Applications of Alginate in Bioseparation of Proteins

Abstract

Alginate is a polysaccharide that is a block polymer consisting of block units of guluronic acid and mannuronic acid. It shows inherent biological affinity for a variety of enzymes such as pectinase, lipase, phospholipase D, α and β amylases and glucoamylase.

Taking advantage of its precipitation with Ca²⁺ and the above-mentioned property, alginate has been used for purification of these enzymes by affinity precipitation, aqueous two phase separation, macroaffinity ligand facilitated three phase partitioning, immobilized metal affinity chromatography and expanded bed affinity chromatography. Thus, this versatile marine resource has tremendous potential in bioseparation of proteins.

Keywords:

• Alginate
• Affinity precipitation
• Three phase partitioning
• Macroaffinity ligand facilitated three phase partitioning
• Aqueous two phase affinity systems
• Expanded bed affinity chromatography

INTRODUCTION

Searching for valuable material from the sea is an important aspect of sustainable technology. In this regard, chitin, the polysaccharide that is the main component of crab shell, is a good illustrative example. Chitin and its partially deacetylated derivative, chitosan, have already proved useful for a variety of areas in science and technology [[1], [2]]. A number of books and reviews provide details of such applications [[3-7]]. Alginate is another marine resource and is a polysaccharide like chitin/chitosan. While alginate has not attracted as much attention as chitin, there is a growing interest in this polysaccharide.

In a way, alginate resembles chitosan more closely, in as much as it is a water-soluble polymer. Both chitosan and alginate can be classified as naturally occurring smart materials since solubility of these polymers can be reversibly altered by changing stimulus [[8], [9]]. It is quite some time since the usefulness of alginate in biotechnology was evaluated [[10]]. This overview provides a snapshot of a variety of applications of this interesting molecule. The focus of this review, however, is on recent applications of this material in bioseparation.

MOLECULAR STRUCTURE OF ALGINATE

Alginates are linear unbranched polymers of α (1 → 4) linked guluronic acid and mannuronic acid. They occur as structural components in brown marine algae (phaeophyceae) and in some micro-organisms, where they are believed to have many multiple functions such as capsular polysaccharides in soil bacteria, Azotobacter vindenadii [[11]]. The proportion of the guluronic acid residues and manuronic acid residues determines the chemical and physical properties of alginate. There are different sequences and composition of these monomers in various available types of alginate. The monomer occurs in the alginate chain in the form of blocks. The regions are referred to as M blocks for poly (mannuronic acid), G blocks for poly (guluronic acid), and MG blocks for poly (mannuronic-alt-guluronic acid) [[12]]. Each of these blocks has different conformational preferences and behavior. D-mannuronic acid residues being ⁴C₁ have diequatorial links between them, whereas L-guluronic acid residues being ¹C₄ have diaxial links between them. The diaxial glycosidic linkage between G residues exhibit large hindered rotation and gives polyG a special zigzag structure [[13]].

The M-block section is flat and the G-block section is buckled. This sectional nature of the alginate polymer confers different backbone chain flexibility to polymers in solution.

EGG BOX MODEL FOR CALCIUM PRECIPITATION OF ALGINATE

Alginate forms gels with most di- and multivalent cations. Alginate gel formation with calcium ions has been of interest in most applications. Monovalent cations and Mg²⁺ ions do not induce gelation, while ions like Ba²⁺ and Sr²⁺ will produce stronger alginate gels than Ca²⁺. The gel strength will depend upon the guluronic content and also on the average number of G-units in the G-blocks. Gelling of alginate occurs when divalent cations take part in the interchain binding between G-blocks giving rise to a three-dimensional network in the form of a gel. To explain the nature of interaction between the calcium ions and alginate, the Egg Box model has been proposed. The diaxially linked guluronic acid residues form cavities that function as binding site for cations. The divalent calcium ion fits into guluronate structures like eggs in an egg box. The diequatorially linked mannuronate acid residues form much flatter structures with more shallow nests for cations to occupy. This explains the instability of these chains to complex with calcium ions except at higher ion concentration. The analogy therefore is that the strength and selectivity of co-operative binding is determined by the comfort with which eggs of a particular size may pack in the box and with which the layers of the box pack with each other around the eggs [[14]].

ALGINATE BASED BIOSEPARATION STRATEGIES

Structurally speaking, there are many facets to this interesting molecule. These different structural features singly or in combination have proved useful in application of this polysaccharide in devising some powerful bioseparation strategies (Table 1).

