Enantioselective transacetylation of (R,S)-β-citronellol by propanol rinsed immobilized Rhizomucor miehei lipase

<p>Abstract</p> <p>Background</p> <p>Use of enzymes in low water media is now widely used for synthesis and kinetic resolution of organic compounds. The frequently used enzyme form is the freeze-dried powders. It has been shown earlier that removal of water molecules from enzyme by rinsing with n-propanol gives preparation (PREP) which show higher activity in low water media. The present work evaluates PREP of the lipase (from <it>Rhizomucor miehei</it>) for kinetic resolution of (<it>R,S</it>)-<it>β</it>-citronellol. The acylating agent was vinyl acetate and the reaction was carried out in solvent free media.</p> <p>Results</p> <p>The PREP, with 0.75% (v/v, reaction media) water, was indeed found to be more efficient and gave 95% conversion to the ester. Using this PREP, with no added water, 90% ee for (<it>R</it>)-(+)-<it>β</it>-citronellyl acetate at 45% conversion (E = 42) was obtained in 4 h. The control with freeze-dried enzyme, with zero water content, gave 78% ee at 30% conversion (E = 13). FT-IR analysis showed that PREP had retained the α-helical content of the enzyme. On the other hand, freeze-dried enzyme showed considerable loss in the α-helical content.</p> <p>Conclusion</p> <p>The results show that PREP may be a superior biocatalyst for enantioselective conversion by enzymes in low-water media.</p

Solid state fluorescence of proteins in high throughput mode and its applications [version 2; peer review: 2 approved]

Direct comparison between fluorescence spectra of a sample in solution and solid state form is valuable to monitor the changes in protein structure when it is “dried” or immobilized on a solid surface (for biocatalysis or sensor applications). We describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory for solid samples in a high-throughput format. The 96-well plate used in our studies, was coated black from all the sides and the excitation and emission paths are identical and are from the top of the well. These two features minimize scatter and provide fairly noise free spectra. Even then the fluorescence intensity may be dependent upon many factors such as the extent of protein aggregation, morphology and sizes of the protein particles. Hence, (changes in) λmax emission may be a more reliable metric in the case of fluorescence spectra of proteins in the solid state. However, any large changes in the intensity could indicate changes in the microenvironment of the fluorophore. The fluorescence emission spectra were blue-shifted (4 to 9 nm), showed an increase in the intensity for different proteins studied upon lyophilization, and were similar to what has been reported by others using available commercial accessories for solid state samples. After validating that our method worked just as well as the dedicated accessories, we applied the method to compare the fluorescence emission spectra of α-chymotrypsin in solution, precipitated form, and the lyophilized powder form. We further examined the fluorescence emission spectra of green fluorescent protein (GFP) in solution and solid form. We also analyzed fluorescence resonance energy transfer (FRET) between tryptophan (Trp57) and the cyclic chromophore of GFP. These findings pointed towards the change in the microenvironment around the cyclic chromophore in GFP upon lyophilization

Enhancement of lipase activity in non-aqueous media upon immobilization on multi-walled carbon nanotubes

<p>Abstract</p> <p>Background</p> <p>Immobilization of biologically active proteins on nanosized surfaces is a key process in bionanofabrication. Carbon nanotubes with their high surface areas, as well as useful electronic, thermal and mechanical properties, constitute important building blocks in the fabrication of novel functional materials.</p> <p>Results</p> <p>Lipases from <it>Candida rugosa </it>(CRL) were found to be adsorbed on the multiwalled carbon nanotubes with very high retention of their biological activity (97%). The immobilized biocatalyst showed 2.2- and 14-fold increases in the initial rates of transesterification activity in nearly anhydrous hexane and water immiscible ionic liquid [Bmim] [PF6] respectively, as compared to the lyophilized powdered enzyme. It is presumed that the interaction with the hydrophobic surface of the nanotubes resulted in conformational changes leading to the 'open lid' structure of CRL. The immobilized enzyme was found to give 64% conversion over 24 h (as opposed to 14% with free enzyme) in the formation of butylbutyrate in nearly anhydrous hexane. Similarly, with ionic liquid [Bmim] [PF6], the immobilized enzyme allowed 71% conversion as compared to 16% with the free enzyme. The immobilized lipase also showed high enantioselectivity as determined by kinetic resolution of (±) 1-phenylethanol in [Bmim] [PF6]. While free CRL gave only 5% conversion after 36 h, the immobilized enzyme resulted in 37% conversion with > 99% enantiomeric excess. TEM studies on the immobilized biocatalyst showed that the enzyme is attached to the multiwalled nanotubes.</p> <p>Conclusion</p> <p>Successful immobilization of enzymes on nanosized carriers could pave the way for reduced reactor volumes required for biotransformations, as well as having a use in the construction of miniaturized biosensensor devices.</p

A novel a-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf ) from Clostridium thermocellum

Articles in International JournalsThe study describes a comparative analysis of biochemical, structural and functional properties of two recombinant derivatives from Clostridium thermocellum ATCC 27405 belonging to family 43 glycoside hydrolase. The family 43 glycoside hydrolase encoding a-L-arabinofuranosidase (Ct43Araf) displayed an N-terminal catalytic module CtGH43 (903 bp) followed by two carbohydrate binding modules CtCBM6A (405 bp) and CtCBM6B (402 bp) towards the C-terminal. Ct43Araf and its truncated derivative CtGH43 were cloned in pET-vectors, expressed in Escherichia coli and functionally characterized. The recombinant proteins displayed molecular sizes of 63 kDa (Ct43Araf) and 34 kDa (CtGH43) on SDS-PAGE analysis. Ct43Araf and CtGH43 showed optimal enzyme activities at pH 5.7 and 5.4 and the optimal temperature for both was 50uC. Ct43Araf and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg21 and 5.0 Umg21, respectively, which increased by more than 2-fold in presence of Ca2+ and Mg2+ salts. This indicated that the presence of CBMs (CtCBM6A and CtCBM6B) did not have any effect on the enzyme activity. The thin layer chromatography and high pressure anion exchange chromatography analysis of Ct43Araf hydrolysed arabinoxylans (rye and wheat) and oat spelt xylan confirmed the release of L-arabinose. This is the first report of a-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both pnitrophenol- a-L-arabinofuranoside and p-nitrophenol-a-L-arabinopyranoside. The protein melting curves of Ct43Araf and CtGH43 demonstrated that CtGH43 and CBMs melt independently. The presence of Ca2+ ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed 48% b-sheets, 49% random coils but only 3% a-helices

Applied Biocatalysis: An Overview

220-22

Alpha chymotrypsin coated clusters of Fe₃O₄ nanoparticles for biocatalysis in low water media

<p>Abstract</p> <p>Background</p> <p>Enzymes in low water containing non aqueous media are useful for organic synthesis. For example, hydrolases in such media can be used for synthetic purposes. Initial work in this area was carried out with lyophilized powders of enzymes. These were found to have poor activity. Drying (removing bulk water) by precipitation turned out to be a better approach. As enzymes in such media are heterogeneous catalysts, spreading these precipitates over a large surface gave even better results. In this context, nanoparticles with their better surface to volume ratio provide obvious advantage. Magnetic nanoparticles have an added advantage of easy separation after the reaction. Keeping this in view, alpha chymotrypsin solution in water was precipitated over a stirred population of Fe₃O₄ nanoparticles in <it>n</it>-propanol. This led to alpha chymotrypsin activity coated over clusters of Fe₃O₄ nanoparticles. These preparations were found to have quite high transesterification activity in low water containing <it>n</it>-octane.</p> <p>Results</p> <p>Precipitation of alpha chymotrypsin over a stirred suspension of Fe₃O₄ nanoparticles (3.6 nm diameter) led to the formation of enzyme coated clusters of nanoparticles (ECCNs). These clusters were also magnetic and their hydrodynamic diameter ranged from 1.2- 2.6 microns (as measured by dynamic light scattering). Transmission electron microscopy (TEM), showed that these clusters had highly irregular shapes. Transesterification assay of various clusters in anhydrous <it>n</it>-octane led to optimization of concentration of nanoparticles in suspension during precipitation. Optimized design of enzyme coated magnetic clusters of nanoparticles (ECCN 3) showed the highest initial rate of 465 nmol min^-1 mg^-1protein which was about 9 times higher as compared to the simple precipitates with an initial rate of 52 nmol min^-1 mg^-1 protein.</p> <p>Circular Dichroism (CD)(with a spinning cell accessory) showed that secondary structure content of the alpha Chymotrypsin in ECCN 3 [15% α-helix, 37% β-sheet and 48% random coil] was identical to the simple precipitates of alpha chymotrypsin.</p> <p>Conclusion</p> <p>A strategy for obtaining a high activity preparation of alpha chymotrypsin for application in low water media is described. Such high activity biocatalysts are useful in organic synthesis.</p

Relevance of chemistry to white biotechnology

Abstract 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.</p