Biochemical and biophysical characterisation of a small purified lipase from Rhizopus oryzae ZAC3

Article abstract of DOI:10.1080/10242422.2021.1883006

The characteristics of a purified lipase from Rhizopus oryzae ZAC3 (RoL-ZAC3) were investigated. RoL-ZAC3, a 15.8 kDa protein, which was optimally active at pH 8 and 55 °C had a half-life of 126 min at 60 °C. The kinetic parameters using p-nitrophenylbuty

Full text of DOI:10.1080/10242422.2021.1883006

Biocatalysis and Biotransformation
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Biochemical and biophysical characterisation of a
small purified lipase from Rhizopus oryzae ZAC3
Zainab A. Ayinla, Adedeji N. Ademakinwa, Richard A. Gross & Femi K.
Agboola
To cite this article: Zainab A. Ayinla, Adedeji N. Ademakinwa, Richard A. Gross & Femi K.
Agboola (2021): Biochemical and biophysical characterisation of a small purified lipase from
Rhizopusꢀoryzae ZAC3, Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2021.1883006
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2021.1883006
RESEARCH ARTICLE
Biochemical and biophysical characterisation of a small purified lipase from
Rhizopus oryzae ZAC3
Zainab A. Ayinlaa, Adedeji N. Ademakinwaa, Richard A. Grossb and Femi K. Agboolaa
b
aDepartment of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile-Ife, Nigeria; Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA
ABSTRACT
ARTICLE HISTORY
Received 25 November 2020
Revised 24 January 2021
Accepted 25 January 2021
The characteristics of a purified lipase from Rhizopus oryzae ZAC3 (RoL-ZAC3) were investigated.
RoL-ZAC3, a 15.8 kDa protein, which was optimally active at pH 8 and 55 ꢀC had a half-life of
126min at 60 ꢀC. The kinetic parameters using p-nitrophenylbutyrate as substrate were
0.19 0.02 mM, 126 5.6 U/ml and 122sꢁ1 for Km, Vmax and kcat respectively. RoL-ZAC3 showed
stability in methanol and isopropanol with Naþ enhancing the activity. p-nitrophenyloleate and
castor oil were the best preferred substrates among the p-nitrophenyl esters and vegetable oils
tested respectively. About 43% residual activity was observed after incubation for 30 min at
75 ꢀC. Circular dichroism thermal scan showed that the lipase displayed intense negative elliptic-
ities even at high temperature. Perturbation of the tertiary structure with increasing temperature
caused the exposure of hydrophobic side chains to the aqueous environment as revealed by
tryptophan fluorescence, with a tꢁTm of 50 ꢀC. Differential scanning calorimetry analysis showed
melting temperature and calorimetric enthalpy of 55.5 ꢀC and 444 kJ/mol respectively. Dynamic
light scattering analysis indicated that the lipase was prone to aggregation upon unfolding at
high temperature. It can be concluded that RoL-ZAC3 possesses promising potential for numer-
ous biotechnological applications.
KEYWORDS
Lipase; Rhizopus oryzae;
tryptophan fluorescence;
circular dichroism;
calorimetric enthalpy
Introduction
substrates. Fungal lipases generally have molecular
weights ranging from less than 20 kDa–60 kDa (Mehta
et al. 2017). Only very few reports exist on small
lipases with molecular weights less than 20 kDa. These
include lipases from microbial strains such as Rhizopus
strain JK-1, 16.25 kDa (Kantak and Prabhune 2012),
Rhizopus arrhizus, 6.9 kDa (Dobrev et al. 2011), Bacillus
thermoleovorans CCR11, 11 kDa (Castro-Ochoa et al.
2005), Fusarium sp. YM-30, 12 kDa (Mase et al. 1995)
and Bacillus thermoleovorans ID-1, 18 kDa (Lee et al.
2001). There are even fewer studies on their complete
purification and characterisation, particularly for fungal
lipases and specifically even less common for Rhizopus
sp. Generally, the low molecular weight property
endows such enzymes with industrial potential and
can easily be exploited for better and wider substrate
specificity because the amino acids are relatively easy
to modify (Kantak and Prabhune 2012), unlike the
modification of larger proteins in which the possibil-
ities of side products and missed modifications
increase with increasing protein size. Also, complete
modification of large proteins may present difficulties
Knowledge of how surrounding environmental factors
such as ionic strength, temperature, solvent compos-
ition and pH influence structure-function relationship
in terms of folding and stability is crucial for improved
enzyme production at the industrial scale and for the-
oretical purpose, as such knowledge provides an
insight into the molecular basis of enzyme stability
(Kishore et al. 2012). Thus, pH denaturation, chemical
unfolding with the use of chaotropic agents (such as
guanidinium chloride and urea) and thermal denatur-
ation can be used for measuring conformational stabil-
ity and enzymatic activity (Rabbani et al. 2012).
Lipases (triacylglycerol acyl hydrolases) speed up
the breakdown of triglycerides at the lipid-water inter-
face and also catalyse the reverse reactions, which
include triglyceride synthesis, esterification, interesteri-
fication, transesterification, aminolysis, acidolysis and
alcoholysis (Salihu and Alam 2012). Catalysis by these
hydrolases occurs in biphasic (polar/non-polar) media,
in which the polar component solubilises the enzyme
while the non-polar component solubilises the
CONTACT Femi K. Agboola
fkagbo@oauife.edu.ng
Department of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile-
Ife, Nigeria
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
Z. A. AYINLA ET AL.
due to the increased chances of higher order structure
and incomplete denaturation (Krusemark et al. 2011).
Previous work reported the isolation of the fungus,
Rhizopus oryzae ZAC3 (RoZAC3), and the enzyme pro-
duction under optimised conditions (Ayinla et al.
2017). This study aims at understanding the stability
and conformational dynamics of the low molecular
weight Rhizopus oryzae ZAC3 lipase (RoL-ZAC3). In this
study, we report the physicochemical properties, as
well as the structural features (folding/unfolding stud-
ies) of RoL-ZAC3. Changes in conformation and stabil-
ity were assessed by far ultraviolet-circular dichroism
(CD), differential scanning calorimetry (DSC), fluores-
cence spectroscopy and dynamic light scattering
(DLS). Far-UV CD was used to study changes in RoL-
ZAC3 secondary structure at different temperature, pH
and guanidinium chloride (Gdm-Cl) concentration. The
tertiary structure of the protein was studied by fluor-
escence spectroscopy while data for tertiary structure
melting temperature (t ꢁ Tm), calorimetric enthalpy
(DHcal) and the changes in excess heat capacity (DCp)
were obtained from DSC measurements.
ZAC3 activity was the amount of enzyme that released
1 mmol of p-nitrophenol from p-NPB in one minute
under the assay conditions.
Production, purification and molecular weight
determination of RoL-ZAC3
R. oryzae ZAC3 lipase (RoL-ZAC3), an extracellular
enzyme, was produced by submerged fermentation (in
medium containing 1% olive oil, 1% xylose, 1% yeast
extract at pH 5 and temperature of 45 ꢀC for 96 h) as
previously reported (Ayinla et al. 2017). The crude
enzyme was concentrated by ultrafiltration and loaded
onto
a
pre-equilibrated DEAE–Sepharose column
(0.5 cm ꢂ 9.6 cm) on an AKTA FPLC system from GE
Healthcare at 4 ꢀC. After successive washes with the
equilibration buffer (50 mM sodium phosphate buffer,
pH 8), bound proteins were eluted with the same buf-
fer using a linear gradient of 0.5 ꢁ 1 M NaCl. Elution
was at a flow rate of 1.9 ml/min and monitored by
absorbance at 280 nm. Peak fractions were assayed for
lipase activity and active fractions were pooled.
Gel filtration on Sephacryl S-200 column (1.6 cm ꢂ
60 cm) was used to determine the native molecular
weight of RoL-ZAC3. The column was calibrated with
the following protein standards of different molecular
weights: bovine serum albumin (66,000 Da), ovalbumin
(45,000 Da), pepsin (35,000 Da), trypsin (24,000 Da) and
lysozyme (14,000 Da). The purity and subunit molecu-
lar weight was ascertained on 15% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
as described by Laemmli (1970). Protein concentra-
tions were determined by the method of Smith et al.
(1985) using the BCA protein assay kit.
Materials and methods
Materials
p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate
(pNPB), p-nitrophenyl laurate (pNPL), p-nitrophenyl
palmitate (pNPP), p-nitrophenyl oleate (pNPO), triton
X-100, isopropanol, sodium dihydrogen phosphate
and disodium hydrogen phosphate were purchased
from Sigma-Aldrich (St. Louis, USA). SimplyBlueTM Safe
Stain (Coomassie G-250) and PierceTM BCA (bicincho-
ninic acid) protein assay kit were obtained from
ThermoFisher ScientificTM (Massachusetts, USA). Bio-
Rad Precision Plus Protein Standards was from Bio-Rad
(Hercules, USA). All the chemicals and reagents used
were of analytical grade.
Effect of temperature on RoL-ZAC3 activity
and stability
The optimal temperature for the purified lipase was
determined by heating the appropriately diluted
enzyme at a specific temperature (ranging from 15 ꢀC
to 85 ꢀC respectively) for 5 min in an Eppendorf
Lipase activity assay
Lipase activity was assayed spectrophotometrically
using p-NPB as substrate according to the method of
Vorderwulbecke et al. (1992) with slight modification.
One hundred microlitres of the enzyme solution was
added to 900 mL of 50 mM sodium phosphate buffer,
pH 8 (containing 1% Triton X-100). The assay was initi-
ated by the addition of 30 mL of pNPB solution (14 mM
in isopropanol) to the diluted enzyme solution. The
absorbance at 405 nm was monitored over 3 min in a
spectrophotometer. The initial reaction rate was then
determined from the linear plot. One unit (1 U) of RoL-
R
thermomixerV, followed by the addition of the pNPB
substrate and determining the lipase activity as
described earlier.
The thermal stability was investigated by incubating
the diluted enzyme at different temperatures
(55–75 ꢀC) for 2 h and withdrawing aliquots (1 ml) of
the enzyme at 30 min intervals. The incubation was
R
carried out in an Eppendorf ThermomixerV at a spe-
cific temperature with constant mixing at 350 rpm.
The withdrawn enzyme was rapidly cooled on ice for

