A novel extracellular cold-active esterase of Pseudomonas sp. TB11 from glacier No.1: Differential induction, purification and characterisation

Article abstract of DOI:10.1016/j.molcatb.2015.07.015

The production, purification, characterization and application of a novel cold active esterase by Pseudomonas sp. TB11 are described herein. A new finding regarding the production of extracellular esterase activity depending upon single cream as an induce

Full text of DOI:10.1016/j.molcatb.2015.07.015

Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
A novel extracellular cold-active esterase of Pseudomonas sp. TB11
from glacier No.1: Differential induction, purification and
characterisation
Juan Donga,c, Wei Zhaoa,b, Mohammed A.A. Gasmallab, Jingtao Sunc, Xiao Huaa,b
Wenbin Zhanga,b, Liang Hanb, Yuting Fanb, Yinghui Fengb, Qiuyun Shenb,
Ruijin Yanga,b,∗
,
a State Key Laboratory of Food Science & Technology, Jiangnan University, 214122, China,
b School of Food Science and Technology, Jiangnan University, 214122, China,
c College of Food Science and Engineering, Shihezi University, 832003, China,
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 May 2015
Received in revised form 30 July 2015
Accepted 30 July 2015
Available online 1 August 2015
The production, purification, characterization and application of a novel cold active esterase by Pseu-
domonas sp. TB11 are described herein. A new finding regarding the production of extracellular esterase
activity depending upon single cream as an inducer used for growth was investigated in this study.
The crude esterase was subjected to a three-step enzyme purification, which resulted in a 15.21-fold
purification and the specific activity of the final purified esterase increased to 1526.2 U/mg protein and
purified EstTB11 had a molecular mass of 65 kDa. The N-terminal sequence of ten amino acids were:
GVYDYKNLTT. Peptide mass finger printing revealed that some peptides showed homologues sequences
(29%) to polyurethanase of Pseudomonas sp. FH4. Furthermore, the enzyme displayed the optimum pH of
8.5 and optimum temperature of 25 ◦C and significantly high stability at 15–35 ◦C for 72 h. The enzyme
was incubated with different metal ions at concentrations of 5 and 10 mM, the activity of esterase was
increased in the presence of K+, Na+ and Mg2+ and decreased with Ca2+, Al3+, Mn2+, and Fe3+. Experiments
indicated that EstTB11 could hydrolyze milk fat to produce short and medium-chain fatty acid and this
result layed the foundation for the application in increased aroma of milk products.
Keywords:
Cold-adapted esterase
Single cream induction
Purification
Characterisation
Hydrolysis
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Moreover, recent researchs particularly on cold-active enzymes,
herald the rapid growth in this burgeoning field [2]. Thus, it is cru-
Glacier No. 1 has an elevation range of 3730–4486 m a.s.l. and is
located at the headwaters of the Urumqi River in Tianshan, China
(43◦05ꢀN, 86◦49ꢀE) [1]. It is a perennially dry and cold zone with
an annual average temperature of approximately −5 ◦C and serves
as a vast bioresource repository of cold-adapted microorganisms.
cial to develop the investigation on microorganism resources of
glacier No. 1, which has received a tremendous amount of atten-
tion in the research of extremophiles [3]. Enzymes produced from
psychrophilic and psychrotolerant microorganisms in permanently
cold habitats could be better adapt to the low temperatures. When
compared to the mesophilic counterparts, cold-adapted enzymes
display a higher catalytic efficiency over a temperature range of
roughly 0–40 ◦C.
Esterases/lipases have been recognised as useful biocatalysts
due to the success of certain applications, including those in
the oleochemical and detergent industries, the production of
biodegradable polymers, food flavouring, waste treatment, oil
biodegradation, and biodiesel production [4,5]. The high catalytic
activity at low temperatures can also be favorable in their appli-
cation [6]. Esterases differ from lipases mainly based on their
substrate specificity and interfacial activation. In fact, lipases pre-
fer water-insoluble substrates, typically triglycerides composed of
Abbreviations: EstTB11, a cold-adapted esterase from psychrotolerant Pseu-
domonas sp. TB11; pNPA, p-nitrophenyl acetate; pNPB, p-nitrophenyl butanoate;
p, NPCp-nitrophenyl caprylate; pNPD, p-rophenyl decanoate; pNPL, p-nitrophenyl
laurate; pNPP, p-nitrophenyl palmitate; p, NPSp-nitrophenyl tearate; ARTP, atmo-
spheric and room temperature plasma; PVA, polyvinyl alcohol; pNP, p-nitrophenol;
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; MALDI-TOF-
MS, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry;
PVDF, polyvinylidene difluoride; SPME, solid-phase microextraction; GC/MS, gas
chromatography/mass spectrometer.
∗
Corresponding author at: State Key Laboratory of Food Science & Technology,
Jiangnan University, 214122, China. Fax: +86 510 85919150.
E-mail addresses: dongjuanvv@126.com (J. Dong), zhaow@jiangnan.edu.cn
(W. Zhao), yrj@jiangnan.edu.cn (R. Yang).
http://dx.doi.org/10.1016/j.molcatb.2015.07.015
1381-1177/© 2015 Elsevier B.V. All rights reserved.

54
J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
long-chain fatty acids, whereas esterases preferentially hydrolyze
short or medium-chain fatty acids.
bottom sediments of west branch of Tianshan glacier No. 1 in Xin-
jiang, China, with an elevation of 3800 m (43◦7ꢀ8ꢀꢀN, 86◦48ꢀ42ꢀꢀE).
The strain was maintained on LB agar slopes containing peptone 1%,
NaCl 1%, yeast extract 0.5% and agar 2.0%. The duplicate screening
medium was used as production medium which contained peptone
1%, K2HPO4 0.2%, MgSO4·7H2O 0.05%, (NH4)2SO4 0.1%, emulsion of
olive oil 20 mL/L. The initial pH of the medium was adjusted to 9.0
with 0.1 M NaOH or HCl prior to sterilization.
