Applications of alginate in bioseparation of proteins.

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, a and ss 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.

An integrated process for separation of major and minor proteins from goat serum.

Analysis of minor proteins in animal sera is of considerable clinical significance. To be able to detect these proteins, depletion of major proteins (albumin and immunoglobulin G [IgG]) is necessary. Many of these proteins are also required in pure form for a variety of biochemical applications. The present work uses goat serum as the system and describes the separation and purification of both major and several minor proteins. This was carried out by judicious adaptation and combination of separation technologies such as immobilized metal ion affinity chromatography (on a somewhat novel matrix), dye affinity chromatography, and lectin affinity chromatography. Albumin, IgG, alpha2-macroglobulin, alpha1-proteinase inhibitor, and transferrin were obtained from the serum. The purified preparations were found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

A microwave-assisted microassay for lipases.

A quick and efficient two-step assay for monitoring and screening lipase activity that uses a microtitre plate is described.

Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed.

A smart bioconjugate of trypsin with alginate.

Alginate is a polymer of guluronic acid and mannuronic acid residues and is an inexpensive, nontoxic polysaccharide of marine origin. Trypsin was immobilized noncovalently on alginate with 100% retention of activity. The enzyme did not leach off the polymer even in the presence of 0.01 M HCl and Triton X-100 (0.2% vv(-1)). The V(max)/K(m) values did not change significantly on immobilization. There was 22% loss of activity in first cycle of pH change and after that the conjugate could be reused upto 4 precipitation cycles without any further loss of activity. This smart bioconjugate was also found to have better operational stability in the presence of casein than free enzyme. Fluorescence studies were carried out to probe structural changes upon immobilization.

Purification of green fluorescent protein overexpressed by a mutant recombinant Escherichia coli.

Green fluorescent protein was purified from sonicated recombinant Escherichia coli and its mutant obtained after exposure to UV light. The latter overexpresses green fluorescent protein. The two-step procedure consisted of a two-phase aqueous extraction with PEG/salt and precipitation of the proteins from PEG phase by free Zn2+. The recoveries of green fluorescent protein were 73 and 83% in the cases of recombinant E. coli and its mutant, respectively. The corresponding fold purifications were 24 and 9, respectively. In both cases, the purified protein showed a single band on SDS-PAGE corresponding to 28 kDa.

Purification of recombinant green fluorescent protein by three-phase partitioning.

The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step. TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts. In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase. When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification. The sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of purified preparation shows single band. The fluorescence excitation and emission spectra agreed with values reported in literature.

Purification of goat immunoglobulin G by immobilized metal-ion affinity using cross-linked alginate beads.

The alginate beads obtained by cross-linking of the polymer by epichlorohydrin were charged with Cu(II). The copper-charged beads could be directly used as a immobilized-metal-affinity-chromatographic medium for purification of goat IgG. The best results in the packed-bed mode were obtained by using beads charged with 95.4 micromol/ml Cu(II). We found that we could recover 97.4% IgG with an 8-fold purification.

Microwave-assisted method for microassay of proteins using bicinchoninic acid.

A microwave-assisted microassay has been developed for estimation of proteins, using bicinchoninic acid (BCA). The protocol described here is rapid with a reaction time of merely 95 sec, requires lesser amounts of BCA and other reagents, and tolerates larger or comparable concentration of various interfering substances.

Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads.

Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu(2+), Zn(2+), and Ni(2+), which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn(2+) directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL(-1), as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity.