A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+ and Fe3+ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions synergized and Zn2+ and Co2+ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards p-nitrophenyl palmitate and showed a Vmax and Km of 0.70 mmol.mg−1.min−1 and 32 mM for hydrolysis of pNPP.
In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the ‘working horses’ in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst’s preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field of non-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.
A purified alkaline thermo-tolerant bacterial lipase from
MTCC 8372 was immobilized on a
-MBAm) hydrogel. The hydrogel showed approximately 94% binding capacity for lipase. The immobilized lipase (2.36 IU) was used to achieve esterification of myristic acid and isopropanol in
-heptane at 65 °C under continuous shaking. The myristic acid and isopropanol when used at a concentration of 100 mM each in
-heptane resulted in formation of isopropyl myristate (66.0 ± 0.3 mM) in 15 h. The reaction temperature below or higher than 65°C markedly reduced the formation of isopropyl myristate. Addition of a molecular sieve (3 Å × 1.5 mm) to the reaction mixture drastically reduced the ester formation. The hydrogel bound lipase when repetitively used to perform esterification under optimized conditions resulted in 38.0 ± 0.2 mM isopropyl myristate after the 3
cycle of esterification.
A wide range of fatty acid esters can be synthesized by esterification and transesterification reactions catalyzed by lipases in non-aqueous systems. In the present study, immobilization of a purified alkaline extra-cellular lipase of
MTCC 8372 by adsorption on diatomaceous earth (celite) for synthesis of ethyl acetate via transesterification route was investigated.
lipase was deposited on celite (77% protein binding efficiency) by direct binding from aqueous solution. Immobilized lipase was used to synthesis of ethyl acetate from vinyl acetate and ethanol in
-nonane. Various reaction conditions, such as biocatalyst concentration, substrates concentration, choices of solvents (
-alkanes), incubation time, temperature, molecular sieves (3Å × 1.5 mm), and water activity(a
), were optimized. The immobilized lipase (25 mg/ml) was used to perform transesterification in
-alkane(s) that resulted in approximately 73.7 mM of ethyl acetate at 55 °C in
-nonane under shaking (160 rpm) after 15 h, when vinyl acetate and ethanol were used in a equimolar ratio (100 mM each). Addition of molecular sieves (3Å × 1.5 mm) as well as effect of water activity of saturated salt solutions (KI, KCl and KNO
) to the transesterification efficiency has inhibitory effect. Batch operational stability tests indicated that immobilized lipase had retained 50% of its original catalytic activity after four consecutive batches of 15 h each.
Authors:Satyendra Kumar, Shweta Pahujani, R. P. Ola, R. P. Ola, S. S. Kanwar, S. S. Kanwar and Reena Gupta
A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55ºC, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70ºC. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55ºC.
Authors:S. S. Kanwar, R. K. Kaushal, H. Sultana and S. S. Chimni
An alkaline thermotolerant lipase of Bacillus coagulans BTS1 was successively purified by ammonium sulfate precipitation and DEAE anion exchange chromatography. The purified lipase immobilized in alginate beads showed an optimal activity at pH 7.5 and 55ºC. A pH of 5.0 or 10.0 completely quenched the activity of immobilized lipase. The alginate-bound lipase retained its activity following exposure to most of the organic solvents including amines, alkanes and alcohols. Chloride salt of Al3+, Co2+, Mg2+ and NH4+ modulated the lipase activity of alginate-immobilized enzyme. The alginate entrapped lipase showed a preferentially high activity towards p-nitrophenyl palmitate (C: 16) and activity of matrix increased following exposure to SDS. Moreover, the immobilized lipase retained more than 50% of its activity after 3rd cycle of reuse.
Authors:S. S. Kanwar, M. Srivastava, S. S. Chimni, I. A. Ghazi, R. K. Kaushal and G. K. Joshi
Lipase (EC 126.96.36.199) is a tri-acylglycerol ester hydrolase, catalysing the hydrolysis of tri-, di-, and mono-acylglycerols to glycerol and fatty acids. To study the effect of adsorption of a lipase obtained from Bacillus coagulans BTS-1, its lipase was immobilized on native and activated (alkylated) matrices, i.e. silica and celite. The effect of pH, temperature, detergents, substrates, alcohols, organic solvent etc. on the stability of the immobilized enzyme was evaluated. The gluteraldahyde or formaldehyde (at 1% and 2% concentration, v/v) activated matrix was exposed to the Tris buffered lipase. The enzyme was adsorbed/entrapped more rapidly on to the activated silica than on the activated celite. The immobilized lipase showed optimal activity at 50ºC following one-hour incubation. The lipase was specifically more hydrolytic to the medium C-length ester (p-nitro phenyl caprylate than p-nitro phenyl laurate). The immobilization/entrapment enhanced the stability of the lipase at a relatively higher temperature (50ºC) and also promoted enzyme activity at an acidic pH (pH 5.5). Moreover, the immobilized lipase was quite resistant to the denaturing effect of SDS.
Authors:S. S. Kanwar, H. K. Verma, S. Pathak, R. K. Kaushal, Y. Kumar, M. L. Verma, S. S. Chimni and G. S. Chauhan
A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa MTCC-4713 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel. The hydrogel-bound lipase achieved 93.6% esterification of ethanol and propionic acid (300 mM: 100 mM) into ethyl propionate at temperature 65oC in 3 h in the presence of a molecular sieve (3 Å). In contrast, hydrogel-immobilized lipase pre-exposed to 5 mM of HgCl2 orNH4Cl resulted in approximately 97% conversion of reactants in 3 h into ethyl propionate under identical conditions. The salt-exposed hydrogel was relatively more efficient in repetitive esterification than the hydrogel- bound lipase not exposed to any of the cations. Moreover, bound lipase exposed Hg2+ or NH4+ ions showed altered specificity towards p-nitrophenyl esters and was more hydrolytic towards higher C-chain p-nitrophenyl esters (p-nitrophenyl laurate and p-nitrophenyl palmitate with C 12 and C 16 chain) than the immobilized lipase not exposed to any of the salts. The later showed greater specificity towards p-nitrophenyl caprylate (C 8).
Authors:R. Selvakumar, S. Aravindh, C. Kaushik, V. Katarani, Vidya Thorat, Prema Gireesan, V. Jayavignesh, K. Swaminathan and Kanwar Raj
The present study involves the screening of silver nanoparticles containing carbonized yeast cells isolated from coconut cell
sap for efficient adsorption of few long lived radionuclides like 137Cs55, 60Co27, 106Ru44, 239Pu94 and 241Am95. Yeast cells containing silver nanoparticles produced through biological reduction were subjected to carbonization (400 °C
for 1 h) at atmospheric conditions and their properties were analyzed using fourier transform infra-red spectroscopy, X-ray
diffraction, scanning electron microscope attached with energy dispersive spectroscopy and transmission electron microscope.
The average size of the silver nanoparticles present on the surface of the carbonized silver containing yeast cells (CSY)
was 19 ± 9 nm. The carbonized control yeast cells without silver exposure (CCY) did not contain any particles on its surface.
The efficiency of CSY and CCY towards the radionuclide adsorption was studied in batch mode at fixed contact time, concentration,
and at its native pH. CSY was efficient in removal of 239Pu94 (76.75%) and 106Ru44 (54.73%) whereas CCY showed efficient removal only for 241Am95 (62.89%). Both the adsorbents did not show any retention with respect to 60Co27 and 137Cs55. Based on the experimental data, decontamination factor and distribution coefficient (Kd) were calculated and, from the values, it was observed that these adsorbents have greater potential to adsorb radionuclides.