BIOCATALYSIS AND BIOTRANSFORMATION
3
5 min and left at room temperature for 10 min, fol-
lowed by the addition of the pNPB substrate solution
and assayed for lipase activity. The residual activities
(a measure of irreversible enzyme activity loss) were
expressed as a percentage of the activity at time zero
(activity when enzyme had not been heated) which
was taken to be 100%. The kd is the deactivation rate
constant. The data from the thermal stability study
were fit to first order deactivation kinetics and the
apparent half-life values (t1/2) estimated using t1/2 ¼ In
2/kd (kd ¼ deactivation rate constant).
The reaction in the absence of metal ions was taken
as control.
Effect of organic solvents on RoL-ZAC3 activity
To determine the effect of organic solvents on RoL-
ZAC3 activity, reaction was performed in the presence
of 20% organic solvents with different polarities (logp
values): methanol (ꢁ0.76), ethanol (ꢁ0.24), acetone
(ꢁ0.23), isopropanol (0.05), t-butanol (0.58), ethylace-
tate (0.64), dichloromethane (1.25) and hexane (3.5).
The relative activity for each organic solvent was
determined as percentages of the activity obtained
with isopropanol (Pimentel et al. 2007).
Effect of pH on RoL-ZAC3 activity and stability
To determine the optimum pH, lipase activity assays
were carried out in the following buffers at the indi-
cated pH values: 50 mM phosphate buffer (pH 3.0–3.5),
50 mM acetate buffer (pH 4.0–5.0), 50 mM citrate buf-
fer (pH 5.5–6.0), 50 mM phosphate buffer (pH 6.0–8.5),
50 mM Tris buffer (pH 7.0–9.5) and 50 mM borate buf-
fer (pH 9–9.5).
The pH stability (at pH 7, 7.5, 8 and 8.5) was deter-
mined by incubating the enzyme in phosphate buffer
(50 mM, pH 7 ꢁ 8.5) for 1 h while withdrawing a 100 mL
aliquot for assaying at 15 min intervals.
Substrate specificity
The effect of several substrates on RoL-ZAC3 was
determined using the following p-nitrophenyl esters
(0.1% w/v) as substrates: pNPA, pNPB, pNPL, pNPP and
pNPO. The standard protocol for pNPB was followed
for all the other substrates using 30 mL aliquot. The
relative activities were ascertained as percentages of
the activity obtained with pNPB (Ekinci et al. 2016).
Effect of RoL-ZAC3 on natural oils
Kinetic studies
The hydrolytic activity of RoL-ZAC3 on various natural
oils (olive oil, castor oil, lemon oil, origanum oil) was
investigated by titrimetric method. The emulsion was
prepared by emulsifying 10% (v/v) oil with 5% (w/v)
gum arabic in 50 mM sodium phosphate buffer, pH 7.
The enzyme (100 mL) was added to the emulsion fol-
lowed by incubation at 37 ꢀC for 30 min. One millilitre
of acetone: ethanol (1:1) solution was used to termin-
ate the reaction and to extract the fatty acid. Titration
with 1 M NaOH was used to estimate the amount of
fatty acids liberated (Sirisha et al. 2010).
The apparent kinetic parameters (Km, Vmax and kcat) of
RoL-ZAC3 for pNPB were determined by varying the
concentration of pNPB between 0.05 and 1.0 mM.
Temperature and pH were maintained at 55 ꢀC and pH
8 respectively. The initial reaction rate was determined
for each concentration of pNPB and the data plotted
according to Lineweaver and Burk (1934) using
GraphPad Prism 7. The kcat was calculated by using
Vmax, molecular weight and concentration of the
enzyme (0.267 mg/ml). The equation is shown
as follows:
Circular dichroism (CD) analysis
kcat ¼ Vmax/[Et]
A JASCO J-815 spectropolarimeter with a Peltier-type
temperature controller coupled to it was used for CD
analysis. The CD spectra were recorded using Spectra
Manager 228 software (JASCO, Easton, USA). All
recorded spectra were an average of 4 accumulations.
Analysis was performed in a 1-mm pathlength cuvette
using 10 mM lipase solutions prepared in (i) 10 mM
sodium acetate buffer at pH 5.0 and (ii) 10 mM sodium
phosphate buffer at pH 8 depending on the pH being
tested. To analyse the protein secondary structure,
wavelength scans were performed in the far-UV
where [Et] is the enzyme concentration in mM.
Effect of metal ions on RoL-ZAC3 activity
The effect of metal ions on RoL-ZAC3 activity was
determined by incubating 100 mL of the enzyme with
5, 10 and 20 mM of chloride salts of different metal
ions (Naþ, Kþ, Ca2þ, Ba2þ, Mg2þ, Mn2þ and Hg2þ
respectively) in sodium phosphate buffer (pH 8) con-
taining 1% triton X-100 for 1 min. Lipase activity was
subsequently assayed as previously described above.

