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Biotechnology Advances 27 (2009) 782–798

Contents lists available at ScienceDirect

Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Methods for detection and characterization of lipases: A comprehensive review

Fariha Hasan, Aamer Ali Shah ⁎, Abdul Hameed
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Microbial lipases are very prominent biocatalysts because of their ability to catalyze a wide variety of
Received 4 March 2009 reactions in aqueous and non-aqueous media. The chemo-, regio- and enantio-specific behaviour of these
Received in revised form 3 June 2009 enzymes has caused tremendous interest among scientists and industrialists. Lipases from a large number of
Accepted 5 June 2009
bacterial, fungal and a few plant and animal sources have been purified to homogeneity. This article presents
Available online 17 June 2009
a critical review of different strategies which have been employed for the detection, purification and
characterization of microbial lipases.
Keywords:
Lipase
© 2009 Elsevier Inc. All rights reserved.
Detection
Purification
Characterization

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
2. Detection and measurement of lipolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
2.1. Screening methods in solid media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
2.2. Egg yolk as substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
2.3. Titrimetric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
2.4. Nephelometry and turbidimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
2.5. Released glycerol from triacylglycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
2.6. Electric conductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
2.7. The Wilhelmy plate method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
2.8. Acoustic wave conductance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
2.9. Colorimetric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
2.10. Fluorometric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
2.11. Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
2.12. Chromatographic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
2.12.1. HPLC methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
2.12.2. Gas chromatographic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
2.13. Interfacial tension monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
2.14. Test kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
2.15. Lipoprotein lipase ELISA kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
2.16. EnzChek lipase substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
2.17. Coumarin-based lipase substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
2.18. MarkerGene™ fluorescent lipase assay kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
2.19. QuantiChromTM lipase assay kit (DLPS-100) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
2.20. Immunological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
2.21. NMR method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
2.22. Atomic force microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
2.23. Infrared spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
2.24. Detection by electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
2.25. Radioactive assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

⁎ Corresponding author. Tel.: +92 51 90643185; fax: +92 51 9219888.

E-mail address: [email protected] (A.A. Shah).

0734-9750/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2009.06.001
 Author's personal copy

F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798 783

2.26. The oil-drop method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

2.26.1. Advantages and disadvantages of various Lipase assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
3. Factors affecting production of lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
3.1. Physical factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
3.2. Biochemical/physicochemical factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
4. Purification of Lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790
5. Characterization of lipase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
5.1. Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794

1. Introduction interface (specific surface) as well as the interfacial parameters (such

as the interfacial tension, surface viscosity, surface potential, etc.).
Lipases, triacylglycerol hydrolases, are an important group of bio- Lipolytic process depends on the “interfacial quality” of the substrate
technologically relevant enzymes and they find immense applications (Small, 1997; Dahim and Brockman, 1998; Panaitov and Verger, 2000).
in food, dairy, detergent and pharmaceutical industries. Lipases are by The emulsification of the water-insoluble substrates, which requires
and large produced from microbes and specifically bacterial lipases the presence at the interface of surface active amphiphiles such as
play a vital role in commercial ventures (Gupta et al., 2004). Lipases detergents, other lipids, proteins, etc., can therefore drastically in-
are also defined as glycerol ester hydrolases that catalyze the hydro- fluence lipase activity measurements (Gargouri et al., 1986).
lysis of triglycerides to free fatty acids and glycerol. Lipases catalyze Lipases with new specificities are needed and the engineering of
esterification, interesterification, acidolysis, alcoholysis and aminoly- cloned enzymes as well as the isolation of new lipases from natural
sis in addition to the hydrolytic activity on triglycerides (Joseph et al., sources is therefore of increasing potential value. Reliable, convenient,
2008). Novel biotechnological applications have been successfully and sensitive assays to detect a true lipase activity in cellular homo-
established using lipases for the synthesis of biopolymers and bio- genates are required. As with all reactions catalysed by enzymes,
diesel, the production of enantiopure pharmaceuticals, agro-chemi- activity measurements can be carried out using various physicochem-
cals, and flavour compounds (Jaeger and Eggert, 2002). ical methods (by monitoring the disappearance of the substrate or the
Taxonomically close strains may produce lipases of different release of the product) (Beisson et al., 2000).
types. There are many microorganisms known to produce different
lipases (Iizumi et al., 1990). Microbial lipases possessing either high 2. Detection and measurement of lipolytic activity
alkalophilic or thermophilic properties have been reported from
Alcaligenes (Kakusho et al., 1982). Pseudomonas fragi (Nishio et al., Several methods have been developed for the measurement of
1987) P. nitroreducens (Watanabe et al., 1977) and other Pseudo- lipase activity in crude or purified lipase preparations. Rates of lipase
monas strains were isolated from soil that produced lipase having reaction can be measured by determining either (a) the rate of dis-
different characteristics. Choo et al. (1998) isolated a psychrotrophic appearance of the substrate, the triglyceride (b) the rate of production
Pseudomonas strain from Alaskan soil, which produced a cold- of fatty acids or (c) the rate of clarification of emulsion (Smeltzer et al.,
adapted lipase at low temperatures. The oily environment (sewage, 1992).
rubbish dump sites and oil mill effluent) may provide a good envi-
ronment for lipolytic microorganisms to flourish and for isolation 2.1. Screening methods in solid media
of lipase producing microorganisms (Rahman et al., 2007).
When screening bacteria for lipase production both culture pH and Lipolysis is observed directly by changes in the appearance of
assay pH are important parameters. The stability depends upon the the substrate such as tributyrin and triolein, which are emulsified
presence of substrate (Andersson et al., 1979; Watanabe et al., 1977). mechanically in various growth media and poured into a petri dish.
Almost all microbial lipases can be regarded as acid lipases or neutral Lipase production is indicated by the formation of clear halos around
lipases if they are classified by their optimum pH value for the lipolytic the colonies grown on tributyrin- containing agar plates (Jaeger et al.,
activity (Iizumi et al., 1990). Quantitative analysis is important to 1994; Kim et al., 2001; Ertuğrul et al., 2007). Bacillus strains were
compare the lipolytic activities of various isolates. There is no single screened for lipolytic activity on agar plates containing tributyrin (1%,
universal method of lipase assay. The choice of a particular method w/v), agar (2%, w/v) in Luria–Bertani medium (Fakhreddine et al.,
will depend on the user's own specific requirements. For the assay of 1998). Lipolytic Bacillus sp LBN 4 was isolated on tributyrate agar
any enzyme, the sensitivity, availability of substrates, and ease of the medium using glycerol tributyrate as substrate (Bora and Kalita, 2007).
procedure have to be considered (Hendrickson, 1994). Visualization of lipolytic activity on solid media can be determined
Growth conditions such as availability of carbon and nitrogen by using dyes such as Victoria blue B, Spirit blue, Nile blue sulfate
sources, the presence of activators, stimulators, inhibitors, surfactants, and night blue (Shelley et al., 1987a). The drop in pH due to the fatty
incubation temperature, pH, and the level and source of inoculum acids released is observed by the change in colour of indicators used
(Hadeball, 1991) and oxygen tension (Chartrain et al., 1993) can in- (Scholze et al., 1999). There is a linear relationship between the
fluence the synthesis of lipase. According to Shelley et al. (1987b) three diameter of the fatty acid diffusion spot and the logarithm of the
factors must work together if a lipase- positive bacterium is to be enzyme concentration. This technique is very convenient for rapidly
detected: (1) the organism must grow; (2) the organism must produce screening of lipolytic microorganisms but acidification of the medium
or release lipase under the prevailing growth conditions; and (3) the due to the generation of acidic metabolism other than free fatty acids
detection method used must be of sufficient sensitivity. (FFAs), which are released by microbial lipases, can give false results.
One important aspect of lipolytic enzymes is the unique physico- Geobacillus zalihae sp. nov. was screened for the production of li-
chemical character of the reactions they catalyse at lipid–water inter- pase by using triolein agar plate. Triolein agar comprising of triolein
faces, involving interfacial adsorption and subsequent catalysis sensu (0.25%), bacteriological agar (1%), nutrient broth (0.8%) and Victoria
stricto. Most of the lipases are water-soluble enzymes acting on water- Blue (0.01%) was adjusted to pH 7.0 (Rahman et al., 2007).
insoluble substrates (supersubstrates). Due to this heterogeneous Fluorescent dye Rhodamine B can also be used in plate assay
character it is difficult to accurately quantitate both the amount of containing emulsified olive oil to detect lipolytic organisms. Substrate
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784 F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798

