A high-detergent-performance, cold-adapted lipase from Pseudomonas stutzeri PS59 suitable for detergent formulation

Article abstract of DOI:10.1016/j.molcatb.2014.01.006

A high-detergent-performance and cold-adapted lipase was purified and characterised from Pseudomonas stutzeri PS59, which was isolated from Daqing oil fields (Heilongjiang, PR China). The lipase was purified to homogeneity using ammonium sulphate precipitation, dialysis, freeze-drying, ion exchange chromatography and gel filtration chromatography. The molecular weight of the lipase was approximately 55 kDa, as measured by SDS-PAGE. The lipase showed optima activity at pH 8.5 and 20 C. The lipase activity was activated by metal ions, such as Ca²⁺ and Mn²⁺, and surfactants, such as Tween 80, Tween 20, sodium dodecyl benzene sulfonate and urea. Oxidising agents, such as H₂O₂ and NaClO, were found to have little effect on the activity of the lipase, and most organic solvents can enhance the activity of the lipase. The broad substrate specificity and the compatibility of the lipase in the presence of surfactants, oxidising agents, and other detergent additives clearly indicate its potential application in the laundry industry. The hydrolysis resolution of (R,S)-ethyl 2-methylbutyrate by P. stutzeri PS59 lipase was carried out with the yield of 31.2% for R-ethyl 2-methylbutyrate, the enantiomeric excess of residual substrate (ee_s) was 85.7%. Thus, the lipase also showed an attractive potency for application in biocatalysis.

Full text of DOI:10.1016/j.molcatb.2014.01.006

Accepted Manuscript
Title: A High-Detergent-Performance, Cold-Adapted Lipase
from Pseudomonas stutzeri PS59 Suitable for Detergent
Formulation
Author: Xiao-Lu Li Yan Shi Wen-Hui Zhang Yu-Jie Dai
Hui-Tu Zhang Yue Wang Hai-Kuan Wang Fu-Ping Lu
PII:
S1381-1177(14)00008-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.01.006
MOLCAB 2863
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
22-8-2013
30-11-2013
1-1-2014
Please cite this article as: X.-L. Li, Y. Shi, W.-H. Zhang, Y.-J. Dai, H.-T. Zhang, Y. Wang,
H.-K. Wang, F.-P. Lu, A High-Detergent-Performance, Cold-Adapted Lipase from
Pseudomonas stutzeri PS59 Suitable for Detergent Formulation, Journal of Molecular
Catalysis B: Enzymatic (2014), http://dx.doi.org/10.1016/j.molcatb.2014.01.006
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A High-Detergent-Performance, Cold-Adapted Lipase from Pseudomonas stutzeri PS59 Suitable for
Detergent Formulation
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Xiao-Lu Li, Yan Shi, Wen-Hui Zhang, Yu-Jie Dai, Hui-Tu Zhang, Yue Wang, Hai-Kuan Wang2*, Fu-
Ping Lu1*,
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Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of
Biotechnology, Tianjin University of Science and Technology, Tianjin, People’s Republic of China
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Abstract
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A high-detergent-performance and cold-adapted lipase was purified and characterised from
Pseudomonas stutzeri PS59, which was isolated from Daqing oil fields (Heilongjiang, PR China). The
lipase was purified to homogeneity using ammonium sulphate precipitation, dialysis, freeze-drying, ion
exchange chromatography and gel filtration chromatography. The molecular weight of the lipase was
approximately 55 kDa, as measured by SDS-PAGE. The lipase showed optima activity at pH 8.5 and 20
°C. The lipase activity was activated by metal ions, such as Ca2+ and Mn2+, and surfactants, such as
Tween 80, Tween 20, sodium dodecyl benzene sulfonate and urea. Oxidising agents, such as H2O2 and
NaClO, were found to have little effect on the activity of the lipase, and most organic solvents can
enhance the activity of the lipase. The broad substrate specificity and the compatibility of the lipase in the
presence of surfactants, oxidising agents, and other detergent additives clearly indicate its potential
application in the laundry industry. The hydrolysis resolution of (R, S)-ethyl 2-Methylbutyrate by P.
stutzeri PS59 lipase was carried out with the yield of 31.2% for R-ethyl 2-Methylbutyrate, the
enantiomeric excess of residual substrate (ees) was 85.7%. Thus, the lipase also showed an attractive
potency for application in biocatalysis.
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Keywords: Pseudomonas stutzeri; Lipase; Purification; Washing performance; Detergent.
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* Corresponding author; E-mail: lfp@tust.edu.cn
Tel: ++86 22 6060 1958; fax: 86 22 6060 2298.
2 ,* Corresponding author; E-mail: hkwang@aliyun.com
Tel: ++86 22 6060 1958; fax: 86 22 6060 2298.
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1. Introduction
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Microbial lipases are one of the largest groups of industrial enzymes and have applications in a
wide range of industrial and household products, including detergents, leather, silk, food and
pharmaceuticals [23,10]. However, the single largest market for their use is in the detergent industry.
For this reason, increasing numbers of lipases have been exploited by researchers in the detergent
industry. Lipases from P. alcaligenes (Lipomax®) and P. mendocina (Lumafast®) are produced as
detergent additives by Genencor [15]. These enzymes have been widely used in the detergent
industry since their introduction in 1913 by Rohm. Various commercial detergent lipases, such as
Lipolase and Polarzyme (Novozymes, Denmark), Lumafast (Genencor, USA), and Lipofast
(Advanced Biochemicals, India) are available in the market [11]. The lipases produced by Candida
cylindracea and Aspergillus niger have been tested to determine their efficiency in an aqueous
solution of lipase with or without surfactants under various conditions for the removal of olive oil
from cotton fabric [7,18]. A novel alkaline lipase from Burkholderia cepacia RGP-10, which is used
in detergent formulations, exhibits better stability toward commercial detergents and oxidants [26].
