Biotechnol. Appl. Biochem.
(1999) 29, 119–131 (Printed in Great Britain)
REVIEW
The realm of microbial lipases in biotechnology
Ashok Pandey *1, Sailas Benjamin†2, Carlos R. Soccol*, Poonam Nigam‡, Nadia Krieger*
and Vanete T. Soccol§
*Laboratorio de Processos Biotecnologicos, Departamento Engenharia Quimica, Universidade Federal do Parana (UFPR), CEP
81531-970 Curitiba-PR, Brazil, †Biotechnology Division, Regional Research Laboratory, Trivandrum-695 019, India,
‡Biotechnology Group, University of Ulster, Coleraine BT52 1SA, N. Ireland, U.K., and
§Departamento de Patologia BaT sica, UFPR, CEP 81531-970, Curitiba-PR , Brazil
In this review, a comprehensive and illustrious survey is
made of the applied aspects of microbial lipases in
modern biotechnological practices. Lipases are the
most versatile biocatalyst and bring about a range of
bioconversion reactions such as hydrolysis, interesteri-
fication, esterification, alcoholysis, acidolysis and am-
inolysis. After a brief description of the microbial
sources of lipases, the pivotal role of lipases in the
processes and products of the food and flavourings
industry is illustrated. An illustration is presented of
biomedical applications. The panorama of lipases in the
manufacture of fine chemicals is depicted with special
emphasis on pharmaceuticals, pesticides, cosmetics,
biosensors and detergents. Widening applications such
as those in waste management and improved tanning
techniques are other novel aspects of lipase utilization
that are discussed in this review.
Introduction
Multi-faceted microbial lipases (glycerol ester hydrolases ;
EC 3.1.1.3) have an unsurpassed role in swiftly growing
modern biotechnology. Lipases are indispensable for the
bioconversion of lipids (triacylglycerols) from one organism
to another and within the organisms. In addition to their
biological significance, lipases have tremendous potential in
areas such as food technology, biomedical sciences and
chemical industries.
Lipases possess the unique feature of acting at an
interface between the aqueous and non-aqueous (i.e. or-
ganic) phase ; this feature distinguishes them from esterases.
The concept of lipase interfacial activation arises from the
fact that their catalytic activity generally depends on the
aggregation state of the substrates. It is believed that
activation involves the unmasking and structuring of the
enzyme’s active site through conformational changes re-
quiring the presence of oil-in-water droplets.
Lipase activity generally depends on the available surface
area. Recent structural studies on several lipases have
119
provided some clues towards the understanding of hy-
drolytic activity, interfacial activation and stereoselectivity
[1]. They catalyse a wide range of reactions, which include
hydrolysis and interesterification specifically ; in addition,
they also catalyse alcoholysis, acidolysis, esterification and
aminolysis. Lipases act under extremely mild conditions.
They can be used in a variety of organic solvents and show
selectivity for a specific reaction type. Recently, Patel et al.
[2] reviewed lipase-catalysed biochemical reactions in novel
media. Gill and Valivety [3] reviewed biotransformation and
the biotechnological applications of polyunsaturated fatty
acids, which involved the unique nature of lipase-catalysed
processes.
Although lipases have been studied for many years and
can be produced on large scale by the growth of micro-
organisms in a fermenter, the use of lipases were confined
mainly to the products and processes of oleo-chemistry and
dairy-based industries [4]. In fact, the last quarter of the
twentieth century has witnessed an unprecedented advances
in lipases into the novel horizons of biotechnology in tandem
with already established fields of pharmaceuticals, pesticides
and, more recently, in the production of single-cell protein
and cosmetics, in waste disposal and in biosensor modu-
lations. The main reason for the steadily growing interest in
lipases, reflected by the appearance of an average of 1000
publications each year, is the biotechnological versatility of
these enzymes [5,12]. In this review we intend to conduct
a comprehensive survey of the applied aspects of lipase
research from bacteria, fungi and yeast. Emphasis is given on
more recent developments of the past decade or so.
Sources of lipases
Although lipases are of widespread occurrence throughout
the Earth’s flora and fauna, they are found more abundantly
in microbial flora comprising bacteria, fungi and yeast [4–6].
1
To whom correspondence should be addressed.
2
Present address : Biotechnology Department, Christian College, Kattakada,
Trivandrum, India.
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120 A. Pandey and others

Table 1 Microbial sources of lipases They are also found in the pancreas of mammals such as pigs
Micro-organism References and humans, and in higher plants such as castor bean (Ricinus
communis) and rapeseed (Brassica napus) [7].
Achromobacter sp. [8]
Acinetobacter sp. [9] In the field of biotechnology, much attention has been
A. calcoaceticus [185] paid to the use of lipases of microbial origin. As is evident
Alcaligenes sp. [8,10,11] from Table 1, although a large number of microbial strains
A. denitrificans [11]
Arthrobacter sp. [8,13–15] have been used for the enzyme production, Candida sp.,
Aspergillus sp. [16,17] Pseudomonas sp. and Rhizopus sp. are the important sources.