hydrolysis causes the formation of orange fluorescent halos around 2.4. Nephelometry and turbidimetry
bacterial colonies visible upon UV irradiation and the quantitation of
lipase activities range from 1 to 30 nkat (Kouker and Jaeger, 1987; Kim The method is based upon a comparison of the intensity of light
et al., 2001). Psychrotrophic bacteria were isolated from tundra soils of scattered by the sample under defined conditions with the intensity
Alaska and Siberia on agar plates of Luria-Bertani (LB) medium (pH of light scattered by a standard reference suspension. The higher the
7.6) at 4 °C. Lipase activity of the Pseudomonas sp. strain B11-1 was intensity of scattered light, the higher the turbidity (O'Dell, 1993). This
detected through the formation of halos around the colonies in three method, which is comparable to those involving the use of colored
kinds of agar plates as follows. The basal medium (1% tryptone, 0.5% indicators in agar plates together with carboxylic esters as the lipase
yeast extract, 0.5% NaCl, pH 7.2) was supplemented with medium A, substrate, consists basically of determining the diameter of the product
CaCl2 · 2H2O and Tween 80; medium B, CaCl2 · 2H2O and tributyrin; or diffusion area. The occurrence of lipolysis will give rise to a clarified zone
medium C, olive oil and rhodamine B. Lipase production on medium C on the agar plate.This optical phenomenon is observable only if the
plates was monitored by fluorescence with UV light at 350 nm (Choo released fatty acids are partly water-soluble. This clarification process
et al., 1998). results from the decrease in the size of the hydrolysed emulsified
Spirit blue agar medium was also used for the detection of lipolytic particles, which in turn causes a decrease in the diffused light. This type
activity of Serratia marcescens (Abdou, 2003). Rose Bengal chloram- of technique can also be applied to monitor the hydrolysis of Tweens by
phenicol agar and Potato Dextrose agar (PDA) media supplemented some lipases (Zinterhofer et al., 1973) and decrease in the absorbance of
with streptomycin (40 μL/mL) and tetracycline (20 μL/mL) with a a TAG emulsion with time (Arzoglou et al., 1982).
concentration of 20 μg/mL of media and incubated at 28 °C for 3– In Nephelometry, triolein (long chain fatty acids) is degraded to
5 days and stored at 4 °C was used for the isolation of lipase producing diglycerides. The degradation of triolein results in decreased turbidity
Rhizopus oryzae KG-5 (Shukla and Gupta, 2007). (Lipase. http://www.diaglab.vet.cornell.edu/clinpath/modules/chem/
lipase.htm).
2.2. Egg yolk as substrate
2.5. Released glycerol from triacylglycerol
The lipase acts on the lipid moiety of lipo-protein (lipovitellenin)
in egg yolk, resulting in alterations in the solubility of lipovitellenin. Direct determination of free glycerol is not commonly performed,
Modified Egg Yolk Agar, based on an egg yolk medium was developed since all three acyl chains of a triacylglycerol molecule are rarely released
by McClung and Toabe (1947) for the isolation and presumptive by a single lipase, and therefore the initial hydrolysis rates cannot be
differentiation of clostridia based on lecithinase and lipase production determined. For estimating the released free glycerol, periodic oxidation
and proteolytic activity. of free glycerol, which leads to the formation of formaldehyde can be
Egg-yolk and lecithin LB-plates were used for screening of E. assayed spectroscopically (Bjoerkhem and Sandelin, 1982).
coli clones expressing lipase variants with phospholipolytic activity
(Kauffmann and Schmidt-Dannert, 2001). Lipase activity of Gardner- 2.6. Electric conductivity
ella vaginalis was detected in 21 of 31 strains tested (68%) using egg
yolk agar but using the 4-methylumbelliferyl-oleate MUO spot test The electric conductivity of the medium increases during the lipase
only 12 of 31 strains tested positive (39%) (Moncla and Pryke, 2009). hydrolysis reaction due to the development of electric charges carried
by the released FFAs (Ballot et al., 1982, 1984). This technique has
2.3. Titrimetric methods numerous drawbacks: the measurements are highly temperature-
dependent, and the sensitivity is really satisfactory only when triacetin
Lipolytic reaction liberates an acid which can be titrimetrically is used as substrate. However, triacetin is not a particularly suitable
assayed. The pH-stat method is generally used as a reference lipase lipase substrate (Verger, 1997).
assay (Brockman 1981, Dellamora-Ortiz et al., 1997). This is not-so- A general method for the direct measure of enzymatic activity of
expensive and convenient technique for characterising lipase activity lipases using an electrochemical technique was described by Valincius
and specificity, as well the interfacial activation phenomenon. With et al. (2005). The method utilizes a solid-supported lipase substrate,
the pH-stat method, lipase activity is measured on a mechanically which is formed by dripping and drying a small amount of an ethanol
stirred emulsion of natural or synthetic triacylglycerides (TAGs) by solution of 9-(5′-ferrocenylpentanoyloxy) nonyl disulfide (FPONDS)
neutralising the FFAs released with time by adding titrated NaOH onto gold modified by a hexanethiol self-assembled monolayer. The
in order to maintain the pH at a constant end point value. The pH-stat redox ferrocene group of FPONDS generates the electrochemical signal,
equipment is commercially available at Radiometer (Copenhagen, the intensity of which is proportional to the number of FPONDS
Denmark) or Metrohm Ltd. (Herisau, Switzerland). It is a quantitative molecules at the interface. Electrochemical and surface-enhanced
method sensitive to within 1 µmol of released fatty acid per min and is infrared absorption spectroscopic data, as well as control experiments
not a reliable means of detecting activity levels lower than 0.1 μmol with an engineered, deactivated mutant enzyme, demonstrate that the
per min (Ferrato et al., 1997). wild-type lipase from Thermomyces lanuginosus is capable of cleaving
Use of olive oil as a substrate for the titrimetric method have been the ester bonds of FPONDS molecules via an enzymatic hydrolysis
reported by many scientists (Pignède et al., 2000; Macedo et al., 1997, mechanism, which includes the adsorption of the lipase onto the
Kashmiri et al., 2006; Fujii et al., 2003; Pospísil and Kabátová, 1976). substrate surface. The hydrolysis liberates the ferrocene groups from
To detect the activity of lipase produced by Bacillus sp LBN 4, the the interface triggering a decay of the electrochemical redox signal.
reaction mixture containing olive oil emulsion (composed of 25 ml The electrostatic properties of the enzyme were calculated and
olive oil and 75 ml 2% polyvinyl alcohol solution, 4 ml of 0.2 M tris validated by isoelectric focusing. It was reported that the broad
buffer, 1 ml of 110 mM CaCl2 and 1 ml enzyme solution) was used isoelectric region of Candida antarctica lipase B is unique as compared
(Watanabe et al., 1977). After the incubation, the enzyme activity to almost all other alpha/beta-hydrolases which have a well-defined
was stopped by adding 20 ml of acetone ethanol (1: 1) mixture and isoelectric point (Trodler et al., 2008).
liberated free fatty acid was titrated against 0.02 M NaoH using The cyclic voltammetric (CV) technique was used to coat a carbon
phenolphthalein as indicator. One unit of lipase was defined as the electrode with a thin layer of polyaniline. This modified electrode
amount of enzyme, which liberates 1 mol of fatty acid/min (1 ml of proved to be highly sensitive and specific to detect lipase between 0
0.02 N NaOH is equivalent to 100 mol fatty acid liberated per minute) and 225 IU/L, which is well within the normal physiological range. The
(Bora and Kalita, 2007). consistency and reliability of this technique proved to be a very
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F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798 785

formidable detector of lipase present in abnormal quantities due to et al., 1997). p-nitrophenyl laurate appears to be stable at elevated
pancreatitis and other pathological disorders (Kumar et al., 2005). temperatures. Results obtained using p-NPL correlated well with
those obtained titrimetrically (Sigurgísladóttir et al.,1993). β-naphthyl
2.7. The Wilhelmy plate method laurate has been used as a substrate for serum lipase (Pereira-Meirelles
et al., 1997).
Among the interfacial tensiometry methods, the monomolecular Berg et al. (1998) have reported a colourimetric assay of Humicola
film technique at the air–water interface has been extensively lanuginosa lipase (HLL) using para-nitrophenyl butyrate, which parti-
developed and used by Ransac et al. (1999). The equipment used in tioned at the interface of small anionic unilamelar vesicles (SUVs)
this method is commercially available at several companies: KSV with a mean diameter of around 40 nm, such as palmitoleoyl-oleoyl-
(Helsinki, Finland), Krüss GmbH (Hamburg, Germany) and Kibron Inc. phosphatidylglycerol (POPG)-SUVs. All the available methods of
(Helsinki, Finland). With this technique, it is possible to measure and measuring lipase activity using nitrophenyl esters therefore need to
control some important interfacial parameters such as the surface be handled with great care, even when pure lipases are used. A series of
pressure (the interfacial free energy) and the molecular area of the p-nitrophenyl esters with variable acyl chains have been frequently
substrate, as well as the surface excess of the water-soluble lipases. used, however, as reported in the literature, to quickly assay the so-
Surface pressure of phospholipase A2 solution in a circular Teflon called chain length specificity of the microbial lipases used in many
trough (4-cm diameter–1-cm depth) was measured using a Wilhelmy biotransformation processes (Rangheard et al.,1989). Lipase activity was
plate attached to a computer-controlled Cahn electrobalance (model assayed by a spectrophotometric method using p-nitrophenyl laurate
C-32) (Wijewickrama et al., 2006). (Salameh and Wiegel, 2007; p-nitrophenyl decanoate as a substrate
Surface pressure was monitored using the Wilhelmy method and (Kouker and Jaeger, 1987; Lee et al., 2004), in P. aeruginosa culture
temperature was held at 24 °C. Lipid films were spread from a hexane- supernatants with p-nitrophenyl-palmitate (Kim et al., 2001; Rashid
ethanol (95:5; v/v) solution until the desired surface pressure was et al., 2001; Salameh and Wiegel, 2007; Winkler and Stuckmann, 1979;
reached. After allowing the lipid monolayer to stabilize, inorganic phos- Urban et al., 2001) methyl or butyl esters of 2-hydroxy octanoic acid
phate was introduced into the stirred aqueous subphase from a micro- (Sakaki et al., 2002), p-nitrophenyl butyrate (Brady et al., 2008; Kim
syringe through a small port in the side of the circular compartment. One et al., 2001).
minute later an aliquot of protein solution was added in the same The formation of 2,4-dinitrophenol (pKa 3.96) can be monitored
manner. Surface pressure was monitored until the monolayer was col- from the increase in the absorbance at 360 nm (Mosmuller et al.,1992).
lected on one side of a hydrophobic filter paper disk, and the extent Since the molar extinction coefficient of a phenolic solution is gen-
protein adsorption was determined (Sumiyoshi et al., 2003). erally very dependent on its ionization state, dinitrophenyl esters are
Synthetic medium-acyl chain lipids were originally mainly used as more convenient substrates than p-nitrophenyl esters when the pH of
substrates with lipolytic enzymes (Momsen and Brockman, 1997). An the assay is around 7–8. The red colour obtained with n-naphthol
alternative method is using a non surface-active agent, β-cyclodextrin, can be monitored after complexing the solution with a diazonium
dissolved in the aqueous subphase in order to trap the long-chain salt (Whitaker, 1973). The use of acyl esters of 5-(4-hydroxy-3,5-
lipolytic products generated by the lipolysis of monomolecular films dimethoxyphenylmethylene)-2-thioxothiazoline-3,5-dimethoxyphe-
of long chain neutral acylglycerols or phospholipids (Ivanova et al., nylmethylene)-2-thioxothiazoline-3-acetic acid as chromogenic lipase
1996). substrates has also been described (Miles et al., 1992). Upon hydrolysis,
these substrates yield an intensely red colour which can be assayed at
2.8. Acoustic wave conductance 505 nm. However, esters of this kind do not in fact seem to be specific
substrates for determining true lipase activities.
A surface acoustic wave sensor system for assaying the activity of Smeltzer et al. (1992) developed a specific spectrophotometric
pancreatic lipase has been proposed by Ge et al. (1995). The assay of assay for the quantitative determination of lipase activity in Staphy-
this enzyme is based on the change in conductance of the solution lococcus aureus by clearance of a tributyrin emulsion. Rawyler and
caused by the release of a fatty acid, using triolein as a substrate. Siegenthaler, (1989) described a continuous spectrophotometric assay
based on the metachromatic properties of the cationic dye safranin.
2.9. Colorimetric methods This method was reported to be two fold higher than the pH-stat
method.
The speed and sensitivity of the assays for FFAs can be increased by Simple, direct and automation-ready procedures for measuring
the use of colorimetric methods (Lowry and Tinsley, 1976). These lipase activity in serum, plasma, saliva, urine and other biological
methods involve the complexation of FFAs in organic solvent with a samples, are very desirable. BioAssay Systems' QuantiChromTM Li-
divalent metal (usually copper) followed by the spectrophotometric pase Assay is based on an improved dimercaptopropanol tributyrate
analysis of the metal in the organic phase. This method was first dev- (BALB) method, in which SH groups formed from lipase cleavage of
eloped by Duncombe (1963). The copper complex was subsequently BALB react with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) to form
estimated spectrophotometrically at λ 440 nm. a yellow colored product. The color intensity, measured at 412 nm, is
Several methods employ a special substrate designed to give a proportionate to the enzyme activity in the sample (http://www.
colored end product after hydrolysis, or one that can be easily con- bioassaysys.com/DLPS.pdf).
verted to a colored product. Lipase activity in bacterial culture supe- Panteghini et al. (2001) evaluated an assay reagent for lipase deter-
rnatants is determined by hydrolysis of p-nitrophenyl esters of fatty mination, based on the use of 1,2-o-dilauryl-rac-glycero-3-glutaric acid-
acids with various chain lengths (NC-10). The hydrolysis of carboxylic (6′-methylresorufin) ester (DGGR) as substrate. DGGR is cleaved by
esters of n-naphthol, p-nitrophenol or 2,4-dinitrophenol leads to the lipase, resulting in an unstable dicarbonic acid ester which is spon-
release of alcohols that can be monitored continuously and quantita- taneously hydrolysed to yield glutaric acid and methylresorufin, a
tively using a spectrophotometric method. The appearance of the bluish-purple chromophore with peak absorption at 580 nm.
yellow coloured p-nitrophenol (pKa 7.15) can be monitored by reading A continuous, sensitive in vitro lipase assay was developed using
the absorbance at 405 nm (Stuer et al., 1986; Vorderwülbecke et al., a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG
1992; Lee et al., 2004) or 410 nm (Winkler and Stuckmann, 1979; was found to provide an efficient substrate for rat adipocyte and
Liebeton et al., 2001. Enzyme activity is expressed in μmoles of p- human recombinant HSL. Ultrasonic treatment applied in the pres-
nitrophenol released per minute. p-nitrophenyl derivatives of fatty ence of phospholipids leads to the incorporation of NBD-MAG into
acids (generally lauric or palmitic acids) have been popular (Becker the phospholipid liposomes and to a concomitant change of its
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786 F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798