4
Z. A. AYINLA ET AL.
(260–200 nm) wavelength range at a scan rate of
50 nm/min at 25 ꢀC. Following this, CD thermal unfold-
ing scans were performed from 10 ꢀC to 80 ꢀC at a
scan rate of 1ꢀCminꢁ1. Ellipticity (h), expressed in
mdeg by the spectropolarimeter, was converted to
mean residue ellipticity, MRE ([h]mrw) in deg cm2
dmolꢁ1. Thermal denaturation of the enzyme was fol-
lowed by continuously monitoring ellipticity changes
at 207 nm as a function of temperature.
Guanidinium chloride (GdmCl)-induced lipase
unfolding was also characterised by CD. Protein solu-
tions (10 mM) were incubated overnight in 10 mM
sodium phosphate buffer with varying concentrations
of GdmCl (1, 3 and 6 M) at 25 ꢀC. Incubating overnight
was necessary to allow enough time for equilibrium
between folded and unfolded states. The lipase-GdmCl
solutions were then subjected to CD wavelength scans
as described above and the ellipticity from 260 to
200 nm for each GdmCl concentration was recorded.
was also run, which was subtracted from the protein
scan. The wavelength that gave the highest fluores-
cence intensity, denoted kmax, was obtained from the
wavelength scan. A temperature scan was run for the
thermal transition analysis. Temperature was increased
at intervals of 5 ꢀC and sample was incubated for
3 min at each temperature. A plot of kmax as a func-
tion of temperature was made and the midpoint of
this thermal transition was taken as the t ꢁ Tm (protein
tertiary structure melting/unfolding temperature).
Dynamic light scattering (DLS) analysis
Aggregation studies were carried out by DLS analysis
to determine the temperature at which aggregates
begin to form as the protein unfolds. DLS measure-
ments were carried out on a Malvern Zetasizer ZSP90
(Malvern, Massachusetts, USA). Thermal scans were
run on 10 mM protein solution from 25 ꢀC to 90 ꢀC at
intervals of 5 ꢀC and equilibration time of 180 s at
each temperature. As the temperature increased, the
scattered light intensity was measured. A sharp rise in
the intensity of scattered light signified the onset/
beginning of aggregation denoted Tagg (aggregation
point) (Shirke et al. 2018).
Differential scanning calorimetry (DSC) analysis
A
NanoDSC differential scanning calorimeter (TA
Instruments, New Castle, USA) with a sample cell vol-
ume of 300 mL was used for the calorimetric measure-
ments of melting temperature (Tm). The enzyme
sample (300 mL) was loaded into the sample capillary
cell and similar volume of 10 mM sodium phosphate
buffer was loaded into the reference capillary cell. The
cells were heated from 20 ꢀC to 100 ꢀC at 3 atm pres-
sure and a rate 1ꢀCminꢁ1. The resulting thermogram
was baseline corrected and NanoAnalyze software was
used to analyse the normalised data. From this, esti-
mates of the melting temperature (Tm) and denatur-
ation enthalpy (DH) were determined.
Results
Estimation of RoL-ZAC3 molecular weight
RoL-ZAC3 was purified 2.5-fold to apparent homogen-
eity by the process of ultrafiltration and DEAE-
Sepharose anion exchange chromatography with a
47% overall recovery (Table 1). A distinct band corre-
sponding to a subunit molecular weight of 15.8 kDa
was obtained on SDS-PAGE (Figure 1). This was con-
sistent with the native molecular weight of 16.2 kDa
determined by gel filtration chromatography on
Sephacryl S-200. This suggests that the lipase exists as
a monomer.
Intrinsic tryptophan fluorescence analysis
Intrinsic tryptophan fluorescence analysis was per-
R
formed on a Spex FluorologV Tau-3 (Horiba, New
Jersey, USA) fluorimeter. Slit width of 3 nm was used
for the excitation and emission beam. A concentration
of 2 mM of the protein solution in 10 mM phosphate
buffer, pH 8 was used for the analysis. Excitation was
at 295 nm and intrinsic tryptophan fluorescence emis-
sion was recorded from 300 to 410 nm (wavelength
scan). For the wavelength scan, a blank buffer scan
Temperature effect and thermal stability of
RoL-ZAC3
The effect of temperature on the lipolytic activity of
RoL-ZAC3 showed an optimum temperature, Topt at
55 ꢀC (Figure 2(A)). Further increase in temperature
Table 1. RoL-ZAC3 purification table.
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Yield (%)
Purification fold
Crude
Ultrafiltration
DEAE-Sepharose
1403
935
660
21.4
10.8
4.1
65.6
86.6
161
100
66.6
47.0
1
1.3
2.5