All of these lipases are used in the detergent industry for the removal of fatty residues in laundry.
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The performance of lipases in detergents depends on various factors, such as the pH of the
detergent, ionic strength, washing temperature, detergent composition, washing procedure, and wash-
water hardness. However, one of the most important factors for the application of detergent enzymes
is the detergency of the enzymes. There is always a need for novel enzymes with novel properties
that can further enhance the washing performance of the currently used enzyme-based detergents;
however, few novel enzymes have been recently exploited based on their detergency.
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To generate a novel lipase with a high washing performance, Lipolase was modified by
novozymes using protein engineering, and Lipolase Ultra was achieved by replacing serine 96 in the
active centre of Lipolase with leucine. In washing experiments, the detergency of Lipolase Ultra was
found to be four-fold higher than that of Lipolase under the same washing conditions [30]. Lipases
with cold activity, thermostability, alkaline stability and resistance to metal ions, organic solvent, and
various detergent additives are attractive to researchers and have been isolated for use in detergents
[21,2,14]. However, the washing performance was usually ignored. The Ralstonia pickettii lipase
retains high activity in the presence of most detergents but achieves only a 39.5% removal rate of
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olive oil from cotton fabric in the same washing test [12]. The removal rates of olive oil from cotton
fabric in the presence of lipase from Cryptococcus sp. S-2 under its optimum conditions was found to
be only 19.6 and 43.1 % at 20 and 37 °C, respectively [31]. Kamini et al. demonstrated that the oil
removal rate from cotton fabric by the lipase produced by Aspergillus niger is approximately 55%
[18]. In this artile, the lipase from Pseudomonas stutzeri PS59 shows about 75% of removal rate of
olive oil. The lipase shows the maximum activtiy at 20°C and keeps high stablity at a wide range of
pH 4-11. Based on these reports, all novel detergent lipases should be evaluated based on their
washing performances. In addition, even though these novel lipases exhibit good properties, it is
obvious that the lipase with superior washing performance is the most desirable lipase to be included
in the formulation of a detergent.
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In present study, a number of bacterial strains were isolated, and the washing performances of
their lipases were evaluated. A strain producing a high-washing-performance lipase was isolated and
identified as Pseudomonas stutzeri. This lipase from the novel isolated bacterium P. stutzeri PS59
was also purified and characterised.
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2. Materials and Methods
2.1. Materials
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p-Nitrophenyl palmitate (p-NPP) was obtained from Sigma (St. Louis, OH, USA). The surfactants
Tween® 80, Tween® 20, Triton® X-100, sodium dodecyl sulphate (SDS), sodium dodecyl sodium
sulphate (SDBS) and sodium lauryl sulphate (IPC-SDS) were obtained from Sinopharm Chemical
Reagent Co. Ltd. All other chemicals were of analytical grade.
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2.2. Screening and isolation of high-detergent-performance and cold-adapted lipase producing strains
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Soil samples were collected from the areas contaminated by crude oil in the Daqing oil fields in PR
China. In the first screen, those microorganisms with high detergent performance were screened using an
enriched medium (1% yeast extract, 0.1% K2HPO4, 0.2% MgSO4•7H2O, and 2% olive oil emulsion, pH=
9.0). The flasks were incubated at 30 °C and 180 rpm for 72 h. The cultures were then acclimated by their
successive transfer to a fresh enrichment medium three times. The enriched culture was spread after its
serial dilution on Victoria blue agar plates (0.1% K2HPO4, 0.3% NaNO3, 0.05% MgSO4•7H2O, 0.001%
FeSO4•7H2O, 2% olive oil emulsion with 0.2% Victoria blue B, and 1.5% agar, pH= 9.0). The screening
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plates were incubated at 30 °C for 48 h. The colonies with dark blue zones were isolated and transferred
to slant medium (1% peptone, 0.5% yeast extract, and 1% NaCl, pH= 8.0).
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In the second screen, those strains identified in the first screen were inoculated into the second
screen medium (4% peptone, 2% sucrose, 0.1% K2HPO4, 0.05% MgSO4•7H2O, and 1% olive oil, pH=
8.0). The flasks were incubated at 30 °C and 180 rpm for 36 h in a rotary shaker. The crude lipases were
obtained by centrifugation at 8000 rpm and 4 °C for 15 min. Cloth washing experiments were then
performed using the crude lipases as the detergent. In particular, the high-detergent-performance lipase
producing strains were screened through repeated washing experiments. The strain producing the lipase
with higher detergent performance was selected for further studies.
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2.3. Assay of lipase activity
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The lipase activity was determined by alkali titration using olive oil as the substrate, as described by
Wang et al. with some modifications [34]. In brief, an assay mixture consisting of 1 ml of olive oil, 3 ml
of 2% polyvinyl alcohol, 5 ml of phosphate buffer (20 mM, pH= 8.5), and 1 ml of suitably diluted culture
broth was incubated at 30 °C and 180 rpm for 15 min. The reaction was terminated by the addition of 15
ml of 95% ethanol, and the amount of liberated fatty acids was titrated with 50 mM NaOH in the presence
of two or three drops of phenolphthalein (0.5%, w/v in ethanol) as the indicator. One unit of lipase
activity was defined as the amount of enzyme required to liberate 1 µmol of free fatty acid per minute
under the experimental conditions.