spectrophotometric properties. The enzymatic release of NBD-FA and cular structure as monitored by ESI-mass spectrometry (Invernizzi
its dissociation from the carrier liposomes is accompanied by the et al., 2009).
recovery of the original spectrophotometric characteristics. The rate
of lipolysis was monitored by measuring the increase in optical den- 2.12. Chromatographic methods
sity at 481 nm, which was found to be linear with time and linearly
proportional to the amount of lipase added (Petry et al., 2005). Various chromatographic techniques can be used to detect lipids as
Extracellular lipase activity of Pseudomonas fluorescens AFT36 well as FFAs released from TAGs, namely florisil columns or silicic acid
was detected after 10 h of incubation using reflectance colorimetry, columns, thin-layer chromatography or gas–liquid chromatography
but 28 h were required using tributyrin agar. Reflectance colorimetry (Christie, 1987; Gunstone et al., 1994).
and chromogenic substrates allowed a rapid, sensitive, and mean-
ingful detection of esterase and lipase activity in milk (Blake et al., 2.12.1. HPLC methods
1996). A high-performance liquid chromatographic (HPLC) assay for
Fluorometric and colorimetric methods have been used for deter- determining lipase activity was developed by Maurich et al. (1991)
mination of triglycerides (triacylglycerols) in serum (Winartasaputra using β-naphtyl laurate, palmitic and lauric esters of p-nitrophenol as
et al., 1980). Samples were incubated with microbial lipase for 10 min, substrates. β-naphthyl laurate is incubated with enzyme and the
and the glycerol released from the triglycerides is oxidized by NAD+ formation of naphthol is quantified by reversed phase HPLC.
in the presence of glycerol dehydrogenase. In the fluorometric method, HPLC method was used for assaying phospholipase A2 (PLA2).
the resulting NADH is in turn oxidized by resazurin as catalyzed by The procedure is based on heptane–isopropanol–H2SO4 extraction
diaphorase to form resorufin, a highly fluorescent compound. In the of fatty acids released by the enzyme in the presence of margaric
colorimetric method, the NADH is oxidized by coupling with a tetra- acid as an internal standard, and precolumn derivatization with 9-
zolium salt/diaphorase system to form formazan, a highly colored anthryldiazomethane. The derivatives of naturally occurring long-
compound. chain fatty acids were accurately determined by reverse-phase HPLC
with ultraviolet detection at 254 nm (Tojo et al., 1993).
2.10. Fluorometric methods HPLC was used for the measurement of the product, (R)- and (S)-
2-hydroxy octanoic acid, when methyl or butyl esters of 2-hydroxy
Jette and Ziomek (1994) described the quantitative fluorescence octanoic acid used as a substrate (Sakaki et al., 2002).
lipase assay based on the interaction of rhodamine B with fatty acids
released during the enzymatic hydrolysis of triglycerides. The meth- 2.12.2. Gas chromatographic methods
od, Rhodamine–Triglyceride–Agarose assay, allows flexibility in the GC may be used to separate and quantify the hydrolytic products
choice of substrate and large number of samples can be assayed of lipase (Louwrier et al., 1996; Patel et al., 1996). This method is
simultaneously, making it practical for assaying lipase activity in generally preferable to HPLC, primarily because HPLC suffers from the
column fractions during purification. low sensitivity of available detectors. Transesterification activity and
Wilton (1991) described as assay by which quantities as low as 20 pg the industrial potential of a novel lipase prepared from Acinetobacter
of purified pig pancreatic lipase (PPL) could be detected. Densyl-phos- ventiatus RAG-1 were evaluated. Production of 1-octyl butyrate by
phatidylethanolamine (Johnson et al., 1980) and NBD-phospholipids lipase-catalyzed transesterification of vinyl butyrate with 1-octanol
(Wittenauer et al., 1984a,b) have been used as a substrate to detect the in hexane was monitored by gas chromatography (Snellman and
phospholipase activity of lipoprotein lipase. These fluorescent phos- Colwell, 2008).
pholipid molecules are not, however, typical and specific substrates for
other lipases. In the case of continuous lipase assays, the fluorophore 2.13. Interfacial tension monitoring
group can be either coumarin, NBD, or pyrene. A first group of fluoro-
genic substrates consists of coumarin fatty acyl esters (De Laborde As lipases are active at interfaces, it is possible to monitor en-
de Monpezat et al., 1990) and lauroyl pyrenemethanol (Nègre et al., zyme activity by following changes in surface pressure. Cleavage of a
1989). These molecules are chemically unrelated to TAGs and can be monomolecular substrate film at the air–water interface causes the
hydrolyzed by non-specific esterases. A second group of fluorogenic substrate to leave the interface, leading to reduced surface density and
substrates consists of dansyl-phosphatidylethanolamine (Johnson et al., reduced surface pressure, which can be quantified (Verger, 1980).
1980) and NBD-phospholipids (Wittenauer et al., 1984a,b) which were
reported as substrate for the phospholipase activity of lipoprotein lipase. 2.14. Test kits
The pyrene group was also used as a fluorophore in continuous assays
of lecithin: cholesterol acyltransferase (Bonelli and Jonas, 1992), choles- Several test kits are available for determination of lipase activity.
teryl ester hydrolase (Joutti et al., 1985), phospholipase C (Thuren and The tests are usually based on the enzyme clearing of an emulsion
Kinnunen, 1991), and phospholipases A (Hendrickson and Rauk, 1981; wherein decreases in absorbance values in the UV range (340 nm) are
Thuren et al., 1988). used to estimate lipase activity.
The main advantages of fluorimetric assays is their sensitivity and The Lipase Assay Kit allows fast and easy measurement of lipase
the fact that they make it possible to continuously monitor the re- activity in vitro, in cell preparations or in vivo using the fluorescent
action kinetics and depend on the sensitivity of the FFA detection, and fatty acid substrate 1,2-dioleoyl-3-pyrenyldecanoyl-rac-glycerol. The
the specific activity of lipase. fluorescent triacylglycerol 1,2-dioleoyl-3-(1-pyren-1-yl) decanoyl-
Surribas et al. (2007) used the green fluorescence protein (GFP) rac-glycerol includes a pyrene decanoic acid as one of the three fatty
fusion partner for monitoring extracellular production of a Rhizopus acyl residues of a natural triacylglycerol which forms excimers in vivo.
oryzae lipase (ROL) in Pichia pastoris by means of 2D-fluorimetric Upon enzyme activity, the pyrenedecanoic acid monomer is cleaved,
techniques. resulting in decreased excimer fluorescence. Pyrene triacylglycerols
are useful for measuring cholesteryl ester transfer, protein-mediated
2.11. Mass spectrometry triacylglycerol transport between plasma lipoproteins, and have also
been found to be excellent substrates for lipoprotein lipase and hepatic
The lipase from Burkholderia glumae (BGL) was incubated at vari- triacylglycerol lipase (http://www.markergene.com/kits.php).
able temperature, pH and concentration of organic solvents, and the Lipase activity of Pseudomonas sp. strain B11-1 was determined
decrease of enzymatic activity was compared to changes in the mole- with a lipase assay kit (Dainippon Pharmaceutical, Osaka, Japan) at
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F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798 787

