Nitin W. Fadnavis

Gelatin Blends with Alginate: Gels for Lipase Immobilization and Purification

Blends of natural polysaccharide sodium alginate (5%) with gelatin (3%) cross‐linked with glutaraldehyde provide beads with excellent compressive strength (8 × 104 Pa) and regular structure on treatment with calcium chloride. Lipases from porcine pancreas, Pseudomonas cepacia, and Candida rugosawere immobilized in such a blend with excellent efficiency. The immobilized enzymes were stable and were reused several times without significant loss of enzyme activity both in aqueous and reverse micellar media. The beads were functionalized with succinic anhydride to obtain beads with extra carboxylic acid groups. These functionalized beads were then successfully used for 7.4‐fold purification of crude porcine pancreatic lipase in a simple operation of protein binding at pH 5 and release at pH 8.5.

Resolution of racemic 2-amino-1-butanol with immobilised penicillin G acylase

Abstract Racemic 2-amino-1-butanol has been resolved to obtain ( S )-2-amino-1-butanol with >99% e.e. via enantioselective hydrolysis of its N -phenylacetyl derivative with penicillin G acylase immobilised on Eupergit C.

Highly Efficient “Tight Fit” Immobilization of α‐Chymotrypsin in Mesoporous MCM‐41: A Novel Approach Using Precursor Immobilization and Activation

The zymogen α‐chymotrypsinogen A is bound to mesoporous silica MCM‐41 with a protein loading of 170 mg/g solid (MCM‐Z) by a simple stirring in aqueous tris‐HCl buffer (pH 7.2). The bound zymogen is then activated with trypsin to obtain α‐chymotrypsin immobilized on MCM‐41 (MCM‐E.I) that displays an effective enzyme activity corresponding to 65 mg protein/g of solid support (3250 BTEE units/g). A direct immobilization of commercially available α‐chymotrypsin (MCM‐E.II) gives lower loading (1250 BTEE units/g). Protein content of the solid support after immobilization is confirmed by thermogravimetric analysis (TGA). The enzyme is tightly bound to the support and can be used over 100 recycles over 1 week in aqueous as well as reverse micellar media. The immobilized enzyme (MCM‐E.I) has been used for resolution of N‐acetyl‐dl‐amino acid esters and racemic trans‐4‐methoxy‐3‐phenylglycidic acid (PGA) methyl ester.

An unusual reversible sol-gel transition phenomenon in organogels and its application for enzyme immobilization in gelatin membranes

An unusual phenomenon is observed for gelatin solutions (1.7−6.8%) in the microemulsion system of 0.3 M bis(2‐ethylhexyl)sulfosuccinate sodium salt in isooctane and 14.5% distilled water. Highly viscous gels obtained at temperatures above 30 °C become free‐flowing liquids at low temperatures (5−10 °C). This reversible temperature‐dependent sol−gel transition phenomenon is used to immobilize several enzymes, such as lipase from Candida rugosa, alcohol dehydrogenase from bakers yeast, mandelonitrile lyase from Sorghum bicolor, and horseradish peroxidase in the gelatin matrix by solubilizing the enzyme in a microemulsion‐based gelatin solution at low temperature (<5 °C) and then cross‐linking with glutaraldehyde. The enzymes retain 70−80% of their activity after immobilization and can be used in biotransformations in organic solvents without any changes in enantioselectivity. This work provides a unique low‐temperature technique for enzyme immobilization in a biocompatible gelatin matrix with a great flexibility of size and shape.

Remote control of stereoselectivity: lipase catalyzed enantioselective esterification of racemic α-lipoic acid☆

Abstract Lipase of Candida rugosa (E.C.3.1.1.3) catalyzes the enantioselective esterification of racemic 6,8-dithioctic acid (α-lipoic acid) with aliphatic alcohols in hexane. Although the reaction centre is four carbon atoms away from the stereogenic centre, (S)-esters with enantioselectivity dependent upon the chain length of alcohol are obtained. A model for the active site of the enzyme is suggested.