A. niger [10,18–22] Candida rugosa has been termed the most frequently used
A. oryzae [22–24,118]
Bacillus laterosporus [25] organism for lipase synthesis [51].
B. sphericus [25] The industrial deployment of lipases can either be in situ
B. thermocatenulatus* [115] by cultivating the desired micro-organism in the medium
B. thiaminolyticus [25]
Candida sp. [16,17,26–28] with a suitable substrate (especially in the food and tanning
C. antarctica [26,27,29–35] industries), or by ex situ application by using purified
C. cylindracea [8,10,21,29,31,32,36–43] commercial lipases, particularly in the making of fine
C. lipolytica [10,22]
C. rugosa [18,20,44–57] chemicals [6,87]. Solid-state or submerged fermentation
Chromobacterium sp. [8,13] technologies are generally employed in the former case,
C. viscosum [21,40,46,58,59] whereas the latter requires the use of immobilized enzymes
Coelomyceles [129]
Cryptococcus laurentii [25] in abundance [51–57,88–91]. Although microbial lipases
Enterococcus faecalis [60] have traditionally been produced in submerged fermen-
Flavobacterium ferruginem [25] tation, much attention has recently been paid to enzyme
Geotrichum candidum [10,29,40]
Glomus versiforme [61] production in solid-state fermentation [92]. Solid-state
Hansenula anomala [22] fermentation offers several advantages over liquid fermen-
Humicola lanuginosa [10,29,36] tation, including higher product titres, simpler growth and
Microthrix parvicella [9]
Mucor javanicus [20,47] production media, concentrated product, simpler isolation
M. miehei [10,30,42,45,62] techniques and minimal generation of liquid waste [93–96].
Mycobacterium chelonae [81]
Neurospora sitophila [63]
Nocardia amarae [9]
Penicillium camembertii [117]
P. candidum [64,65] Industrial applications of lipases
P. citrinum [66–70]
P. cyclopium [117]
P. expansum [117]
Lipases are an excellent alternative to classical organic
P. roquefortii [80] techniques in the selective transformation of complex
P. simplicissimum [117] molecules. They possess many features that favour their use
P. solitum [117]
P. urticae [123]
as an excellent biocatalyst. They impart specificity to a
Protaminobacter alboflavus [25] reaction in which a chemical process is typically more non-
Pseudomonas sp. [8,13,16,17,21,27,29,40,71–73] specific. In addition, the use of enzyme can decrease side
P. aeruginosa [11,74]
P. cepacia [20,31,49,75,76]
reactions and simplify post-reaction separation problems.
P. fluorescens [13,20,31,46,49,77] Lipase-catalysed processes offer cost-effectiveness too, in
P. fragi [78] comparison with traditional downstream processing in
P. pseudoalcaligenes [74]
Rhizomucor miehei [44,46,109]
which energy consumption and toxic by-products might
Rhizopus sp. [16,46,47] often present problem [97]. Table 2 enumerates a few of the
R. arrhizus [47,62,79,80] most significant industrial applications of microbial lipases.
R. delemer [16,17,21,40,82]
R. javanicus [37]
R. oligospora [63]
R. oryzae [10,43,83]
R. nigricans [22] Lipases in the food industry
Rhodococcus rubra [9] Lipases have become an integral part of the modern food
Rhodopseudomonas sphaeroides [25] industry. The use of enzymes to improve the traditional
Rhodotorula glutinus [25]
Saccharomycopsis fibuligera [25] chemical processes of food manufacture has been developed
Saccharomyces cerevisae [135] in the past few years. Nowadays industrial enzymes, es-
Staphylococcus hyicus [84] pecially lipases, are commonly used in the production of a
S. warneri [85]
S. xylosus [85] variety of products, ranging from fruit juices, baked foods
Streptomyces sp. [128] and vegetable fermentation to dairy enrichment [80,98].
Ustilago maydis [75]
Yarrowia
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Portland Press Ltd [86]
* Expressed in E. coli.