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A different lipase assay was performed using spectrophotography, as described previously by Wang
et al. with some modifications [33]. The substrate solution was prepared by dissolving 30 mg of pNPP in
10 ml of isopropanol. The assay mixture consisted of 100 µl of suitably diluted enzyme, 810 µl of buffer,
and 90 µl of the substrate solution. The mixture was incubated at 30 °C for 15 min, and the amount of
liberated p-nitrophenol was determined at 410 nm. One unit of enzyme activity was defined as the amount
of enzyme that liberated 1 µmol of p-nitrophenol per minute under the assay conditions.
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2.4. Evaluation of enzymes for detergent performance
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To determine the efficiency of the employed lipases, which were produced from the strains identified
in the first screen, for use as a bio-detergent additive, 10 X 5 cm white cotton fabric cloths were stained
with olive oil and washed in phosphate buffer (pH = 8.0) in the presence of the studied lipases.
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The cotton cloths were previously degreased in boiling chloroform to ensure that all lipids were
removed from the cotton cloths prior to soiling. Both sides of the cotton cloth were impregnated with 0.5
ml of olive oil in acetone solution (100 mg/ml) and dried for 15 min at room temperature. The stained
cloth pieces were incubated for 1 h at 30 °C on a rotary shaker (180 rpm) in 250 ml Erlenmeyer flasks
containing 100 ml of the washing solution, which consists of phosphate buffer (pH= 8.0) and the crude
lipases (total of 100 U).
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The weight of the cotton cloths was measured before and after the addition of olive oil. The weight
of the stained cotton cloth was also measured after washing. The efficiency of olive oil removal, ω, was
calculated taking into account the weight of the cotton cloths before and after the addition of olive oil and
the weight of the stained cotton cloths after washing and is expressed by the following equation:
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Wb −Wa
Wb −Wc
%ω =
×100
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Where Wa and Wb represent the weights of the cotton cloth before and after the addition of olive oil,
respectively, and Wc is the weight of the stained cotton cloth after washing. All of the washing tests were
performed three times, and the standard error was also calculated [31,18,9].
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2.5. Bacterial strain identification
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Strain PS59 was identified through 16S ribosomal DNA (rDNA) analysis. The 16S rDNA sequence
of the isolated strain was determined by Shanghai Sunny Biotechnology Co, Ltd. A homology search
using the reference strains registered in DDBJ/EMBL/GenBank was performed using NCBI BLAST.
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2.6. Enzyme production medium and culture condition
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P. stutzeri strain PS59 was cultured in the seed medium (Luria-Bertani broth medium) containing
(w/v) 1% peptone, 0.5% yeast extract, and 0.5% NaCl (pH= 8.0). A 2% inoculum of the seed medium
was then transferred into the lipase-producing medium (1% peptone, 0.8% sucrose, 0.1% K2HPO4, 0.05%
MgSO4•7H2O, 0.02% CaCl2, and 1% olive oil, pH= 8.5). The cultivations were performed in 500 ml
Erlenmeyer flasks containing 60 ml of medium on a rotary shaker (180 rpm/min) for 36 h at 30 °C. The
crude lipase was obtained by centrifugation at 8000 rpm and 4 °C for 15 min.
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2.7. Purification of the lipase
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The cell-free supernatant (800 ml) was precipitated overnight at 60% saturation with (NH4)2SO4 at 4
°C, and the pellet was collected after centrifugation. The precipitate was dissolved in 120 ml of phosphate
buffer (pH= 8.0) and dialysed in deionised water for two days. The removal of ammonium sulphate was
monitored with BaCl2. A partially purified lipase powder was obtained by freeze-drying the lipase
solution on a vacuum freeze drier. The partially purified lipase was then subjected to ion exchange
chromatography. Approximately 5 ml of the partially purified enzyme was loaded onto a Hitrap Q HP
column (5 X 5 cm) pre-equilibrated with 20 mM phosphate buffer (pH= 7.0). The fractions were eluted
from the column using a linear NaCl gradient (0-1 M). Fractions of 1 ml each were collected at a flow
rate of 1 ml min-1. The active fractions were pooled and concentrated by ultrafiltration. The concentrated
fraction was then chromatographed on a Superdex 200 column pre-equilibrated with 20 mM phosphate
buffer (pH= 8.0) at a flow rate of 0.5 ml min-1. The fractions were monitored continuously at 280 nm for
protein and also assayed individually for lipase activity. The active fractions were stored at -20 °C. The
purity of the fractions showing maximum activity were further analysed by electrophoresis.
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2.8 Sodium dodecyl sulphate polyacrylamide gel electrophoresis
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SDS-PAGE was conducted to determine the purity and molecular weight of the enzymes using 5%
(w/v) stacking and 12% (w/v) separating gels. The samples were prepared by mixing the purified
enzymes (v/v) with distilled water containing 10 mM Tris-HCl (pH 8.0), 2.5% SDS, 10% glycerol, 5% β-
mercaptoethanol and 0.002% bromophenol blue. The samples were heated at 100 °C for 5 min before
electrophoresis. After electrophoresis, the gels were stained with 0.25% Coomassie Brilliant Blue R-250
in 45% ethanol/ 10% acetic acid and destained with 10% ethanol/ 10% acetic acid. The molecular weights
of the purified lipases were estimated using a low-molecular-weight calibration kit as the marker.
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2.9 Effects of pH on the activity and stability of the lipase
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The optimum pH of the lipase was measured by varying the pH of the assay reaction mixture using
the following buffers (0.1 M): sodium phosphate (pH 6-9) and glycine NaOH (pH 9.0-10.5). The stability
of the lipase was analysed after the lipase was pre-incubated for 20 min, 40 min, and 1 h in the various
buffer solutions (pH 4-11). The residual enzyme activity was then measured under the standard assay
conditions.