BIOCATALYSIS AND BIOTRANSFORMATION
5
Table 2. Half-life (in hours) of RoL-ZAC3 at differ-
kDa
250
150
ent temperatures.
Temperature (oC)
Half-life, t1/2 (h)
55
60
65
70
75
2.5
2.1
1.1
0.6
0.5
100
75
50
37
(A)
Phosphate Buffer
100
80
60
40
20
0
25
20
Acetate Buffer
Citrate Buffer
Phosphate Buffer
Tris Buffer
15.8 kDa
15
10
Borate Buffer
1
2
M
Figure 1. SDS-PAGE analysis of RoL-ZAC3. Lane 1 & 2, purified
RoL-ZAC3; lane M, protein molecular weight markers (Precision
Plus ProteinTM unstained standards, BioRad).
3
4
5
6
7
8
9
10
pH
(A)
100
80
(B)
100
50
0
60
40
pH 7
20
pH 7.5
pH 8
pH 8.5
0
10 20 30 40 50 60 70 80 90
Temperature (oC)
15
30
45
60
Time (min)
(B)
Figure 3. Effect of pH on RoL-ZAC3 activity and stability. (A)
Optimal pH. (B) pH stability. The values shown represent the
average from triplicate experiments. Error bars represent the
standard deviation.
100
80
60
at 65 ꢀC and this dropped further to 22% after 120 min
incubation. The apparent half-life (t1/2) of RoL-ZAC3 at
different temperatures is summarised in Table 2. Plot
of Ln (Et/Eo) vs time is available in supplemen-
tal material.
40
20
0
55oC
60oC
65oC
70oC
75oC
0.5
1.0
Time (h)
1.5
2.0
Figure 2. Effect of temperature on RoL-ZAC3 activity and sta-
bility. (A) Optimal temperature. (B) Thermal stability. Error bars
represent deviations from triplicate experiments. The half-life
values are summarised in Table 2.
pH effect and pH stability of RoL-ZAC3
RoL-ZAC3 showed an optimum pH of 8 (Figure 3(A)).
The broad pH optimum exhibited by the enzyme
extends well into the alkaline range (7–9.5). Very low
activity was seen below pH 6 and no activity was seen
below pH 3.5. At pH 7.5, 8.5 and 9, relative activities
were about 84%, 95% and 90% respectively. The pH
stability of the enzyme was also investigated over a
pH range of 7 ꢁ 8.5 (Figure 3(B)). Over 100% residual
beyond 55 ꢀC led to a decline in enzyme activity, and
the enzyme almost completely lost its activity at 85 ꢀC.
Thermostability profile of RoL-ZAC3 is shown in
Figure 2(B). RoL-ZAC3 showed about 72% residual
activity after incubation at 65 ꢀC for 30 min. Almost
55% activity was recovered after 90 min of incubation

6
Z. A. AYINLA ET AL.
Table 3. Summary of kinetic parameters for RoL-ZAC3.
Table 5. Effect of organic solvents on activity of RoL-ZAC3.
Km
(mM)
Vmax
(U/ml)
kcat
Organic solvents
log P
Relative activity (%)
Substrate
(sꢁ1
)
Methanol
Ethanol
Acetone
Isopropanol
t-butanol
Ethylacetate
Dichloromethane
n-Hexane
ꢁ0.76
ꢁ0.24
ꢁ0.23
0.05
0.58
0.64
86.2 1.6
69.6 7.8
65.5 8.8
100 3.1
23.3 4.5
5.4 0.6
pNPB
0.19 0.02
126 5.6
123
Km: Michaelis Constant; Vmax
over number.
:
maximum reaction rate; kcat
:
turn-
1.25
3.5
2.0 0.1
27.1 5.6
Table 4. Effect of metal ions on activity of RoL-ZAC3.
Metal ions
5 mM
10 mM
20 mM
The buffer in the reaction mixture was replaced with 20% solution of the
organic solvent. The percentage activity for each organic solvent was
determined relative to the activity obtained with isopropanol. log P ¼
logarithm of partition coefficient. Values are means SD (n ¼ 3).
Naþ
137 6.1
129 30.4
46.3 2.3
93.7 11.0
88.7 18.1
38.8 0.9
44.6 1.6
151 3.3
107 21.3
81.7 10.8
92.6 8.6
77.9 33.5
17.8 1.2
48.2 0.4
136 11.4
79.2 1.3
95.1 5.8
100 0.9
36.2 1.6
0
Kþ
Ca2þ
Ba2þ
Mg2þ
Mn2þ
Hg2þ
solvent than hexane (log p value of 3.5), the most
hydrophobic solvent used. Appreciably high relative
activities were also observed in all other hydrophilic
solvents tested (methanol, log p ꢁ.76; ethanol, log p
ꢁ.24; Acetone, log p ꢁ.23). On the other hand, ethyl
acetate and dichloromethane almost completely inhib-
ited lipase activity (Table 5).
52.8 0.4
The chloride salt of each metal ion was added to final concentrations of
5, 10 and 20 mM prior to assay of enzyme activity. The percentage activ-
ities were determined relative to the control (without any metal ion).
Values are means SD (n ¼ 3).
activities were also observed at pH 8 and 8.5 after
30 min and 60 min incubation respectively.
Substrate specificity
Determination of kinetic parameters
Chain length specificity of RoL-ZAC3 evaluated using
p-nitrophenyl esters with acyl chain lengths ranging
from C2 to C18 showed that p-nitrophenyl oleate
(pNPO), a long chain monounsaturated fatty acid ester
(C18), had the highest relative activity reaching about
130% (Figure 4) as the lipolytic activity decreased
towards saturated fatty acid chains.
The hydrolysis of pNPB by RoL-ZAC3 followed the
Michaelis-Menten kinetics. The kinetic study showed
that the Michaelis-Menten constant (Km) was
0.19 0.02 mM and the hydrolysis of pNPB occurred at
a maximum reaction rate (Vmax) of 126 5.6 U/ml of
enzyme, as estimated from the Lineweaver Burk plot.
The turn-over number (kcat), which is a measure of the
number of substrate molecules converted to product
per unit time was 123 sꢁ1 (Table 3).
Effect of RoL-ZAC3 on natural oils
RoL-ZAC3 showed broad fatty acid specificity as indi-
cated by its varied rate of hydrolysis of different nat-
ural triacylglycerols. Castor oil showed the highest
hydrolysis rate, followed by olive oil. In comparison,
lemon and origanum oils were hydrolysed more slowly
(Figure 5).
Effect of metal ions on RoL-ZAC3 activity
RoL-ZAC3 activity was inhibited by chloride salts of
Mn2þ and Hg2þ even at low concentrations of 5 and
10 mM. Poor activity was also observed with Mg2þ at
20 mM concentration (Table 4). Mn2þ completely
inhibited the enzyme activity at 20 mM. Of all the
metal ions tested, Naþ significantly enhanced lipase
activity at all three concentrations (5, 10, 20 mM), giv-
ing percent relative activity as high as 150%. Kþ
enhanced lipase activity at 5 and 10 mM but slightly
inhibited the activity by about 25% at a high concen-
tration of 20 mM. The enzyme displayed more activity
at higher Ca2þ concentrations of 10 and 20 mM than
it did at 5 mM (Table 4).
Circular dichroism (CD) analysis
The probe of the global conformational stability was
first attempted through CD study via its secondary
structure stability analysis. The CD wavelength scan in
the far UV region (200–260 nm) at pH 8 revealed a
curve with two negative bands (double minima) typ-
ical of an a/b-protein (Figure 6). As the wavelength
reduced from 260 nm to 200 nm, there was an
increase in the negative ellipticity, which is typical of
intact secondary structure elements. suggesting an
increase in secondary structure. At pH 5, positive ellip-
ticity was seen around 222 nm and a reduction in the
negative molar ellipticity of the double minima which
Effect of organic solvents on RoL-ZAC3 activity
In this study, the highest activity was observed in iso-
propanol (log p value of .05), a more hydrophilic

BIOCATALYSIS AND BIOTRANSFORMATION
7
-4
-4.2
-4.4
-4.6
-4.8
-5
150
100
50
-5.2
-5.4
0
10
20
30
40
50
60
70
80
90
NPL
NPP
NPA
NPB
NPO
p
p
p
p
p
Temperature (o C)
Figure 4. Substrate specificity of RoL-ZAC3. This was done
using 0.1% of the respective p-NP ester dissolved in isopropa-
nol and then following the standard lipase assay. The percent-
age activities were determined relative to the reaction rate
obtained with pNPB.
Figure 7. CD thermal scan (10–80 ꢀC) of RoL-ZAC3 run at a
wavelength of 207 nm and at pH 8 to illustrate thermal
unfolding of the enzyme.
(A)
3000
2000
1000
0
150
100
50
-1000
-2000
-3000
-4000
-5000
0M GdmCl
1M GdmCl
3M GdmCl
6M GdmCl
0
200
220
240
260
Olive Oil
Wavelength (nm)
Castor Oil
Lemon Oil
Origanum Oil
Figure 5. Effect of RoL-ZAC3 on natural oils. This was done by
titrimetric method. The reaction medium in 50 mM sodium
phosphate buffer, pH 7 contained 10% (v/v) oil, 5% (w/v) gum
arabic and 100 mL of the enzyme. This was incubated at 37 ꢀC
and terminated after 30 min. The released fatty acids were
estimated by titration with 1 M NaOH. Data was expressed as
percentage of value obtained for olive oil.
(B)
1
0.8
0.6
0.4
0.2
0
2000
1000
0
0
2
4
6
-1000
-2000
GdmCl Conc (M)
pH 5
pH 8
Figure 8. (A) CD spectra of RoL-ZAC3 at 207 nm in the pres-
ence of guanidinium chloride of concentrations 0 M, 1 M, 3 M
and 6 M. (B) Guanidinium chloride-induced unfolding of
RoL-ZAC3.
-3000
-4000
-5000
signifies a dramatic decrease in structure. For the CD
thermal scan at a temperature range of 10–80 ꢀC,
there was not much difference between the negative
ellipticity recorded at 10 ꢀC and that recorded at 80 ꢀC
(Figure 7) which shows that very little unfolding
occurred. Transforming the ellipticity (in mdeg) to
200
210
220
230
240
250
260
Wavelength (nm)
Figure 6. CD wavelength scan spectra at the far UV for RoL-
ZAC3 at pH 5 (white) and pH 8 (black). All scans were per-
formed at 200–260 nm at a temperature of 25 ꢀC. MRE, Mean
residue ellipticity.

8
Z. A. AYINLA ET AL.
120
70
(A)
500000
450000
400000
350000
300000
250000
200000
150000
100000
50000
0
20
20
30
40
50
60
70
80
90
100
Temperature (oC)
300
320
340
360
380
400
420
Figure 9. DSC thermogram of RoL-ZAC3 at pH 8. The thermo-
dynamic parameters are summarised in Table 6.
Wavelength (nm)
25C
30C
35C
60C
85C
40C
65C
45C
70C
50C
75C
55C
80C
Table 6. Thermodynamic parameters of RoL-ZAC3 deduced
from DSC analysis.
tꢁTm (oC)
55.5
DHcal (kJ/mol)
DCp (kJ/mol.K)
RoLZAC3
444
19.4
(B)
338
337
336
335
334
333
332
331
330
329
fraction unfolded showed that there was barely any
change in the ellipticity or fraction unfolded even as
temperature approached 80 ꢀC (Figure 7).
GdmCl caused unfolding in RoL-ZAC3 (Figure 8(A))
as evidenced by changes in the structure of the CD
spectra. At the 6 M GdmCl concentration, the double
minima signature of secondary structure elements had
completely disappeared. A low level (about 20%) of
structure disruption was observed in the 1 M GdmCl –
induced unfolding (Figure 8(A,B)) thus reflecting the
stability of the enzyme at this concentration of GdmCl.
At higher concentrations of GdmCl (3 M and 6 M),
there is a considerable reduction of the regular con-
formation. This indicates that RoL-ZAC3 secondary
structure is not preserved at these higher concentra-
tions of the denaturant.
25
45
65
85
Temperature (oC)
Figure 10. Temperature-induced unfolding of RoL-ZAC3. (A)
Fluorescence intensity as a function of temperature showing
wavelength of maximum intensity (kmax) at different tempera-
tures thus revealing fluorescence of inner tryptophan residues.
(B) temperature scans to identify thermal-induced unfolding of
RoL-ZAC3 and to determine the tꢁTm.
Differential scanning calorimetry (DSC) analysis
fluorescence intensity (Figure 10(A)) as well as the sig-
nificant shift in kmax (Figure 10(B)), as temperature
increased. There was a shift in kmax from 332 nm at
25 ꢀC to 337 nm at 55 ꢀC and it remained at 337 nm
even at 85 ꢀC with concurrent decrease in fluorescence
intensity.
The DSC thermogram (Figure 9) show baseline sub-
tracted data of heat capacity plotted as a function of
temperature. RoL-ZAC3 exhibited single endothermic
transition during unfolding and had a t ꢁ Tm (tertiary
structure unfolding temperature) of 55.5 ꢀC. The pro-
tein heat capacity (Cp) was seen to be linear from
20 ꢀC to 45 ꢀC. The summary of the thermodynamic
parameters is shown in Table 6.
Dynamic light scattering (DLS) analysis
Figure 11 shows the aggregation temperature (Tagg
)
Intrinsic tryptophan fluorescence analysis
which indicates the onset of aggregation. The mole-
cules have a likelihood to begin to aggregate at this
temperature. The Tagg was 50 ꢀC for RoL-ZAC3. The
aggregates were not physically observed likely
Similar to DSC thermal scan that gave a t ꢁ Tm of
55.5 ꢀC, t ꢁ Tm measured by fluorescence thermal scan
was 50 ꢀC (Figure 10(A,B)). There was a change in

BIOCATALYSIS AND BIOTRANSFORMATION
9
800
700
600
500
400
300
200
100
0
The thermostability exhibited by RoL-ZAC3 (Figure
2(B)) is quite promising. In comparison with other
lipases purified from various Rhizopus species, RoL-
ZAC3 is relatively stable and appears to show better
thermostability than some reported lipases. In thermal
stability study of Rhizopus chinensis lipase by Sun et al.
(2009), residual activity reduced to 55% after incuba-
tion at 50 ꢀC for 60 min. Although, Rhizopus oryzae lip-
ase by Hiol et al. (2000) retained about 65% of its
activity after incubation at 45 ꢀC for 30 min, however,
all activity was lost following incubation at 50 ꢀC for
30 min. At a specific operating temperature, a high
value of the half-life is desired and beneficial for
industrial applications (Shirke et al. 2016). Thus, the
higher the t1/2, the less prone the enzyme is to ther-
mal denaturation. The high t1/2 of RoL-ZAC3 (Table 2)
bears more relevance when compared with other fun-
gal lipases at the same operating temperature. Free
RoL-ZAC3 displayed a t1/2 of 2.1 h at 60 ꢀC, unlike free
Candida rugosa lipase which exhibited a half-life of
18 min at 60 ꢀC while its immobilised form on cellulose
nanocrystals had a half-life of 2 h at 60 ꢀC (Kim
et al. 2015).
RoL-ZAC3 retained higher activity at slightly alkaline
pH (Figure 3(A)). Enzyme activity and stability is
strongly influenced by pH. Changes in pH cause
changes in amino acid protonation state, which in
turn greatly alter the spatial arrangements within the
protein structure, as well as electrostatic interactions
(Horng et al. 2005). As there are shifts in pH, it leads
to changes in the state of protonation of active site
residues, thus having effects on secondary and tertiary
structure stability and leading not only to conform-
ational changes but also changes in protein structure
and activity. The enzyme was stable in slightly alkaline
pH range of 7–8.5 over the 1 h incubation period
(Figure 3(B)). Similar stability profile was observed in
Rhizopus oryzae strain isolated from palm fruit, pH
7–8.5 (Hiol et al. 2000). Alkaline lipases, such as
obtained in this study, offer promising prospect in
bio-based industries such as detergent and tex-
tile industries.
25
35
45
55
65
75
85
95
105
Temperature (oC)
Figure 11. DLS analysis showing thermal induced aggregation
of RoL-ZAC3.
because they were at the nanoscale and could not be
observed physically and/or only a small population of
the protein molecules were involved. The Tagg
obtained in the DLS study was close to and correlates
the Topt and the t ꢁ Tm obtained by fluorescence and
DSC analyses.
Discussion
RoL-ZAC3 was extracellularly produced by the fungus,
Rhizopus oryzae ZAC3, which was isolated from a palm
oil contaminated soil, and the enzyme was purified by
ultrafiltration and ion exchange chromatography. The
distinct band from SDS-PAGE (Figure 1) ascertained
the purity of the enzyme. With a molecular weight of
15.8 kDa, RoL-ZAC3 is considered a very small lipase.
Complete purification and characterisation of very low
molecular weight fungal lipases, such as this, is not
very common.
Temperature-activity studies provide an insight into
the active site stability. Disruptions to the local active
site structure can lead to loss of enzyme activity
(Shirke et al. 2018). The Topt value of 55 ꢀC (Figure
2(A)) for the substrate (pNPB) is a good indicator of
the stability of the active site or a decrease in the
rigidity of the active site, improving traffic into and
out of the active site and/or flexibility necessary for
catalysis. The decline in enzyme activity beyond 55 ꢀC,
and the almost complete loss in activity at 85 ꢀC were
likely due to thermal-induced denaturation. Lipases
purified from other sources showed optimum tem-
perature around 35–55 ꢀC; Rhizopus oryzae, 35 ꢀC (Hiol
et al. 2000), Rhizopus strain JK-1, 40 ꢀC (Kantak and
Prabhune 2012) and Streptomyces rimosus R6-554W,
The kinetic parameters tell the performance charac-
teristics of the enzyme. The Km gives an indication of
the affinity of RoL-ZAC3 for pNPB (Table 3).
Comparison with other small lipases in literature may
not be simple because the kinetic parameters were
expressed in different units and in most cases, differ-
ent substrates were used. Lipase from Geobacillus
stearothermophilus AH22 showed a Km and Vmax of
0.02 mM and 1.03 U/mg respectively when pNPB was
used as substrate (Ekinci et al. 2016) while Rhizomucor
ꢀ
ꢀ
55 C (Abramic et al. 1999).
The thermal inactivation observed at very high tem-
perature is a form of local unfolding (initiated as tem-
perature rises above the Topt and/or as time increases)
which precedes the eventual loss of tertiary structure.

10
Z. A. AYINLA ET AL.
miehei lipase had a Km and Vmax of 1.13 mM and
86.2 mM/min respectively (Tako et al. 2017) when p-
nitrophenyl palmitate (pNPP) was used as substrate.
While Km obtained in this study (0.19 0.02 mM) was
higher than that obtained for Geobacillus stearother-
mophilus AH22 lipase which also utilised pNPB as sub-
strate, the Km was however about six times lower and
Vmax was about 1.28 times higher than values
obtained for Rhizopus oryzae NRRL 1526 lipase, which
utilised pNPP as substrate (Tako et al. 2017). A low Km
and high Vmax indicates that the enzyme has a rela-
tively higher affinity for its substrate and hydrolyses it
better than its counterparts with a higher Km.
microbial lipase activity. Literature review has revealed
that certain lipases, such as that from Burkholderia glu-
mae (El-Khattabi et al. 2003) have a Ca2þ-binding site.
Two conserved aspartate residues very close to the
active site form this binding site. According to
Kambourova et al. (2003) lipase activity enhancement
by Ca2þ is mainly because during hydrolysis, insoluble
ion salts of fatty acids form, thus product inhibition is
avoided. Alternatively, the increase in enzyme activa-
tion observed as Ca2þ ion concentration increased
may be as a result of a higher Ca2þ ion concentration
inducing a conformational change of the enzyme
structure to a more active form. The Ca2þ ions may
form salt bridges between two COO- groups of acidic
amino acids that might have better stabilised the
enzyme structure and caused the increase in activ-
ity observed.
All organic synthesis processes require enzymes to
remain active and stable in most organic solvents.
Enzyme stability in aqueous media largely differ from
its stability in organic media mainly because organic
media has low water content and that leads to
increased conformational rigidity of the enzyme. RoL-
ZAC3 displayed varying stabilities in the different
hydrophobic and hydrophilic organic solvents tested
(Table 5). The log p value is used to distinguish
organic solvents on the basis of their hydrophilic or
hydrophobic nature. The higher the log p value, the
more hydrophobic the organic solvent and vice-versa
(Imanparast et al. 2018). According to Doukyu and
Ogino (2010), microbial lipases show greater activity in
hydrophobic solvents than in hydrophilic solvents,
even though the contrary was observed in this study.
Similar result to what was obtained in this study was
observed in lipase from Actinomadura sediminis UTMC
2870 which showed stability in hydrophilic solvents
with low log p values (Imanparast et al. 2018).
Likewise, in Rhizomucor miehei NRRL 5282 and
Rhizopus oryzae NRRL 1526 (Tako et al. 2017), the lip-
ase remained stable in 20% (v/v) methanol and etha-
nol. A possible explanation for the higher activity
observed in hydrophilic solvents (known to strip cru-
cial water off enzyme molecules) is that the enzyme is
able to retain high water content (high enough to
remain catalytically active) probably because the
essential water/hydration shell is tightly bound to the
enzyme molecule (Klibanov 1989). It is also possible
that activity varied due to the loss of hydrophobic
pressure in protein folding and likely less structure
than rigidity. On the other hand, Doukyu and Ogino
(2010) put forward a number of factors to explain
enzyme activity loss seen in certain organic media.
If a heavy metal such as Hg2þ causes significant or
complete inactivation of an enzyme, it may be an
implication that thiol groups are necessary for
adequate enzyme function (Tako et al. 2017). It is
important to state that buffers have an influence on
the chemical speciation of certain metals and heavy
metal phosphates generally have very low solubility in
aqueous medium and may sometimes precipitate out.
Heavy metals react with sulfhydryl groups of enzymes
and may cause inhibition of enzyme catalysis if an
essential cysteine residue is involved (Reynolds et al.
1993). This correlates the poor lipase activity observed
with Hg2þ in this study (Table 4). However, because
only about 50% inhibition was observed at all three
concentrations of Hg2þ, it suggests that the cysteine
involved in the heavy metal binding may not be an
essential residue that is key to catalysis. Similar to
what was observed in this study, Naþ ions showed
similar stimulatory effects on lipase from Rhizomucor
miehei (Tako et al. 2017). According to Noel and
Combes (2003), the enzyme conformation is stabilised
by the Naþ ions by strengthening the integrity of the
active site and thereby enhancing enzyme activity.
The possibility that the 50 mM sodium phosphate buf-
fer had an additive effect on the high activity
observed with Naþ ions cannot be ruled out. Woehl
and Dunn (1995) proposed two possible mechanisms
to explain the role of monovalent metal ions in
enzyme catalysis: either the metal ion plays a static
structural role whereby it activates and stabilises the
catalytically active conformation of the enzyme by
simply binding to it or the metal ion plays a dynamic
role whereby it binds selectively to ensure that protein
conformational transition takes place which is neces-
sary for the complementarity between the structure of
an activated complex and the enzyme site. The high
activity at increased Ca2þ concentrations of 10 and
20 mM correlated the finding demonstrated by
Kambourova et al. (2003) that Ca2þ ions enhance