Table 1 Bioseparation strategies based on properties of alginate

Property	Bioseparation strategy	Reference
It is a stimuli-sensitive polymer as its solubility changes in presence of Ca^++ in a reversible fashion	Smart carrier for affinity precipitation	[[25]]
Shows affinity for pectinase, starch degrading enzymes, phospholipse D, lipase	Versatile macroaffinity ligand in affinity precipitation	[[17], [18], [20-24]]
Partitions into PEG phase of PEG-salt ATPS	Allows development of a new version of ATPS for numerous enzymes	[[59], [62], [63]]
Precipitates as an interfacial layer in TPP	Suitable as macroaffinity ligand in MLFTPP	[[64-66]]
Can be converted into alginate beads, which shows good fluidization behavior	Useful in EBC for pectinase, pullulanse, etc.	[[19], [48], [67]]
Can be converted into Cu-alginate beads/Zn alginate beads	Can be used as a novel IMAC matrix	[[40], [42]]
Can entrap magnetite iron oxide material	Can be used in magnetic separation	[[68-70]]

The following provides an overview of some of these strategies and how alginate has played a role in specific applications of these methods.

Affinity Precipitation

Precipitation has been extensively used in protein isolation/purification. The common precipitating agents are salts, organic solvents, and polymers [[15]]. The reason behind the continued popularity of precipitation is its simplicity. It is also economical and an easily scalable technique. Used early in purification protocol, it reduces feed volume and gets rid of colored impurities. For many purposes, when high purity of the protein is not required (such as in the case of industrial enzymes), this step alone is sufficient to give the final protein product. Wherever higher purity is required, precipitation is generally followed by chromatographic steps.

Affinity precipitation is a technique that combines the selectivity of affinity chromatography with the advantages of precipitation methods [[16]]. It is primarily based upon the concept of smart polymers. These polymers, as indicated earlier, can exist in a soluble form in a water rich medium. If an affinity ligand is conjugated to such a polymer by normal coupling procedures, it does not alter the smartness of such polymers. Such a bioconjugate would thus be a smart macroaffinity ligand. When added to a crude mixture of proteins, it would selectively complex with the target protein. Again, it has been found that such a complex responds to the stimulus in a way similar to the smart polymer. Thus, the complex can be precipitated by an appropriate stimulus. In the case of alginate, alginate bioconjugates and their complexes with target proteins, addition of Ca²⁺ is a convenient and appropriate stimulus. Once such an affinity complex is isolated by low speed centrifugation, the enzyme/protein can be recovered by a suitable dissociation step, which is generally easily available from affinity chromatography data obtained with the same affinity ligand/protein affinity pair.

Unlike affinity chromatography, affinity precipitation is a single plate process. However, surprisingly, in many cases it has yielded fairly pure protein in the single step. Generally, these purity levels compare to a single band on SDS-PAGE [[17], [18]].

While smartness of the alginate itself would make this a good carrier in affinity precipitation, there have been some surprising observations that make alginate still more useful in affinity precipitation in particular and bioseparation in general. Quite some time back, there was an observation that alginate beads bind pectinase [[19]]. This was considered an example of pseudoaffinity since the enzyme could be eluted off the beads by a moderate concentration of NaCl. These observations formed the basis for purification of pectinase from several sources by affinity precipitation with alginate [[20], [21]]. Recently, Mondal et al. used microwave treated alginate for purification of pectinase from a commercial preparation of Aspergillus niger [[21]]. Microwave pretreatment of alginate at 75°C led to enhanced selectivity of alginate in affinity precipitation of pectinase resulting in higher purification of the enzyme (20-fold purification) as compared to untreated alginate (10-fold purification).

This simple protocol has a few interesting implications: firstly, the possibility of using alginate itself without the necessity of conjugating with an affinity ligand makes the process simple, economical and more acceptable to regulatory agencies. One always has to worry about the affinity ligand slowly releasing from the matrix and contaminating the product. Also, conjugation chemistry invariably uses aggressive chemicals, which also worries regulatory agencies. Secondly, this observation motivated investigations about looking for similar recognition of the polymer by other enzymes. It was found later on that alginate can selectively pick (from the crude broths) α-amylase [[17]], β-amylase [[18]], glucoamylase [[22]], lipase [[23]], and phospholipase D [[24]]. The affinity precipitation in these cases has given excellent recoveries and fold purification (Table 2). The early observation that alginate does not precipitate quantitatively (and hence may not be an ideal carrier) was unduly pessimistic [[25]]. It is likely that network formation (leading to precipitation) is more extensive in the case of alginate-enzyme complex as compared to the case of alginate alone. It is also interesting that for a single theoretical plate process, affinity precipitation gives rather high fold purification. In several cases, the purified product obtained by the single step of this technique gave a single band on SDS-PAGE [[17], [18], [23]]. Thus, this combination of precipitation with the selectivity of affinity interactions taking place in solution shows unexpectedly high power of separation.