Although several studies have been performed deep in the sea
[7], the Antarctic [8,9], the western Himalaya mountains [10], lit-
tle is known with respect to cold-adapted esterases in glacier
resources, especially in the Tianshan Mountains. An extracellu-
lar esterase, EstK, was previously purified from a cold-adapted
Pseudomonas mandelii. The enzyme EstK was active at low tem-
peratures and showed substrate preference for short-chain fatty
acids, especially p-nitrophenyl acetate (pNPA) [11]. A psychrotol-
erant bacterium, named strain T1-39 was obtained by carrying out
extensive screening for esterases producer from the low temper-
ature microorganisms of tundra soils and glacier melt warters in
glacier No. 1, which was identified as a Pseudomonas sp. (accession
number: KP780205). Strain TB11 was a mutant of Pseudomonas sp.
T1-39 by atmospheric and room temperature plasma (ARTP) muta-
tion. However, an esterase, EstT1-39, produced from Pseudomonas
sp. T1-39 is prefer to hydrolyse esters of glycerol with short-chain
fatty acids, especially p-nitrophenyl butanoate (pNPB). This kind
of esterase lay a foundation for further application by hydrolyz-
ing milk products to increase its aroma [12]. Previous work also
confirmed the finding that crude EstTB11, produced from strain
TB11, was stable at room temperature and highly active at low tem-
peratures, and its characteristics made it potentially important for
industrial applications.
2.3. Inoculum preparation and esterase production
Cells of strain TB11 were inoculated in 5 ml LB liquid medium
and allowed to grow on a rotary shaker with shaking at 200 rev./min
and at 15 ◦C for 18 h. For EstTB11 production, conical flasks (250 ml)
containing 25 ml fermentation medium were inoculated with 2%
inoculum and incubated on a rotary shaker at 200 rev./min at 15 ◦C
for 32 h. The samples were removed after certain time intervals to
determine esterase activity and soluble protein. The culture were
collected by centrifugation at 9727 × g at 4 ◦C for 20 min and the cell
free supernatant was used as the crude esterase for determination
of enzyme activity under standard assay conditions.
2.4. Effect and consumption of different inducers on esterase
production
However, at present, this poses a problem because the prop-
erties of each esterase may be different, they may exhibit different
activity, stability or selectivity, and they may be affected in different
ways by the experimental conditions. Moreover, the results may
change between different batches of the enzyme. Therefore, pure
esterase preparations are required to provide complete control and
understanding of the processes. Hence, purification and character-
isation are important for the production of highly active and stable
esterases. Against this background, this study was focused on the
production, purification,characterisation and application of a novel
EstTB11 from Pseudomonas sp. TB11.
The effect of different inducers by replacing olive oil in the
production medium with rape seed oil, soybean oil, single cream,
sunflower oil and triolein were used to establish the substrate
selectivity of the enzyme. Compound emulsion was obtained at a
ratio of 1:3 with these oils and 2% polyvinyl alcohol (PVA). The
optimal inducer was determined by the relative activity, and on
this basis, different additive amounts of the optimal inducer were
experimented to determin the optimum amount.
2.5. Enzyme assay and protein estimation
Esterase activity in culture supernatants was assayed by mea-
suring the absorbance of liberated p-nitrophenol at 410 nm using
p-nitrophenyl butanoate (p-NPB) as a substrate by Margesin [13]
with some modifications.
2. Material and methods
2.1. Material
One esterase unit activity was defined as the amount of enzyme
that produced 1 ␮moL of p-nitrophenol (pNP) per min. 100 ␮L
culture supernatant was added to 1.9 mL substrate buffer, which
containing two parts, one part was dissolved p-NPB (3 mg/mL) and
another part were 50 mmol/L Tris–HCl buffer (pH 8.5), 0.1% gum-
acacia powder, 0.6% Triton X-100, then the two parts were mixed
at a ratio of 1:9 [14]. The mixture was incubated at 30 ◦C for 15 min,
then 1 mL of 95% ethanol was added to terminate the reaction. Inac-
tive enzyme sample as a control group was treated with boiling
water for 10 min. The absorbance of the samples was determined
spectrophotometrically at 410 nm. Then the esterase activity of the
culture broth supernatant (centrifuged at 9727 × g for 20 min) was
measured.
Peptone, yeast extract, glucose and agar were purchased from
Thermo Fisher Oxoid. Rape seed oil, soybean oil, olive oil, single
cream, sunflower oil samples were obtained from local market.
Triton X-100, gum-acacia powder, polyvinyl alcohol and triolein
were obtained from Biosharp. Sodium dodecyl sulphate (SDS),
p-nitrophenyl acetate (pNPA), p-nitrophenyl butanoate (pNPB), p-
nitrophenyl caprylate (pNPC), p-nitrophenyl decanoate (pNPD),
p-nitrophenyl laurate (pNPL), p-nitrophenyl palmitate (pNPP) and
p-nitrophenyl stearate (pNPS) were obtained from Sigma Chem-
ical Co. (China). Palatase 20,000 L was offered with Novozyme.
HiTrap Q Sephrose FF ion exchange column and Superdex 75
10/300 GL gel filtration column were obtained from GE Healthcare
(Co., Ltd., Hong Kong). Mini double-side vertical electrophoresis
apparatus (DYCZ-24DN, Beijing, China). SCIONSQ-456-GC gas chro-
matograph, Bruker, with an DB-WAX fused silica capillary column
(30 m length × 0.25 mm i.d. × 0.25 ␮m film thickness; Agilent Inc.,
USA). All other chemicals in the investigation were of analytical
grade and were purchased from Sinopharm chemical reagent fac-
tory (Co., Ltd., PR China).
Protein concentration was estimated by Bradford method with
bovine serum albumin as a standard [15]. During the chromato-
graphic purification steps, protein concentration was measured as
a function of its absorbance at 280 nm.