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2.10 Effects of temperature on the lipase activity and stability
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The optimal temperature of the lipase was evaluated by measuring the lipase activity at different
temperatures (0-50 °C) in 0.1 M phosphate buffer (pH= 8.0). The effect of temperature on the lipase
stability was determined by measuring the residual activity of the lipase after its incubation in 0.1 M
phosphate buffer (pH 8.0) at temperatures ranging from 30 to 80 °C for 15 min, 30 min, and 60 min.
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2.11 Effect of various metal ions, surfactants, and oxidising agents on the stability of the lipase.
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The effects of different metal ions (Zn2+, Cu2+, Fe2+, Ba2+, Mn2+, K+, Ca2+, and Mg2+) and EDTA
were investigated by pre-incubating the lipase with 1 and 10 mM solutions of these ions and EDTA for 4
h at 30 °C, and aliquots were withdrawn at half-hour time intervals and analysed for residual lipase
activity. The enzyme activity was determined as a percentage of the relative activity compared to that of
the control (without metal ions and EDTA), which was considered 100%.
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To evaluate the stability of the lipase in the presence of additives, the lipase preparation was
incubated in the presence of various surfactants and oxidising agents at a concentration of 1% (v/v) in
assay buffer (0.1 M phosphate buffer, pH= 8.0). Bile salt, sodium deoxycholate, SDS, CTAB, and SDBS
were used at a concentration of 0.1% (w/v), and sodium perborate was used at concentration of 0.1% and
0.5%. The experiment was performed on a rotary shaker (150 rpm) for 4 h to simulate laundry-washing
conditions. Lipase samples were collected at 30 min intervals to analyse the lipase activity by the pNPP
method. The control test was performed under the same conditions without the additives. The residual
activity in each sample was calculated with respect to that of the control, which was considered 100%.
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2.12 Effect of organic solvents on the lipase activity
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The effects of organic solvents at a concentration of 15% (v/v) on the activity of the lipase were
investigated. The lipase was mixed with the organic solvents (DMSO, acetone, glycerol, ethanol,
isopropanol, dichloromethane, butyl alcohol, caprylic alcohol, and caproic acid) and incubated at 30 °C
and 150 rpm on a shaker. The residual lipase activity with respect to that of the control, which was
considered as 100%, was measured at appropriate time intervals over a period of 4 h under the optimised
assay conditions.
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2.13 Analysis of the substrate specificity of the P. stutzeri PS59 lipase
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An assay mixture consisting of 1 ml of liquid ester or 1 g of solid ester, 3 ml of isopropanol, 5 ml of
phosphate buffer (pH= 8.0), and 1 ml of the lipase solution was incubated for 15 min at 30 °C with
stirring at 180 rpm. The reaction was terminated by the additon of 95% ethanol, and the amount of
liberated fatty acids after incubation was determined by titrating with 50 mM NaOH in the presence of
two drops of phenolphthalein solution as the indicator. The control experiment was performed under the
same conditions with the addition of 95% ethanol prior to the reaction. One unit of lipase activity was
defined as the amount of enzyme required to liberate 1 µmol of free fatty acid per minute under the
experimental conditions.
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2.14 Bioresolution of (R, S)-ethyl 2-Methylbutyrate by P. stutzeri PS59 lipase
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The kinetic resolution of (R, S)-ethyl 2-Methylbutyrate was carried out in 10 ml phosphate buffer
(50mM) containing 2 mM (R, S)-ethyl 2-Methylbutyrate catalyzed by 10 mg P. stutzeri PS59 lipase at 30
°C with shaking at 180 rpm.
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The amounts of optical isomers were determined by GC using Agilent-β-DEX-120-Chiral
cyclodextrin capillary GC column (30 m × 0.25 mm × 0.25 μm). The chromatographic conditions
employed were: carrier gas, hydrogen at 60 kPa; the flow rate, 2.0 ml/min split ratio, 1:100; injection
volume, 1ul; injector temperature, 250 °C; detector temperature, 300°C; oven temperature program,
initially at 40 °C and increased at 10 °C/min to 120 °C; total time of analysis, 10 min.
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2.14 Statistical analysis
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The experimental results are expressed as the means ± standard deviation (S.D.). In this study, all of
the experiments were performed in triplicate, and the samples were analysed in duplicate or triplicate. The
Origin8.0 software (OriginLab, USA) was used for the statistical evaluations.
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3. Results and Discussions
3.1 Isolation of high-washing-performance-lipase-producing bacteria
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The first screen analysed a total of 278 lipase-producing bacterial strains that were isolated from the
soil samples collect for this study. Forty-five of these strains, which produced larger fading rings, were
isolated and subjected to the washing performance test. In the first washing experiment, the lipases of the
tested eight strains showed superior washing performance (Fig. 1a). Of these eight strains, five lipase-
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producing strains exhibiting high washing performance were used for the repeated washing test (Fig. 1b),
which revealed that strain PS59 secreted an extracellular lipase with high washing performance at low
temperature and high alkaline conditions. Thus, this strain was therefore selected for the subsequent
experiments.
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Fig. 1.
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Strain PS59, which produces a high-washing-performance, cold-adapted lipase, was identified as
Pseudomonas stutzeri based on an analysis of its 16S rDNA sequences, which revealed 100% homology.
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3.2 Enzyme purification
Table 1
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The lipase from P. stutzeri PS59 was purified to homogeneity, and the results are shown in Table 1.
