Lipase-catalysed production of biodiesel fuel from some Nigerian lauric oils.

Fatty acids esters were produced from two Nigerian lauric oils, palm kernel oil and coconut oil, by transesterification of the oils with different alcohols using PS30 lipase as a catalyst. In the conversion of palm kernel oil to alkyl esters (biodiesel), ethanol gave the highest conversion of 72%, t-butanol 62%, 1-butanol 42%, n-propanol 42% and iso-propanol 24%, while only 15% methyl ester was observed with methanol. With coconut oil, 1-butanol and iso-butanol achieved 40% conversion, 1-propanol 16% and ethanol 35%, while only traces of methyl esters were observed using methanol. Studies on some fuel properties of palm kernel oil and its biodiesel showed that palm kernel oil had a viscosity of 32.40 mm2/s, a cloud point of 28 degrees C and a pour point of 22 degrees C, while its biodiesel fuel had a viscosity of 9.33 mm2/s, a cloud point of 12 degrees C and a pour point of 8 degrees C. Coconut oil had a viscosity of 28.58 mm(2)/s, a cloud point of 27 degrees C and a pour point of 20 degrees C, while its biodiesel fuel had a viscosity of 7.34 mm2/s, a cloud point of 5 degrees C and a pour point of -8 degrees C. Some of the fuel properties compared favourably with international biodiesel specifications.

A spectrophotometric assay for the enzymatic demethoxylation of pectins and the determination of pectinesterase activity.

A rapid spectrophotometric method for the determination of pectinesterase activity is presented. In this assay, methanol released from pectin by pectinesterase is oxidized with alcohol oxidase to form hydrogen peroxide and formaldehyde. Hydrogen peroxide is then quantitated with peroxidase and the chromogen 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Since both reactions exhibit the same pH optimum it was possible to couple the methanol assay directly to the action of pectinesterase for the real-time determination of this enzyme. The assay is reliable and sensitive, being capable of quantitating a minimum pectinesterase activity of 0.0625 unit (1 unit = 1 microM methanol released per minute). It is also capable of detecting the enzymatic demethoxylation of galactopyranosyl uronic acid methyl esters of pectin down to a minimum concentration of 1.56 nM of methanol per milliliter using a pectin substrate with a methoxy content of 10% (w/w) at a concentration of 0.5 microgram/ml.

Alteration of chain length selectivity of a Rhizopus delemar lipase through site-directed mutagenesis.

The coding sequences of the Rhizopus delemar lipase and prolipase were altered by oligonucleotide-directed mutagenesis to introduce amino acid substitutions. The resulting mutant enzymes, synthesized by the bacterial host Escherichia coli BL21 (DE3), were tested for their ability to hydrolyze the triglycerides triolein (TO), tricaprylin (TC) and tributyrin (TB). Mutagenesis and lipase gene expression were carried out using plasmid vectors derived from previously described recombinant plasmids [Joerger and Haas (1993) Lipids 28, 81-88] by introduction of the origin of replication of bacteriophage f1. Substitution of threonine 83 (thr83), a residue thought to be involved in oxyanion binding, by alanine essentially eliminated lipolytic activity toward all substrates examined (TB, TO and TC). Replacement of thr83 with serine caused from two- to sevenfold reductions in the activity toward these substrates. Introduction of tryptophan (trp) at position 89, where such a residue is found in closely related fungal lipases, reduced the specific activity toward the three triglyceride substrates. For the mutagenesis of residues in the predicted acyl chain binding groove, mutagenic primers were designed to cause the replacement of a specific codon within the prolipase gene with codons for all other amino acids. Phenylalanine 95 (phe95), phe112, valine 206 (val206) and val209, were targeted. A phenotypic screen was successfully employed to identify cells producing prolipase with altered preference for olive oil, TC or TB. In assays involving equimolar mixtures of the three triglycerides, a prolipase with a phe95-->aspartate mutation showed an almost twofold increase in the relative activity toward TC. Substitution of trp for phe112 caused an almost threefold decrease in the relative preference for TC, but elevated relative TB hydrolysis. Replacement of val209 with trp resulted in an enzyme with a two- and fourfold enhanced preference for TC and TB, respectively.

Overexpression of a Rhizopus delemar lipase gene in Escherichia coli.

A cloned complementary deoxyribonucleic acid encoding the precursor polypeptide of an extracellular lipase from the fungus Rhizopus delemar was altered by site-directed mutagenesis to generate deoxyribonucleic acid fragments that specifically code for the polypeptides of the proenzyme and the mature form of the lipase. Attempts to produce these polypeptides in enzymatically active form in Escherichia coli revealed toxic effects toward the host. Therefore the polypeptides were expressed as inactive and insoluble forms in the cytoplasm of E. coli BL21 (DE3) cells using plasmid vector pET11-d. With this tightly regulated high-level expression system, lipase and prolipase polypeptides were produced to estimated levels of up to 21% and 15%, respectively, of total cellular protein. The insoluble polypeptides were solubilized in 8 M urea. Refolding into active forms was achieved by treatment with the redox system cystine/cysteine and dilution. Refolded mature lipase was purified to homogeneity by affinity and ion exchange chromatography. The enzyme had a specific activity comparable to that of lipase from the fungal culture. The quantities of pure enzyme obtained from a 1-L culture of E. coli exceeded those obtained from the fungal culture by a factor of at least 100. Refolded recombinant prolipase was purified essentially to homogeneity and had a specific activity similar to that of the mature enzyme. Its pH optimum was 7.5, rather than the pH 8 determined for recombinant mature lipase and for the enzyme purified from the fungal culture. Recombinant prolipase retained activity after 15 min incubation at 65 degrees C, while mature lipase retained activity only up to 45 degrees C.