Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity

Article abstract of DOI:10.1016/j.ab.2018.03.010

This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λ_ex 330 nm and λ_em 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process.

Full text of DOI:10.1016/j.ab.2018.03.010

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AnalyticalBiochemistry549(2018)26–28
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Fluorescent microplate assay method for high-throughput detection of lipase
transesterification activity
Jianyong Zheng, Wei Wei, Xing Lan, Yinjun Zhang, Zhao Wang∗
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hang Zhou 310014,
Zhejiang, People's Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Lipase
Transesterification activity
4-Methyl umbelliferone
Fluorescent
This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification
activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol
in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction
was determined by detecting the fluorescence intensity at λex 330 nm and λem 390 nm. Several lipases were used
to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was
calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the
assay method include high sensitivity, inexpensive reagents, and simple detection process.
tert-Butanol
Lipases (EC 3.1.1.3) can catalyze the hydrolysis, esterification, and
transesterification of triacylglycerols or other water-insoluble esters
[1–3]. Given their unique “lid” structure and interface activation
properties, lipases are suitable for transesterification in organic media
[4,5]. Lipase catalysis has been applied in food, perfume, cosmetics,
pharmaceutical, and bioenergy industries in the past two decades
[6–9]. High-throughput methods are critically important for the con-
struction of large libraries in enzyme engineering, which usually con-
tain 104–106 mutants to be identified [10,11]. Most of the detecting
methods for lipase activity are commonly based on the hydrolytic ac-
tivity, which can be observed by using pH-stat assay, UV spectro-
photometry, fluorimetry, and chromatography [12–15]. Enzymes
identified by these hydrolytic approaches are not suitable for the de-
sired transesterification reactions [16]. Few reports were available on
high-throughput screening methods for the transesterification activity
of lipases. Therefore, a rapid and efficient method is needed for the
screening of lipases (or lipase mutants) for transesterification. A pre-
vious high-throughput-screening method was used to determine hy-
drolase synthetic activity; in this method, the released acetaldehyde is
determined by fluorescence measurements [17].
substance is usually used as the solvent in lipase-catalyzed reaction
[20,21], and Bu-4-Mu and 4-Mu have good solubility in this solvent.
Lipase-catalyzed transesterification of Bu-4-Mu (no fluorescence) and
methanol in tert-butanol was conducted to produce the free fluorescent
4-Mu, the content of which was determined based on fluorescence de-
tection. The concentration of 4-Mu was detected in 96-well microplate
with a microplate reader (Molecular Devices, Spectramax M5).
To determine the optimal emission wavelength (λem) and excitation
wavelength (λex) for 4-Mu detection in tert-butanol, we scanned the λex
(250–360 nm) and λem (350–550 nm) of 0.1 mM 4-Mu solution in tert-
butanol. The fluorescence spectrum of 4-Mu in Fig. 1 indicated that the
maximum detection wavelength of 4-Mu was at λex 330 nm and λem
390 nm, which is in accordance with the results by Hadjmohamadi et al.
[22]. The effects of detection temperature on the fluorescent test were
evaluated and the results (as shown in Supplementary figure 1) in-
dicated that the solution of 0.1 mM 4-Mu maintained a high and con-
sistent fluorescence value in the temperature range of 30 °C–35 °C.
Based on the current results, the standard curve of fluorescence in-
tensity relative to the concentration of 4-Mu in tert-butanol was es-
tablished and is shown in Fig. 2. A good linear relationship was found
between fluorescence intensity and 4-Mu concentration from 0.01 mM
to 0.08 mM with coefficient (R2) of 0.99. A further increase in 4-Mu
concentration resulted in a decrease in correlation.
In this study, we develop a fluorescent high-throughput screening
method to determine the lipase transesterification activity. The trans-
esterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and
methanol in organic media was selected as the model reaction (Scheme
1). 4-Methyl umbelliferone (4-Mu) and its derivatives are a common
and effective compound used in enzyme activity detection [18,19]. tert-
Butanol is the most suitable solvent for this assay method because this
Five commercially available lipases were used to investigate the
accuracy and efficiency of the proposed method. Lipozyme 435 (CAL-B,
lipase
B
from Candida antarctica immobilized on macro-
porouspolyacrylate resin), Lipozyme RM IM (RML, Mucormiehei
∗
Corresponding author.
E-mail address: hzwangzhao@zjut.edu.cn (Z. Wang).
https://doi.org/10.1016/j.ab.2018.03.010
Received 29 January 2018; Received in revised form 9 March 2018; Accepted 12 March 2018
Availableonline13March2018
0003-2697/©2018ElsevierInc.Allrightsreserved.

