were then equilibrated with 15 ml of 1M maltose at 4 °C for 4 h. The alginate microparticles were then magnetically separated and the eluate was dialyzed extensively against the respective assay buffers (24 h at 4°C). The eluate was then checked for enzyme activity.

3. Results

Microemulsion procedure with the organic solvent partially miscible with water (1-pentanol) as the organic phase made it possible to prepare spherical magnetic alginate microparticles having the diameters in the range 25-60 (xm. The diameter of the prepared microparticles varied slightly with the time of vortexing the emulsion. The magnetic phase was homogeneously distributed throughout the particle volume (see Fig. 1). The character of the organic phase (1-pentanol) enabled simple washing of the prepared microparticles with calcium chloride solution which is a very cheap and safe procedure. The magnetic alginate microparticles are easily dispersible in water based solutions. The alginate magnetic microparticles also bind bacterial and porcine pancreatic a-amylase activity just like the bigger magnetic alginate particles (Teotiaand Gupta, 2001). The main advantage of using smaller-sized particles is expected to be higher binding capacity. In earlier work with alginate macroparticles, the binding capacity of 4.7 U ml"¹ (settled volume of magnetic particles) had been reported with a commercial preparation of B. amyloliquefaciens a-amylase (BAN 240 L). The alginate microparticles showed the binding capacity ca. seven times higher, i.e. 36 U ml^-1 (Fig. 2). Similar high capacity for porcine pancreatic a-amylase (38Uml^-1) was also observed (Fig. 3).

Fig. 1. Optical microscopy of magnetic alginate microparticles (magnification: 400 x).

Fig. 2. Binding capacity of bacterial a-amylase on magnetic alginate microparticles. Binding capacity was measured by equilibrating 1 ml of different concentrations of the enzyme (0.5-1 ml of the original enzyme solution diluted up to a total volume of 1 ml) with 1 ml of the settled magnetic alginate beads overnight at 25 °C.

Fig. 3. Binding capacity of porcine pancreatic a-amylase on magnetic alginate microparticles. Binding capacity was measured by equilibrating 1 ml of different concentrations of the enzyme (0.4-1 ml of the original enzyme solution diluted up to a total volume of 1 ml) with 1 ml of the settled magnetic alginate beads overnight at 25 °C.

Table 1

Summary of the purification of a-amylase from BAN 240 L (a bacterial source) using magnetic alginate microparticles

Steps	Activity (U)	Protein (mg)	Activity yield (%)	Specific activity (Umg ')	Purification factor
Crude Wash Eluate (1 M maltose)	580 1.5 511	8.3 6.8 0.8	100 0.3 88	70 0.2 638.7	1 - 9

The settled magnetic alginate microparticles (16 ml) were equilibrated with 20 ml a-amylase at 25 °C for 1 h, washed with the assay buffer and then equilibrated with 15 ml of 1 M maltose at 4°C for 4h. After magnetic separation the eluate was dialyzed against the assay buffer (24 h at 4°C) and checked for enzyme activity.

Table 2

Summary of the purification of a-amylase from porcine pancreatic extract using magnetic alginate microparticles

Steps	Activity (U)	Protein (mg)	Activity yield (%)	Specific activity (Umg ')	Purification factor
Crude Wash Eluate (1 M maltose)	578.8 0.0 557.5	2.5 2.1 0.2	100 0 96	231.5 - 2787.5	1 - 12

The settled magnetic alginate microparticles (16ml) were equilibrated with 20ml a-amylase at 25 °C for 1h, washed with the assay buffer and then equilibrated with 15 ml of 1 M maltose at 4°C for 4h. After magnetic separation the eluate was dialyzed against the assay buffer (24 h at 4°C) and checked for enzyme activity.

The magnetic microparticles could be used to purify a-amylases from both these sources. It has been earlier shown that maltose specifically elutes a-amylase from alginate beads (Sardar and Gupta, 1998). Table 1 summarizes the purification process for BAN 240 L. The observed 88% recovery of enzyme activity with 9-fold purification using alginate magnetic microparticles is much better than 72% recovery with 3.6-fold purification reported earlier using magnetic alginate macroparticles (Teotia and Gupta, 2001). Table 2 shows the purification of a-amylase from crude porcine pancreatic acetone powder extract when 96% enzyme activity could be recovered with 12-fold purification. The specific activities indicate that the isolated enzymes are of high purity (Mondal et al., 2003).

4. Discussion

The modified emulsion procedure using organic solvents partially miscible with water as the oil phase (e.g. 1-pentanol) enables to prepare magnetic alginate microparticles in a very simple way, without the need of harsh organic solvents to remove the organic phase from the prepared beads. This is a very important simplification of the existing approach. Magnetic material is homogenously dispersed through the beads in sufficient amount enabling efficient magnetic separation on a standard magnetic separator.

Prepared alginate microparticles were used for the purification of selected a-amylases. The new magnetic alginate microparticles constitute much improved affinity media for purification of a-amylases than larger particles. While only the purification from mammalian and bacterial sources has been described here, it is possible that just like the earlier macroparticles, these will also be useful for isolation of a-amylases from other sources. Also, alginate has been shown to constitute a macroaffinity ligand for other starch-degrading enzymes (Teotia and Gupta, 2002), pectinase (Gupta et al., 1993), lipase (Sharma and Gupta, 2001) and phospholipase D (Sharma et al., 2000). Thus, the magnetic alginate microparticles described here promise to be a multipurpose affinity media for numerous enzymes of biochemical and biotechnological importance.

Acknowledgements

The research is a part of ILE Research Intention No. AV0Z6087904. The experimental work was supported by the Ministry of Education of the Czech Republic (Grant Project No. OC 523.80) and Grant Agency of the Czech Academy of Sciences (Project No. S6087204). The partial supports provided by Council for Scientific and Industrial Research (CSIR) (Extramural Division and Technology Mission on Oilseeds, Pulses and Maize) and Department of Science and Technology, both Government of India organizations, are gratefully acknowledged.

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