Journal o f Microbiological Methods 9 (1989) 51- 56
Elsevier
51
JMM 00286
A plate assay for primary screening of lipase activity
M o h d Yusof A. Samad 1, C. Nyonya A. Razak 1, A b u Bakar Salleh 1,
W.M. Zin Wan Yunus 2, K a m a r u z a m a n A m p o n I and Mahiran Basri 2
l Jabatan Biokimia dan Mikrobiologi and 2Jabatan Kimia, Fakulti Sains dan Pengajian Alam Sekitar,
Universiti Pertanian Malaysia, Serdang (Malaysia)
(Received 14 March 1988) (Revised version received 26 July 1988) (Accepted 1 August 1988)
Summary
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 intensificationincreased 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.
Key words: Plate assay; Lipase; Tween
Introduction
Our laboratory is engaged in a screening programme for lipolytic microorganisms.
The underlying principle employed in detection of microbial lipases has always been
based on the estimation of free fatty acids liberated from triglycerides after a suitable
incubation time. Lipases (triacylglycerol acylhydrolase; EC: 3.1.1.3) are produced by
various organisms either alone or together with esterase (carboxylic ester hydrolases;
EC: 3.1.1.1). The uncertainties in the type of substrates have always been a problem in
lipase studies. These uncertainties are also reflected in the screening techniques proposed. Screening of lipase producers on agar plates using tributyrin as the substrate
has been reported [1-3]. Tributyrin is not considered a lipase substrate, but it does
Correspondence to: Abu Bakar Salleh, Jabatan Biokimia dan Mikrobiologi, Fakulti Sains dan Pengajian
Alam Sekitar, Universiti Pertanian Malaysia, 43400 UPM Serdang, Malaysia.
0167-7012/89/$ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
52
disperse easier in water. The clearance zone produced on tributyrin plate due to tributyrin hydrolysis is, however, unclear, particularly with low lipase producers. Another
method utilises triolein. Getting triolein to stay in suspension is a problem and emulsifiers may be required. A method has been considered with triolein as substrate and
with rhodamine as indicator [4]. The fluorescent effect on rhodamine B indicated
lipid hydrolysis. We found that this technique is more difficult in practice. The fluorescent effect is inconsistent even when tested with known lipase producers.
Tween has also been used as a substrate with Nile blue as indicator [5]. Initial tests
showed that clearance is unclear. However, we found that this technique could be improved and a new method for rapid plate assay using Tween and Victoria Blue is
presented in this paper.
Materials and Methods
Preparation of agar plate
The composition comprised 2.5o70 agar, 2°7o Tween 2 0 - 8 0 (or other substrates) and
0.01°70 Victoria Blue B (or other indicators). A circular well (1 cm diam.) was made
in each quadrant of the plate.
Enzyme solution
Lipase (Candida cylindraceae, Sigma Chemical Co.) was suspended in water at the
required concentration.
Growth of lipolytic organism and preparation of media
A Rh&opus sp. isolated in our laboratories was grown in selected media, M1, for
specified growth periods at 30 °C. The media M1 comprised 5 o70peptone, 0.5 °70glucose
bran, 0.1o70 NaNO 2, 0.1°70 KH2PO 4 and 0.1°70 MgSO4. The growth media was separated from the mycelia and the supernatant used without purification. Lipolytic microorganisms were plated out from maintenance culture.
Results
Studies using Tween 80 as substrate were conducted. The results are presented in
Table 1. Positive results were obtained using either Victoria Blue B, methyl red or
rhodamine B as indicators. In all cases, it was observed that initially (up to 18 h) a
zone of clearance could be seen around the well containing active enzyme preparation.
This zone of clearance was rather vague, but after 24 h there was a colour intensification which was clearly demarcated. It was also observed that the composition of the
media could affect the intensity and diffusion of the colour formed, as observed when
Tween was used together with beef extract and peptone in water.
Studies using Tween 20 and 60 with Victoria Blue B gave similar results, as shown
in Table 1. In comparison, tributyrin and glycerol trioleate were also tested.