25 °C. One unit of enzyme activity was defined as the amount of 2.19. QuantiChromTM lipase assay kit (DLPS-100)
enzyme that catalyzes the formation of 1 μmol of 2,3-dimercaptopro-
pan-1-ol from 2,3-dimercaptopropan-1-ol tributyl ester per min Rapid Colorimetric Determination of Lipase Activity at 412 nm
(Kurooka et al., 1977; Choo et al., 1998). BioAssay Systems' QuantiChromTM Lipase Assay is based on an im-
Kit for testing the ability of a compound to simulate lipolysis in proved dimercaptopropanol tributyrate (BALB) method, in which
cultured human adipocytes contains a plate of human adipocytes, SH groups formed from lipase cleavage of BALB react with 5,5′-
controls, buffers and reagents for detecting glycerol and non-esteri- dithiobis(2-nitrobenzoic acid) (DTNB) to form a yellow colored pro-
fied free fatty acids (http://www.biocompare.com/itemdetails.asp? duct. The color intensity, measured at 412 nm, is proportionate to the
itemid=493883). enzyme activity in the sample (http://www.bioassaysys.com/Search.
asp?Key=enzyme).
2.15. Lipoprotein lipase ELISA kit
2.20. Immunological methods
In acute pancreatitis, lipase levels can rise 5 to 10-fold within 24
to 48 h. BioAssay Systems' colorimetric lipase assay is based on an Immunological methods are widely used to quantitate the
improved dimercaptopropanol tributyrate (BALB) method, in which SH presence of lipases in biological media, independently from their
groups formed from lipase cleavage of BALB react with 5,5′-dithiobis(2- lipolytic activity (Beisson et al., 2000). Immunoprecipitation was
nitrobenzoic acid) (DTNB) to form a yellow colored product. The color combined with Western blot analysis to detect lipoprotein lipase
intensity, measured at 412 nm, is proportionate to the enzyme activity in (LPL), a secretory asparagine-linked glycoprotein that is synthesized
the sample. The assay can be directly applied to serum, plasma, saliva, by a variety of tissues (Bensadoun, 1991; Braun and Severson, 1992).
urine and other biological samples (Lipase QuantiChrom™ Assay Kit The analysis of LPL protein by techniques such as Western blotting
from BioAssay Systems) (http://www.biocompare.com/itemdetails. is particularly important, as its maturation to an active lipase is de-
asp?itemid = 889933). pendent on co-and post-translational steps that can be most easily
monitored by examining changes in migration of the protein during
2.16. EnzChek lipase substrate SDS PAGE (Ben-Zeev et al., 1992; Ben-Zeev et al., 1994). Method
that uses immunoprecipitation to enrich and concentrate LPL prior
The triacylglycerol-based EnzChek lipase substrate (E33955) offers to its detection by Western blotting has been reported far superior to
higher throughput and better sensitivity than chromogenic (TLC or detection by immunoblotting alone (Doolittle et al., 1998).
HPLC) assays, and a visible detection of pyrene-based fluorescent sub- ELISA (enzyme-linked immuno sorbent assay) forms a highly sen-
strates. In the presence of lipases, the nonfluorescent EnzChek lipase sitive and specific system for detecting and quantifying pure lipases
substrate produces a bright, green-fluorescent product for the accurate (Dati and Grenner, 1984), e.g., lipoprotein lipase (LPL) (Villela and Joven,
and sensitive detection of lipase activity in solution. The green- 1998), HPL (Caro et al.,1999), HGL (Ge et al.,1995), hepatic lipase (Villela
fluorescent product of the EnzChek lipase substrate exhibits pH- and Joven, 1998) and for the serological diagnosis of staphylococcal
insensitive spectra in the physiological pH range. infections (Colque-Navarro et al., 1998) for detecting and quantitating
LPL in tissues or in cell culture lysates (Kawamura et al., 1994). These
2.17. Coumarin-based lipase substrates immunological methods can be used to detect both the active and
inactive forms of a given lipase. A sandwich ELISA was developed using
The fluorogenic lipase substrate O-pivaloyloxymethyl umbellifer- canine gastric lipase (cGL) purified from canine stomachs and polyclonal
one (C-POM) was developed to deliver optimal performance in assays antibodies directed against cGL, raised in rabbits and purified by affinity
of lipase activity. Enzymatic conversion of the essentially nonfluor- chromatography (Steiner and Williams, 2004).
escent C-POM yields a bright blue-fluorescent reaction product. C- Immunoblot analysis, combination of gel electrophoresis with an
POM has been shown to serve as a substrate for a variety of lipases and immunochemical method of detection is used for determining the
displays excellent stability in solution, making it an ideal substrate for molecular mass of lipases, as well as the specificity of antibodies for a
specific lipases. given lipase. Western blotting analysis using monoclonal antibodies has
Unlike lipase substrates that are esters of 7-hydroxy-4-methyl- been widely used to discriminate between sequential and conforma-
coumarin (-methylumbelliferone, H189; Section 10.1) – a dye that is tional epitopes.
not appreciably fluorescent at neutral pH – 6,8-difluoro-4-methy- Using various immunohistochemical techniques, mammalian
lumbelliferyl octanoate (DiFMU octanoate) can be used for the lipases such as gastric lipase (Moreau et al., 1990), LPL (Casaroli-
continuous in vitro assay of lipases at a pH greater than or equal to 6; Marano et al., 1996; Sambandam et al., 1999) hepatic lipase (Breedveld
the blue-fluorescent hydrolysis product of DiFMU octanoate, 6,8- et al., 1997), hormono-sensitive lipase (Kraemer et al., 1993), and
difluoro-7-hydroxy-4-methylcoumarin (DiFMU, D6566; Section 10.1) microsomal triacylglycerol hydrolase (Lehner et al., 1999) have been
is determined (http://probes.invitrogen.com/handbook/sections/ mapped by many scientists. Several immunological assays for deter-
1006.html). Lipase activity was also determined as described by mining the pancreatic lipase concentration in sera have been devel-
Briselden and Hillier (1990), using 4-methylumbelliferyl-oleate oped and used in the clinical diagnosis of pancreatitis (Ingen and
(Fluka 75164, from the Sigma Aldrich Chemical Co., St. Louis, MO.) Sanders, 1992).
as a substrate in spot test (Briselden and Hillier, 1990; Moncla et al., Other immunoassays and commercial kits have been developed
1990). to quantitate immunoreactive LPL (Antikainen et al., 1996; Kimura
et al., 1999) as well as hepatic lipase in human serum and carboxyl
2.18. MarkerGene™ fluorescent lipase assay kit ester lipase (Bensadoun, 1996). A radioimmunoassay has also been
developed for determining the concentration of HPL in the urine
Lipase activity, ubiquitous among most cells, can be monitored using (Sternby and Akerstrom, 1984) and in the duodenal contents (Sternby
the new fluorescent substrate 1,2-dioleoyl-3-(pyren-1-yl)decanoyl-rac- et al., 1991).
glycerol (Product M0258) contained in the kit. Upon cleavage, the
fluorescent fatty acid pyrenedecanoic acid (Product M0274) is released 2.21. NMR method
and activity measurements are easily obtained either in vitro, in cell
preparations, or in vivo (http://www.markergene.com/ProductDetails. NMR could be used for quantitative analysis of lipase activity in
php/M0612). biphasic macroemulsions (O' Connor et al., 1992).
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788 F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798

2.22. Atomic force microscopy separation of radioactive substrate from the labeled products and
involve safety concern.
Nielsen et al. (1999) suggested that atomic force microscopy 3- Titrimetric assays are continuous and quite straight forward.
(AFM) to be helpful in providing the picture of the kinetic of lipid Natural or synthetic substrates can be used, but they suffer from
degradation by lipases. The enzymatic hydrolysis of mixed bilayers of low sensitivity and are subject to conditions that could alter the
acylglycerols/phospholipids by Humicola lanuginosa lipase (HLL), was amount of free hydrogen ion released.
also investigated using AFM (Balasheva et al., 2001). 4- The assays using fluorescent probes are tedious and time-
consuming.
2.23. Infrared spectroscopy
All the issues have to be carefully considered before choosing a
A continuous assay for measuring lipase-catalyzed hydrolysis of method best suited to the enzyme: the particular information that is
TAGs in reverse micelles using Fourier transform infrared spectro- to be obtained, and the availability of substrates, probes, and instru-
scopy (FTIR), was developed by Walde and Luisi (1989) for measuring mentation (Hendrickson, 1994).
the lipolysis of various substrates (trioctanoylglycerol, vegetable oils).
3. Factors affecting production of lipases
2.24. Detection by electron microscopy
Variety of conditions have been described which stimulate or
Fatty acids released in animal tissues by lipases can be detected by repress the production of lipases by bacteria. The most important
electron microscopy. This technique involves the incubation of tissues stages in a biological process are modeling and optimization to
with triolein and then exposed to lead salts to form insoluble soaps, improve a system and increase the efficiency of the process without
and finally processed for electron microscopy. Larger precipitates have increasing the cost (Baş and Boyaci, 2007). There are certain
been obtained by using Tweens instead of triolein, but the former are environmental factors that play significant role during enzyme
not specific lipase substrates (Menon et al., 1992). production and metabolic activities.

2.25. Radioactive assays 3.1. Physical factors

The methods involving the use of TGAs containing radiolabelled acyl When screening bacteria for lipase production both culture pH and
chains (Briquet-Laugier et al., 1999) are specific and very sensitive lipase assay pH are important parameters. The stability depends upon the
assays but they cannot be monitored continuously and are time- presence of substrate (Talon et al., 1995). Other than pH, temperature,
consuming. A method based on the binding of radiolabelled 63Ni to FFAs age and size of inoculums, agitation and time of incubation are also
extracted in organic solvents has been described (Huang et al., 1989). very important factors to be considered (Table 1).
The estimated quantity of 63Ni bound to oleic acid was found to be linear Handelsman and Shoham, (1994) showed that the production of
up to 100 nmol of fatty acid, and quantities as small as 1 nmol of FFA can lipase from thermophilic Bacillus sp. continued during the stationary
be detected per assay. However, this method is also time-consuming. phase for over 24 h and optimum time required for the maximum
In radiometric assays, activity of lipase purified from rice bran was production of lipase was 36 h. Whereas, the lipolytic activity of Bacillus
measured using either [9,10-3H]triolein or [2-palmitoyl-9,10-3H]PC as a cereus and Bacillus coagulans reached a maximum level after incubation
substrate (2.5 mM) (Bhardwaj et al., 2001). for two days with continuous agitation (El-Shafei and Rezkallah, 1997).
Shake flasks are usually used for the production of extracellular
2.26. The oil-drop method enzymes by aerobic organisms as it enhances the aeration rate.
Aeration has been found essential for lipase production (air: medium
Nury et al. (1987) established a method to measure variations in ration 4:1) and produced the highest activity. The lipolytic activity
the oil–water interfacial tension as a function of time during lipase from Bacillus sp. reached a maximum level after incubation for two
hydrolysis by adapting the well-known “hanging-drop method”. This days with continuous agitation (El-Shafei and Rezkallah, 1997). Aer-
method is used to monitor lipase activities on natural long-chain TAGs ation increased the growth and lipase production in Pseudomonas
at a closely controlled oil–water interface. The interfacial tension is aeruginosa KKA-5 (Sharon et al., 1999). Shaking enhanced the
measured here by automatically analysing the oil drop profile on-line, production of intra- and extracellular lipase from thermophilic Rhi-
using the Laplace–Young equation. The accumulation of tensioactive zopus oryzae (Salleh et al., 1993). Penicillium cyclopium, grown in
hydrolysis products at the surface of an oil drop is responsible for the stationary culture, produces a type I lipase specific for triacylglycerols
decrease in the interfacial tension, which in turn is correlated with while, in shaken culture, it produces a type II lipase only active
changes with time in the drop profile (Grimaldi et al., 1991). Fully on partial acylglycerols (Chahinian et al., 2000). Whereas the best
automated oil drop tensiometers based on this principle are now production of lipase by the psychrophile Acinetobacter O16 and
commercially available at Interfacial Technology Concept SARL Geobacillus sp. strain ARM (DSM 21496 = NCIMB 41583 occurred in
(France) and have been found to have many advantages (Labourdenne standing cultures as reported by Breuil and Kushner, 1975) and
et al., 1997). Ebrahimpour et al. (2008). Various researchers have reported dif-
There exists no single universal method of lipase assay, but rather a ferent rates of agitation for optimum production of lipase (Table 1).
whole range of different techniques. The choice of a particular method Addition of 1.5 ml of B. licheniformis B-42 culture to 20 ml medium
will depend on the user's own specific requirements. gave the maximum lipase production. On the other hand, the volumes
of inoculum below or above 1.5 ml gave gradually decreased values as
2.26.1. Advantages and disadvantages of various Lipase assays compared to the optimal one. The maximum lipase yield reached up to
1- Natural triglycerides are used in chromatographic (Ergan and 251.78 U/ml by G. stearothermophilus B-78 at an inoculum size of
Andre, 1989), titrimetric (Charton and MacRae, 1992) and mono- 2.5 ml/ 20 ml (Bayoumi et al., 2007).
layer assays (Verger and Pattus, 1982), but the higher sensitivity is
obtained with chromogenic (Chapus et al., 1976) and fluorogenic 3.2. Biochemical/physicochemical factors
(Negre et al., 1985) synthetic substrates.
2- Radiometric assays are the most sensitive, but they require use of Various biochemical/physicochemical parameters must be optimized
expensive radiolabeled substrates, are discontinuous and require in order to maximize the production of lipase. Various compositions of
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F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798 789

