Renu Tyagi

Polarity Index: The Guiding Solvent Parameter for Enzyme Stability in Aqueous-Organic Cosolvent Mixtures

Enzyme catalysis in aqueous–organic cosolvent mixtures has wide applications. However, inadequate attention has been paid to the issue of stability of enzymes in such media. The results with polyphenol oxidase, peroxidase, acid phosphatase, and trypsin show that solvents with polarity indexes of 5.8 and above are “good” solvents. These solvents when used as cosolvents in aqueous–organic solvent media do not denature the enzymes irreversibly. Enzyme(s) exposed to these solvents retain most of their activity even after 48 h of exposure, whereas solvents with polarity indexes of <5.1 denature the enzyme completely within 0–4 h in most of the cases studied. It appears that at higher concentrations (50% and above) cosolvents effectively compete with the water layer around the enzyme. Fluorescence spectroscopy shows that, although the presence of all the organic cosolvents cause conformational changes in the enzyme molecule at a concentration of 50% (v/v), these changes were completely reversible (when the concentration of organic solvent is diluted with aqueous buffer) in case of solvents having polarity indexes of 5.8 and above. In cases of the solvents having polarity indexes of 5.0 and below, the exposure at 50% concentration changed the conformation of the enzymes irreversibly. Thus, a simple parameter, viz. polarity index, may help in medium engineering of enzyme catalysis in nonaqueous surroundings.

Amorphous enzyme aggregates : Stability toward heat and aqueous-organic cosolvent mixtures

Abstract Chemical aggregates of four enzymes, i.e., polyphenol oxidase, acid phosphatase, β-glucosidase, and trypsin have been prepared by chemical cross-linking with glutaraldehyde. These aggregates were characterized for their stability under stress conditions such as high temperature and exposure to organic cosolvents. All the enzyme aggregates exhibit enhanced thermal stability. The concentration of cosolvents required to inactivate the enzymes by 50% was determined for these aggregates in acetonitrile, dimethylformamide, tetrahydrofuran, and dioxane. In most of the cases, the aggregates showed greater stability in the presence of organic cosolvents. Both in the case of free enzymes as well as the aggregates, the irreversible inactivation is less in organic cosolvents when acetonitrile and dimethylformamide are used (i.e., the solvents with polarity index greater than 5.8); however, all aggregates do not show better stability as compared to free soluble enzyme in organic cosolvent mixtures. Scanning electron micrographs (SEM) of polyphenol oxidase aggregates after exposure to 50% (v/v) dimethylformamide or tetrahydrofuran and after incubation at 60°C for 3 h were recorded. The extent of irreversible inactivation correlated well with the morphological changes observed in the case of these aggregates. The correlation of the nature of the cosolvent, loss of biological activity, and different appearance as seen by SEM may be useful in understanding the effect of nonaqueous microenvironment on enzyme structure.

Influence of immobilization on enzyme activity in aqueous-organic cosolvent mixtures

Four enzymes viz. polyphenol oxidase, trypsin, acid phosphatase and peroxidase were adsorbed on eudragit-S100, chitin and chitosan. The enzyme activities of both free as well as immobilized enzymes were assayed in the presence of varying concentrations of some organic cosolvents viz. acetonitrile, tetrahydrofuran, dimethyl formamide and dioxane. In many cases, the enhanced activity observed in the presence of low concentration of organic cosolvent (upto 20%) were greater in the case of immobilized enzymes. In some cases, immobilization led to greater retention of enzyme activity in the presence of higher concentrations of an organic cosolvent. It was found that influence of the matrix upon co-solvent tolerance varies with the solvent but did not correlate with a solvent parameter such as log P. It is shown that adsorption is a good approach while preparing immobilized enzymes for working in media where concentrations of organic cosolvents do not exceed about 20%.