2.6. Purification of esterase TB11
2.6.1. Ammonium sulphate precipitation and dialysis
The crude esterase was purified by ammonium sulphate precip-
itation followed by a series of column chromatography separations
at 4 ◦C. The cell free supernatant was used for esterase purifi-
cation and ground ammonium sulphate was slowly added with
constant stirring up to 60% saturation. This solution was allowed
2.2. Bacterial strain and culture conditions
Strain TB11 used in this study was a mutant of Pseudomonas
sp. T1-39, which was isolated from frozen soil, sourced from the

J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
55
to stand overnight at 4 ◦C. The precipitate was collected by cen-
trifugation (9727 × g for 30 min at 4 ◦C) and resuspended in 50 mM
Tris–HCl buffer, pH 8.5. Then resuspension solution was transferred
into dialysis tubing, having 8–14 kDa cut off value, and dialyzed to
remove excess salts against the same buffer at 12 h intervals. After
24 h, enzyme solution was taken out and was used for next step of
purification.
selected and subjected to MS/MS. The Mascot MS/MS Ion search
with MS/MS data from the SwissProt database was used.
2.9. N-terminal sequence determination
To determine the N-terminal sequence the purified esterase
was electroblotted with a semi-dry transfer unit on polyvinylidene
difluoride (PVDF) membrane (RPN303F, GE) after electrophoresis
on a 12% SDS–PAGE gel. After blotting, the PVDF membrane was
stained with Coomassie Blue R-250. The esterase band was cut
out and N-terminal Edman sequencing was performed on a PPSQ-
30 Analysis 10 micro sequencer connected to a PPSQ-33A Protein
Sequencer (SHIMADZU) and a PPSQ-30 Data Processing software
was used.
2.6.2. Ion exchange chromatography purification
Esterase activity of each fraction was determined. The most
active components were loaded onto a HiTrap Q-Sepharose Fast-
Flow column (1.6 × 2.5 cm, GE Healthcare, USA), which had been
equilibrated previously with buffer A (50 mM Tris–HCl buffer, pH
8.5), at a flow rate of 5 mL/min. Elution curves were monitored at
280 nm. Unbound protein was washed out with buffer A. Retained
protein was eluted using stepwise ionic strength gradients com-
posed of buffer A and buffer B (50 mM Tris–HCl buffer, 1 M NaCl,
pH 8.5): 0–0.4, 0.4–0.60 and 0.60–1.0 M NaCl. Tubes corresponding
to each fraction were pooled separately, desalted and their enzyme
activities and protein contents were determined. Active fractions
containing high esterase activity were pooled and concentrated
using a 10 kDa Amicon Ultra tube (Millipore, USA). The concen-
trated protein was applied to a Superdex 75 10/300 GL gel-filtration
column (1.0 × 30 cm, 23.562 mL, GE Healthcare, USA). Buffer C
(50 mM Tris–HCl, pH 8.5) was used for column pre-equilibration,
sample loading and elution, and the flow rate was 0.8 mL/min. The
active fractions were pooled and assayed for next characterisa-
tion. The esterase preparations (crude and purified fractions) were
tested on SDS-PAGE gels.
2.10. Characterisation of purified esterase
2.10.1. Substrate specificity
Substrate specificity of the esterase was determined by using p-
nitrophenyl esters of varying acyl chain lengths from C2 to C18. The
following substrates were used: pNPA (C2:0), pNPB (C4:0), pNPC
(C8:0), pNPD (C10:0), pNPL (C12:0), pNPP (C16:0) and pNPS (C18:0).
The substrates were dissolved in isopropanol at a final concen-
tration of 100 ␮M. Reaction was carried out under standard assay
conditions.
2.10.2. The effect of pH and temperature on activity and stability
The effect of pH and temperature on purified EstTB11 was
investigated by using p-NPB as the substrate. The optimum pH of
the purified enzyme was determined over a pH range of 3–10.6
at constant molarity (50 mM) in different buffers: sodium citrate
buffer (pH 3.0–5.0), phosphate buffer (pH 6.0–8.0), Tris–HCl buffer
(pH 8.0–9.0), glycine–sodium hydroxide buffer (pH 9.0–10.6). The
assays were performed at 30 ◦C. Buffers were pre-incubated at
30 ◦C, then we added the substrate and immediately mixed before
adding aliquots of the enzyme. The pH stability was studied by incu-
bating the purified EstTB11 in selected buffers of pH range 3–10.6
for 4 h at 30 ◦C. The residual enzyme activity was measured by
spectrophotometric assay at 30 ◦C, pH 8.5.
The optimum temperature of the purified EstTB11 was deter-
mined by the enzyme activity at various temperatures (0–50 ◦C)
in 50 mM Tris–HCl buffer, pH 8.5. Buffers were pre-incubated for
at least 5 min at the temperature of the assay before initiating the
reaction. Thermostability was determined by incubating purified
esterase in 50 mM Tris–HCl buffer, (pH 8.5) at various temper-
atures (15–55 ◦C) for 12 h and residual activity was analyzed by
spectrophotometric assay at 25 ◦C, pH 8.5.
2.7. Molecular mass determination by SDS-PAGE and
zymography
The purity of the isolated protein and molecular weight of pure
enzyme were determined by SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) on a discontinuous gel con-
taining 5% stacking gel and 12% resolving gel of pH 6.8 and 8.8,
respectively. Electrophoresis was performed with 120 mV fixed
voltage. After electrophoresis the gel was stained with Coomassie
Brilliant Blue and the relative mobility of the purified enzyme
was compared with standard proteins of known molecular weight
marker (TaKaRa BIO INC). The protein marker ranging from 14.3
to 97.2 kDa was used as a standard marker for determination of
molecular weight.
Zymograms allowed rapid detection of active proteins in
polyacrylamide gels, hydrolase activity was also detected by a
deferred assay where native polyacrylamide gels immerseed into
1% TritonX-100 for 1 h and were rinsed four times with 50 Mm
Tris–HCl (pH 8.5). Incubation in substrate buffer solution contain-
ing the substrate pNPB at 30 ◦C for 1 h, proteins diffused from the gel
and hydrolyzed the substrate, resulting in a clear yellow band at the
position of the protein migration by Tamilarasan with some mod-
ifications [16]. In this method, the activity could only be detected
when samples were not treated with ␤-mercaptoethanol prior to
electrophoresis.