Table 2 Purification of proteins using alginate

Purification protocol	Enzyme/protein purified	Affinity ligand	Yield	Purification fold	Reference
Affinity precipitation
	Trypsin	Soybean trypsin inhibitor	45	–	[[25]]
	Pectinase from
	1. Trichoderma viride	None	92	10	[[20]]
	2. Aspergillus niger		83	20	[[21]]
	α-amylase (wheat germ)	None	72	68	[[17]]
	β-amylase (sweet potato)	None	80	44	[[18]]
	Glucoamylase from
	1. Aspergillus niger	None	81	28	[[22]]
	2. Bacillus amyloliquefaciens		78	38
	Lipase from
	1. Chromobacterium viscosum	None	87	2	[[23]]
	2. Porcine pancreas		75	5
	3. Wheat germ		62	35
	Phospholipase D (peanut)	None	61	34	[[24]]
	Peanut lectin	Guar gum	46 mg per 100 g seeds		[[71]]
Aqueous two phase extraction
	Phospholipase D
	1. From peanuts	None	82	78	[[63]]
	2. From carrots		85	17	[[63]]
	Pullulanase (Bacillus acidopullulyticus)	None	85	44	[[62]]
	α-amylase (wheat germ)	None	92	42	[[59]]
	β-amylase (sweet potato)	None	90	43	[[59]]
Macroaffinity ligand facilitated three phase partitioning
	α-Amylase	None
	1. Wheat germ		77	55	[[65]]
	2. Porcine pancreas		92	10
	3. Bacillus amyloliquefaciens		74	5.5
	Glucoamylase (Aspergillus niger)	None	83	20	[[64]]
	Pullulanase (Bacillus acidopullulyticus)	None	89	38	[[64]]
	Pectinase	None	96	13	[[66]]
Immobilized metal affinity chromatography
	Soybean trypsin inhibitor	Zn^2+	90	18	[[40]]
	IgG (from goat serum)	Beads crosslinked with epichlorohydrin and charged with Cu^2+	97.4	8	[[42]]
Expanded bed affinity chromatography
	Pectinase	None	55		[[19]]
			90	211	[[48]]
	α-amylase (wheat germ)	None	90	58	[[49]]
	Phospholipase D	None	78	317	[[41]]
	Pullulanase	None	99	59	[[67]]
	Jacalin	Guargum crosslinked to alginate using epichlorohydrin	88	50	[[72]]
Magnetic separation
	α-amylase from
	1. Wheat germ		70	48	[[68]]
	2. Bacillus subtilis		73	3.6	[[70]]
	3. Porcine pancreas		96	12
	4. Bacillus amyloliquefacines		88	9
	β-amylase (sweet potato)		86	37	[[69]]
	Glucoamylase (Aspergillus niger)		87	31
	Pullulanase (Bacillus acidopullulyticus)		95	49

The conjugation chemistry with alginate has been described by several workers [[26], [27]]. Thus in principle, it should be possible to use alginate as a carrier smart polymer for designing smart macroaffinity ligands.

Alginate as a Macroaffinity Ligand in Aqueous Two Phase Separation (ATPS)

Aqueous two-phase systems consist of two water rich liquid phases and proteins can be partitioned into these phases. The polyethylene glycol (PEG) -dextran (polymer-polymer) and PEG-potassium phosphate (polymer-salt) phase systems are the most common ones, although several others have been described. While effects of salts and temperature have been evaluated for influencing the partitioning of the target protein, use of an affinity ligand in PEG phase by conjugating the ligand to PEG gives the most dramatic results.The affinity partitioning is described by

Some well-known examples of successful applications of affinity partitioning are purification of lactate dehydrogenase (PEG conjugated to Procion yellow HE-3G was the macroaffinity ligand) [[28]], formate dehydrogenase (PEG-conjugated to Procion red HE-3B was used) [[29]], and phosphofructokinase (Cibacron blue 3G-A/PEG was the ligand) [[30]]. The large-scale affinity extractions (upto 30 l scale or 220 kg of phase systems) have been described in the case of lactate dehydrogenase and formate dehydrogenase, respectively [[31], [32]]. The main difficulties perceived in more widespread use of aqueous two-phase affinity extraction have been:

• Cost of PEG, affinity ligand and conjugation.