BIOCATALYSIS AND BIOTRANSFORMATION
11
The enzyme deactivation in hydrophobic organic
media occurs when the protein molecule hydrophobic
core is disrupted due to a change of medium hydro-
phobicity. Unlike water, anhydrous solvents may ren-
der enzymes less active due to restricted and
decreased conformational flexibility causing rigidity of
the enzyme conformation (Doukyu and Ogino 2010).
As hydrophobic solvents do not strip off essential
water on the enzyme molecules which is crucial for
the maintenance of enzyme structure and function,
they sometimes help to promote enzymatic reactions
in anhydrous solvents (Klibanov 1997).
There was a decrease in the rate of hydrolysis as
chain length of the fatty acid esters increased from 4
to 12 to 16 carbon atoms. However, p-nitrophenyl ole-
ate, pNPO (C18:1), a monounsaturated fatty acid ester
was an exception and did not follow this pattern. It
had the highest relative activity as the lipolytic activity
decreased towards saturated fatty acid chains (Figure
4). Since hydrolysis by hydrolases such as lipases occur
in heterogeneous systems, this exception by pNPO
could be due to the orientation of the molecule at the
oil/water interface (which would influence enzyme-
substrate association) or due to enzyme-substrate spe-
cificity or due to both reasons. Chain length specificity
varies generally between C8 and C18 acids for lipases
from filamentous fungi while lipases from zygomycete
show maximum activity towards medium (C6–C12) or
long chain acids (C14–C20) though high reaction spe-
cificity towards C2 to C6 acids is displayed by some of
them (Yu et al. 2009).
RoL-ZAC3 activity towards different natural triglycer-
ides (vegetable oils) further revealed some important
preferences in terms of substrate specificity. Plant oils
are a mixture of triacylglycerols with different fatty
acid chain lengths. The highest hydrolysis rate was
seen in castor oil, followed by olive oil (Figure 5).
Castor oil is composed majorly of about 85–90% of
ricinoleic acid, a monounsaturated fatty acid with a
hydroxyl functional group which makes it more polar
than its counterparts. Olive oil, on the other hand is
composed of 55–83% oleic acid (McKeon 2016), which
is also monounsaturated and having 18 carbon atoms
just as ricinoleic acid. This is consistent with the chain
length specificity results obtained in this study in
which pNPO gave the highest hydrolytic activity. The
results here further confirm the enzyme has prefer-
ence for long chain, monounsaturated fatty acid
esters. Its ability to hydrolyse both natural triglycerides
and synthetic aryl esters shows that it has potential
application in large scale fat hydrolysis which can be
further investigated.
The CD structure of RoL-ZAC3 at pH 5 resembles
that of a random coil (Figure 6). RoL-ZAC3 retained
only about 50% of its structure at pH 5 relative to pH
8 as evidenced by the reduced signal intensity. This
implies that some degree of its original secondary
structure had been lost at the acidic pH of 5. As
denaturation sets in, some secondary structure ele-
ments should normally deplete. This correlates the
result from the effect of pH (Figure 3(A)) where over
90% of RoL-ZAC3 activity had been lost at pH 5 com-
pared to the activity at optimum pH 8. This appears
reasonable because according to Shashidhara and
Gaikwad (2010), at very acidic pH, all side chains that
are ionisable become protonated resulting in
a
charge-charge repulsion that causes loosening of side
chain packing and eventually hydrophobic residues
become exposed to the solvent leading to loss of sec-
ondary structure.
The CD thermal scan should normally reveal the
melting/unfolding temperature (Tm), which is usually
the midpoint of the thermal transition curve. During
the thermal unfolding monitored by CD in this study,
no clear thermal-induced transitions occurred up to
80 ꢀC (Figure 7), thus the apparent Tm could not be
ascertained from the CD. Since CD (at far UV wave-
lengths) assesses majorly secondary structure ele-
ments, the implication of this result is that RoL-ZAC3
displayed substantial secondary structure stability.
Similar finding was reported by Shirke et al. (2018) in
which no thermal transition was observed in glycosy-
lated leaf and branch compost cutinase (LCC) up to
95 ꢀC following thermal unfolding studies by CD. One
possible explanation for Tm not observed in CD ana-
lysis is that as temperature increased up to and above
the Topt, some structural changes attributed to
denaturation might have occurred close to the active
site, which would normally not be reflected in the CD
spectrum. CD spectra only reflect secondary structure
stability or otherwise. The Topt recorded in this study
was 55 ꢀC, yet Tm was not ascertained even at 80 ꢀC
by CD. Sulaiman et al. (2012) also reported a Topt of
50 ꢀC and Tm of 86 ꢀC for non-glycosylated leaf and
branch compost cutinase (LCC-NG) which was consid-
ered a very wide gap. With the large difference
between the Topt (an indication of local active site sta-
bility) and Tm (an indication of global conformational
stability), another unfolding experiment technique was
necessary to further probe the global conformational
stability via DSC and Intrinsic tryptophan fluorescence
analysis on the tertiary structure. It is important to
state that one limitation of CD analysis is that it may