 Microbial lipases 121

Table 2 Some important applications of microbial lipases in biotechnological processes and products

Biomedical applications Waste management

Source [reference] Pesticide [reference] [reference]

Acinetobacter – – Heating oil/furnace oil [185]

calcoaceticus
Arthrobacter sp. – Pyrethroids [168] –
Aspergillus sp. Prostaglandins [131] – –
A. oryzae – – Waste hair [202]
Bacillus subtilis Cephalosporin [130] – –
Candida sp. Pyrolidinedione derivatives [139] – –
C. cylindracea Racemic naproxen [38] – –
C. rugosa Naproxen [140], ibuprofen [122], Ivermectin [136], diazole [41], cyclohexyl –
Aconitium alkaloids [137] acetate [162], tetrahydrofuran [164],
triazole/morpholine [165], 2,4-D [170]
Chromobacterium Vitamin D [126], Verlukast [134] – –
viscosum
Coelomyceles FO-2546 [129] – –
Microbial Antitumour lighans [138] Venturicidin [10] De-watered sludge [186], polymer waste
[191], waste fat [192], waste water
[193], biofilm deposits [197], waste
edible oil [198], slurry [201]
M. miehei Ketoprofen [133] Geraniol [160] –
Penicillium urticae Patuloide A [123] – –
Pseudomonas sp. Pyrolidinedione derivatives [139], (k)- Triazole/morpholine [165] Petroleum-contaminated soil [90],
Indolmycin [72] poisonous gas [202]
P. cepacia Rapamycin-42 [132], Nikkomycin-B [121] Pyrenophorin [159], fenpropimorph –
[161], racemic vinylglycine [163], racemic
morpholine [166], cyanohydrin acetate
[167], pyrethroids [167],
dicyclopentadiene [169]
P. fluorescens Hydantoins [124], Lamivudine (3TC) Tetraconazole [171] –
[127], racemic 2-tetradecyloxirane-
carboxylate [31]
Rhizopus delemer Aryglyceral derivatives [125] – –
R. oryzae – – Palm oil mill waste [199]
Saccharomyces cerevisae Hair tonic [135] Racemic morpholine [165] –
Streptomyces sp. Penicillins [128] – –
Yeast (lipophilic) – – Food waste water [196]

Microbial lipases have been used for the production of
desirable flavours in cheese and other foods, and for the
interesterification of fats and oils to produce modified
acylglycerols, which cannot be obtained by conventional
chemical interesterification [99].
Fats, oils and allied compounds are the main targets of
lipases in food technology. During storage, one of the most
important changes that occur in the lipid fraction is the
hydrolysis of triacylglycerols, catalysed by lipase retaining
non-esterified fatty acids, which are very important for the
characteristic flavour of these products [100]. This aspect
can be well exploited in the laboratory by using commercial
lipases. Similarly, the desired moiety of the triacylglycerol
can be deleted or even added under controlled esterification
and transesterification reactions by specific immobilized or
free lipases [99]. Yoneda et al. [71] have patented a process
on Pseudomonas lipase, which was claimed to be useful in, for
example, food processing and oil manufacture.
Buisman et al. [29] used immobilized lipases from C.
antarctica (CAL-B), C. cylindracea AY30, Humicola lanuginosa,
Pseudomonas sp. and Geotrichum candidum for the esteri-
fication of functionalized phenols for synthesis of lipophilic
antioxidants to be used in sunflower oil [129]. Cao et al. [26]
reported a lipase-catalysed solid-phase synthesis of sugar
fatty acid esters. There are other reports also describing
lipase-catalysed esterification, which might have potential for
food applications [33,44,46,71,78]. Gelo et al. [35] reported
lipase-catalysed esterification in dry medium under focused
microwave irradiation, which were advantageous in terms of
yields and\or purity of the products.
Accurate control of lipase concentration, pH, tem-
perature and emulsion content is required to maximize the
production of flavour and fragrance [80,100]. A survey on
major commercial lipases revealed that Aspergillus lipases
were highly selective for short-chain acids and alcohol ; C.
rugosa (formerly C. cylindracea) lipase was more selective for
propionic acid, butyric acid, butanol, pentanol and hexanol
[101]. Mucor miehei and Rhizopus arrhizus lipases were more
selective for long-chain acids and acetates. This enzymic
catalysis was a valuable method for the production of flavour
ester [102]. Immobilized lipases from M. miehei and C.
antarctica were used for synthesis of short-chain flavour

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122 A. Pandey and others
thioester in solvent-free medium [30]. The production of
flavour esters by lipases of Staphylococcus warneri and S.
xylosus has also been described by Talon et al. [85]. M. miehei
lipase was used by Karra et al. [62] to prepare geraniol esters
in a solvent-free system, which are important components of
fragrances.
Zalacain et al. [98] studied the impact of the addition of
bacterial lipase to fermented sausages on the lipolytic
process. Results showed that the quantity of saturated non-
esterified fatty acids was more than that of polyunsaturated
non-esterified fatty acids. Alcoholysis of cod liver oil for the
production of ω-3 polyunsaturated fatty acids was invest-
igated by using Pseudomonas lipase [103]. Initially, triacylgly-
cerols were converted to diacylglycerols and monoacylgly-
cerols ; non-esterified fatty acids and fatty acid propan-2-yl
esters were obtained in succeeding steps. It has been
reported that fragrance development in dairy products was
dependent on the release of volatile fatty acids. When cattle,
sheep and goat milk fats were treated with different microbial
lipases, C. rugosa lipase yielded large amounts of volatile
branched-chain fatty acids non-selectively ; only small quan-
tities of branched-chain fatty acids were produced. Lipase
from Penicillium roquefortii, Aspergillus niger and R. arrhizus
yielded large amounts of medium chain fatty acids [80,104].