• Separation of target protein from PEG phase.

Kamihara et al. described an interesting approach that overcomes both of these two constraints [[33]]. The approach exploited the concept of smart macroaffinity ligand. Its incorporation in PEG phase resulted in higher selectivity common to all such affinity-based ATPS. At the same time, application of the appropriate stimulus separated the ligand-target protein from the PEG phase. From this point onwards, the method was identical to affinity precipitation. This allowed reuse of PEG. The smart macroaffinity ligand could also be recycled. This approach could be adapted for converting all experiments with alginate in affinity precipitation protocols to devising combined two-phase affinity extraction systems. Table 2 describes these results.

Unlike affinity precipitation, two phase affinity extraction can be directly applied to crude turbid suspensions without any preclarification steps. This is a very important feature from the viewpoint of economics in large-scale separations. The successful partitioning of alginate in PEG phase is the cornerstone of this approach. Again in principle, the method has limitless possibilities since alginate can be conjugated to numerous affinity ligands and one is not limited to target proteins/enzymes, which show fortuitous affinity to this unconjugated polysaccharide.

Macroaffinity Ligand Facilitated Three Phase Partitioning (MLFTPP)

A few years back it was observed that an alginate solution (in aqueous buffer), when mixed with ammonium sulfate and tert-butanol, led to formation of three phases: upper tert-butanol rich phase, lower water rich phase, and an interfacial precipitate of about 95% alginate [[34]]. This was similar to the so-called three phase partitioning (TPP) reported for proteins/enzymes [[35], [36]]. The experience with another smart polymer, Eudragit S-100, had shown that in such a three phase partitioning of the water soluble polymer, the polymer could simultaneously affinity capture the target protein from a crude mixture of proteins [[37]]. That is, if TPP of the polymer was carried out in the presence of a crude mixture of proteins, the polymer formed an affinity complex with the target protein before forming precipitate at the interface. This allows separation of the target protein as the protein can be dissociated from the complex after dissolution of the precipitate. This process was called MLFTPP and is recognized as one of the potentially useful nonchromatographic techniques [[38]]. Again, the versatile molecular recognition trait of alginate towards several interesting enzymes can be exploited in this technique as well (Table 2). A recent work from our own lab describes the successful application of affinity precipitation and MLFTPP for refolding and simultaneous purification of urea denatured pectinase using alginate [[39]]. In this approach, alginate functions as a pseudochaperonin and pushes the enzyme towards the correct folding pathway.

Immobilized Metal Ion Affinity Chromatography

Immobilized metal ion affinity chromatography (IMAC) exploits the affinity of metal ions like Cu²⁺, Zn²⁺, Ni²⁺, etc., for certain amino acids (histidine, cystine and tryptophan) present on the protein surface via electron donor groups. In the most frequently used design, a chelating ligand (most frequently used is iminodiacetic acid) is covalently bonded to agarose and charged with the metal ion. Gupta et al. used Zn-alginate beads directly as IMAC media for purification of the soybean trypsin inhibitor (STI) from crude extract of soybean flour [[40]]. Zn-alginate beads were prepared by dropping alginate solution (2%) in a zinc chloride (0.1 M) solution using a syringe needle. These metal alginate beads require 6 mM metal ion in working buffers throughout the separation process to maintain the integrity of beads. However, even 6 mM concentration of metal was found to be enough to precipitate a large number of proteins (e.g. β-galactosidase, β-amylase, catalase, α-chymotrypsin, etc.). This limitation can be overcome by using crosslinked alginate beads (obtained by crosslinking alginate with epichlorohydrin) [[41]]. As epichlorohydrin crosslinked hydroxyl groups present on alginate, carboxyl groups were still free and available for charging with metal ions. The metal ion charged beads were used for purification of goat IgG as an illustrative and biochemically useful application [[42]]. The use of metal alginate beads eliminates the step of covalent coupling of the chelating ligand. Apart from elegance in design and making the process more economical, this approach circumvents the problems of traces of chelating ligand and coupling reagents ending up in the product by slow leaching. IMAC has become an important technique in the context of purification of recombinant proteins in the form of fusion proteins with polyhistidine tag [[43], [44]]. IMAC has also been used for protein refolding [[45]]. This new generation IMAC design still needs to be evaluated for such applications. However, on the basis of existing successful applications in bioseparation, it looks quite promising to be used for purification of recombinant proteins and protein refolding.