Adsorption Induced Enzyme Denaturation: the Role of Polymer Hydrophobicity in Adsorption and Denaturation of α-Chymotrypsin on Allyl Glycidyl Ether (AGE)-Ethylene Glycol Dimethacrylate (EGDM) Copolymers

Effects of changes in hydrophobicity of polymeric support on structure and activity of alpha-chymotrypsin (E.C. 3.4.21.1) have been studied with copolymers of allyl glycidyl ether (AGE) and ethylene glycol dimethacrylate (EGDM) with increasing molar ratio of EGDM to AGE (cross-link density 0.05 to 1.5). The enzyme is readily adsorbed from aqueous buffer at room temperature following Langmuir adsorption isotherms in unexpectedly large amounts (25% w/w). Relative hydrophobicity of the copolymers has been assessed by studying adsorption of naphthalene and Fmoc-methionine by the series of copolymers from aqueous solutions. Polymer hydrophobicity appears to increase linearly on increasing cross-link density from 0.05 to 0.25. Further increase in cross-link density causes a decrease in naphthalene binding but has little effect on binding of Fmoc-Met. Binding of alpha-chymotrypsin to these copolymers follow the trend for Fmoc-methionine binding, rather than naphthalene binding, indicating involvement of polar interactions along with hydrophobic interactions during binding of protein to the polymer. The adsorbed enzyme undergoes extensive denaturation (ca. 80%) with loss of both tertiary and secondary structure on contact with the copolymers as revealed by fluorescence, CD and Raman spectra of the adsorbed protein. Comparison of enzyme adsorption behavior with Eupergit C, macroporous Amberlite XAD-2, and XAD-7 suggests that polar interactions of the EGDM ester functional groups with the protein play a significant role in enzyme denaturation.

Chemoenzymatic synthesis of (4S,5R)-5-hydroxy-γ-decalactone

Abstract Reduction of ethyl 2-hydroxy-3-oxooctanoate with immobilised bakers yeast at pH 4.0 yields anti -2 R ,3 R -dihydroxy ester with high diastereoselectivity and enantioselectivity (de 70%, ee 80%) which is conveniently converted to (4 S ,5 R )-5-hydroxy-γ-decalactone.

Lipase catalyzed regio- and stereospecific hydrolysis: chemoenzymatic synthesis of both (R)- and (S)-enantiomers of α-lipoic acid

Abstract Native lipase of Candida rugosa (EC 3.1.1.3) enantioselectively and regiospecifically hydrolyses the n-butyl ester of 2,4-dithioacetyl butanoic acid either at the carboxylic acid terminus or at the α-thioacetate to provide enantiomerically pure ( R )-2,4-dithioacetyl butyric acid and ( S )-butyl 2-thio-4-thioacetyl butyrate (ee >98%) while the lipase modified by treatment with diethyl p-nitrophenyl phosphate attacks only the α-thioacetate giving enantiomerically pure ( S )-butyl 2-thio-4-thioacetyl butyrate. These enantiomerically pure intermediates can be used as chiral building blocks to obtain both ( S )- and ( R )-enantiomers of α-lipoic acid and their analogues.

Biotransformations with baker's yeast: pH effects on diastereoselectivity during α-hydroxy-β-ketoester reductions and carboncarbon bond cleavages

Abstract Bakers yeast mediated reduction of α-substituted-β-ketoesters lead to reduction of the carbonyl group with high enantiospecificity and diastereoselectivity at low pH (4.0–5.0, e.e.>99%, d.e.>90%) but cleavage of the CC bond is observed at higher pH (>8.0). Similar carbon-carbon bond cleavages are observed in the reactions of α-acetamido-β-ketoesters and acetamidocinnamic acid.

Baker's yeast mediated enantiospecific synthesis of anti-(2R,3R)-p-chloro-3-hydroxytyrosine: an α-amino-β-hydroxy acid of vancomycin

Abstract Bakers yeast mediated reduction of α-azido-β-keto ester lead to reduction of the carbonyl group with high enantiospecificity and diastereoselectivity at low pH (4.0, e.e. >99%, d.e. 79%). At pH ∼7, although the enantioselectivity is maintained, the diastereoselectivity is lost.