Chromobacterium viscosum lipase showed good potential for
the instant generation of aroma and flavour compounds and
could be stored at least for 1 month ; the lipase activity was
immediately regenerated on dehydration [59].
Considerable efforts have been made to commercialize
lipase-catalysed transesterification and interesterification for
the production of relatively valuable food products [80,105].
Kanisawa et al. [106] investigated the production of dairy
flavours in concentrated milk and creams by using various
microbial lipases. Organoleptically, each lipase developed a
characteristic flavour. C. rugosa lipase, however, emerged as
the most suitable candidate. Ester exchange for vegetable oil
modification involving microbial lipases suggested that lipases
converted triolein in vegetable oil to a monoleate and it
could be used for improving inexpensive vegetable oil with
high industrial qualities. Unilever obtained a series of patents
for the interesterification of fats and acylglycerols [107]. This
process afforded interesterified fats suitable for use in
emulsions and other fat-based food products such as
margarine, artificial creams and ice creams. A patent was
obtained by Suisancho-Chokan for the efficient synthesis of
lipid containing highly unsaturated fatty acids in high content.
The method uses one or a mixture of lipases and phos-
pholipases. The resultant lipids are used in health foods
[108]. The activity and stability of lipases are dependent on
the reaction medium components such as solvents and other
substances such as surfactants and detergents [109].
Fermented foods prepared from fruits, vegetables,
cereals, root crops, legumes and oilseeds are found all over
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the world in baked and cooked forms [4,80,110]. An
important result of the fermentation of fruits and vegetables
was that the products obtained could be stored for a long
time as a food supply during off-seasons. The bioconversion
resulting from fermentation contributed greatly to the
character and organoleptic properties of the fermented
products. In many cases fermentation also contributed to
the digestibility and nutritional value of the products. This
can be well illustrated with an example. Soybeans could be
stored relatively well in the dry state but they were still not
suitable for consumption, even after cooking. When con-
verted to tempe (Indonesian food), they formed a base
material for many delicious, easily digestible and nutritious
food preparations, providing a good number of human
populations with a valuable and affordable source of protein.
In addition to tempe, kenkey and mave (African food) coupled
with fermented vegetables and salads should be noted in this
context [111].
From a food safety point of view, it has been suggested
that fermented foods might have considerable potential
against enteric pathogens such as enterotoxic Escherichia coli
and Shigella flexnei. Volatile fatty acids including aromatics
could be preserved during processing by subjecting them to
dilution [110]. It is important that the lipases produced by
microbial sources do not exhibit any toxicity when used in
food applications. The evaluation of safety involves mainly
testing for acute, subacute and subchronic oral toxicity and
mutagenic potential. Coenen et al. [83] subjected the lipase
derived from Rhizopus oryzae to a series of toxicological
evaluations to establish its safety as a food additive. An
extensive literature search as well as their own experiments
revealed no safety concern for lipase preparation derived
from R. oryzae under controlled fermentation conditions.
Though microbial lipases are best utilized for food
processing, a few, especially psychrotrophic bacteria of
Pseudomonas sp. and a few moulds of Rhizopus sp. and Mucor
sp. cause havoc with milk and dairy products and soft fruits
[83,112]. In a review, Stead [112] described the role of
lipases in psychrotrophs in food spoilage and their control. A
bacterial strain of Pseudomonas fluorescens B52 was identified
as a major cause to such spoilage. Lipases have also been
used as index for the rapid identification of Clostridium
botulinum and botulinus toxin in food [113].
The lipase mediation of carbohydrate esters of fatty
acids offers a potential market for use as emulsifiers in food,
pharmaceuticals and cosmetics [4,5,80,111]. Asai Electro-
chemical produced maltose- and lactose-like sugar fatty acid
esters by using microbial lipase [1]. The production of these
esters involved reacting the disaccharide or its analogue
(with at least one primary alcohol group) and a C8–C22 fatty
acid in the presence of a hydrolase. Suitable fatty acids
include myristic acid, stearic acid, linolenic acid and hydroxy-
stearic acid. The sugars include acetyl or amino or acetyl-

amino maltose or lactose. Nakano et al. [114] formulated an
industrially feasible method for glycero-glycolipid production
from a mixture containing glycosides and fatty acids by using
Penicillium camembertii lipase.
Lipases have been used for the quantification of
triacylglycerols. The thermoalkalophilic lipase of Bacillus
thermocatenulatus, expressed in E. coli BL321, cleaved each of
the three ester bonds of trioleylglycerol [115]. VanVeld-
hoven et al. [116] developed a protocol for the quantitative
measurement of the amount of triacylglycerols in extracts
from cultured cells or tissues. Ibrik et al. [117] compared
triacylglycerol lipases from several species of Penicillium.
Lipase from P. cyclopium showed no similarity to P. camem-
bertii, which was specific for monoglycerols and diacyl-
glycerols. P. cyclopium lipase showed high similarity to lipase
produced by P. expansum and P. solitum but low similarity to
that of P. simplicissimum. A triacylglycerol lipase from
Aspergillus oryzae showed a high specificity towards triacylgly-
cerols of middle-chain saturated fatty acids [118]. The
enzyme hydrolysed monoacylglycerols, diacylglycerols and
triacylglycerols.
Recently, microemulsions have emerged as a tool for
the regioselective lipase-catalysed esterification of aliphatic
diols [36]. The direct conversion of alkane diols into their
monoesters and diesters has strong implications for the food
and pharmaceutical industries, because the products have
non-ionic surfactant activity and can be used as monomeric
units in cross-linking in polymerization.
Lipases in biomedical applications
Because of their excellent capability for specific regio-
selective reactions in a variety of organic solvents with broad
substrate recognition, lipases have emerged as an important
biocatalyst in biomedical applications. Recently, Parmar et al.
[119] reviewed a variety of substrates accepted by hydrolytic
enzymes, including lipases, to produce compounds in high
enantiomeric excess, which can be used as chiral building
blocks for the synthesis of compounds of pharmaceutical
interest. There are other reports on the application of
microbial lipases to the hydrolysis of racemic esters, to
transesterification and to racemization in situ to yield
optically pure enantiomers for the manufacture of chiral
synthons [120–122]. In addition to racemization in situ,
lipases are also capable of catalysing synthetic reactions,
which lead to the production of life-saving drugs. Efficient
kinetic resolution processes are in vogue for the preparation
of optically active homochiral intermediates for the synthesis
of nikkomycin-B, non-steroidal anti-inflammatory drugs
(naproxen, ibuprofen, suprofen and ketoprofen), the po-
tential anti-viral agent lamivudine (which can be used against
HIV) and for the enantiospecific synthesis of anti-tumour
agents, alkaloids, antibiotics and vitamins [123–140].
Microbial lipases 123
Conventional chemical synthesis of drugs containing a
chiral centre generally yields equal mixtures of enantiomers
[120]. During the past decade, many studies have shown that
racemic drugs usually have the desired therapeutic activity
residing mainly in one of the enantiomers and the other
enantiomers might interact with different receptor sites,
which can cause unwanted side effects. Cui et al. [38]
reported lipase-catalysed esterification in organic solvent to
resolve racemic naproxen. Naproxen is a non-sterol anti-
inflammatory drug whose (S)-enantiomer is 28-fold more
active than that the corresponding (R)-enantiomer. Chang
and Tsai [18] also reported a facile process for the
preparation of (S)-naproxen ester prodrug in organic
solvents in which lipases from various microbial sources
were used. Akita et al. [72] performed enzymic hydrolysis in
organic solvents for the kinetic resolution of water-insoluble
α-acyloxy esters with immobilized lipases to produce chiral
intermediates for the synthesis of the calcium antagonist
diltiazem hydrochloride and the antibiotic (k)-indolmycin.
Chiral masked glycerol derivatives, which are potential
building blocks for pharmaceuticals, having an additional alkyl
subsituent at the chiral centre, were synthesized chemically
and resolved enzymically using lipases [32].
A. niger and Penicillium urticae lipases were highly
enantiospecific (E l 100), which frequently cleaved un-
desired enantiomers ; C. rugosa lipase, however, was highly
(S)-specific [97,123,131]. Lipases from species of Mucor and
Rhizopus were also (S)-specific with low enantiospecificity (E
l 2–3) [120,125]. Pseudomonas cepacia lipase was found to
be equally active towards both isomers [121]. Enantio-
selective interesterification and transesterification have
great significance in pharmaceuticals for selective acylation
and deacylation. Thus although this field is still in its formative
stage it has already shown considerable academic interest
with expanding industrial applications. Chillemi et al. [141]
performed the chemoenzymic synthesis of lysophosphatidyl-
nucleosides, which involved lipase-catalysed transacylation.
The synthesis was also applied to the preparation of O-(1-O-
palmitoyl-sn-glycero-3-phosphoryl) conjugates of Acyclovir
and AZT, which are of potential pharmacological interest.
A method was developed by Jimenez et al. [31] to
synthesize methyl (R)- and (S)-2-tetradecyloxiranecarboxyl-
ate through sequential kinetic resolution catalysed by lipases.