2.10.3. Effect of metal ions on enzyme activity
For determining the effect of metal ions on enzyme activity,
enzyme assays were performed in presence of various metal ions
such as Al3+, K+, Ca2+, Fe3+, Na+, Cu2+, Mn2+ and Mg2+ with a final
concentration of 5 mM and 10 mM using pNPB as substrate. After
60 min incubation at 25 ◦C, the residual esterase activities were
assayed. Residual esterase activity was calculated as a percentage
of that, without effectors.
2.8. Peptide mass finger printing of esterase analysis and protein
identification
2.11. Determination of kinetic parameters
The EstTB11 band in the SDS-PAGE gel was excised and submit-
ted for in-gel digestion with trypsin. Eluted proteins were subjected
to matrix-assisted laser desorption/ionisation time-of-flight mass
spectrometry (MALDI-TOF-MS) to obtain their peptide mass fin-
gerprint. Peptide sequences were matched to the sequence of the
native proteins with bioinformatics tools (Matrix Science) to con-
firm the identity of the proteins. Peptides mass fingerprints were
The maximum reaction rate (Vmax) and Michaelis–Menten con-
stant (Km) values of the enzyme were determined at temperature
(25 ◦C) and pH (8.5) using different concentrations 0.1–10 mM of
pNPB substrate. Enzyme kinetics reaction was started by adding
4030 U/L purified enzyme in the substrate solution. The constant
values were calculated by fitting data to linear regression using
Lineweaver-Burk equation plot. All the assays were performed by

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J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
standard assay conditions. Kinetic constants were calculated using
Lineweaver–Burk plot (1).
is not favourable to the production of esterase because the yield of
other proteins is promoted by a high concentration of lipids, which
appears be the main cause of the inhibition of esterase produc-
tion. As a result, the optimal addition of single cream was found to
enhance esterase production in flask experiments.
1
V
Km
1
[S]
1
Vmax
=
×
+
(1)
Vmax
where [S] is the substrate concentration and V is the initial reaction
rate of the enzyme.
3.2. Purification of extracellular EstTB11
2.12. SPME-GC/MS analysis on hydrolysates of lipolytic
EstTB11and Palatase 20,000L
After 32 h of culture, the cells were separated from the
fermented medium by centrifugation. The crude esterase was sub-
jected to a three-step enzyme purification process as shown in
Table 1. In this study, EstTB11was purified by using ammonium
sulphate precipitation (Fig. 2A) followed by HiTrap Q-Sepharose
FF anion Exchange (Fig. 2B) and Superdex 75 10/300 GL gel chro-
matography (Fig. 2C). By this purification procedure, the enzyme
was purified 15.21-fold with an overall yield of 11.78%, and the spe-
cific activity of the final purified esterase increased to 1526.2 U/mg
protein (Table 1). The purified enzyme preparations were stored at
−20 ◦C and were used for further studies to evaluate its properties.
Single cream was hydrolyzed by EsteraseTB11 and Palatase
20,000L (Novozyme) on the conditions of enzymolysis temperature
at 25 ◦C and 50 ◦C, respectively, enzymolysis pH 8.0, the additive
amount of enzyme 150 U/g, substrate concentration 50% and enzy-
molysis time 5 h. SPME-GC/MS technology was used for extracting
and analyzing the volatile compounds among the enzyme products.
Five grams of enzymatic hydrolysates were placed in a 20 mL
vial. The sample was stirred for 30 min at 55 ◦C to accelerate
the equilibrium of headspace volatile compounds between the
hydrolysates matrix and the headspace. Then, volatile compounds
were extracted by placing a 50/30 ␮m divinylbenzene/carboxen/
polydimethylsiloxane (DVB/CAR/PDMS, Supelco Inc. Bellefonte,
PA) SPME fibre into the vial and exposing it to the headspace
for 30 min at 55 ◦C. Analysis of the volatile compounds in the
hydrolysates was performed using an SCIONSQ-456-GC gas chro-
matograph coupled with a mass detector. An DB-WAX fused silica
capillary column (30 m length × 0.25 mm i.d. × 0.25 ␮m film thick-
ness) was used. The analysis was performed using helium as the
carrier gas with a flow rate of 0.8 ml/min in a splitless injection
mode. The oven temperature was held at 40 ◦C for 3 min, rose at
5 ◦C/min to 90 ◦C, then ramped to 230 ◦C at a rate of 10 ◦C/min, and
maintained at 230 ◦C for 7 min. GC–MS was performed with an ion
source temperature of 200 ◦C, an ionization voltage of 70 eV, and
a mass range of 1–2000 amu. All the SPME/GC–MS measurements
were conducted in triplicate on different samples.
3.3. Gel electrophoresis and zymography
The homogeneity of the eluted protein was confirmed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. The purified
esterase showed a single protein band of a relative molecular
mass of approximately 65 kDa (Fig. 3A, lane 4). The zymogram of
the purified esterase was obtained after performing Native Page
in chromogenic substrate. Distinct yellow bands were observed
against the white background within 30 min, as shown in Fig. 3B.
3.4. Peptide mass fingerprinting and sequencing
Gel bands were manually excised and subjected to automated
in-gel chemical modification of cysteine residues with dithio-
threitol and iodoacetamide followed by tryptic digestion and the
digestion mixture was analysed directly by MALDI-TOF-MS. This
mass spectrometric peptide mapping was compared with the the-
oretical maps of known proteins (Table 2); 10 peptides in the
spectrum had a mass that was homologous for peptides from
polyurethanase (Esterase lipase) of Pseudomonas sp. FH4, result-
ing in a sequence coverage of 29% (178 aa for a total size of
615 aa for the polyurethanase). Seven peptides matched with
polyurethanase Esterase lipase) of Pseudomonas fluorescens, two
peptides are homologous to the glutamine synthetase of P. fluo-
rescens (fragments).
The volatile compounds were identified by matching the mass
spectra with those in the Wiely7.0 and NIST 2012 Library of MS
spectra and the rentention index (RI) was calculated with a homol-
ogous series of n-alkanes (C4–C28).
