A plate assay for primary screening of lipase activity

Abstract

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.

Probiotic Potential of Yeasts Isolated From Traditional Indian Fermented Foods

“…Inoculated plates were incubated at 37ºC for 48 hrs. and were then observed for change in color from red to yellow around the yeast colony [29].…”

Probiotic potential of indigenous yeasts isolated during the fermentation of table olives from Northeast of Portugal

“…Lipase activity was performed according to the method described by Samad et al (1989), with few modifications. The Yeast Extract Agar medium (YEA, tryptone 6.0 g/L, yeast extract 3.0 g/L and agar 15 g/L), supplemented with Tween 80 (1%) and phenol red (0.01%, w/v) at pH 7.3-7.4, was inoculated with different isolates of yeasts and incubated at 37°C for 48 h. Lipase activity was observed by the colour change of the medium (red to yellow).…”

Microbial Lipases: A Prospect for Biotechnological Industrial Catalysis for Green Products: A Review

“…Tributyrin is not considered a lipase substrate, but it does disperse easier in water. The clear zone produced on trybutyrin plate due to trybutyrin hydrolysis is however unclear particularly with low lipase producers [36].…”

“…The underlying principle employed in detection of microbial lipase has always been based on the estimation of free fatty acids liberated from triglycerides after suitable incubation time [36]. The uncertainties in the type of substrates have always been a problem in lipase studies.…”

“…[25] Thermomyces lanuginosus [22] Aeromonas sp [26] Acinetobacter baylyi [27] Pseudomonas aeruginosa Ps-X [28] Pseudomonas aeruginosa EF2 [29] Bacillus sp RS-12 [30] Streptococcus lactis [11] Chromobacterium viscosum [31] Aspergillus niger [32] Aspergillus nidulans [33] Candida rugosa [34] Streptomyces cinnamomeus [35] Another method utilizes triolein. Getting triolein to stay in suspension is a problem and emulsifiers maybe required [36]. A method has been considered with triolein as substrate and with rhodamine as indicator [38].…”