The lipase was precipitated using ammonium sulphate (60% saturated) to yield an active fraction with a
93% yield and a 3.75-fold increase in its specific activity. The precipitate was dissolved in double
distilled H2O, and the enzyme solution was dialysed using a MD77 bag to remove the salt. During
dialysis, a yield of 88.7% was achieved with 3.64-fold purification. The lipase solution was freeze-dried
to yield the crude lipase with a loss of approximately 5.0% of the enzyme activity. The crude lipase
powder was used as a starting material for further purification. After ion exchange chromatography (Q-
Sepharose), a specific activity of 330.6 U/mg was obtained with 11.7-fold purification. The purification
using ion exchange chromatography resulted in a peak of the active fraction at 60% NaCl. Further
purification using gel filtration chromatography resulted in two peaks, and the first peak contained the
active fraction with a specific activity of 631 U/mg and 22.45-fold purification. SDS-PAGE analysis of
the first peak obtained through gel filtration chromatography clearly revealed a single protein band
corresponding to a molecular mass of approximately 55 kDa (Fig. 2). The SDS-PAGE analysis indicated
that the first peak exhibited a high purity of the active lipase fraction.
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Fig. 2
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The lipases of Pseudomonas were mainly classified into three groups [15]. Group lipases have a
higher molecular mass (approximately 55 kDa) than the other lipases. Thus, based on the molecular mass
of the lipase investigated in our study, the lipase produced by P. stutzeri PS59 is a group lipase.
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3.3 Effects of pH on the lipase activity and stability
Fig. 3.
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The lipase characterised herein was found to be functionally active in the pH range of 7.0 to 11.0.
The lipase showed optimal activity at pH 8.5 (100%) and showed approximately 80% residual activity at
pH 9.0 (Fig. 3a). The P. stutzeri PS59 lipase was found to be active and stable over a wide pH range of 4
to 11, which indicates its potential applicability in the laundry industry (Fig. 3b). These findings are in
accordance with several earlier reports. For example, the lipase from Ralstonia pickettii was stable at high
pH [12], and a novel lipase from the fungus Talaromyces thermophilus was stable in a wide pH range of 9
to 11 [2].
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3.4 Effects of temperature on the lipase activity and stability
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The activity of the lipase was determined at a wide range of temperatures (0-50 °C). The optimum
temperature for lipase activity was determined to be 20 °C. The lipase activity was almost constant
between 20-30 °C and gradually declined at temperatures below 15 °C and beyond 35 °C (Fig. 3c).
Similar cold-active lipases with optimal temperatures ranging from 15 to 30 °C have been identified from
other microbial strains, such as Yarrowia lipolytica NCIM 3639 (25 °C), Acinetobacter sp. strain no 6 (20
°C), and Geotrichum sp. SYBC WU-3 (two cold active lipases, 20 and 15 °C) [35,29,3]. Moreover, a
cold-active lipase of marine Antarctic origin exhibited maximum activity at 20 °C [24]. A lipase from
Aeromonas sp. LPB 4 was described to be highly active at 10 °C [20]. In general, fat stains are not easy to
remove at low temperatures using conventional detergents; therefore, it is necessary to identify cold
active lipases that can be used in detergent formulations. The cold active lipases used in detergent
formulations can reduce the energy consumption as well as the wear and tear on textile fibres to maintain
the texture and quality of fabrics [6]. The lipase investigated in this study was also found to be stable at a
broad temperature range (30-60 °C) (Fig. 3d). The activity and stability profile of the lipase was better or
comparable with that of other cold active lipases that have been reported to be compatible with detergent
formulations [17,35].
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In most developing countries like China and India, cold water was usually adopted for cloth washing.
Therefore, a detergent enzyme, which exhibits high activity at low temperature, will be more practical
than those thermophilic ones. In this article, a high-detergent-performance and cold-adapted lipase was
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isolated and characterized. The lipase shows high specific activity of 631 U/mg, which is comparable to
the commercialized Lipase TL (http://www5.mediagalaxy.co.jp/meito/kaseihin/lipase/data/lip_tl.html).
But, compared with lipase TL, the newly isolated lipase exhibits the optimum activity at lower
temperature of 20 °C and it shows high stability at a wide pH range from 4 to11. According to our
research, the P. stutzeri PS59 lipase is more suitable for washing at low temperature than lipase TL.
These superior properties make the lipase an ideal candidate for application in detergent industry.
7
8
3.5 Stability of the lipase in the presence of various metal ions, surfactants, and oxidising agents
Fig. 4.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
The compatibility of the P. stutzeri PS59 lipase with various concentrations of metal ions,
surfactants, and oxidising agents was investigated. Of the metal ions tested at concentration of 1 mM and
10 mM, the P. stutzeri PS59 lipase showed high activity and stability in the presence of Ca2+ and was
inhibited by EDTA (Fig. 4a and 4b). In the initial phase, Ca2+ addition activated the lipase (up to 24.1%
and 19% compared with the control at 1 mM and 10 mM, respectively). It suggests that the enzyme needs
calcium as a catalytic activator. The activity of most lipases from Pseudomonas has been found to be
enhanced by Ca2+ [37,1,19,4]. This increase in the activity of the lipase is observed because calcium plays
a vital role in the construction of a stable catalytic enzyme structure, which is a result of the calcium ions
binding to the internal structure of the enzyme. At metal ion concentrations of 1 mM and 10 mM, the
lipase was also slightly activated by Zn2+, Mn2+ and K+ during the first half-hour and repressed by Cu2+,
Fe2+, Ba2+, and Mg2+ (Fig. 4a and b). After 30 min of incubation with 1 mM and 10 mM Cu2+, Fe2+, Ba2+,
and Mg2+, the lipase retained approximately 80 and 70% residual activity, respectively. Two hours later,
the lipase exhibited more than 50% residual activity in the presence of most metal ions (except 10 mM
Cu2+ and Fe2+). The results shown in Fig. 4a and 4b reveal that Ca2+, Zn2+, and Mn2+ can enhance the
enzyme activity, whereas Cu2+, Fe2+, Ba2+, and Mg2+ inhibited the enzyme activity. K+ had little effect on
the enzyme activity. Most Pseudomonas lipases are inhibited by heavy metal ions such as Zn2+, Hg2+,
Cu2+, Ni+, Cd+, Fe2+, and Co2+, and activated by Ca2+ and Mg2+ [28]. An alkaline lipase from the newly
isolated Pseudomonas mendocina PK-12CS was found to be inhibited by Hg2+, Fe2+, Zn2+ and activated
by Mg2+ [16]. The activity of the lipase produced by Pseudomonas gessardii was found to be significantly
enhanced by Ca2+, slightly less enhanced by Mg2+, and inhibited by other metal ions, such as Zn2+, Fe2+
and Cu2+ [25].