2.13. Statistical analysis
Statistical analysis was performed on the datas using the anal-
ysis of variance analysis (ANOVA) with Duncan’s multiple range
tests. A p value of 0.05 or less was considered to be statistically sig-
nificant. The analyses were conducted by SPSS 17.0 software (SPSS
Inc., Illinois).
3.5. N-terminal sequence of EstTB11
N-terminal sequencing of the polyvinylidene fluoride (PVDF)
transferred band from an electrophoretic gel allowed the identi-
fication of 10 amino acid residues: GVYDYKNLTT. This sequence
was compared with the sequences of known esterases/lipases
(Table 3). It exhibited significant similarity (80–90%) to the N-
terminal sequence of other source esterases/lipases. The first 10
amino acids of EstTB11 were found to be identical to the 2–11 amino
acids of a putative lipase identified from an ORF in the genome
sequence of Pseudomonas chlororaphis O6 (genebank accession No.
EIMI14990). It is likely that this ORF codes for EstTB11 as an esterase
sequence that is preceded by a leader (targeting) sequence.
3. Results
3.1. Effect of different inducers on EstTB11 production
Six lipids were used to select the optimal inducer. The results,
presented in Fig. 1A, showed that the relative activity of single
cream significantly different (p < 0.05) which was obviously higher
than those of the other five oils. Hence, the esterase production
was induced by single cream, which could specifically hydrolyze
milk fat to promote the formation of aroma substances. Without
the addition of any inducer, its relative activity was as high as 42.8%,
which showed that Pseudomonas sp. TB11 could secrete esterase by
itself, however, its enzyme activity was lower.
3.6. Characterisation of purified the enzyme EstTB11
The quantity of enzyme production can be effectively improved
by induction under a suitable physiological state of the bacteria. As
shown in Fig. 1B, the activity of esterase was the highest on single
cream with consumption of 60 ml/L. Overloading with single cream
3.6.1. Substrate specificity of purified EstTB11
The purified EstTB11 was active against a wide range of p-
nitrophenyl esters of fatty acids with higher hydrolytic activity
towards p-NPB and p-NPC, which were significantly different

J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
57
Fig. 1. (A) Effect of six inducers and without any inducer on production of extracellular esterase. (B) the optimum single cream consumption was detemined by the relative
activity under the different addition amounts (5, 10, 20, 40, 60, 80, 100, 140, 180 mL/L).
Table 1
Summary of steps of purification of extracellular esterase from Pseudomonas sp.TB11.
Purification steps
Toalactivity (IU)
Totalprotein(mg)
Specific activity(IU/mg)
Foldpurification
Yield(%)
Culture enzyme
Ammonium sulphate precipitation
Q Sepharose FF
7644
76.2
9.62
1.80
0.59
100.31
597.83
815.82
1526.2
1.00
5.96
8.13
100
5751.2
1468.48
900.46
75.24
19.21
11.78
Superdex G75
15.21
(p < 0.05) from the other p-nitrophenyl esters (Fig. 4). The results
showed a preference of the enzyme for short and medium-chain
fatty acids, indicating that it was more closely related to esterase. It
was reported that a cold active lipase from Pseudomonas sp. Strain
KB700A showed substrate preference towards medium acyl chain
[21] and the other lipase from Yarrowia lipolytica had maximum
activity towards p-NP caprylate [22].
3.6.2. Effect of pH and temperature on the esterase activity and
stability
The EstTB11 characterised here were found to be functionally
active in a pH range of 3.0–10.6 and showed optimum activity at
pH 8.5 (Fig. 5A). It was found to be active and stable over a pH range
of 7.0–10.0 at 30 ◦C for 240 min, which indicates residual activity
up to 60% or more (Fig. 5B). The residual activities of EstTB11 was
about 50% with incubation at pH 5.0 for 90 min. When the pH value

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J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
Fig. 2. Purification diagrams of EstTB11. (A) Relative of ammonium sulphate precipitation fractions; (B) Elution curve of HiTrap Q Sepharose Fast Flow chromatography; (C)
Elution curve of Superdex 75 10/300 GL chromatography.
was below 5, the change in the relative enzyme activity increased
until little enzyme activity remained. Thus, the pH has a obvious
influence on EstTB11 activity, which is stable in both neutral and
alkaline conditions.
Temperature not only influences the stability of proteins but also
the maximum reaction rate of enzymes by influencing the enzyme
and the substrate. The influence of temperature on esterase activity
is shown in Fig. 5C and D. As can be seen in Fig. 5C, maximum activ-

J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
59
Table 2
MALDI-TOF-MS and MS/MS analysis of esterase LipTB11 obtained by chemical modification of cysteine residues with dithiothreitol and iodoacetamide and tryptic digestion.
Protein
Organism
Region name
Observed
Mr (expt)
Mr (calc)
Peptide
Polyurethanase
Pseudomonas sp. FH4
Esterase lipase
1021.4891 1020.4819 1020.4659 DGQGGISMTR
1216.6217 1215.6144 1215.5996 ANTWVQDLNR
1471.7900 1470.7828 1470.7678 DSTIIVANLSDPAR
1531.6914 1530.6841 1530.6740 NTFLFDGDFGQDR
1551.7927 1550.7854 1550.7729 VLNIGYENDPVYR
1568.8226 1567.8153 1567.7955 DVTHSVTDQGLLAGR
1591.8227 1590.8154 1590.8002 NVEVANDGAGTLYIR
1641.8531 1640.8458 1640.8311 WLFGLDGNDHLIGGK
1840.9289 1839.9216 1839.9075 GNDVLVGGAGNDLLEAGGGR
1591.8227 1590.8154 1590.8002 NVEVANDGAGTLYIR
1840.9289 1839.9219 1839.9075 GNDVLVGGAGNDLLEAGGGR
2255.1029 2254.0956 2254.0729 LVFMGVEGVDGQYNYLDHAK
1720.9245 1719.9172 1719.9018 LVPGFEAPVMLAYSAR
2884.3655 2883.3582 2883.2995 IYSEQGSWMSDQDVEGGNHGHRPGIK
1471.7900 1470.7828 1470.7678 DSTIIVANLSDPAR
Polyurethanase
Pseudomonas fluorescens Esterase lipase
Pseudomonas fluorescens Gln-synt N
Glutamine synthetase
Putative calcium-binding protein Pseudomonas sp. GM48
Esterase lipase
Table 3
N-terminal sequence comparison of LipTB11 with homologous esterase/lipase.