By varying the enzyme concentration, the diameter o f the zones of intensification
increased with increasing concentration and could be clearly observed after 24 h. Figure 1 shows an agar plate with Tween 80 and Victoria Blue B with the wells filled with
different concentrations of enzyme. A linear relationship was obtained when log en-
2
2
2
2
2
2
1
1
1
% Agar
Victoria Blue B 0.01%
Victoria Blue B 0.01%
Methyl red
Rhodamine B
Victoria Blue B 0.01%
Victoria Blue B 0.01 070
Victoria Blue B 0.01%
Chromogen
H20
H20, beef extract 0.3% and peptone 107o
H20
H20
H20
H20
Phosphate 0.05 M, pH 8.0
Phosphate 0.05 M and NaC1 pH 8.0
H20
Media/buffer
4- 4- 4- = high intensity, high diffusion; 4- 4- = moderate intensity, high diffusion; + = low intensity, low diffusion
Tween 80, 2%
Tween 80, 2%
Tween 80, 2%
Tween 80, 2°70
Tween 60, 2%
Tween 20, 2070
" Tributyrin, 0.375%
Triolein, 0.078%
Triolein, 0.078%
Substrate
EFFECT OF TWEEN-CHROMOGENS AND O T H E R SUBSTRATE SYSTEMS FOR DETECTION OF LIPASE A C T I V I T Y
TABLE 1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Effect
t~
t~
54
Fig. 1.
Agar plate with Tween 80 as substrate and Victoria Blue B as indicator. The wells were filled with
different concentrations of enzymes.
20 ¸
E
c-
.£
1~-
03
C
Q;
.c
"6 10
t'-
J
E
c~
5
I
I
I
0.5
1.0
1.5
Log enzyme concentration
I
2.0
Fig. 2. Effect of enzyme concentration and time of incubation on the diameter of the zone of intensification. The diameter of this zone is total diameter of the zone minus the diameter of the well. The incubation
times are 24 h ( o ) , 48 h ( • ) , 72 h ( • ) and 92 h (z~).
55
zyme concentration was plotted against the diameter of the zone of colour intensification (Fig. 2). The diameter of the zone increased with the time of incubation (Fig. 2).
It seemed that the zone stabilised after about 90 h.
Similar results were obtained when media from the growth of lipolytic fungus was
placed in the wells.
Lipolytic bacteria (Pseudomonas spp.) and fungi (Rhizopus sp.; Aspergillus sp.) isolated in our laboratory were plated out on a Tween-Victoria Blue plate. The zone of
intensification could be clearly observed around the colonies and mycelia. Casual observation of the size of the zone of intensification could give some indication of the
lipolytic activity, but no quantitative measurement was made.
Discussion
Glycerol triolein is considered the true substrate for lipases [6]. However, triolein
is insoluble in water and a stabiliser is needed to keep the substrate in homogenous
suspension. Tributyrin is usually favoured as the substrate in plate assay techniques
[1 - 3, 6, 7]. However, the zone of hydrolysis on the tributyrin plate is not always clear.
We experimented with Tween as the substrate, as developed by Karnetova and colleagues, and Nile Blue as the indicator [5]. We found that the initial clearance zone
produced on Tween plates against the background colour of all indicators used in our
studies was also hazy. This initial clearance is probably due to the immediate effect
of free acid released onto the agar. On further incubation the clearance zone turned
a shade darker than the background colour, as the acid acted on the indicator.
We called the darkened areas zones of intensification. The boundary of the zone
of intensification and the background colour was clearly demarcated. Although all
the indicators tested showed a similar response, our preference for the indicator was
Victoria Blue B as used by Fryer et al. [2] and Alford and Steinle [8] owing to the intense
darkening of the blue colour.
Jensen [7] in his review stated that Tween is not a suitable substrate for various organisms, but we showed that it was comparable with tributyrin and triolein with the enzymes and microorganisms tested. Thus, the use of Tween as an emulsifier, reviewed
by Jensen [7], is also questionable as we showed that it can act as a substrate.
When log enzyme concentration was plotted against the diameter of zone of intensification, a linear relationship was obtained, as presented by Lawrence et al. [9].
Although no quantitative measurement was made of the growing microorganisms, intense colour development can be observed around plated lipase-producing colonies.
Therefore, we propose that this technique is a simple and reliable technique for screening lipolytic activity.
Acknowledgements
This work is part of the project for the degree o f M.S. for MYAS. The project is
financed by Majlis Penyelidikan dan Kemajuan Sains Negara (MPKSN) Malaysia.
56
References
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organisms. J. Dairy Sci. 50, 477-484.
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5 Karnetova, J., Mateju, J., Rezanka, T., Prochazka, P., Nohynek, M. and Rokos, J. (1984) Estimation
of Lipase activity by the diffusion Plate method. Folia Microbiol. 29, 346-347.
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lipolysis. J. Appl. Bacteriol. 30, 488-494.
9 Lawrence, R. C., Fryer, T. E and Reiter, B. (1967) Rapid method for quantitative estimation of microbial
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