11
Page 11 of 33

1
Fig. 5.
2
3
The industrial applications of surfactants and oxidising agents are mainly related to detergents.
These chemicals can destroy the activity of a lipase by changing the lipase’s tertiary structure. However,
these additives may preserve lipase activity and stability by inhibiting lipase aggregation. Hence their
addition may enhance lipase activity. As shown in Fig. 5a, the lipase investigated in the present study was
stable in the presence of most ionic and non-ionic surfactants. The lipase activity can be promoted by the
presence of Tween 20, Tween 80, SDBS and urea by 35%, 47%, 27% and 33%, respectively for the first
15 min of incubation, and the lipase retained approximately 80% or more residual activity after longer
incubation period. In addition, the lipolytic activity was inhibited by the presence of SDS, sodium
deoxycholate, bile salt, IPC-SDS, and CTAB, whereas Triton X-100 had little effect on the lipase activity.
It was obvious that the ionic interaction between most ionic surfactants (SDS, IPC-SDS, and CTAB) and
the enzyme plays an important role in the inactivation of globular proteins [27,4]. In contrast, the absence
of nonionic surfactants can reduce the harmful electrostatic interactions to preserve the activity of the
enzyme. The activation and promotion of the enzyme by some nonionic surfactants such as Tween 80 (up
to 126%), was due to the increase in the conformation flexibility of the active site, which is a result of the
formation of hydrogen bond between the surfactant molecules and the enzyme [36]. As previously
reported, Lipolase® maintains 68.5%, 52.3%, 31.6% and 26.7% residual activity after one hour of
incubation in the presence of Triton X-100, Tween 80, Tween 20 and SDS [21], respectively, whereas the
P. stutzeri PS59 lipase maintained 78.2%, 126%, 80.1%, and 40.8% residual activity, respectively.
Compared with commercial Lipolase®, some properties of the P. stutzeri PS59 lipase suggest that it can
potentially be applied in the detergent industry. In addition, the choice of surfactants is notably important
in detergent formulations containing lipases. The activity of the lipase produced from indigenous
Pseudomonas aeruginosa was found to be strongly inhibited by Tween 80 [9], whereas Tween 80 was
found to significantly promote the activity of the lipase of P. stutzeri PS59 in our study.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Furthermore, the lipase produced by P. stutzeri PS59 was evaluated in the presence of oxidising
agents (Fig. 5b). Stability of lipases in the presence of oxidising agents is an important property of lipases
in detergent formulations and has been achieved through protein engineering or site-directed mutagenesis
[8]. Compared with Lipolase® (Novozyme, Denmark), which maintains 82.5% and 46.3% residual
activity after one hour of incubation in the presence of H2O2 and NaClO, respectively, the lipase produced
12
Page 12 of 33

1
2
3
4
by P. stutzeri PS59 retained 77 and 56.6% residual activity. In addition, a low concentration (1%) of
sodium perborate can promote the activity of the lipase during the first 1.5 h of incubation, the activity of
the lipase declined to approximately 80% residual activity after 1.5 h. Moreover, the activity of the lipase
was inhibited by a high concentration (5%) of sodium perborate.
5
6
3.6 Effect of organic solvents on the lipase
Fig. 6.
7
8
The hydrolysation of water-insoluble substrates by lipases must take place at an interface. Mixtures
of water and organic solvents contribute to the bioconversion of water-insoluble substrates by lipases.
Although organic solvents damage the activity of enzymes, the activity of enzymes in mixtures of water
and organic solvents has been reported [5]. In general, enzymes would lose their activity in the presence
of organic solvents at concentrations of 10-20%. The activity and stability of the enzymes in organic
solvents depend not only on the properties and concentration of the organic solvents but also on the nature
of the enzyme [32]. For this reason, the effects of various organic solvents at a concentration of 15% (v/v)
on the lipase from P. stutzeri PS59 were determined, and the results are presented in Fig. 6.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
The P. stutzeri PS59 lipase retained more than 100% residual activity (containing 100%) in the
presence of isopropanol, ethanol, butyl alcohol, glycerol, DMSO, and caprylic alcohol during the first half
hour of incubation and retained 139.1%, 126%, and 107.5% relative activity after 1 h of incubation in the
presence of caprylic alcohol, glycerol, and DMSO, respectively (Fig. 6). However, the lipase activity
declined to 69 and 58.2% after one hour of incubation in the presence of ethanol and butyl alcohol.