Both the enantiomers are a potent anti-diabetic and anti-
oxidant agent. Hernaiz et al. [48] reported two isoenzymes,
A and B from C. rugosa, that were stereoselective. Although
both isoenzymes mainly esterified (S)-ibuprofen, lipase-B
was more stereoselective. Goto et al. [20] also studied the
enzymic resolution of racemic ibuprofen by surfactant-
coated lipases in organic media. Enzymic esterifications were
performed in dry homogeneous organic media, and the
effects of lipase origin (M. miehei, C. rugosa, P. fluorescens, P.
cepacia and A. niger), alcohol alkyl chain length and reaction
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124 A. Pandey and others
medium reactivity were investigated. Coating with a non-
ionic surfactant enhanced lipase activity in organic media.
Several lipases were used in a process development for
production of the (S)-acid precursor of a novel elastase
inhibitor, L-694458, an experimental drug in the treatment
of cystic fibrosis [142]. A preparation of optically active
amines that were intermediate in the preparation of
pharmaceuticals and pesticides have been described by Smidt
et al. [27], which involved reacting stereospecific N-
acylamines with lipases, preferably from C. antarctica or
Pseudomonas sp. In an attempt to determine the substrate
specificity for lipases, alkyl esters of 2-arypropionic acids, a
class of non-steroidal anti-inflammatory drugs, were hydro-
lysed with C. rugosa lipase. All transformations were highly
enantioselective [50].
A new method was developed for preparing triacyl-
glycerols containing highly unsaturated fatty acids in large
amounts, which involved treating one or a mixture of
monoacylglycerols and\or diacylglycerols containing highly
unsaturated fatty acids (e.g. docosahexaenoic acid) with one
or more lipases. The product has improved learning
functions, activates the immune system and has anti-
arteriosclerotic, anti-tumor and anti-allergic activities [40].
Lipases were also used to produce an enzymically active
precursor of the CC-1065\duocarmycin pharmacophore
[143,144].
Lipases as biosensors
Sensing lipids and lipid-binding proteins are a developing
technology [145]. In the fat and oil industry, in food
technology and in clinical diagnosis, the quantitative de-
termination of triacylglycerols is of great importance
[146,147]. Chemical methods for the analysis are rather
costly and time-consuming. A promising new method
involves the manipulation of microbial lipases as a biosensor.
Biosensors can be of a chemical, biochemical or electronic
nature. Biochemical biosensors can have enzymes, anti-
bodies, other proteins, large organs, cells or cell extracts,
immobilized or linked to suitable signal producers (trans-
ducers) [148]. Besides potentiometric (ion-selective) am-
perometric arrangements, transducers can involve optic
fibres, piezoelectric and heat-toning systems. These can be
minimized in suitable ceramic, piezo or optical elements,
which are compatible with microelectronics, which permits
the direct linking of signal production to the sensors. If
electrical systems are unsuitable, an optic fibre system can be
applied.
Danilov and Egorov [149] reviewed the data on the
creation of biosensors with bacterial bioluminescence in
medicine. An important analytical use of lipases in the
determination of lipids was for clinical purposes [150]. The
basic concept was to utilize a lipase to generate glycerol
from the triacylglycerol in the analytical sample and to
# 1999 Portland Press Ltd
quantify the released glycerol (or alternatively the non-
esterified fatty acids) by a chemical or enzymic method. As
far as physicians were concerned, this principle enabled
them to diagnose the patients with cardiovascular complaints
very precisely. Shoemaker et al. [147] developed a promising
method, which involved the use of microbial lipase, in which
the glycerol liberated during lipid hydrolysis was oxidized by
glycerol dehydrogenase. NADH formed during the reaction
was measured by fluorescence spectroscopy. Non-specific
lipases, especially that of C. rugosa, with high specific activity
have been selected to allow the rapid liberation of glycerol.
Lipase biosensors are in vogue not only in the diagnosis
of clinical samples : they have found new horizons such as the
food and drinks industry, pollution analysis, especially
pesticide contamination, and the pharmaceutical industry
[151]. A C. rugosa lipase biosensor from C. rugosa has been
developed as a DNA probe by Pittner et al., which optimally
conjugates to a biorecognition group in DNA [152]. Schoe-
maker et al. [147] developed a lipoxygenase biosensor, which
was useful in the determination of essential fatty acids in
foods.
Krawczyk [153] reviewed analytical applications of the
inhibition of enzymic reactions in the determination of
environmental pollutants. Numerous enzymes in immobil-
ized form such as biosensors were used, which included
lipase for the determination of fluoride. Wei et al. [154,155]
developed a method for the enzymic determination of
organophosphorous pesticides with a surface-acoustic-wave
impedance sensor, which was based on lipase-catalysed
hydrolysis. It was successfully applied to the determination
of dichlorovos residues in the root, stem and blade of
Chinese cabbage.