Table 1 Table 2
Physical factors. Biochemical/physicochemical parameters.

pH and temperature Growth media

Bacillus sp. FH5 pH 8, 37 °C Hasan et al. (2006) Candida rugosa 0.1% glucose, 4.0% olive oil, 0.3% Song et al.
Bacillus thermoleovorans IHI-91 pH 6.0, 65 °C (Markossian et al., 2000; Becker NH4NO3, 1.2% K2HPO4 and 0.4% (2001)
et al., 1997) MgSO4. 7H2O
Bacillus coagulans pH 8.5, 55 °C Kumar et al. (2005) Streptomyces flavoriseus 0.8% castor oil as the carbon source Elwan et al.
BTS-3 thermophilic in Dox's liquid medium (1983)
Bacillus clausii SKAL-16 pH 8–10 Lee and Park (2008) supplemented with 0.3% yeast
Bacillus cereus 30–33 °C pH 8.0 Dutta and Ray (2009) extract
for 24 h Bacillus circulans Dox's liquid medium supplied with Elwan et al.
Bacillus coagulans BTS-3 55 °C and pH 8.5 Kumar et al. (2005) 2 and/or 30% sucrose (1983)
Salinivibrio sp. SA-2 pH 8, 35 °C Amoozegar et al. (2008) Bacillus sp. 0.5% soybean flour and 0.1% stearyl Kambourova
Lactobacilli 30 °C, Papon and Talon (1988) glycerol esters or natural fats et al. (1996)
Lactobacillus plantarum, DSMZ pH of 5.5 Lopes et al. (1999) Bacillus THL027 2% (v/v) buffer solution (7.3% (w/v) Dharmsthiti
12028 (thermophilic) Na2HPO4, 3.2% (w/v) KH2PO4, pH and Luchai
Acinetobacter radioresistens pH 7, 30 °C Chen et al. (1998) 7.2, 40 μg/ ml FeSO4 and 40 μg/ ml (1999)
Enterobacter agglomerans pH 7.0, 30 °C for Zhen-qian and Chun-yun (2009) MgSO4, 0.1% (w/v) (NH4)2SO4
48 h supplemented with 3% NaCl, 0.1%
Pseudomonas sp. 7323, pH 9.0, 30 °C Zhang and Zeng (2008) glucose, 1.0% rice bran oil and 0.5%
Penicillium candidum pH 9, 35 °C Ruiz et al. (2001) (w/v) rice bran
Candida rugosa pH 6.5, 30 °C Song et al. (2001) Bacillus sp. FH5. Na2HPO4.2H2O 7, K2HPO4 3, NH4Cl 1, Hasan et al.
Cryptococcus sp. S2 pH 5.6, 25 °C Kamini et al. (2000) NaCl 0.5, Glucose 4, MgSO4 0.25, CaCl2 (2006)
0.02, casamino acid 2.5, olive oil 10
Effect of incubation time Bacillus sp. 10 g/l yeast extract, 15 g/l NH4Cl, Heravi et al.
Bacillus sp. (thermophilic) 36 h Handelsman and Shoham (1994) 3 g/l K2HPO4, 1 g/l KH2PO4, 0.1 g/l (2008)
Bacillus cereus and Bacillus 24 h El-Shafei and Rezkallah (1997) MgSO4 MgSO4 × 7H2O, 2 g/l glucose,
coagulans 0.6 mM MgCl2 and 15 ml/l olive oil
Geobacillus sp. strain ARM 24 h Ebrahimpour et al. (2008)
(DSM 21496 = NCIMB 41583 Effect of substrates
Bacillus sp. FH5 48 h Hasan et al. (2006) Staphylococcus hyicus Phospholipids Simons et al.
Pseudomonas strain N 72 h Sarkar et al. (1998) (1998)
Candida rugosa 60 h Song et al. (2001) Salinivibrio sp. SA-2 Triacylglycerols Amoozegar
Cryptococcus sp. S-2. 120 h Kamini et al. (2000) et al. (2008)
Rhizopus oryzae 4 days Hiol et al. (2000) Thermoactinomyces vulgaris Presence and absence of tributyrin Elwan et al.
Penicillium wortmanii 7 days Costa and Peralta (1999) (1983)
Bacillus subtilis168 p-nitrophenyl esters and Lesuisse et al.
Effect of age of inoculum triacylglycerides esters with C8 acyl (1993)
Bacillus sp. 6h Sidhu et al. (1998) groups
Bacillus sp. FH5 24 h Hasan et al. (2006) Bacillus thermoleovorans Olive oil (1.5%, w/v) Lee et al.
Thermoactinomyces vulgaris 18 h Elwan et al. (1978) ID-1 (1999)
Bacillus sp. LBN4 Olive oil and yeast extract Bora and Kalita
Effect of Size of inoculum (2007)
Bacillus sp. Tributyrin–Tween 80, Olive olive oil Heravi et al.
Bacillus thermoleovorans ID-1, 1% Lee et al. (1999)
(2008)
Bacillus sp. FH5 5% Hasan et al. (2006)
Staphylococus sp. Water-soluble and insoluble Falk et al.
Geobacillus sp. strain ARM 1% Ebrahimpour et al. (2008)
triglycerides as well as polyoxythylene (1991)
(DSM 21496 = NCIMB 41583
sorbitan fatty acids (Tween)
Acinetobacter baumannii p-NP caprate (C10) Park et al.
Effect of shaking and oxygen availability
BD5 (2009)
Candida rugosa 180 rpm Song et al. (2001)
Acinetobacter calcoacetius LP009 200 rpm Pratuangdejkul and Dharmsthiti
Effect of NaCl and bile salts
(2000)
Salinivibrio sp. SA-2 150 rpm Amoozegar et al. (2008) Candida rugosa NH4NO3 Song et al.
Geobacillus sp. strain ARM Static condition Ebrahimpour et al. (2008) (2001)
(DSM 21496 = NCIMB 41583 Penicillium roqueforti Stimulated by NaCl Larsen and
Jensen (1999)
Bacillus sp. FH5. 1.0 M NaCl. sodium taurocholate Hasan et al.
bile salts inhibited the lipase (2006)
media have been reported by many researchers that can enhance the production

production of lipase (Table 2). Effect of carbon and nitrogen sources

The lipolytic enzymes are constitutive as well as inductive in
Bacillus sp. FH5 Yeast extract, Salicin Hasan et al.
nature. Substrate in the form of lipids must be present in the medium (2006)
that enhances the production of lipase. It was shown that the physical B. licheniformis B-42 Fructose, glucose, ammonium Bayoumi et al.
state of substrates is influenced by temperature and in turn, influences G. stearothermophilus B-78 molybdate (2007)
Bacillus thermoleovorans Yeast extract Markossian
the rates of substrate hydrolysis (Sugiura and Isobe, 1975). Substrate
IHI-91 et al. (2000)
specificity may depend on the chemical structure of substrate mole- Geobacillus sp. strain ARM Peptone, yeast extract, olive Ebrahimpour
cules and the physical properties of the emulsion or other surface (DSM 21496=NCIMB 41583) oil and gum arabic et al. (2008)
(Espostio et al., 1973). Triglycerides containing unsaturated or Lactobacilli strains and Low glucose concentration Papon and
medium-chain saturated fatty acids (C8–C12) were good substrates Brochothrix thermosphacta Talon (1988)
Lactobacillus delbrueckii Wheat bran, glucose El-Sawah et al.
for lipase, although those containing longer or shorter-chain fatty
subsp. Bulgaricus (1995)
acids were poorly hydrolyzed. While long-chain fatty acid methyl Rhizopus oryzae Peptone, tryptone, tryptic soy digest, Salleh et al.
esters were well hydrolyzed. The rate of hydrolysis increased with polypeptone, corn steep liquor (1993)
increasing reaction temperature, especially for triglycerides of long- Enterobacter agglomerans Lactose, beef extract and yeast Zhen-qian and
chain fatty acids. This resulted in the substrate specificity at 60 °C extract Chun-yun
(2009)
differing greatly from that at 30 °C. Physical state of substrates
(continued on next page)
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790 F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798

