Smita Raghava

Purification and properties of the alkaline lipase from Burkholderia cepacia A.T.C.C. 25609

A Burkholderia cepacia (bacteria) strain, A.T.C.C. 25609, which had been isolated from the bronchial washings of a cystic fibrosis patient, was used to produce lipase. The presence of sodium alginate at an optimal concentration of 8 mg·ml−1 in the growth medium nearly doubled the production of extracellular lipase activity. The enzyme could be purified with 38‐fold purification and 96% activity recovery using a two‐step purification protocol. The molecular mass of the purified lipase determined by SDS/PAGE was shown to be 28 kDa. The pH optimum of the purified enzyme was 9 and it was stable up to 12 h at pH 9 and 10. The enzyme has a temperature optimum of 40 °C and its half‐life (t1/2) values were 54 and 46 min at 50 and 60 °C respectively. The lipase was found to be stable in the presence of the detergents Tween 20 and Triton X‐100. The secondary‐structure analysis of lipase by CD spectroscopy showed 52% α‐helix, 7.7% β‐sheet, 12.6% β‐turn and 27.8% random structure. The lipase was cloned and overexpressed in Escherichia coli. The gene sequence of the cloned lipase was determined and compared with other lipases.

Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies.

Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three‐phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP‐refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.

Enzyme Stabilization via Cross-Linked Enzyme Aggregates

Extensive cross-linking of a precipitate of a protein by a cross-linking reagent (glutaraldehyde has been most commonly used) creates an insoluble enzyme preparation called cross-linked enzyme aggregates (CLEAs). CLEAs show high stability and performance in both conventional aqueous media as well as nonaqueous media. These are also stable at fairly high temperatures. CLEAs having more than one kind of enzyme activity can be prepared and such CLEAs are called combi-CLEAs or multipurpose CLEAs. Extent of cross-linking often influences their morphology, stability, activity, and enantioselectivity.

Strategy for purifying maltose binding protein fusion proteins by affinity precipitation

The maltose binding protein (MBP) affinity tag has been extensively used for protein purification. A commercial grade cationic starch could precipitate MBP or an MBP-tagged protein quantitatively by simultaneous addition of 10% (w/v) polyethylene glycol (PEG) and 50 mM calcium chloride. The precipitated MBP or MBP-tagged protein could be selectively dissociated by suspending the precipitate in 1 M NaCl. In the case of a soluble MBP fusion with a fragment of human immunodeficiency virus protein gp120, 38% of the contaminating proteins could be removed by precipitation with PEG/CaCl(2) and 100% of the fusion protein was recovered. In all cases, the purified proteins showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expected changes in fluorescence emission spectra upon binding to maltose.

Relevance of chemistry to white biotechnology

White biotechnology is a fast emerging area that concerns itself with the use of biotechnological approaches in the production of bulk and fine chemicals, biofuels, and agricultural products. It is a truly multidisciplinary area and further progress depends critically on the role of chemists. This article outlines the emerging contours of white biotechnology and encourages chemists to take up some of the challenges that this area has thrown up.

Nanoparticles of unmodified titanium dioxide facilitate protein refolding

Titanium dioxide (TiO2) nanoparticles (∼10 nm) were found to effectively assist refolding of thermally denatured proteins α-chymotrypsin, RNase A and papain. The isoelectric points (pI) of the enzymes and elution of the refolded enzymes from the nanoparticles after about one hour incubation with salt show that the protein-nanoparticle interaction was predominantly electrostatic in nature. The refolded enzymes regained nearly 100% activity in all the three cases and their CD spectra were similar to corresponding CD spectra of these enzymes in their native form. Dynamic light scattering (DLS) shows that complexes between TiO2nanoparticles and denatured proteins reached their maximum sizes in the same time period (i.e., 1 hour) which was optimum for regaining the biological activity during nanoparticle assisted refolding.

Tuning permeabilization of microbial cells by three-phase partitioning

Three-phase partitioning of cells was carried out by mixing t-butanol and ammonium sulfate with aqueous suspension of cells. Permeabilized cells formed the interface between aqueous and alcohol layers. A preincubation step in which cells were exposed to just t-butanol was found to tune the selectivity of permeabilized cells of Thermus thermophilus,Saccharomyces cerevisiae, and Escherichia coli. Smaller proteins (green fluorescent protein and lipase with molecular weights of 29 and 34 kDa, respectively) were released with preincubation of 15 min, and penicillin G acylase ( approximately 85 kDa) was released with preincubation of 30 min. The high-molecular-weight proteins (alcohol dehydrogenase from S. cerevisiae and T. thermophilus with molecular weights of 150 and 170 kDa, respectively) were retained even after preincubation of 60 min. The specific activities and electrophoretic analysis of some of the proteins obtained reflected their high purity.

Preparation and properties of thermoresponsive bioconjugates of trypsin

Covalent attachment of enzymes and other proteins to the smart polymer, poly(N-isopropylacrylamide) [poly (NIPAAm)], has been widely used as a method for the preparation of thermosensitive protein conjugates. In the present study, reversible soluble-insoluble polymer-enzyme conjugates were prepared by conjugating a copolymer of NIPAAm with 5-mol % of 6-acrylaminohexanoic acid to trypsin by the carbodiimide-NHS (N-hydroxysuccinimide) coupling method. Four bioconjugates with different units of enzyme coupled to the matrix were prepared. Increased enzymatic activity in terms of high effectiveness factor (in the range of 3–5) was found in the conjugates. Kinetic parameters for the immobilized and free enzyme were determined. The Vmax/Km value of the enzyme significantly increased on immobilization by the factors in the range of 12–28. The immobilized enzyme also showed stability to autolysis at 50°C.

Purification and characterization of an alcohol dehydrogenase with an unusual specificity towards glycerol from Thermus thermophilus

The purification and characterization of an NAD(+)-dependent and zinc containing alcohol dehydrogenase (ADH) from Thermus thermophilus (TTHADH) is described. The enzyme could be purified with 25-fold purification and 68% yield using a single chromatographic step. The enzyme was found to be a tetramer (170 kDa) of identical subunits. The pH optimum of the purified enzyme was 8.8 and the temperature optimum was found to be 80 degrees C. Thermal denaturation curves were determined by monitoring the CD values at 222 nm and the T(m) was found to be 89 degrees C. The enzyme showed much higher activity towards glycerol as compared to short chain primary and secondary alcohols. This thermostable enzyme was also highly stereospecific in oxidation of glycerol and converted glycerol into d-glyceraldehyde. The enzyme which converts glycerol into a chiral molecule like d-glyceraldehyde opens up several synthetic opportunities.

The Interface Between Applied Biocatalysis and Environmental Management

The early thrust of applied biocatalysis was in the traditional areas of fermentation and food processing. Slowly, as enzymology developed, the applications of enzymes (or whole cells) extended to numerous other areas like textile, detergent, leather and oil and fat industries (Godfrey and West, Industrial enzymology. Macmillan Press Ltd., London, 634 p, 1996; Roy and Gupta, J Biochem Biophys 39:220–228, 2002; Polaina and MacCabe, Industrial enzymes: Structure, functions and applications. Springer Verlag, Dordrecht, 2007). Given their twin virtues of higher rates and specificity, it was natural that biocatalysts started being used in environmental management. The concepts, techniques and some illustrative applications (showing the interface between applied biocatalysis and environment management) form the theme of this chapter. To start with, broad areas wherein applied catalysis has been relevant are discussed.