Lipase mediated upgradation of dietary fats and oils

In the present scenario, fats and oil modification is one of the prime areas in food processing industry that demands novel economic and green technologies. In this respect, tailored vegetable oils with nutritionally important structured triacylglycerols and altered physicochemical properties have a big potential in the future market. In this context, it is well established that lipases especially microbial lipases, which are regiospecific and fatty acid specific, are of immense importance and hence could be exploited for retailoring of vegetable oils. Further, of the bulk available, cheap oils could also be upgraded to synthesize nutritionally important structured triacylglycerols like cocoa butter substitutes, low calorie triacylglycerols, PUFA-enriched and oleic acid enriched oils. It is also possible to change the physical properties of natural oils to convert them into margarines and hard butter with higher melting points or into special low calorie spreads with short or medium chain fatty acids. Today, by and large, fat and oil modifications are carried out chemically following the method of directed inter-esterification. The process is energy intensive and non-specific. Lipase mediated modifications are likely to occupy a prominent place in oil industry for tailoring structured lipids since enzymatic modifications are specific and can be carried out at moderate reaction conditions. However, as a commercial venture, lipases are yet to be fully exploited. Once the technologies are established, the demand of lipases in oil industry is expected to increase tremendously in the near future for specific modifications of fats and oils to meet the changing consumers' dietary requirements

Microwave-assisted rapid characterization of lipase selectivities.

A rapid screening procedure for characterization of lipase selectivities using microwaves was developed. The rate of reaction of various commercial lipases (porcine pancreas, Mucor miehei, Candida rugosa, Pseudomonas cepacia) as well as lipases from laboratory isolates-Bacillus stearothermophilus and Burkholderia cepacia RGP-10 for triolein hydrolysis was 7- to 12-fold higher in a microwave oven as compared to that by pH stat. The esterification of sucrose/methanol and ascorbic acid with different fatty acids was also achieved within 30 s in a microwave using porcine pancreas, B. stearothermophilus SB-1 and B. cepacia RGP-10 lipases. The relative rates and selectivity of the lipases both for hydrolytic and synthesis reactions remains unaltered. However, the rate of reaction was dynamically enhanced when exposed to microwaves. Microwave-assisted enzyme catalysis can become an attractive procedure for rapid characterization of large number of enzyme samples and substrates, which otherwise is a cumbersome and time-consuming exercise

Interaction between PCNA and Diubiquitinated Mcm10 Is Essential for Cell Growth in Budding Yeast

The minichromosome maintenance protein 10 (Mcm10) is an evolutionarily conserved factor that is essential for replication initiation and elongation. Mcm10 is part of the eukaryotic replication fork and interacts with a variety of proteins, including the Mcm2-7 helicase and DNA polymerase alpha/primase complexes. A motif search revealed a match to the proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box in Mcm10. Here, we demonstrate a direct interaction between Mcm10 and PCNA that is alleviated by mutations in conserved residues of the PIP box. Interestingly, only the diubiquitinated form of Mcm10 binds to PCNA. Diubiquitination of Mcm10 is cell cycle regulated; it first appears in late G(1) and persists throughout S phase. During this time, diubiquitinated Mcm10 is associated with chromatin, suggesting a direct role in DNA replication. Surprisingly, a Y245A substitution in the PIP box of Mcm10 that inhibits the interaction with PCNA abolishes cell proliferation. This severe-growth phenotype, which has not been observed for analogous mutations in other PCNA-interacting proteins, is rescued by a compensatory mutation in PCNA that restores interaction with Mcm10-Y245A. Taken together, our results suggest that diubiquitinated Mcm10 interacts with PCNA to facilitate an essential step in DNA elongation

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-D-galactoside and 1-octyl-beta-D-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50 mM lactose for at least a week. (C) 2004 Elsevier B.V. All rights reserved

Damage-specific modification of PCNA

Okazaki fragment processing is an integral part of DNA replication. For a long time, we assumed that the maturation of these small RNA-primed DNA fragments did not necessarily have to occur during S phase, but could be postponed to late in S phase after the bulk of DNA synthesis had been completed. This view was primarily based on the arrest phenotype of temperature-sensitive DNA ligase I mutants in yeast, which accumulated with an almost fully duplicated set of chromosomes. However, many temperature-sensitive alleles can be leaky and the re-evaluation of DNA ligase I-deficient cells has offered new and unexpected insights into how cells keep track of lagging strand synthesis. It turns out that if Okazaki fragment joining goes awry, cells have their own alarm system in the form of ubiquitin that is conjugated to the replication clamp PCNA. Although this modification results in mono- and poly-ubiquitination of PCNA, it is genetically distinct from the known post-replicative repair mark at lysine 164. In this Extra View, we discuss the possibility that eukaryotic cells utilize different enzymatic pathways and ubiquitin attachment sites on PCNA to alert the replication machinery to the accumulation of single-stranded gaps or nicks behind the fork