Isopropanol had no effect on the lipase activity. In addition, the lipase activity suffered some inhibition in
the presence of acetone, dichloromethane and caproic acid. The P. stutzeri PS59 lipase maintained high
activity and stability in the presence of caprylic alcohol, glycerol, DMSO, butyl alcohol, ethanol, and
isopropanol. The stability of the lipase in these organic solvents is due to the fact that these solvents play
a key role in stabilising the enzymatic activity in the solution. However, the inhibition of the lipase by
acetone, dichloromethane, and caproic acid may be due to the adsorption of the solvents on the substrate
and the resulting repression of the enzyme’s interaction with the substrate [22]. In addition, the residual
activity of the lipase after one hour of incubation in several other organic solvents was listed in table 2.
28
Table 2
13
Page 13 of 33

1
2
3.7 Substrate specificity of the lipase
Fig. 7.
3
4
In general, an oil stain consists of many different long-chain fatty acid esters. The washing
performance of a lipase depends on the hydrolytic ability of a lipase. The substrate specificity of the
lipase to various esters was detected through alkaline titration. As shown in Fig. 7, the lipase from P.
stutzeri PS59 can hydrolyse most long-chain fatty acid esters. To study the substrate specificity of the
lipase, the activity for triolein was considered to be 100%. The lipase showed 89.3%, 83.3%, and 78.6%
relative activity for stearin, palmitin, and gallicin, respectively. In addition, the activity of the lipase for
ethyl palmitate, ethyl linoleate, and ethyl oleate was 74.7%, 58.4%, and 53%, respectively. In addition,
the lipase showed 72.6%, 51.6%, 33.1%, 31.0%, and 38.1% relative activity for methyl palmitate, methyl
stearate, methyl oleate, methyl linoleate, and methyl laurate, respectively. Based on these data, we
determined that the lipase can effectively hydrolyse triglycerides and has a broad specificity for other
esters. Furthermore, the lipase can be potentially used for the removal of oil stains, as reflected by the
directly measured washing performance of the lipase.
5
6
7
8
9
10
11
12
13
14
15
3.8 Bioresolution of (R, S)-ethyl 2-Methylbutyrate by P. stutzeri PS59 lipase in an aqueous system
16
17
18
19
20
21
22
23
(R, S)-ethyl 2-Methylbutyrate is one of the typical flavor compounds used in food. The (R, S)-ethyl
2-Methylbutyrate was hydrolyzed by P. stutzeri PS59 lipase in an aqueous system, and the P. stutzeri
PS59 lipase was found to be selective towards (S)-ethyl 2-methylbutyrate (Fig.8). The time course of
resolution of (R, S)-ethyl 2-Methylbutyrate showed that the 66.4% of substrate was conversed, and the
residual substrate, (R)-ethyl 2-methylbutyrate, remained with the yield of 31.2% after 12 h of reaction,
and the enantiomeric excess of (R)-ethyl 2-methylbutyrate reached 85.7%, giving an E-value over 5
(Fig.9), which is higer than other lipases such as lipase (E=3.6) produced from Candida cylindracea[13].
Thus the P. stutzeri PS59 lipase exhibited a relatively high enantioselectivity for ethy 2-Methylbutyrate.
24
25
26
Fig.8.
Fig.9.
4 Conclusions
14
Page 14 of 33

1
2
3
4
5
6
7
8
In conclusions, a high-detergent-performance, cold-adapted extracellular lipase from the P. stutzeri
PS59 was purified and identified. The lipase has an optimum temperature and pH of 20 °C and 8.5,
respectively. Moreover, the analysis of the stability of the enzyme in the presence of various metal ions,
surfactants, oxidising agents, and solvents, as well as its broad substrate specificity, demonstrates that the
enzyme exhibits potential as a commercial additive in detergents. And the lipase showed good
enantioselectivity towards (R, S)-ethyl 2-Methylbutyrate. These indicated that the lipase can be a very
attractive enzyme for potential application in biocatalysis. Further research is needed to study the
mechanism of the high-washing performance of the P. stutzeri PS59 lipase.
9
Acknowledgments
10
11
12
Financial support was provided by the Hi-Tech Research and Development Program of China (863
Planning: Grant number: 2013AA102803; Task number: 2013AA102803C) and the program for
Changjiang Scholars and Innovative Research Team in University (IRT1166).
13
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35. Yadav KNS, Adsul MG, Bastawde KB, Jadhav DD, Thulasiram HV, Gokhale DV (2011)
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37. Zhang A, Gao R, Diao N, Xie G, Gao G, Cao S (2009) Cloning, expression and
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1
Figure captions
2
3
4
5
6
Fig. 1. (a) The washing performance of eight lipases analysed in the second screen of the isolated strains.
(b) Repeated assay of five lipases that exhibited the highest washing performance in the second screen.
Fig. 2. SDS-PAGE of the purified lipase. Band 1: standard protein markers (Da); Band 2: crude lipase
solution (20 mg/ml); Band 3: the lipase purified by Q-sepharose; Band 4: the lipase purified by Superdex
200.
7
8
Fig. 3. (a) Effect of pH on lipase activity. The lipase activities were evaluated at 20 °C in the pH range of
6.0 to 10.5 using buffers with different pH values. The maximum activity was obtained at pH 8.5
(considered 100%). (b) pH stability of the lipase. The lipase activity is determined after 20 min, 40 min,
and 60 min of incubation at 30 °C in the presence of sodium phosphate buffer (pH 3.0-8.0) and glycine
NaOH buffer (pH 9.0-11). (c) Effect of temperature on the activity of the lipase. The lipase activity was
measured at various temperatures. The activities are shown relative to that measured at 20 °C, which was
considered 100%. (d) Temperature stability of the purified lipase. The lipase activity was measured after
15 min, 30 min, and 60 min of incubation at pH 8.0 at the different temperatures. The activity of the
lipase incubated at 30 °C was considered 100%.