Definition (accession No.)
Source/Organism
Region name
Sequence
Identities(%)
100
Reference
LipTB11
Polyurethanase A(EIM14990)
Pseudomonas
Esterase/lipase
Esterase lipase
Lipase 3
Lipase 3
Esterase lipase
GVYDYKNLTT (1–10)
GVYDYKNLGT (2–11)
GIYDYKNLGTT (2–12)
GIYDYKNLGT (2–11)
GVYDYKNFST (2–11)
This study
[17]
[18]
[19]
[20]
Pseudomonas chlororaphis O6
Pseudomonas brassicacearum
Pseudomonas fluorescens
Uncultured bacterium
90
82
LipA
LipA
LipB
(AAF87594)
(WP 014338392)
(AAP76489)
80
80
Note: Non-matching amino acid residues are underlined and highlighted in bold.
Fig. 3. SDS-PAGE of purified EstTB11 and zymogram analysis. (A) Lane 1: stan-
dard proteins; lane 2: culture supernatant; lane 3: culture supernatant precipitated
with 60% ammonium sulphate and dialyzed; lane 4: purified protein after HiTrap Q
Sepharose FF and Superdex 75 10/300 GL chromatography. (B) Zymogram from an
Native-PAGE of purified EstTB11 analyzed for activity by pNPB incubation (left) and
stained with silver nitrate (right). Purified EstTB11 (lanes 1–3).
Fig. 4. Hydrolytic ability of EstTB11 towards pNPA, pNPB, pNPC, pNPD, pNPL, pNPP
and pNPS as substrates. Set the one with the highest relative activity as 100%.
the enzyme activity by 9–60% (Fig. 6). Mg2+ ions at a 5 mM concen-
tration enhanced the enzyme activity by 15% more than the control;
however, Mg2+ ions at 10 mM had no effect on esterase activity.
Cu2+ and Ca2+ ions at 5 and 10 mM separately inhibited enzyme
activity, but at 10 mM, Cu2+ ions enhanced the enzyme activity by
10% more than the control. The presence of Al3+ and Mn2+ ions at
5 mM and 10 mM concentration drastically reduced the levels of
enzyme activity, which were 40–50% less than the control.
ity of EstTB11 was occurred at 25 ◦C while the activity of enzyme
decreased gradually at temperatures below 20 ◦C beyond 30 ◦C, but
it still retained more than 90% and 38% of its maximal activity at
35 ◦C and 0 ◦C, respectively.
The EstTB11 stability at 15–35 ◦C was stable, at around 90%
at 15–25 ◦C and of about 80% at 35 ◦C for 720 min. However, the
EstTB11 activity at 45 ◦C remained above 65% for 60 min and began
to sharply decline after 90 min of incubation, with residual activity
of less than 30% after 120 min. At 55 ◦C for 15 min, approximately
8% residual activity was observed for EstTB11, whereas it was inac-
tivated after 240 min (Fig. 5D).
3.7. Enzyme kinetic parameters
The kinetic parameters Km and Vmax were calculated for the
purified esterase from the Michaelis–Menten plot (Fig. 7). The puri-
fied esterase showed a lower Km value (0.698 mmol−1) and a higher
Vmax value (22.93 mmol/min·mg) than the esterase/lipase from the
other Pseudomonas sp. reported in the literature [23,24]. The lower
Km value represents a higher affinity between enzymes and sub-
3.6.3. Effect of metal ions on EstTB11 activity
The presence of metal ions at 5 mM of K+ and Na+ promoted
esterase activity for 60 min, that was about 12% to 35% greater than
the control, whereas 10 mM concentration of K+ and Na+ enhanced

60
J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
Fig. 5. (A) Effect of pH on enzyme activity by assaying in pH values(3–10.6) at 25 ◦C for 15 min. pH 3–5 (citric acid and sodium citrate buffer), pH 6–8 (phosphate buffer), pH
8–9 (Tris–HCl buffer), pH 9–10.6 (glycine–sodium hydroxide buffer); (B) the stability of pH was measured by adding the esterase in different pH (3–10) at 25 ◦C for 30, 60,
90, 120, 150, 180, 210, 240 min, respectively.
(᭿) pH 3, (᭹) pH 4, (ꢀ) pH 5, (ꢁ) pH 6, (ꢁ) pH 7, (ꢂ) pH 8, (ꢀ) pH 9, (ꢂ) pH 10; (C) Effect of temperature on EstTB11 activities by measuring relative activity at 0–50 ◦C (0, 4,
10, 15, 20, 25, 30, 35, 40, 45, 50 ◦C) in 50 mM Tris–HCl buffer (pH 8.5) for 15 min; (D) thermostability was measured by incubating the esterase in 50 mM Tris–HCl (pH 8.5) at
15 ◦C (᭿), 25 ◦C (᭹), 35 ◦C (ꢀ), 45 ◦C (ꢁ) and 55 ◦C (ꢀ).
Fig. 6. The effects of different metal ions were investigated by pre-incubating the
Fig. 7. Lineweaver–Burk plot of purified esterase from Pseudomonas sp.TB11 using
esterase with 5 mM and 10 mM final concentration of these ions at pH 8.5, temper-
p-nitrophenyl butanoate as a substrate.
ature 25 ◦C for 30 and 60 min respectively. Without metal ions as the control, which
was considered 100%.

J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
61
Fig. 8. Chromatograms obtained by the SPME-GC/MS analysis of the investigated compounds in two hydrolysates from EstTB11 and Palatase 20,000 L under the given
conditions in paper. (A) The hydrolysate chromatograms of EstTB11 and (B) The hydrolysate chromatograms of Palatase 20,000 L.
strates and Vmax represents the higher catalytic efficiency of lipase
[25].
hydrolysis degree of the two enzymes was near to equivalence.