Carbodi-imide coupling of enzymes to the reversibly soluble insoluble polymer Eudragit S-100

The coupling of proteins and enzymes to soluble–insoluble polymers by carbodi‐imide can be performed by using numerous variations of the protocol. This protocol has been investigated for the coupling of five different enzymes, namely wheatgerm acid phosphatase, β‐glucosidase, β‐galactosidase, trypsin and xylanase, to an enteric methacrylate polymer Eudragit S‐100. The following results were found. (1) The activity of the bioconjugate was critically dependent on the physical state of the polymer and the pH of the coupling reaction. For example, in the case of wheatgerm acid phosphatase, the activity of the bioconjugate was 49% when coupling was performed at pH 7·2 and 67% when coupling was performed at pH 4·5. With β‐galactosidase the corresponding values were 57% and 23% and with β‐glucosidase they were 57% and 52% respectively. (2) In some cases, such as β‐glucosidase and β‐galactosidase, it might be necessary to remove excess carbodi‐imide before the addition of the enzyme to the activated matrix. (3) In most of the cases investigated, a sig‐nificant amount of the enzyme (more than 90%) could be bound to the matrix merely by adsorption. (4) More importantly, after the carbodi‐imide coupling procedure, a sufficient fraction of the bound enzyme could be eluted off the matrix, indicating that this was merely adsorbed and not covalently coupled.

Purification and Immobilization of Aspergillus Niger Pectinase on Magnetic Latex Beads

A commercial preparation of pectinase pectinex was purified with the help of alginate-magnetite beads. The purified pectinase was immobilized an magnetic latex beads via carbodiimide coupling. The maximum efficiency of the immobilized preparation was calculated as 81% of the total activity bound to the beads. This was at the lowest level of coupling tried. The pH optimum (pH 4.5 for both free as well as immobilized enzyme) and km (0.7 mg/ml for free enzyme; 1 mg/ml for immobilized enzyme) did not vary significantly upon immobilization. While the half life of free enzyme was calculated as 9 min., the immobilized preparation remained stable up to 3 h at 60d`C.

Stability of enzymes in water/organic co-solvent mixtures

The half lives of polyphenol oxidase and trypsin at 50% (v/v) co-solvent (water miscible) were from 23 to 176 h when polarity index of the co-solvent was >5.8 or if the co-solvent was an alcohol. Polyphenol oxidase, trypsin, chymotrypsin and β-glucosidase lost only 10% activity in such water/organic co-solvent mixtures when the organic co-solvents were present in the concentration ranges of 55–90%, 60–78%, 15–70% and 7–25% respectively.

Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution.

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, α–amylase, β–glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70°C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X‐ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so‐called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu‐96, Ile‐107, and Leu‐133. The development of such a hydrophobic environment at the recognition site introduces a striking conformation change in Ile‐107 by rotating its side chain about CαCβ bond by 180° to bring about the δ‐methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change has earlier been observed in proteinase K when it is complexed to a substrate analog lactoferrin fragment. Proteins 2000;39:226–234.

Preparation of trypsin free chymotrypsin

Chitosan/chitosan-magnetite beads were prepared and binding of different enzymes were done on these beads. The difference in extent of binding of trypsin and chymotrypsin to chitosan-magnetite beads is used for the separation of these enzymes.

Purification of peanut lectin using guar gum as an affinity ligand

Abstract Affinity precipitation is a convenient purification approach which combines the advantages of precipitation with selectivity of affinity interactions. The main limitation of the technique at present is the cost of affinity ligands and matrix. In this work, peanut lectin was isolated by affinity precipitation using guar gum linked alginate. Both matrix as well as affinity ligand were inexpensive in nature. The recovered lectin (46 mg per 100 g seeds) purified by this single-step procedure appeared as a single band of M r 29000 on SDS-polyacrylamide gel electrophoresis.

Noncovalent and reversible immobilization of chemically modified amyloglucosidase and beta-glucosidase an DEAE-cellulose

Abstract The acylation of amino groups of proteins by pyromellitic dianhydride (PMDA) leads to an increase in negative charges on the protein surface by 4 units for every amino group modified. This reaction has been used to modify enzymes for the purpose of adsorption on DEAE-cellulose. The 49% amino groups of amyloglucosidase were modified with 90% residual enzyme activity. Whereas the native enzyme bound to DEAE-cellulose and could be eluted out with 0·1 m sodium chloride, the modified enzyme exhibited stronger binding and could only be eluted with 0·25 m sodium chloride. In the case of adsorbed native enzyme, heating at 60°C for 1 h led to desorption of 97% protein. In the case of the modified enzyme, the immobilized preparation retained full enzyme activity under similar conditions. beta-Glucosidase (which did not bind to DEAE-cellulose) upon modification with PMDA was found to bind to DEAE-cellulose and could be eluted out with 97% protein recovery by washing with 0·2 m sodium chloride. Thus, chemical modification with PMDA may be a useful and general strategy for obtaining enzyme derivatives for reversible adsorption on anion exchangers.