12
Z. A. AYINLA ET AL.
not detect minute structural changes that can cause
activity loss (Shirke et al. 2017).
(calorimetric enthalpy) of 444 kJ/mol and DCp (change
in heat capacity at constant pressure) of 19.4 kJ/mol.K
(Table 6). DCp is a measure of the extent to which
non-polar groups are exposed to water (Privalor 1979).
A positive DCp implies solvation of the unfolded polar
groups of the protein (Richardson and Makhatadze
2004) and solvation of the polar groups may mean dif-
ficulty in the refolding of groups back to the tertiary
structure. The thermal unfolding of lipase from
Pseudomonas fluorescens had a DCp of 4 kJ/mol.K
(Makhzoum et al. 1993) which is comparatively lower
than 19.4 kJ/mol.K obtained for RoL-ZAC3. A high Cp
means that the process of unfolding is a slow one.
While CD gives information on 2ꢀ structure unfold-
ing, DSC and fluorescence thermal analysis give infor-
mation on 3ꢀ structure unfolding. Temperature was
used to perturb and monitor changes in the 3ꢀ struc-
ture of RoL-ZAC3 via intrinsic tryptophan fluorescence
analysis. Hydrophobic side chains are exposed when
unfolding or partial loss of 3ꢀ structure occurs and this
exposure may sometimes contribute to aggregation.
Tryptophan residues (selectively excited at 295 nm)
when buried, show an emission maxima (kmax) around
330 nm, which on solvent exposure, shifts to a longer
wavelength, sometimes greater than 340 nm. The
fluorescence intensity change (Figure 10(A)) and the
significant shift in kmax (Figure 10(B)) as temperature
was raised, was an indication that a change had
occurred in the local tryptophan environment of RoL-
ZAC3 suggesting thermal unfolding at the 3ꢀ structure
level. The red shift in kmax from 332 nm at 25 ꢀC to
337 nm at 55 ꢀC with simultaneous decrease in fluores-
cence intensity suggests that some of the tryptophan
residues of the enzyme had been exposed to the solv-
ent of the surrounding environment (Castro-Ochoa
et al. 2005). In other words, the tryptophan residues
must have moved to a more polar environment from
the non-polar interior. The decrease in fluorescence
intensity implied that the fluorescence of the trypto-
phan residues in the denatured enzyme had been
greatly quenched. Upon unfolding, a protein loses
many of the interactions that maintain its native struc-
ture. Decrease in the rigidity of the side chains also
set in as unfolding occurs. As the CD thermal scan did
not reveal unfolding of the protein, it may imply that
Trp residues are not directly involved in maintaining
the protein secondary structure.
Prior to analysis by fluorescence, it was important
to confirm the secondary structure stability implied
from the CD results. Samples were incubated over-
night with different concentrations of guanidinium
chloride (GdmCl) followed by CD analysis. As the con-
centration of GdmCl was increased from 1 M to 3 M,
transition from native to unfolded state was observed
(Figure 8(A)). The GdmCl-denaturation data is an indi-
cation that RoL-ZAC3 underwent complete loss of its
a-helical component and changes in other secondary
structural elements at 6 M GdmCl. The concentration
at the transition midpoint (Cm) is the GdmCl concen-
tration where 50% of the protein is unfolded (He et al.
2009). This was about 2.2 M for RoL-ZAC3. Brito e
Cunha et al. (2019) studied the secondary structural
stability of commercial (CalB) and recombinant (LipB)
lipase B from Candida Antarctica in the presence of
urea and GdmCl at different concentrations. A gradual
loss of secondary structure began above 1 M and 3 M
urea concentration for LipB and CalB respectively.
Some secondary structural elements were still main-
tained even at a high concentration of 8 M by both
enzymes. When GdmCl was used as the chaotropic
agent, a prominent structure loss was observed in
both LipB and CalB, even at a low GdmCl concentra-
tion of 1 M. At 3 M GdmCl, complete denaturation of
both enzymes was observed (Brito e Cunha et al.
2019). At 1 M GdmCl, RoL-ZAC3 still maintained a sig-
nificant percentage of its native structure, at 3 M
GdmCl, only about 28% of its structure is preserved
while at 6 M GdmCl there was complete loss of struc-
ture (Figure 8(A)). Literature has proposed that chaot-
ropic agents may act by interacting directly with the
polar side chains and the protein backbone or by
interacting with water causing perturbations in the
water solvation (O’Brien et al. 2007). Studies have also
shown that lower GdmCl concentrations than urea are
needed to reach the protein unfolding midpoint
because GdmCl is more efficient in protein solvation
causing salt bridges and hydrogen bonds to rupture
(O’Brien et al. 2007).
For the DSC analysis (Figure 9), the linear heat cap-
acity from 20 ꢀC to 45 ꢀC implied that no domains or
regions displayed any significant degree of unfolding
before the commencement of the actual thermal tran-
sition/heat denaturation at around 50 ꢀC. Loss of ter-
Aggregation studies were carried out using DLS
analysis. Protein aggregation sets in after unfolding,
following exposure of more hydrophobic surface area
(Yan et al. 2003). As proteins aggregate, the aggre-
gates scatter light to different intensities, depending
tiary structure is
a cooperative process usually
associated with a large enthalpy and entropy change.
The t ꢁ Tm obtained from the analysis was 55.5 ꢀC,
which is very close to the Topt of 55 ꢀC; DHcal

BIOCATALYSIS AND BIOTRANSFORMATION
13
on the sizes of the aggregates. Scattered light inten-
sity was monitored as a function of temperature. A
sharp increase in scattered light intensity, as tempera-
ture increased, marked the onset of aggregation
(Figure 11). Aggregation tendencies are most likely
due to enhanced hydrophobic interactions following
partial disruption/loss of tertiary structure. With a Topt
of 55 ꢀC (local active site unfolding temperature),
t ꢁ Tm of 55 ꢀC (measured by DSC) and 50 ꢀC (meas-
ured by fluorescence analysis), Tagg of 50 ꢀC (measured
by DLS thermal scan) and CD Tm > 80 ꢀC (a clear ther-
mal-induced 2ꢀ structure unfolding was not observed
up to 80 ꢀC), it can be seen that these observations
are in agreement with a proposed unfolding mechan-
ism by Shirke et al. (2018) that tertiary structure loss
starts at the fluorescence-determined unfolding
temperature.
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Conclusion
An unusually low molecular weight lipase from
Rhizopus oryzae ZAC3 was purified and extensively
characterised. RoL-ZAC3 showed optimum activity at
55 ꢀC and pH 8. It exhibited enzymatic activity on a
broad range of substrates and was stable over a wide
range of temperature and pH. Its stability in organic
solvents with low logP values such as methanol and
ethanol suggest its potential applications in transester-
ification reactions. It also displayed impressive second-
ary structure stability. Based on the characterisation
results and the benefits inherent in low molecular
weight lipases, RoL-ZAC3 has interesting potential in
industrial applications.
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Acknowledgements
Structural studies were carried out at the Center for
Biotechnology and Interdisciplinary Studies (CBIS), Rensselaer
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Disclosure statement
Kantak J, Prabhune A. 2012. Characterization of smallest
active monomeric lipase from novel Rhizopus strain: appli-
cation in transesterification. Appl Biochem Biotechnol.
166(7):1769–1780.
No potential conflict of interest was reported by
the author(s).
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Yung-Hun JK, Kan YE, Kim YH, Lee SH. 2015.
Biocompatible cellulose nanocrystals as supports to immo-
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ORCID
Femi K. Agboola
http://orcid.org/0000-0001-6023-2872

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