Therefore, EstTB11 has a great advantage in enzymolysis temper-
ature and its hydrolysates are better use to produce milk essence
seasoning to improve milk flavors. Chromatograms obtained by the
SPME-GC/MS are were shown in Fig. 8.
3.8. Analysis of volatile compounds of EstTB11hydrolysates
The two hydrolysates produced by EstTB11 and Palatase
20,000 L were analyzed by SPME-GC/MS. The results showed that
68 volatile compounds in hydrolysates of EstTB11 and 39 volatile
compounds in that of Palatase 20,000 L were detected. The volatile
components hydrolyzed from EstTB11were much more than those
from Palatase 20,000 L, which was consistent with the result of sen-
sory evaluation. 25 volatile compounds were selected to compare
from the two hydrolysates as main contributing components in
milk flavors improvement.
The chemical name of 25 volatile compounds, CAS number,
RI and relative contents are shown in Table 5. As shown in
Table 5, EstTB11 and Palatase 20,000 L obvious prefer for short
and medium-chain fatty acids, the contents of butanoic acid, hex-
anoic acid, heptanoic acid and octanoic acid were all increased,
but tetradecanoic acid and 9-octadecenoic acid were not change
or decreased. The results were consistent with the conclusion of
substrate specificity of purified EstTB11. In Table 5, the volatile
compounds from EstTB11 (25 ◦C) had higher contents and quanti-
ties than those from Palatase 20,000 L (50 ◦C), which indicated that
low temperature could better keep the flavor substances when the
4. Discussion
Microbial esterases/lipases are mostly extracellular, and their
production is greatly influenced by the medium compositions,
however, an inducer is one of them. The esterase productivity of
Pseudomonas sp.TB11 was improved to different degrees by induc-
tion with adding rapeseed oil, soybean oil, olive oil, single cream
and sunflower oil. Therefore, EstTB11 is an induction-type esterase;
however, its productivity was sharply inhibited on triolein as an
inducer. At present, reports are available regarding the hydrolysis
of olive oil [26], butter oil [27], beef tallow [28] and other animal fats
[29] using lipase, but data on the use of single cream inducing Pseu-
domonas for the production of esterase have not been reported. In
this study, the experiment proved successfully improving EstTB11
activity by induction of Pseudomonas sp.TB11 with single cream.
This esterase acts preferentially on short-chain fatty acids, which
are commonly used to generate desirable flavours in dairy prod-
ucts [12]. The use of single cream as an inducer was found to have
a significant correlation with esterase production for strain TB11.

62
J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
Table 4
Temperature and pH properties of some cold-adapted lipases from different microorganisms.
Microorganism
Optimum temperature(◦C)
Relative enzyme activity(temperature)
Optimum pH
References
Pseudomonas sp.TB11(esterase)
Pseudomonas sp.strain B11-1 (esterase)
Psychrobacter celer 3Pb1(est12)
Pseudoalteromonas sp. wp27
Pseudoalteromonas haloplanktis TAC125
Pseudomonas fragi strain no. IFO3458
Pseudomonas sp. KB700A
Pseudomonas sp. 7323
Geotrichum sp. SYBC WU-3 (Lipase-A)
Psychrobacter sp.
25
45
35
30
40
29
35
30
20
20
25
36% (0 ◦C)
17% (0 ◦C)
41% (0 ◦C)
60% (4 ◦C)
15% (10 ◦C)
59% (10 ◦C)
13% (0 ◦C)
20% (0 ◦C)
60% (0 ◦C)
Not shown
40% (5 ◦C)
8.5
8.0
7.5
7-8
8.5
8
8-8.5
9
9.5
8.0
5.0
This study
[35]
[33]
[37]
[38]
[36]
[21]
[7]
[31]
[8]
[22]
Yarrowia lipolytica NCIM 3639
In previous studies, the available literatures on the purifica-
tion of cold-adapted esterase were not adequate. A salt-tolerant
esterase was purified 42.7-fold with a 6.4% rate of recovery (spe-
cific activity, 569.2 U/mg protein) by precipitation with ammonium
sulphate followed by anion and cation exchange chromatography
[30]. An extracellular lipase from Y. lipolytica was purified by Q-
Sepharose anion exchange chromatography and Sepharose CL-4B.
This resulted in 195 U/mg specific activity and 13.9-fold purifi-
cation with an 11.14% rate of recovery [22]. Two cold-adapted
lipases (Lipase-A and Lipase-B) of mesophilic Geotrichum sp. SYBC
WU-3 were purified by using (NH4)2SO4 fractionation, chromatog-
raphy separation on a DEAE-cellulose-32 column and a Sephadex
G100 column. The overall level of purification recovery was 20.4%
(11.2% for Lipase-A and 9.2% for Lipase-B) [31]. Compared to these
cold-adapted lipases, EstTB11was purified 15.21-fold, with a high
specific activity (1526.2 U/mg), using a three-step purification pro-
tocol. The purified esterase showed a molecular mass of 65 kDa.
In contrast, an esterase (PsEst1) of the psychrotrophic bacterium
Pseudomonas sp. B11-1 has been found to possess a molecular
mass of 69 kDa [32]. A novel esterase Est12 has been reported with
an estimated molecular mass of 35.15 kD [33]. The EstA esterase
has an apparent molecular mass of about 23 kDa [34]. A lipase
from Pseudomonas stutzeri PS59 with high detergent performance
and cold-adapted possessed a molecular weight of approximately
55 kDa [5]. The molecular mass of the purified esterase in this study
is comparatively higher than those reported elsewhere, which indi-
cated that it was a novel enzyme from Pseudomonas.