Table 2 (continued) are obtained for different temperatures depending on the substrate
Effect of carbon and nitrogen sources (c) for a given substrate, the activation by a low concentration of bile
Serratia marcescens SM-6 glycogen or pectin B Winkler et al. salts depends on the temperature. It increases with increase in
(1978) temperature (d) this temperature effect does not seem to be due to a
Serratia marcescens polysaccaharides; glycogen, Winkler and
modification of the physical parameters of the interface as measured
hyaluronate, laminarin, pectin B and Stuckmann
gum arabic (1979)
by the interfacial tension.
Pichia lynferdii Nrrl Y-7723. Sucrose, yeast extract Chang-Ho et al. Carbon and nitrogen are the fundamental substances for the
(2003) growth of the bacteria. Normally they are supplied from natural
sources (Pimentel et al., 1994). The effect of different carbon sources
Effect of substrate concentration
and trace elements added to basal medium was observed with the aim
Bacillus licheniformis B-42 1.5 g/l Bayoumi et al.
of improving enzyme production of lipase from a Brazilian strain of
Geobacillus stearothermophilus 2.5 g/l (2007)
B-78 Fusarium solani FSI. When the production medium was supplemented
with trace elements using olive oil, corn oil and sesame oil, the lipase
specific activity was optimum (Maia et al., 2001). A thermophilic
is influenced by temperature and in turn, influences the rates of isolate Bacillus coagulans BTS-3 produced an extracellular alkaline
substrate hydrolysis (Sugiura et al., 1975). lipase with refined mustard oil as carbon source and a combination of
Regio-specific and non-regio-specific lipases from mammals and peptone and yeast extract (1:1) as nitrogen sources (Kumar et al.,
microorganisms catalyse the hydrolysis of short, medium and long- 2005). The best results in the production of lipase from Candida
chain fatty acid anhydrides (Pieroni and Fourneron, 1990). rugosa, were obtained with the use of olive oil as the carbon source in
Chander et al. (1979a) have reported that short chain fatty acids the presence of nitrogen sources. Biomass concentration was also high
stimulate lipase production in case of Streptococcus faecalis but longer in the presence of olive oil (Fadiloglu and Erkmen, 2002).
chain, unsaturated fatty acids, such as oleic acid, strongly inhibit it. The superior carbon and nitrogen sources for production of lipase
With additional olive oil supplementation, the volumetric productivity from Bacillus cereus were starch and ammonium sulfate (nitrogen
of lipase from Acinetobacter radioresistens could be improved; however, level 21.2 mg/100 ml), peptone (nitrogen level 297 mg/100 ml), and
the lipase yield decreased with increasing concentrations of olive oil. The urea (nitrogen level 46.62 mg/100 ml) in combination, respectively
increase in the rate of lipase formation by olive oil in the presence of n- (Dutta and Ray, 2009).
hexadecane was attributed to its enhancement of the uptake of n- When Penicillium citrinum was cultured in the simple medium
hexadecane; the hydrolytic products of olive oil promoted the emulsifica- (1.0% olive oil and 0.5% yeast extract), using olive oil as carbon source
tion of n-hexadecane (Chen et al., 1998). Sardine oil, soy bean oil and in the inocula, the enzyme extracted showed maximum activity. In
triolein were effective inducers for lipase production. Cryptococcus sp. S2 addition, decrease of yeast extract concentration also reduces the
(Kamini et al., 2000). Maximum production of lipase by Bacillus sp. FH5, lipase activity. When yeast extract was replaced by ammonium sulfate,
was shown in the presence of castor oil (Hasan et al., 2006). no activity was detected (Pimentel et al., 1994). To improve production
Lipase(s) produced by Streptomyces flavogriseus is (at least partly) of extracellular lipase by Rhizopus delemar industrial grade carbon and
constitutive in its (their) nature and can be produced in the absence of nitrogen sources were used. Reduced olive oil concentration and
lipids, however, its production is stimulated in their presence addition of Tween 80 to the medium achieved maximum production. It
(Mostafa and Ali, 1979). was shown that it is necessary to modify the carbon source-nitrogen
A suggestion is given of a regulatory system of lipase synthesis, source ratio (Cruz et al., 1993). The optimum Carbon/Nitrogen ratio for
involving the supply of starch to initiate growth, allowing induction of lipase and biomass production in case of Candida rugosa, was found to
lipase by the inductive substrate. There appears to be no common rule be 6–6.5 and 9–9.5 respectively (Rao et al., 1993).
about what kinds of lipids are effective for producing microbial lipases The yield of lipase production could further be improved by
(Elwan et al., 1978). Espinosa et al. (1990) suggested a double effect of including additives, such as hexadecane, Triton X-100, or Tween 80, in
Tweens that can serve as both inducers because of their chemical the growth medium. Hexadecane stimulates the production of lipase
nature similar to that of some natural substrates, as well as the primarily at the posttranslational level, most likely by enhancing its
surfactants, stimulating the enzyme release. Song et al. (2001) found secretion. Although Triton X-100 and Tween 80 increased lipase
that the surfactants could be helpful to the lipase production. production to an extent similar to that of hexadecane, these compounds
Tween 80 or ethanol, to the normal complex medium, inhibited are less suitable for inclusion in large-scale production because of
lipase production from Acinetobacter O16. Sodium acetate, oleic acid, contamination of the culture supernatant with cellular proteins, which
olive oil, or Tween 20 added to synthetic media did not affect lipase affects the downstream processing of lipase (Boekema et al., 2007).
production (Breuil and Kushner, 1975). The induction of production of In case of Bacillus pumilus SG2, the best carbon source for enzyme
lipolytic activities by Tween 80 has been reported for Bacillus sp. CM7 production was glucose and the best nitrogen sources were yeast
(Emanuilova et al., 1993). Tween 80 has been found to increase the extract and casein. Addition of additives like SDS, Triton X-100 and
lipolytic activity of Bacillus sp. (Sidhu et al., 1998) as well as that of Tween-20 influenced enzyme production. Castor oil, gingelly oil and
other microorganisms such as Rhizopus delemar (Espinosa et al., 1990) olive oil were the best substrates for lipase production. Organic nitro-
and Bacillus stearothermophilus (Gowland et al., 1987). gen sources were used efficiently by Pseudomonas sp for production
Inorganic ions such as Na+, PO3− 4 and Ca2+ are essential for of both protease and lipase. Casein was the best nitrogen source for
the growth of bacteria. It was found that addition of NaCl to cultures lipase production. Inorganic nitrogen sources were not as efficient as
of Bacillus natto enhanced lipase production and that the lipase pro- organic nitrogen sources (Sangeetha et al., 2009).
ducing period became shorter with increasing NaCl concentration Olive oil, sesame oil and tea oil as raw materials were catalyzed
(Ohkuro et al., 1978). to biodiesel by the lipase of Enterobacter agglomerans. This strain can
Beside their inhibitory effect upon lipase adsorption, bile salts at potentially serve as a promising alternative lipase for biodiesel production
low concentration (around 0.2 mM) enhanced lipolysis slightly with raw materials containing water (Zhen-qian and Chun-yun, 2009).
because of stabilization of the adsorbed lipase brought about by low
concentrations of bile salts (Granon and Semeriva, 1980). This 4. Purification of Lipases
hypothesis of Granon and Semeriva relies on the following observa-
tions; (a) for a given temperature, the activation by bile salts depends Interest on lipases from different sources (microorganisms, ani-
on the substrate (b) in the absence of bile salts, the optimal activities mals and plants) has markedly increased in the last decade due to the
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F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798 791

potential applications of lipases in industry and in medicine. Microbial Table 3

and mammalian lipases have been purified to homogeneity allowing Effect of pH and temperature on the activity of lipase.

the successful determination of their primary amino acid sequence pH Temperature

and, more recently, of the three-dimensional structure. The X-ray (°C)
studies of pure lipases will enable the establishment of the structure- Aspergillus oryzae RIB128 5.5 40 Toida et al. (1998)
function relationships and contribute for a better understanding of the Aeromonas sobria LP004 6.0 45 Lotrakul and Dharmsthiti (1997)
Acinetobacter sp. CR9 8.0 40 Kasana et al. (2008)
kinetic mechanisms of lipase action on hydrolysis, synthesis and
Salinivibrio sp. SA-2 7.5 50 Amoozegar et al. (2008)
group exchange of esters (Taipa et al., 1992). Enterococcus faecalis 8.0 60 Kar et al. (1996)
Liquid–liquid extraction of biomolecules using reversed micelles Cryptococcus sp. S-2 7.0 37 Kamini et al. (2000)
is a promising method when the traditional techniques with organic Pseudomonas aeruginosa 8.0, 55 Chartrain et al. (1993)
solvents are limited by protein denaturation and solubilization (Hatton, MB 5001
Bacillus thermoleovorans ID-1 7.5 70–75 Lee et al. (1999)
1989; Castro and Cabral, 1988). Rhizopus oryzae 7.5. 35 Hiol et al. (2000)
Two forms of lipases were obtained from Geotrichum candidum
has been purified by ethanol precipitation and chromatographies on
Sephacryl-200 HR, high resolution anion-exchange (mono Q) and
Polybuffer exchanger 94 purified 35-fold with a 62% recovery in Stepaniak, 1983). The substrate specificity Cryptococcus sp. S-2
activity and 94-fold with a 18% recovery in activity, respectively towards simple triglycerides and oils was broad and the enzyme
(Veeraragavan et al., 1990). The crude enzyme preparation containing was regarded as a partial glyceride hydrolase. The enzyme has good
the lipases was obtained with fractional precipitation with ethanol potential for the hydrolysis of vegetable oils, which are industrially
(50–80%) from the culture broth of Chromobacterium viscosum. High and economically important for the production of biodiesel (Kamini
molecular weight (lipase A) was purified from the crude extract by et al., 2000). Partially-purified lipase from Pseudomonas aeruginosa
chromatography using Amberlite CG-50 and Sephadex G-75. The KKA-5 was found to hydrolyze 81% castor oil (Sharon et al., 1999).
enzyme was purified about 23-fold with a recovery of 2.8% from the Pseudomonas aeruginosa strain MB 5001 lipase was also found to be
crude extract (Sugiura et al., 1974). C. viscosum, lipase B, was purified more active on short-chain versus long-chain triglycerides, and hy-
through precipitation with ammonium sulfate followed by chromato- drolyzes C18-unsaturated fatty acid esters more efficiently than it
graphy on Sephadex G-100, CM-cellulose and DEAE- Sephadex and hydrolyzes C18-saturated fatty acid esters (Chartrain et al., 1993).
homogeneity of the preparation was demonstrated by disc electro- Lipases of P. fluorescens GK13 hydrolyzed triolein. Polyesters contain-
phoresis (Sugiura and Isobe, 1974). The enzyme was purified using 60% ing side chains in the polymer backbone such as poly (3-hydro-
ammonium sulfate precipitation and sephadex G200 column chroma- xybutyrate) and other poly(3-hydroxyalkanoates) were not or were
tography (Abada, 2008). only slightly hydrolyzed by the lipases tested (Jaeger et al., 1995).
A novel lipase of Staphylococcus aureus 226 was purified by Metal ions generally form complexes with ionized fatty acids,
ammonium sulfate precipitation followed by successive chromato- changing their solubility and behavior at interfaces. Release of fatty
graphies on hydroxyapatite, Sephadex G-200 and Sephadex G-150. acids to the medium is rate determining and could be affected by
This method gave 385-fold purification of the enzyme from the cul- metal ions. However, the effects of metal ions depend on particular
ture medium in a yield of 25%. The purified enzyme appeared homo- lipase. The effects of metal ions on activity can be more directly
geneous on polyacrylamide gel electrophoresis (Muraoka et al., 1982). addressed with bacterial lipases than with pancreatic lipase which
The purification of lipase from yeast Trichosporon asteroides strain requires a cofactor protein. This is a very complex phenomenon as the
LP005 included (NH4)2SO4 precipitation at 70% saturation and gel reaction takes place in heterogenous system.
filtration on Sephadex G-200. The molecular mass of the lipase was The activity of lipase from Rhizopus japonicus NR400 was not
37 kDa as determined by SDS/ PAGE (Dharmsthiti and Ammaranond, affected by the addition of 1 mM metal ions or bile salts. Stimulation of
1997). Gilbert et al. (1991) purified extracellular lipase from a Tween the lipase activity was observed upon addition of albumin to the
80-limited continuous culture of Pseudomonas aeruginosa EF2 by ul- reaction mixture (Suzuki et al., 1986). Monovalent ions had little effect
trafiltration of the culture supernatant followed by anion-exchange on lipolytic activities of both enzymes, Lipase I and II from Geotrichum
and gel-filtration FPLC. candidum, while divalent ions at concentrations above 50 mM in-
By the application of ion exchange chromatography with DEAE- hibited the lipase activities in a concentration-dependent manner
Sepharose CL-6B, two protein peaks exhibiting lipase activities were (Veeraragavan et al., 1990). Kar et al. (1996) found that phosphates
resolved in the fractions eluted by buVer containing 0–1 M NaCl and common surfactants have no or very little inhibitory effects on the
Pseudomonas monteilii TKU009 (Wang et al., 2009). The lipase purified activity lipase from Enterococcus faecalis, whereas, bile salts were
from Bacillus sp. FH5, on SDS-PAGE, revealed two bands, a larger band found to be inhibitory to the enzyme activity. The purified lipase of
with molecular weight approximately 63 kDa and a smaller band of Bacillus coagulans BTS-3 was found to be inhibited by Al3+, Co2+,
24 kDa approximately (Hasan et al., 2006). Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions
enhanced the enzyme activity; Na+ ions have no effect on enzyme
5. Characterization of lipase activity activity (Kumar et al., 2005). The lipase of an Alaskan psychrotroph
Pseudomonas sp. B11-1 was strongly inhibited by Zn2+, Cu2+, Fe3+,
The effect of various factors of the activity and stability of purified and Hg2+ but was not affected by phenylmethylsulfonyl fluoride and
and crude lipases have been studied by many researchers. Factors such bisnitrophenyl phosphate. Various water-miscible organic solvents,
as pH, temperature, effect of metal ions, organic solvents, detergents/ such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30%
surfactants, and other inhibitors can enhance or suppress the activity (vol/vol) activated the enzyme (Choo et al., 1998).
of lipases (Tables 3 and 4). Characterization of lipase would determine The activity of the lipase purified from Aspergillus terreus was
the suitability of lipase in different environments and industries etc. inhibited by ionic detergents, whereas, non-ionic detergents stimu-
Maximum lipolytic activity was shown when the lipase gene from lated enzyme activity. The enzyme was unaffected by the metal
the thermophile Bacillus thermocatenulatus into E. coli DH5α at pH chelator EDTA or by 2-mercaptoethanol and potassium ferrocyanide.
8.0–9.0 with tributyrin and olive oil as substrates and at 60–70 °C At a concentration of 100 μM, 3,4-dichloroisocoumarin caused weak
with p-NPP and olive oil as substrates (Schmidt-Danert et al., 1996). inhibition with 40% loss of activity, but diethyl p-nitrophenyl phos-
The purified Pseudomonas fluorescens strain AFT 36 lipase showed phate at the same concentration strongly inhibited enzyme activity
maximum activity on tributyrin at pH 8.0 and 35 °C (Fox and (98.12% loss of activity), confirming that the Aspergillus terreus lipase
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792 F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798

Table 4
Effect of various factors on activity of lipase.