Expanded Bed Affinity Chromatography

It has already been mentioned that it is very useful to be able to directly deal with crude suspensions. In fact, in this respect, packed bed chromatography has a great disadvantage since turbid suspensions clog the column. Expanded bed chromatography is a brilliant strategy that circumvents the problem. This technique uses a fluidized bed in which the crude is fed from the bottom of the column in an upward direction. The bed captures the target protein but allows impurities (soluble or insoluble) to pass through the space between chromatographic media beads [[46]]. An expanded bed is a fluidized bed (liquid-solid fluidization) in which the flow of the liquid is as turbulence-free as in the packed bed [[47]]. This is achieved by the designer chromatography media, which shows this kind of fluidized behavior.

Quite a few years back, alginate beads were found to perform reasonably well in fluidized bed chromatography for affinity chromatography of pectinase [[19]]. The same concept could be extended for affinity capture of various other enzymes by using a variety of alginate beads (Table 2). It has been found that better fluidization behavior can be obtained by choosing alginate beads of the appropriate size [[48]].

The alginate beads, strictly speaking, do not exhibit ideal expanded bed behavior [[49]]. However, the results obtained (Table 2) prove that these are adequate substitutes for hugely more expensive commercial media [[50]]. In any case, no single commercial media shows the versatile affinity behavior of alginate beads!

In fact, many protocols for preparation of alginate beads of various sizes have been described in the literature (Table 3). Obviously, there is scope for design of better fluidized beds by more extensive studies.

Table 3 Different methods of preparation of alginate beads

Method of preparation	Size	Reference
1. Using rotating atomizer	200–1200 µm	[[73]]
2. Crosslinking using epichlorohydrin		[[41]]
3. Using rotating flat disc atomizer	1–3 mm	[[74]]
4. Paramagnetic alginate beads	10–100 µm	[[75]]
5. Depolymerization, suspension in organic phase, dispersion using cellulose acetate butyrate	220 µm	[[76]]
6. Gluteraldehyde crosslinking	1.2–1.6 mm	[[77]]
7. Laminar jet break–up technique	250–1 mm	[[78]]
8. By suspension in nonaqueous phase (kerosene, n-butanol, vegetable oil)	1–500 µm	[[79]]
9. Emulsification/internal gelation of alginate sol dispersed within vegetable oil	50–1000 µm	[[80]]
10. Electrostatic atomization	100 µm (mean diameter)	[[81]]
11. Electrostatic generation of beads	0.3–2 mm	[[82]]
12. Chitosan alginate beads using tripolyphosphate	440–660 µm	[[83]]
13. Using CO2 gas-forming agents	1.1–2.2 mm	[[84]]
14. Aerosol decomposition (spray pyrolysis)	1- 100 nm	[[85]]
15. Alginate filler beads	4.35±0.08 mm	[[86]]
16. Spray dried alginate beads	212–425 µm	[[48]]

Magnetic Separations

Magnetic materials offer fast and efficient ways of dealing with viscous and turbid crude feed in the area of bioseparation. Some excellent reviews in this area are available [[51], [52]]. Alginate beads entrapping Fe₃O₄ can be used to affinity capture the various enzymes for which alginate shows affinity. The results are summarized in Table 2. As magnetic beads have formed a larger number of applications in analysis [[51], [53]] rather than separation per se, it may be possible in future to conjugate affinity ligands such as antigens/antibodies, etc., to such alginate beads and extend the application of such beads to the analytical area.

CONCLUSION

The use of enzymes to replace “fire and sword” chemistry [[54]] is the underlying concept in the development of green chemistry or white biotechnology, which can introduce environment-friendly industrial practices. A major limitation has been the perception (often true) that enzymes are costlier catalysts (as compared to chemical catalysts). A major percentage of overall production cost is due to downstream processing steps [[55]]. In the past, bioseparation of proteins has often been carried out by multistep protocols [[56]]. Also, it has been felt that chromatographic tools, though costly, are unavoidable [[57]]. Lately, the trend has been to use fewer but highly selective separation steps for protein purification [[58]]. Most of these strategies involve use of an affinity material [[17], [37], [59]].

Again, the cost of affinity material has been a major factor that has held back cost-cutting in protein production. Alginate, a cheap and easily available material, in this respect constitutes a unique opportunity. Also, alginate is used widely in processed foods [[60], [61]]. Hence its use, especially as such (in unconjugated form) in bioseparation, makes it an ideal choice for protein purification in the commercial sector where acceptability by regulatory agency is a major issue. Hopefully, this overview makes an adequate case for wider exploitation of this interesting molecule in bioseparation.