The EstTB11 showed optimal activity at pH 8.5 and was found to
have a higher stability over a pH range of 7.0–10.0 at 30 ◦C. A cold
active lipase from Y. lipolytica NCIM 3639 showed stability over a
pH range of 4–6, with complete loss of activity at pH 7.0 [22]. The
P. stutzeri PS59 lipase was found to be functionally active in the pH
range of 7.0 to 11.0 and displayed optimal activity at pH 8.5 and
showed approximately 80% residual activity at pH 9.0 [5]. In addi-
tion, the enzyme EstTB11 exhibited maximum activity at 25 ◦C, and
it still retained more than 38% of its maximal activity at 0 ◦C, com-
paring with the reported, the relative activity of EstTB11 exhitbited
ice-adapted enzyme of extremophiles (Table 4). A list of similar
cold-adapted esterases/lipases producing strains are presented in
Table 4 to make a comparison with EstTB11. The optimal tempera-
Table 5
25 volatile compounds in the hydrolysates of EstTB11and Palatase 20,000 L identified by SPME-GC/MS.
No.
Compounds
CAS No.
Ricala
IDb
RIc
RT (min)
Relative contents (%)
EstTB11
Palatase2000 L
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Hexanal
2-Heptanone
2-Nonanone
Nonanal
1-Octene
Acetic acid
Benzaldehyde
2-Nonenal, (E)-
2-Undecanone
Butanoic acid
Pentanoic acid
2-Dodecanone
Hexanoic acid
Heptanoic acid
Octanoic acid
Nonanoic acid
2H-Pyran-2-one, tetrahydro-6-pentyl-
n-Decanoic acid
9-Decenoic acid
Undecanoic acid
-Dodecalactone
2H-Pyran-2-one, 6-Heptyltetrahydro-
Dodecanoic acid
Tetradecanoic acid
9-Octadecenoic acid (Z)-
66-25-1
1072
1181
1385
1392
1427
1449
1513
1528
1597
1624
1732
1809
1841
1953
2061
2165
2215
2273
2336
2379
2393
2440
2487
2713
3180
RI
RI
RI
RI
MS
RI
RI
RI
RI
RI
RI
MS
RI
RI
RI
RI
RI
RI
RI
RI
MS
RI
RI
RI
RI
1072
1180
1387
1390
–
1448
1508
1530
1598
1627
1736
–
1850
1954
2060
2164
2203
2279
2335
2369
–
2458
2490
2714
3173
7.236
10.001
15.360
15.440
16.107
15.475
17.646
17.876
18.798
19.130
20.534
21.440
21.784
22.938
24.045
25.066
25.499
26.051
26.597
26.991
27.094
27.604
27.983
30.645
31.447
0.122 0.002
3.572 0.08
1.862 0.019
0.007 0.001
0.028 0.004
0.031 0.004
0.007 0.001
0.019 0.002
0.459 0.014
5.659 0.153
0.111 0.043
0.131 0.009
30.07 1.071
1.089 0.066
30.97 0.444
0.311 0.052
0.082 0.006
17.22 0.866
1.34 0.004
0.104 0.03
0.017 0.004
0.051 0.006
1.958 0.128
0.404 0.056
0.038 0.008
0.007 0.003
0.304 0.007
0.353 0.011
nd
110-43-0
821-55-6
124-19-6
111-66-0
64-19-7
nd
0.101 0.031
nd
100-52-7
18829-56-6
112-12-9
107-92-6
109-52-4
6175-49-1
142-62-1
111-14-8
124-07-2
112-05-0
705-86-2
334-48-5
14436-32-9
112-37-8
2305-5-7
713-95-1
143-07-7
544-63-8
112-80-1
0.003 0.001
0.27 0.011
8.573 0.168
0.147 0.011
0.118 0.178
34.32 1.204
0.978 0.032
29.41 0.636
0.323 0.029
0.071 0.010
17.67 0.570
1.318 0.036
0.111 0.05
0.014 0.002
nd
3.676 0.094
1.424 0.088
0.089 0.005
nd: not found.
a
Linear retention indices calculated of volatile compounds on an DB-WAX capillary column (30 m × 0.25 mm × 0.25 ␮m) with a homologous series of n-alkanes (C4–C28).
b
Identification method: MS, identification by comparing mass spectra in the Wiley7.0 and NIST 2012 library search system; RI, identification by retention indexes with
literature data.
c
From the flavornet database (http://flavornet.org, accessed June 2007), and NIST atomic spectra database.

J. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 53–63
63
ture of an esterase PsyEst was found to be around 35 ◦C [4]. A novel
Acknowledgments
cold-adapted esterase Est12 from a psychrotrophic bacterium Psy-
chrobacter celer 3Pb1 displayed the optimal activity at 35 ◦C and
was unstable at temperatures above 40 ◦C [33]. EstTB11 was quite
stable at 15 ◦C to 35 ◦C,around 90% of maximum activity at 15–25◦C
for 720 min and the relative activity at 45 ◦C remained above 65%
for 60 min, which displayed significantly high thermostability. The
purified cold-adapted lipase from Y. lipolytica NCIM 3639 exhibited
maximum activity at 25 ◦C and retained 40% of its activity at 5 ◦C.
The activity decreased sharply at temperatures above 30 ◦C. The
enzyme was found to be stable at 10 ◦Cand 30 ◦C. It lost about 40%
of its activity after 6 h of incubation at 35 ◦Cand was completely
inactive at 45 ◦C after 4 h of incubation [22]. Two new cold-adapted
lipases from mesophilic Geotrichum sp. SYBC WU-3 have good sta-
bility at room temperature, for they can retain 80% of their activity
after incubation at 25 ◦C for 72 and 48 h, respectively [31]. How-
ever, some other lipases from psychrotrophic and psychrophilic
microorganisms did not show good heat stability. For example, the
half-life of lipase from Pseudomonas sp. 7323 at 30 ◦C is only 4.5 h
[7]; lipase from Pseudomonas sp. B11-1 retains 92% of its activity
after incubation at 30 ◦C for 60 min [35]; half-life of the lipase from
Pseudomonas fragi is about 5 h at 27 ◦C [36].
The authors gratefully acknowledge Tianshan Glaciological Sta-
tion, CAS to provide help for sampling. This work was supported by
the National Natural Science Foundation of China (31401529), Key
Project of Chinese Ministry of Education (210250) and the Excellent
Young Program of Shihezi University in Xinjiang (2013ZRKXYQ15).
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