Levanon et al. [156] performed the isolation and
screening of micro-organisms from the Amazon rainforest in
Brazil. The aim of the programme was to screen for
thermostable lipases and other enzymes of biotechnological
significance for use in biosensors.
With recent advances in biosensor industry, it seems
that a rational strategy for achieving particular enzyme might
be within reach. Fagin and Kennedy [157] have reviewed the
range of option in constructing such biosensors. The major
objectives are that substrate has to be presented in a manner
that meets the interfacial construction of lipase activity and
also to facilitate the repeated use of the biocatalyst by
immobilization, owing to its cost effectiveness. In fact, the
immobilization of lipase in CLIEC was found to be very useful
in analysis in a fluid. Furthermore, the advent of modern
computers has also revolutionized biosensor technology,
from the oldest electrode to modern miniaturized optically
active models through calorimetric determination [148].
Much remains to be done by enzyme technologists and
biomedical engineers towards achieving developments in
this area.

Lipases in pesticides
Fine and intermediate chemicals makers emphasize new
products and processes for the pesticide industry via lipases,
in view of its potential for decreasing costs and environ-
mental contamination [158]. To make it cost-effective,
lipases are used mostly in immobilized form without loss of
viability for longer periods [157]. A variety of pesticides
(insecticides, herbicides, fungicides or their precursors)
made with the application of lipases are currently in use
[159–171]. The most important application of lipases has
been in the organic synthesis of pesticides for the production
of optically active compound [120]. Generally, these com-
pounds were produced through the resolution of racemic
mixtures of alcohol or carboxylic esters ; stereospecific
synthesis reactions were also employed. Akita et al. [121]
described a highly stereospecific synthesis of the versatile
chiral synthon possessing two stereogenic centres, which
was subsequently converted into a homochiral intermediate
for the synthesis of the biologically active potent pesticide
nikkomycin-B.
Mitsuda et al. [13] reported the preparation of (k)-α-
ethynyl alcohol moieties of pyrethroid insecticides by lipase-
catalysed enantioselective hydrolysis. Hirohara et al. [15]
also reported the enzymic preparation of optically active
alcohols related to synthetic pyrethroid insecticides. A
patent was granted on the production of an optically active
vinylglycine compound by the enzymic resolution of a
racemic vinylglycine in aqueous and also in two-phase
systems, which was an intermediate in the production of
various compounds with a broad spectrum of activities as a
herbicide, fungicide, or anti-viral agent [163]. In an another
patent, the synthesis of a new ivermectin fatty acid ester
derivative esterified by lipase has been reported. Apart from
its usefulness in human and veterinary medicines, ivermectin
was found to be active against many pests [136]. A new
method was described for the production of the optically
active malate β-monoalkyl ester or optically active isoserine,
which involved treating the ester with a lipase and esterase.
Optically active isoserine is used as a starting material for the
cost-effective and efficient production of medicines and
pesticides [25].
In the field of pesticide biotechnology, much attention
has been focused on the use of lipases as an enantioselective
biocatalyst in organic media. The restricted flexibility of
microbial lipases in organic solvents paved the way for new
possibilities in the production of enantiomeric substances
for the pesticide industry [10]. Mitsuda and Nabeshima [77]
studied five microbial lipases for the enantioselective trans-
esterification of a racemate (R,S)-4-methyl-1-heptyn-4-en-3-
ol, a component of the insecticide S-2852, in which P.
fluorescens lipase yielded the highest reaction rate. Mitsuda et
al. [8] examined the lipase-catalysed enantioselective hy-
drolysis of the acetic acid ester of racemic α-cyano-3-
Microbial lipases 125
phenoxybenzyl alcohol (CPBA) for the production of (S)-
CPBA, an active insecticidal stereoisomer. Lipases were
derived from several bacterial, yeast and fungal sources.
They proposed a chemico-enzymic process for the prep-
aration of (S)-CPBA. By a combination of the enzymic
resolution with a chemical inversion of the (R)- alcohol,
Mitsuda et al. [14] also developed an efficient process for the
total conversion of racemic 4-hydroxy-3-methyl-2-(2-propy-
nyl)-cyclopent-2-enone (HMPC) to the (S)- form, an im-
portant intermediate in the preparation of an insecticidally
active synthetic pyrethroid. The enzyme involved was a
lipase from a microbial source.
The lipase-catalysed resolution of 2-(4-propan-2-
yloxy)-phenyl propionic acid, an intermediate in the synthesis
of a chiral acaricide, was described by Bosetti et al. [41],
using lipase from yeast, fungus and bacteria. The stereo-
selective enantio-discrimination of C. rugosa lipase yielded
optically pure propionic acid derivative in the (S)- form. The
(S)- form was then converted to the corresponding (R)-
form, which was found to be very effective as an ovicide
against the pest Tetranychus urticae [42]. Avdagic et al.
reported the production of enantiomerically pure (S)-(-)-
fenpropimorph, a systematic fungicide with P. cepacia lipase-
mediated kinetic resolution [161]. Lipases have also been
used in the synthesis of the carbinol stereoisomers of
8-methyl-2-decanol esters, which are used by several
Diabrotica (rootworm) species that are serious pests of
maize [172].