Effect of metal ions Activator Inhibitor

Bacillus thermoleovorans ID-1 Ca2+ or Zn2+ Lee et al. (1999)
Bacillus natto Na+, PO3−
4 and Ca2+ Ohkuro et al. (1978)
2+ 2+
Pseudomonas fluorescens No. 33 Zn and Hg Kumura et al. (1993)
Pseudomonas aeruginosa MB 5001 CaCl2 taurocholic acid ZnSO4 Chartrain et al. (1993)
Pseudomonas putida 3SK CaCl2 taurocholic acid ZnSO4 Lee and Rhee (1993)
Staphylococcus aureus 226 Mg2+ and Ca2+ Mn2+ Muraoka et al. (1982)
Staphylococcus aureus Ca2+ Mn2+, Cobalt and zinc Talon et al. (1995)
Pseudomonas strain Alaskan soil Zn2+, Cu2+, Fe3+, and Hg2+ Choo et al. (1998)
Pseudomonas lipase Ferric ions Sugiura et al. (1977)
Rhizopus oryzae Fe3+, Cu2+, Hg2+ and Fe2+ Hiol et al. (2000)
Aspergillus terreus Mg2+ and Ca2+ Co2+, Cu2+, Ni2+ and Fe3+ Yadav et al. (1998)
Serratia marcescens 345 Mg2+ Ni2+, Zn2+, Fe2+ Cu2+ Hg2+ Bachkatova and Severina (1980)
Bacillus sp. FH5 Ba2+, Fe2+, Mn2+, Ca2+, Mg2+ and Hg2+ Cu2+, Co2+, Na+ and Zn2+ Hasan et al. (2006)
Acinetobacter sp. CR9 Cu2+, Mo2+, Mg2+, Zn2+, Ca2+ Kasana et al. (2008)
Acinetobacter baumannii BD5. Ca2+, Mg2+, and Mn2+ Zn2+ and Cu2+ Park et al. (2009).
Pseudomonas sp. 7323,. Ca2+. Zhang and Zeng (2008)
psychrotroph Pseudomonas sp. B11-1 Zn2+, Cu2+, Fe3+, and Hg2+ Choo et al. (1998)

Effect of inhibitors
Acinetobacter sp. CR9 PMSF, DTT, 2-ME EDTA Kasana et al. (2008)
Bacillus thermoleovorans ID-1 SDS Lee et al. (1999)
Pseudomonas putida 3SK SDS Lee and Rhee (1993)
Geotrichum candidum SDS Veeraragavan et al. (1990)
Bacillus sp. FH5 PMSF, SDS, EDTA Hasan et al. (2006)
Acinetobacter baumannii BD5. Tween 20 SDS and Triton X-100 Park et al. (2009).
psychrotroph Pseudomonas sp. B11-1 methanol and dimethyl sulfoxide Choo et al. (1998)

Surfactants and others

Pythium ultimum 144 Taurocholic acid, Triton X-100, n-octyl glucoside, Mozaffar and Weete (1993)
n-dodecyl-beta-d-maltoside, 3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS),
3-[-cholamidopropyl)
dimethylammonio]-2-hydroxy-1-propanesulfonate
Lactobacillus delbrueckii subsp. Bulgaricus Acacia gum, na-deoxycholate El-Sawah et al. (1995)
Penicillium cyclopium Triton X-100, SDS. Ibrik et al. (1998)
Rhizopus oryzae Sodium cholate or taurocholate Triton X-100, SDS. Hiol et al. (2000)
Pseudomonas strain Methanol and dimethyl sulfoxide, Choo et al. (1998)
Bacillus sp. Acetone n-hexane Sugihara et al. (1991)
Acinetobacter radioresistens n-hexadecane Benzene Chen et al. (1998)
Bacillus sp. FH5 Ethanol, isopropanol and acetone. Isopropanol, Hasan et al. (2006)
Staphylococcus haemolyticus L62 Tributyrin, tripropionin, and trimyristin Oh et al. (1999)
Aspergillus terreus Pig fat, groundnut oil Kusum oil Yadav et al. (1998)
Aspergillus oryzae RIB128 Triacylglycerols monoacylglycerols and diacylglycerols. Toida et al. (1998)
Penicillium cyclopium Tributyrin, trioctanoin, olive oil Acylglycerols Ibrik et al. (1998)
(monooctanoin and dioctanoin)
and methyl esters
Rhizopus oryzae, Glycerol olive oil, triolein, and oleic acid Salleh et al. (1993)
Rhizopus oryzae Saturated fatty acid chains (C8–C18) Hiol et al. (2000)
Fusarium-heterosporum Middle-chain fatty acid triglycerides (6 less-than-or-equal-to Shimada et al. (1993)
C less-than-or-equal-to 12)
Bacillus subtilis 168 p-nitrophenyl esters and triacylglycerides preference for triacetyl glycerol Lesuisse et al. (1993)
esters with C-8 acyl groups
Botrytis cinerea Umbelliferone esters Commenil et al. (1995)
Pseudomonas sp. 7323 p-nitrophenyl caprate and tributyrin Zhang and Zeng (2008)

is a serine hydrolase (Yadav et al., 1998). EDTA also did not affect phoorganic compound. It was suggested from these observations that
lipolytic activity of the enzyme, Trichosporon asteroides strain LP005, the lipases belong to “serine” enzymes containing sulfhydryl groups
it was not a metalloenzyme (Dharmsthiti and Ammaranond, 1997). (Lobyreva and Marchenkova, 1980).
PMSF and bis-nitrophenyl phosphate did not affect the activity of the The hydrolysis of olive oil by lipases is inhibited by the addition of
lipase from psychrotrophic Pseudomonas strain from Alaskan soil emulsified organic solvents. This inhibition is competitive and is
(Choo et al., 1998). Lotrakul and Dharmsthiti, (1997) also showed that observed with any emulsified organic solvent. It is independent of the
PMSF and 2-mercaptoethanol, a reducing agent, did not affect the chemical structure of the solvent or that of the emulsifiers and the
enzyme activity in case of lipase from Aeromonas sobria LP004. Com- proteins are not chemically modified (Sugiura, 1984). The Cryptococ-
mon surfactants except Triton X 100 and cetyltrimethylammonium cus sp. S-2 enzyme was also stable in different organic solvents at a
bromide have no or very little inhibitory effects on activity of lipase concentration of 50% (v/v) for 60 min (Kamini et al., 2000).
produced by Bacillus cereus. The enzyme was found to be stable in The lipolytic activity of Bacillus cereus and Bacillus coagulans was
presence of oxidizing agents and protease enzyme (Dutta and Ray, elevated by the presence of either olive oil or tributyrin and with lesser
2009). extent in the presence of castor oil. The enzyme levels were drastically
The lipases from Penicillium roqueforti 141 were inhibited by a reduced in the presence of animal fat, cotton seed oil, margarine or
broad spectrum of reagents for sulfhydryl groups and by a phos- glycerol (El-Shafei and Rezkallah, 1997). Chander et al. (1979a) have
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F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798 793

