Joyeeta Mukherjee

Combi-protein coated microcrystals of lipases for production of biodiesel from oil from spent coffee grounds

BackgroundReplacing chemical catalysts with biocatalysts is a widely recognized goal of white biotechnology. For biocatalytic processes requiring low water containing media, enzymes for example commercial preparations of lipases, show low catalytic efficiencies. Some high activity preparations for addressing this concern have been described. Protein coated microcrystals (PCMC) constitute one such preparation. The present work describes a Combi-PCMC for synthesis of biodiesel from the oil extracted from spent coffee grounds.ResultsDifferent lipases were screened for biodiesel synthesis from crude coffee oil out of which Novozym 435 gave the best conversion of 60% in 4 h. Optimization of reaction conditions i.e. % water, temperature and purification of coffee oil further enhanced conversion upto 88% in 24 h. A mixture of Novozym 435 and a cheap commercially available 1,3-specific lipase RMIM (from Mucor miehei) was used in different ratios and 1:1 was found to be the best trade-off between conversion and cost. The commercial preparations then were replaced by a novel biocatalyst design called Combi-Protein coated microcrystals (Combi-PCMC) wherein CAL B and Palatase were co-immobilized with K2SO4 as the core and this performed equivalent to the commercial preparations giving 83% conversion in 48 h.ConclusionCoffee oil extracted from spent coffee grounds could be used for the synthesis of biodiesel by using appropriate commercial preparations of lipases. The expensive commercially immobilized preparations can also be replaced by a simpler and inexpensive immobilization design called combi-PCMC which synergizes the catalytic action of a nonspecific lipase CAL B and a free form of 1,3-specific lipase from Mucor miehei.

Activation of Alpha Chymotrypsin by Three Phase Partitioning Is Accompanied by Aggregation

Precipitation of alpha chymotrypsin in the simultaneous presence of ammonium sulphate and t-butanol (three phase partitioning) resulted in preparations which showed self aggregation of the enzyme molecules. Precipitation with increasing amounts of ammonium sulphate led to increasing size of aggregates. While light scattering estimated the hydrodynamic diameter of these aggregates in the range of 242–1124 nm; Nanoparticle tracking analysis (NTA) gave the value as 130–462 nm. Scanning electron microscopy and gel filtration on Sephadex G-200 showed extensive aggregation in these preparations. Transmission electron microscopy showed that the aggregates had irregular shapes. All the aggregates had about 3× higher catalytic activity than the native enzyme. These aggregates did not differ in λmax of fluorescence emission which was around 340 nm. However, all the aggregates showed higher fluorescence emission intensity. Far-UV and near-UV circular dichroism also showed no significant structural changes as compared to the native molecule. Interestingly, HPLC gel filtration (on a hydroxylated silica column) gave 14 nm as the diameter for all preparations. Light scattering of preparations in the presence of 10% ethylene glycol also dissociated the aggregates to monomers of 14 nm. Both these results indicated that hydrophobic interactions were the driving force behind this aggregation. These results indicate: (1) Even without any major structural change, three phase partitioning led to protein molecules becoming highly prone to aggregation. (2) Different methods gave widely different estimates of sizes of aggregates. It was however possible to reconcile the data obtained with various approaches. (3) The nature of the gel filtration column is crucial and use of this technique for refolding and studying aggregation needs a rethink.

Increasing importance of protein flexibility in designing biocatalytic processes

Enzymes require some flexibility for catalysis. Biotechnologists prefer stable enzymes but often this stabilization comes at the cost of reduced efficiency. Enzymes from thermophiles have low flexibility but poor catalytic rates. Enzymes from psychrophiles are less stable but show good catalytic rates at low temperature. In organic solvents enzymes perform poorly as the prior drying makes the enzyme molecules very rigid. Adding water or increasing reaction temperature improves flexibility and catalytic rates. In case of hydrolases, flexibility and enantioselectivity have interdependence. Understanding the complex role of protein flexibility in biocatalysis can help in designing biotechnological processes.

Lipase catalyzed transesterification of castor oil by straight chain higher alcohols.

Biolubricants from Castor oil were produced enzymatically by transesterification with higher alcohols using a lipase mixture of immobilized Mucor miehei (RMIM) and immobilized Candida antarctica lipase B (Novozym 435) under low water conditions. The conversions were in the range of 80-95% under the optimized conditions.

Lipase coated clusters of iron oxide nanoparticles for biodiesel synthesis in a solvent free medium.