9
10
11
12
13
14
15
16
17
Fig. 4. Percentage of residual lipolytic activity as a function of time in the presence of (a) 1 mM metal
ions and (b) 10 mM metal ions.
18
19
Fig. 5. Percentage of residual lipolytic activity as a function of time in the presence of (a) various
surfactants and (b) various oxidising agents.
20
21
Fig.6. Percentage of residual lipolytic activity as a function of time in the presence of 15% organic
solvents.
22
23
Fig.7. Substrate specificity of the lipase. The activity of the lipase toward various esters was determined,
and the activityof the lipase for triolein was considered 100%.
24
25
Fig.8. Kinetic resolution of (R, S)-ethyl 2-Methylbutyrate catalyzed by P. stutzeri PS59 lipase in an
aqueous system
26
Fig.9. Time course of bioresolution of (R, S)-ethyl 2-Methylbutyrate catalyzed by P. stutzeri PS59 lipase.
20
Page 20 of 33

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3
Table 1
Summary of the steps used to purify the extracellular lipase produced by Pseudomonas stutzeri
Enzyme
Purification step activity (U
ml-1)
Specific
activity
(U mg-1)
Protein
Total activity
(U)
Yield
(%)
Fold
(mg ml-1)
purification
Cell-free
culture
43
1.53
2.05
28.1
27950
26004
100
93
1
supernatant
Ammonium
sulphate 60%
saturation
216.7
105.4
3.75
3.64
Dialysis
173.4
-
0.59
-
102.3
-
24800
23400
88.7
83.7
Freeze drying
Purified by Q-
sepharose
334
120
1.01
0.19
330.6
631
16200
12040
57.9
43.1
11.77
22.45
Purified by
Superdex 200
4
5
6
7
8
9
21
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5
6
Table 2
Effect of 15% organic solvents on the activity of the lipase from P. stutzeri PS59
Organic solvents
Methyl benzene
Cyclohexane
Residual activity (%)
98±3.5
Organic solvents
Acetic acid
Isoamylol
Residual activity (%)
79±3
102±2.5
109±3.8
114±5
Tetrahydrofuran
n-Hexane
108±5
Glycol
93±8.5
Diacetone
Diethyl ether
65±3
Ethyl acetate
±
84 1
±
77 3.6
7
8
22
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1
2
3
4
100
80
60
40
20
a
0
100
80
60
40
20
0
61-2 59-11 61-1 59-14 61-9 59-10 61-4 61-3 Lipex
lipases
first test
second test
b
59-11
59-14
61-9
59-10
61-4
Lipex
lipases
5
6
Lipex® 100L is produced by Novozymes*
Fig. 1.
7
8
9
10
11
12
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3
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5
6
7
8
Fig. 2.
9
10
11
12
13
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5
6
7
8
9
10
11
12
13
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17
18
19
20
21
a
100
80
60
40
20
0
100
80
60
40
20
0
c
6
7
8
9
10
11
0
10
20
T(℃)
30
40
50
pH
70
60
50
40
30
20
10
pretreatment of 15 min
pretreatment of 30 min
pretreatment of 60 min
d
100
80
60
40
20
0
b
pretreatment of 20 min
pretreatment of 40 min
pretreatment of 60 min
2
4
6
8
10
12
30
40
50
T(℃)
60
70
80
pH
Fig. 3
25
Page 25 of 33

1
2
3
4
a
Zn2+
Ba2+
Ca2+
Cu2+
Mn2+
Mg2+
Fe2+
K+
140
120
100
80
EDTA
60
40
20
0
0
30
60
90
120
150
180
210
240
Time(min)
5
Zn2+
Ba2+
Ca2+
Cu2+
Mn2+
Mg2+
Fe2+
K+
140
120
100
80
b
EDTA
60
40
20
0
0
30
60
90
120
150
180
210
240
Time(min)
6
7
8
Fig. 4.
9
10
26
Page 26 of 33

1
2
3
4
5
160
140
120
100
80
Tween 20
TritonX-100
sodium deoxycholate
CTAB
Tween 80
Bile salt
a
SDS
urea
SDBS
IPC-SDS
60
40
20
0
0
30
60
90
120
150
180
210
Time(min)
6
140
120
100
80
sodium perborate 0.1%
sodium perborate 0.5%
H2O2
b
NaClO
60
40
20
0
0
30
60
90
120
150
180
210
Time(min)
7
8
Fig. 5
9
10
11
27
Page 27 of 33

1
2
3
4
5
6
7
8
9
10
180
160
140
120
100
80
isopropanol
dichloromethane
DMSO
ethanol
butylalcohol
caprylic alcohol
acetone
glycerol
caproic acid
60
40
20
0
0
30
60
90
120
150
180
210
240
Time(min)
11
12
13
14
15
16
17
18
19
20
21
Fig. 6.
28
Page 28 of 33

1
2
3
4
5
6
7
8
phenylacetic ally(benzene)
ethyl palmitate(C16)
ethyl linoleate(C18,2n)
ethyl oleate(C18,1n)
methyl laurate(C12)
methyl palmitate(C16)
methyl oleate(C18,1n)
methyl linoleate(C18,2n)
methyl stearate(C18)
gallicin(benzene)
palmitin
stearin
Triolein
0
20
40
60
80
100
relative activtiy (%)
9
10
Fig. 7.
11
12
13
14
15
29
Page 29 of 33