reported that short chain fatty acids stimulate lipase production in case emulsification of tributyrin or triolein. Bile salts may be used, but
of Streptococcus faecalis but longer chain, unsaturated fatty acids, such their action is complex.
as oleic acid, strongly inhibit it. In case of Bacillus coagulans BTS-3, In case of lipases in mainly organic reaction mixtures, the precise
optimum lipase activity was obtained when refined mustard oil was level of water remaining has major effects on their catalytic activity.
used as carbon source and a combination of peptone and yeast extract Different biocatalysts require different levels of water to exhibit
(1:1) as nitrogen sources (Kumar et al., 2005). substantial activity. Many lipases require only relatively low water
When the effects of organic solvents were tested on the lipase from levels (Valivety et al., 1992). This could be due to the differences
solvent resistant strain Pseudomonas putida 3SK, isooctane was found in water uptake by the organic phase, or in its binding by other
to be most suitable for enzymatic fat hydrolysis in the two-phase components present in the catalyst, such as protein impurities, salts,
system (Lee and Rhee, 1993). Lipase activity of Brazilian strain of Fu- sugars or a support.
sarium solani FSI increased in the presence of increasing concentra- Lipases are active and stable under alkaline conditions and over
tions of hexane and toluene. In contrast, incubation of this enzyme a broad temperature range, which makes them ideal for incorpora-
with water-soluble solvents decreased its activity after 10% concen- tion in various applications (Table 5). Lipases are also stable in
tration (v/v) of the solvent (Maia et al., 2001). detergents containing protease and activated bleach system (Gormsen
The addition of DMSO and diethyl ether to the assay mixture in the et al., 1991). The purified Aspergillus terreus lipase showed excellent
range of 0–10% (v/v) stimulated the enzyme Cryptococcus sp. S-2 temperature tolerance (15–90 °C) and was highly thermostable,
significantly (Kamini et al., 2000). The solvent-tolerant lipase from retaining 100% activity at 60 °C for 24 h. It showed good pH tolerance
Fusarium-heterosporum was not inactivated even after incubation at (3.0–12.0) and was stable over a pH range of 4.0–10.0 for 24 h (Yadav
30 °C in 50% solvent such as dimethylsulfoxide (DMSO), hexane, et al., 1998).
benzene and ether for 20 h (Shimada et al., 1993). It was observed The stability of this enzyme at elevated temperatures makes it very
Hasan et al. (2006) that the organic solvents were not good stim- useful. Activity of lipase II from Penicillium cyclopium (Chahinian et al.,
ulators of lipolytic activity in case of Bacillus sp. FH5. 2000) and Penicillium wortmani (Costa and Peralta, 1999) is stable at
When the gene coding for an extracellular lipase in Bacillus pH 7.0 and 40–45 °C. Activity is lost at temperatures above 50 °C. The
stearothermophilus L1 was cloned in Escherichia coli, the lipase produced lipases from Penicillium roqueforti 141 were stable to heating (up to
was found to have highest activity toward p-nitrophenyl caprylate 55 °C) and within a wide pH range. Penicillium cyclopium lipase was
among the synthetic substrates and tripropionin among the triglycer- also found to be stable at 35 °C for 60 min and has maximal activity
ides. It hydrolyzed beef tallow and palm oil more rapidly than olive oil at in a pH range of 8–10 (Ibrik et al., 1998). The optimum temperature
50 °C (Kim et al., 1998). Bacillus sp. SKAL-16 grew on tributyrin and for enzyme activity of lipases from a Brazilian strain of Penicillium
glycerol, but could not grow on acetate and butyrate. This strain excreted citrinum was also found in the range of 34–37 °C. However, after
butyric acid during growth on tributyrin, and selectively ingested 30 min at 60 °C, the enzyme was completely inactivated. The enzyme
glycerol during growth on a mixture of butyric acid and glycerol (Lee also showed optimum at pH 8.0 (Pimentel et al., 1994).
and Park, 2008). The Brazilian strain of Fusarium solani FSI lipase activity was stable
Partially purified lipase from Acinetobacter sp. CR9 hydrolyzed below 35 °C but above this temperature activity losses were observed
wide range of short chain fatty acid esters of p-nitrophenyl. The (Maia et al., 2001). The optimum pH of the solvent-tolerant lipase
organism CR9 also hydrolyzed tributyrin, Tween 80, soybean oil, from Fusarium heterosporum at 40 °C and optimum temperature at pH
mustard oil and olive oil (Kasana et al., 2008). The purified lipase of 5.6 were 5.5–6.0 and 45–50 °C, respectively, when olive oil was used
Bacillus coagulans BTS-3 also showed a variable specificity/hydrolytic as the substrate. The lipase was stable over a pH range of 4–10 at 30 °C
activity towards various 4-nitrophenyl esters (Kumar et al., 2005). for 4 h, and up to 40 °C at pH 5.6 for 30 min (Shimada et al., 1993).
Baillargeon et al. (1989) studied lipase from Geotrichum candidum The activity of the crude and partly purified enzymes was studied
which is of industrial interest for the production of special chemicals in relation to pH and temperature optima. Crude lipase from the
from fats and oils because it has a unique specificity for unsaturated psychrophilic Acinetobacter O16 was more temperature-stable than
fatty acids and the esters of these fatty acids are hydrolyzed more the purified enzyme (Breuil and Kushner, 1975). Alkaline Lipase
rapidly than most others. produced by Acinetobacter radioresistens had an optimum pH of 10
Purified Aeromonas sobria LP004 (Lotrakul and Dharmsthiti, 1997) and was stable over a pH range of 6–10; it could have great potential
and Rhizopus oryzae (Hiol et al., 2000) lipases could be classified as a for application in the detergent industry. The optimal temperature and
1,3-position specific enzymes. Similarly, the Aspergillus oryzae RIB128 pH for lipase fermentation were 30 °C and 7, which were related to
lipase was also found to have 1, 3-positional specificity (Toida et al., cell growth, and stability and reactivity of lipase (Chen et al., 1998).
1998). The substrate specificity of lipase from Bacillus sp., towards
simple triglycerides was broad and 1- and 3-positioned ester bonds
were hydrolyzed in preference to a 2-positioned ester bond (Sugihara
et al., 1991). Berger and Schneider (1991) developed a facile system for Table 5
pH and temperature stability of lipases.
the quantitative determination of lipase regioselectivities in organic
solvents towards the 1 (3)-position of glycerides. Their study showed pH and temperature stability
that the lipases from Mucor meihei and Pseudomonas fluorescens does Aspergillus oryzae RIB128 pH 4.0–7.5 at 30 °C for 24 h, Toida et al. (1998)
not display the high 1 (3)-specificities. The Aspergillus terreus lipase and pH 5.5 at 30 °C for 1 h
exhibited 1,3-regiospecificity (Yadav et al., 1998). Bacillus sp. FH5 98.6% and 93% for 1 h at 40 Hasan et al. (2006)
and 45 °C
It has been found that not only the degree of dispersity of an Aeromonas sobria LP004 pH 6.5–10.0 at b 40 °C Lotrakul and Dharmsthiti
emulsion influenced the activity of lipase with respect to a lipid (1997)
being emulsified, but also the nature of an emulsifier affected hy- Cryptococcus sp. S-2 pH 5.0 and 9.0 at 50 °C Kamini et al. (2000)
drolysis of solid fats by microbial lipases (Lobyreva and Marchen- Chromobacterium pH 5.0–8.0 Sugiura et al. (1974)
Chromobacterium lipase B pH 4.0–11.0 Sugiura and Isobe (1974)
kova, 1979). Tributyrin and triolein have been most commonly
Staphylococcus pH 6.0–8.0 Chander et al. (1979b)
employed as substrates. If the substrate is not emulsified or the Pseudomonas pH 5.0–10.5 Sugiura et al. (1977)
emulsification is inadequate, the rate of shaking becomes an im- Bacillus sp. LBN4 80 °C for 10 min Bora and Kalita (2007)
portant factor that affects the rate of hydrolysis (Wills 1961). Egg Rhizopus oryzae 4.5 to 7.5 Hiol et al. (2000)
albumin and gelatin were originally proposed as good emulsifiers. Pseudomonas sp. 7323,. N 25 °C Zhang and Zeng (2008)
Bacillus coagulans BTS-3 pH 8.0 and 10.5 to 70 °C Kumar et al. (2005)
Desnuelle et al. (1955) suggested the use of 10% arabic gum for the
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794 F. Hasan et al. / Biotechnology Advances 27 (2009) 782–798

Temperature and pH stabilities of new lipase OBase from Acinetobac- 1993). The thermostable lipase produced from Bacillus sp. showed the
ter nov. sp. strain KM109, were at or lower than 35 °C and in a range of optimal pH at 30 °C and optimal temperature at pH 5.6 were 5.5–7.2,
pH 6–8, respectively (Mitsuhashi et al., 1999). and 60 °C respectively, when olive oil was used as the substrate
Maximal enzyme activity of pancreatic lipase purified from human (Sugihara et al., 1991). Mostly bacterial lipases are stable at the neutral
pancreatic juice, occurred at pH 9.5 in glycine buffer (0.1 mol/L) pH range but for Bacillus lipase the optimum pH was observed to be
containing deoxycholate (19 mmol/L), colipase (3 mg/L) and triolein 3.8 (Elwan et al., 1977). The extracellular lipase produced, when the
(0.3 mmol/L) (Iizuka et al., 1991). lipase gene of Bacillus stearothermophilus L1 was expressed in E. coli,
The purified extracellular lipase of Staphylococcus warneri was was found to be active at 60–65 °C and in alkaline conditions around
found to have an optimum pH of 9.0 and an optimum temperature of pH 9–10 (Kim et al., 1998). The lipase of the thermophile Bacillus
25 °C. The enzyme was stable within the range pH 5.0–9.0 (Talon et al., thermocatenulatus when expressed into E. coli DH5α showed high
1995). The optimum temperature of the purified lipase Staphylococcus stability at pH 9.0–11.0 (Schmidt-Danert et al., 1996).
aureus 226 enzyme was 60 °C with triolein (Muraoka et al., 1982). The maximum activity of lipase purified from Bacillus sp. FH5 was
Optimum temperature and pH for the hydrolysis of olive oil by Sta- observed at pH 10. As the pH level was increased from 10 to 11, the
phylococcus haemolyticus L62 was found to be 28 °C and pH 8.5, lipolytic activity decreased percentage stability decreased. At pH 9 the
respectively. The enzyme was stable up to 50 °C in the presence of Ca2+ enzyme was 74% stable, followed by decrease in the lipolytic activities
and over the pH range 5–11 (Oh et al., 1999). The Staphylococcal lipase and percentage stabilities at pH 8, 7, 6, 5 and 4 respectively (Hasan
preparation was found to be stable within pH 5.0 to 9.0 and exhibits et al., 2006).
maximal activity at pH 8.0. The optimal temperature for the enzymatic
reaction was established at 55 °C (Tyski et al., 1983). 5.1. Kinetics
At 55 °C, the Pseudomonas aeruginosa LP602 lipase had the highest
activity but not stability (Dharmsthiti and Kuhasuntisuk, 1998). An The Km value of the lipase of psychrotrophic Pseudomonas strain,
alkaline lipase from Pseudomonas fluorescens AK 102 (Shimada et al., for p-nitrophenyl butyrate increased with increases in the assay
1993) and Pseudomonas putida 3SK (Lee and Rhee, 1993) were found temperature (Choo et al., 1998). Pseudomonas fluorescens strain AFT
to have pH stability in the range 4–10 and optimum pH 8–10. The pH 36 lipase had a Km on tributyrin of 3.65 mM and was inhibited by
dependencies of activity and stability may depend on culture concentrations of substrate greater than approximately 17 mM. (Fox
conditions. According to Watanabe et al. (1977) if the organisms are and Stepaniak, 1983). Km of lipase from Brazilian strain of Fusarium
grown at alkaline pH, the lipase produced may have an alkaline pH solani FSI using pNPP (p-nitrophenylpalmitate) as substrate, was
optimum. The lipase purified from Pseudomonas aeruginosa EF2 was 1.8 mM with a Vmax of 1.7 μmol/min/mg protein (Maia et al., 2001).
very stable to freezing and thawing (Gilbert et al., 1991). Pseudomonas The Km and Vmax values of the lipase purified from Bacillus sp. FH5,
species, KWI-56, extracellular thermostable lipase resisted more than were calculated by Lineweaver and Burk (1934) plot. Purified lipase
90% of the enzyme activity after 24 h of heat treatment at 60 °C was incubated with different concentrations of substrate and activity
(Nakamura et al., 1992). The lipase produced by Bacillus sp. LBN4 had was measured after one hour. It was observed that the lipase has the
temperature optima of 65 °C. Over 80% of its peak activity was at the Km value as 5.05 mM and Vmax as 0.416 μmol/ml/min (Hasan et al.,
pH range of 7–8, with an optimum of 8.0. The enzyme was found to be 2006). The Michaelis–Menten constant of lipase purified from Serratia
most stable at 80 °C for 10 min. Almost 80% of the residual activity was marcescens was 1.35 mM on tributyrin (Abdou, 2003). The values of
retained at 80 °C (Bora and Kalita, 2007). Km and Vmax of a lipase from Aspergillus niger F044 calculated from
The lipase from psychrotrophic Pseudomonas sp. (Choo et al., 1998) the Lineweaver–Burk plot using pNPP as hydrolysis substrate were
and Pseudomonas fluorescens strain AFT 36 (Fox and Stepaniak, 1983) 7.37 mmol/L and 25.91 micromol/(min × mg), respectively (Shu et al.,
were found to be stable between pH 6 and 9. Pseudomonas sp. enzyme 2007).
was found to be stable between pH values 6 and 12 and below 60 °C
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