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J. Zheng et al.
AnalyticalBiochemistry549(2018)26–28
Scheme 1. Model reaction for the determination of lipase transes-
terification activity.
O
O
Lipase
R
OH
+
+
OR
tert-Butanol
O
O
O
HO
O
O
Bu-4-Mu
4-Mu
Fig. 1. Excitation and emission spectrum of 4-Mu in tert-butanol.
Fig. 3. The conversion of lipase catalyzed transesterification of 0.1 mM Bu-4-Mu and
0.5 mM methanol in tert-butanol using several lipases. The reactions were performed by
the general procedure.
Fig. 2. The standard curve of 4-Mu by fluorescence detecting at λex 330 nm and λem
390 nm.
immobilized on ionic resin), and Lipozyme TL IM (TLL,
Thermomyceslanuginosus immobilized on silica) were supplied by
Novozymes A/S (Bagsvaerd, Denmark). Lipase PS immo (PCL, lipase
from Pseudomonas cepacia) was purchased fromPurolite Ltd (Wales,
UK), and Lipase AK (PFL, Pseudomonas fluorescens lipase) was purchased
from Sigma-Aldrich (Steinheim, Germany). The fluorescence measure-
ment in the microplate reader was performed as follows: 150 μl of
0.5 mM Bu-4-Mu in tert-butanol and 150 μl of 2.5 mM methanol in tert-
butanol with 3 mg of lipase were dispensed into each well of the 96-well
microplates. The mixture was incubated at 30 °C with a shaking speed
of 400 rpm for 10 min. The fluorescence intensity of each reaction was
detected at λex 330 nm and λem 390 nm both at 30 °C. All experiments
were conducted in triplicate, and the relative error value of the parallel
results was less than 10%. Control experiment was conducted without
adding the enzyme. Fig. 3 shows a typical time course of this model
reaction by using different lipases as detected by fluorescence mea-
surements. The progress of the reaction can be easily and sensitively
monitored.
Fig. 4. Comparision of fluorescence analysis (Gray) and HPLC analysis (LT Gray) method
for determining the transesterification activity of the testing lipases.
performance liquid chromatography (HPLC) to verify the proposed
fluorescent microplate assay method. The reaction conditions were in
accordance with the aforementioned methods. Conversions were con-
ducted by Waters E2695 HPLC with a DAD detector. Analysis was
performed using the Welchrom C18 column (4.6 × 250 mm, 5 μm), and
a mixture of acetonitrile and ultrapure water 80:20 (v/v) was used as
the mobile phase. Flow rate was 1.0 mL/min, and the retention times
for 4-Mu and Bu-4-Mu were 3.0 min and 4.4 min, respectively. The
standard curve of 4-Mu was analyzed by HPLC to determine 4-Mu
concentration and calculate the conversion of the reaction (as shown in
Supplementary figure 2). As shown in Fig. 4, the results of the con-
version with the fluorescence detection method are correlated with
those by HPLC analysis, and the variance of the ratio of the data was
0.0118. Among the enzymes, PCL exhibited the highest transester-
ification activity in the tested lipases.
The lipase-catalyzed transesterification was also studied by high
27

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J. Zheng et al.
AnalyticalBiochemistry549(2018)26–28
advantages of this method include simple detection process, high sen-
sitivity, inexpensive reagents, and low requirement for instruments.
This assaying method is useful in high-throughput-screening formats, in
dealing with a large sample size for enzyme activity identification, and
in optimizing catalytic condition.
Acknowledgements
This research was financially supported by National Natural Science
Foundation of China (Grant Nos. 31600639 and 31660247).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.ab.2018.03.010.
References
Fig. 5. Lineweaver-burk plots of CAL-B, PCL and TLL.
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The major advantages of this method are high efficiency and cost
effectiveness. For previous methods without post-processing and deri-
vative reaction, the test time was only 10 min per assay. Given that 96
samples can be analyzed at one time in a 96-well microplate, the pro-
posed assay can examine approximately 9200 samples per day.
Furthermore, the test cost of substrate is relatively low (2.5 × 10−4
€
per sample), and the price of 1 g of Bu-4-Mu is only 20 € (Shanghai
aladdin Biochemical Technology Co., Ltd). Compared with the method
reported in Reference [16], this method has obvious advantages such as
high detection accuracy and low reagent cost.
In summary, we have developed a novel high-throughput-screening
method for assaying lipase transesterification activity. This method is
based on fluorescence detection of 4-Mu concentration. The major
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