Methyl or ethyl esters of long chain fatty acids are called biodiesel. Biodiesel is synthesized by the alcoholysis of oils/fats. In this work, lipase from Thermomyces lanuginosus was precipitated over the clusters of Fe3O4 nanoparticles. This biocatalyst preparation was used for obtaining biodiesel from soybean oil. After optimization of both immobilization conditions and process parameters, complete conversion to biodiesel was obtained in 3h and on lowering the enzyme amount, as little as 1.7U of lipase gave 96% conversion in 7h. The solvent free media with oil:ethanol (w/w) of 1:4 and 40°C with 2% (w/w) water along with 20% (w/w) silica (for facilitating acyl migration) were employed for reaching this high % of conversion. The biocatalyst design enables one to use a rather small amount of lipase. This should help in switching over to a biobased production of biodiesel.

Decarboxylative aldol reaction catalysed by lipases and a protease in organic co-solvent mixtures and nearly anhydrous organic solvent media

Abstract The effects of the choice of lipase, reaction medium, immobilization, presence of additives and temperature on conversion and stereoselectivity during a lipase catalysed decarboxylative aldol reaction were examined. It was shown that Candida antarctica lipase B (CALB) catalysed a decarboxylative aldol reaction between 4-nitrobenzaldehyde and ethyl acetoacetate in a 60% acetonitrile–40% aqueous buffer co-solvent mixture. Interestingly, free and immobilized forms of CALB showed opposite enantioselectivity in this media. The addition of 30 mol% imidazole increased the reaction rate from 8.5 to 55.7 μM min− 1 mg− 1. A 98% conversion could be achieved in 14 h (instead of 168 h) by adding imidazole. Other lipases also catalysed this reaction in different reaction media to a varying extent. With Mucor javanicus lipase in 30% DMSO, 20% enantiomeric excess (ee) of the (R)-product was observed. CALB also catalysed this reaction in nearly anhydrous acetonitrile. In the presence of cross-linked protein coated microcrystals of CALB, 90% conversion was obtained in this media in 24 h. A commercially available protease, alcalase, was also found to catalyse this reaction. While low water media gave poor conversion, the reaction in aqueous–60% acetonitrile co-solvent mixture gave 99% conversion in 72 h, provided imidazole was used as an additive.

Protein aggregates: Forms, functions and applications

Protein aggregation is implicated in diverse biochemical phenomena which include formation of inclusion bodies and amyloids. In recent years, inclusion bodies of many enzymes have been found to be catalytically active. Enzyme precipitates and their crystalline aggregates have found extensive applications in Biocatalysis in low water media. Protein aggregates also play a useful role in processed food. Enzymes are incorporated in detergents in the form of granulates. This review also looks at the various techniques which are used for characterizing protein aggregates.

The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A.

CtCBM6 of glucuronoxylan-xylanohydrolase (CtXynGH30) from Clostridium thermocellum was cloned, expressed and purified as a soluble ~14 kDa protein. Quantitative binding analysis with soluble polysaccharides by affinity electrophoresis and ITC revealed that CtCBM6 displays similar affinity towards decorated and undecorated xylans by binding wheat- and rye-arabinoxylans, beechwood-, birchwood- and oatspelt-xylan. Protein melting studies confirmed thermostable nature of CtCBM6 and that Ca(2+) ions did not affect its structure stability and binding affinity significantly. The CtCBM6 structure was modeled and refined and CD spectrum displayed 44% β-strands supporting the predicted structure. CtCBM6 displays a jelly roll β-sandwich fold presenting two potential carbohydrate binding clefts, A and B. The cleft A, is located between two loops connecting β4-β5 and β8-β9 strands. Tyr28 and Phe84 present on these loops make a planar hydrophobic binding surface to accommodate sugar ring of ligand. The cleft B, is located on concave surface of β-sandwich fold. Tyr34 and Tyr104 make a planar hydrophobic platform, which may be inaccessible to ligand due to hindrance by Pro68. Site-directed mutagenesis revealed Tyr28 and Phe84 in cleft A, playing a major role in ligand binding. The results suggest that CtCBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans.

Alpha chymotrypsin coated clusters of Fe 3 O 4 nanoparticles for biocatalysis in low water media

BackgroundEnzymes 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 n-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 n-octane.ResultsPrecipitation 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 n-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.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.ConclusionA 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.

Dual bioimprinting of Thermomyces lanuginosus lipase for synthesis of biodiesel

Graphical abstract