A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphatepolyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co2+ and Hg2+, whereas Mg2+, Fe2+, Cu2+, Zn2+ and Sr2+ had little or no inhibitory effect. However, Mn2+ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t 1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca2+.

A method for primary plate assay to determine lipase activity was developed. Tween 80 was used as the substrate with either Victoria Blue B, methyl red or rhodamine B as the indicator. Lipolytic activity was determined by the formation of the zone of intensification of the indicator colour after 24 h. Similar results were obtained using Tween 20 and 60 as substrates. Intensity of the colour is greater than that of the trioleindye system and clearer than the hydrolysis zone of tributyrin plate. Tests using a commercial enzyme preparation and growth media with lipolytic activity showed that the zone of intensification increased with increased lipolytic activity. A linear relationship can be seen when log enzyme concentration is plotted against the diameter of zone of intensification. Using this technique, primary screening of lipolytic microorganisms can be conducted using the formation of zones of intensification around the colonies and mycelia.

No abstract

Lipase fromCandida rugosa was modified with several hydrophobic modifiers before being adsorbed onto organic polymer beads. The effects of different enzyme modifiers, supports, solvents, reaction temperatures, fatty acids, and alcohols on the activity of the immobilized enzyme were investigated. The immobilized lipases were good biocatalysts for esterification reactions in organic solvents. They exhibited high activities in all solvents tested, including polar solvents. The activity seemed to depend on the type of support rather than on the modifier of the enzyme. The medium polar support, XAD7, appeared to be the best for the modified lipases. The immobilized lipase favored the medium‐chain fatty acids rather than the long‐chain fatty acids as acyl donors. The alcohol selectivity of the enzyme was unchanged upon immobilization. The native and immobilized lipases favored the short‐chain and terpene alcohols as nucleophiles.

Lipase from Candida rugosa was immobilized on three different supports, i.e. Amberlite XAD7, poly(methylmethacrylate) (PMMA) and celite. With the conditions tested, maximum adsorption can be achieved after 30 min. The activities of the immobilized lipases were determined by the esterification reaction of oleic acid and butanol. The immobilized lipases were found to be very effective in the esterification reaction. The immobilized activities generally were high in apolar organic solvents with log P values from 2•0 to 4•0. The preference for fatty acids as acyl donors differed in all cases of immobilized lipases. Lipase immobilized on XAD7 and PMMA exhibited high preference of acyl donors (fatty acids) with chain lengths 12-18 and 8-18, respectively. Lipase immobilized on celite, however, showed high activity in all cases of fatty acids. The nucleophile (alcohol) selectivity studies showed that lipase immobilized on XAD7 and celite was more accessible to alcohols of chain lengths 3-12. However, lipase immobilized on PMMA showed a significant preference towards alcohols of chain lengths from 3 to 10.

A mycelial lipase from Rhizopus rhizopodiformis was prepared in fragment form. The lipase was examined to catalyze the alcoholysis of palm oil mid-fraction (PMF) in organic solvents. High percentage conversions of PMF to alkyl esters were achieved when methanol or propanol was used as acyl acceptor. Of the two most prevalent fatty acids in PMF, palmitic acid seemed to be preferred over oleic acid in the formation of methyl and propyl esters. The optimal ratio of oil to methanol in the alcoholysis reaction is 1 to 2 moles. The lipase exhibited high alcoholysis activities in nonpolar solvents (log P > 2), such as hexane, benzene, toluene, and heptane. The enzyme showed exceptionally high thermostability. JAOCS 74, 113-116 (1997).

1993. Extra-and intracellular lipases from a thermophilic Rhizopus oryzae and factors affecting their production. Can. J. Microbiol. 39: 978-981.A thermophilic Rhizopus oryzae was isolated, and parameters affecting its production of extra-and intra-cellular lipases were investigated. All carbon sources tested with the exception of sucrose generally inhibited the production of extracellular lipase, but enhanced the production of intracellular lipase. Peptone was the best substrate for extracellular enzyme production, but for intracellular lipase production other substrates such as tryptone, tryptic soy digest, polypeptone, and corn steep liquor gave comparable results. Among lipid substrates, glycerol was the only stimulator of extracellular enzyme production, whereas olive oil, triolein, and oleic acid had very positive effects on intracellular enzyme production. Shaking enhanced the production of both types of enzymes; the temperature optima were 45 and 37°C for extra-and intra-cellular lipases, respectively. A pH of 5.0 was optimal for production of both enzymes. intracellular lipases from a thermophilic Rhizopus oryzae and factors affecting their production. Can. J. Microbiol. 39 : 978-98 1.Nous avons isole une souche de Rhizopus oryzae et etudit les parametres qui peuvent influencer la production de ses lipases extra-et intra-cellulaires. A ['exception du sucrose, chacune des sources de carbone verifiees inhibaient generalement la lipase extracellulaire mais favorisait la production de la lipase intracellulaire. Les peptones etaient le meilleur substrat pour la production de I'enzyme extracellulaire mais pour la lipase intracellulaire d'autres substrats comme la tryptone, les polypeptones, le produit de digestion du soja par la trypsine et une infusion de m a~s ont donne des resultats comparables. Parmi les substrats lipidiques, seule le glycerol stimulait la production de I'enzyme extracellulaire alors que I'huile d'olive, la trioleine et I'acide oleique avaient un effet t r b positif sur la production de I'enzyme intracellulaire. L'agitation favorise la production des deux types d'enzymes. La temperature optimale est de 45°C pour la lipase extracellulaire et de 37°C pour la lipase intracellulaire. Un pH de 5.0 est optimal pour la production des deux enzymes.

Two strains ofRhizopus rhizopodiformis that produced lipases in broth culture were isolated. Maximum lipase production (23 U/ml) was obtained after 72 h culture. Both the crude lipases were stable at 50°C for 30 min and at 45°C for 24 h. Maltose was the best carbon source and peptone the best nitrogen source for the production of lipases. Only glycerol and lecithin stimulated lipase production further.