Lipases in detergents
Biological detergent making is a fast-growing technology
[173]. Lipase, protease, amylase and cellulase enzymes are
added to the detergents where they catalyse the breakdown
of chemical bonds on the addition of water. Lipases split fats
(triacylglycerols), proteases split protein, amylases split
starch, and cellulases split cotton-fluff. Lipases are generally
added to the detergents primarily in combination with
proteases and cellulases. However, there might be other
enzymes such as amylases, peroxidases and oxidases [174].
At present they are in extensive use in household detergents
and industrial cleaners, and in leather processing [74]. Lipases
have also been used in the formulations prepared to clean
drains clogged with food and\or non-food plant-material-
containing deposit [175]. Here they are used in association
with pectinase.
Removal of oil\fatty deposits by lipase is attractive
owing to its suitability under washing conditions. To be a
suitable additive in detergents, lipases should be both
thermophilic and alkalophilic and capable of functioning in
the presence of the various components of washing powder
formulations [4,5].
Most lipases have been reported to be optimally active
at neutral pH in the temperature range 35–48 mC [4]. Misset
# 1999 Portland Press Ltd
126 A. Pandey and others
et al. tailored Pseudomonas alkaligenes lipase for elevated
activity at washing conditions, such as alkaline pH (7–11),
high temperature (up to 60 mC) and the presence of non-
ionic and anionic detergents [176]. This enzyme was also
found to be stable during storage in the surfactants and in the
washing machine. The Novo group has reported a highly
alkaline, positionally non-specific lipase from a strain of
Streptomyces sp. that was useful in laundry and dish-washing
detergents, industrial cleaners, for example ; in addition
thermophilic lipases have been reported from many bacterial
species [5,177,178]. The stability and activity of detergent
enzymes were crucial factors, which were greatly improved
by the so-called ‘ surfactant lipases ’ [179]. Lipolase, which
was introduced by Novo, was a pioneer in this regard. It was
developed by genetic and protein engineering of the Humicola
lanuginosa lipase gene into the A. oryzae genome for its
overproduction [180]. Unilever, Cosmo Oil and Procter &
Gamble are major companies working on recombinant DNA
technology for surfactant lipases [5]. As a whole, the present
status of lipase-mediated detergent promises a high-tonnage
use with the prospects of recombinant enzymes.
Lipases in the leather industry
Leather processing involves the removal of subcutaneous fat,
dehairing and stuffing. An enzyme preparation that contained
lipases in combination with other hydrolytic enzymes such as
proteases would open a new avenue in leather processing. A
new enzymic process for the production of hides and skins,
ready for tanning, involved steps of soaking, washing,
dehairing and bathing in aqueous baths, where each bath had
a pH of 8–13 and contained an alkalophilic lipase [4,181]. In
this process, no tenside was used in various stages of
soaking, washing, dehairing and bathing and involved minimal
use of the detergent. A Russian patent described an
alternative technology for sheep skin that improved the
quality of intermediate sheep skin products by increasing the
strength and elasticity of the skin and decreasing rigidity,
with a decreased use of surfactants [182]. Tanning processes
are usually performed in an alkaline environment, so
alkalophilic microbes ought to be better for exploration.
Many Bacillus sp. strains, which grew successfully under
highly alkaline conditions, were found to be useful in leather
processing [183]. Alkaline lipases might be used in com-
bination with alkaline or neutral proteases and\or a sur-
factant and\or sequestrant [184].
Lipases in environmental management
Bioremediation for waste disposal is a new avenue in lipase
biotechnology. Oil spills during rigging and refining, oil-wet
night soils and shore sand, and lipid-tinged wastes in lipid
processing factories and restaurants could be well handled
by the use of lipases of different origins [185–204]. For
# 1999 Portland Press Ltd
instance, lipases have been used extensively (ex situ or in situ)
in wastewater treatment [186,193]. Dauber and Boehnke
devised a technology to convert dewatered sludge in the
factories to biogas, in which an enzyme mixture including
lipase was used [186]. A method was proposed for the
treatment of waste by the direct cultivation of lipophilic
microbes into the waste with controlled cost [196]. Another
important application of lipases has been reported in the
degradation of polyester waste into useful products, es-
pecially for the production of non-esterified fatty acids and
lactones [189,191,194]. The broadening use of lipases in
bioremediation has achieved more importance with its
successful application to the removal of biofilm deposits
from cooling water systems [197], to the manufacture of
liquid soap [198], to the upgrading of waste fat [199], to
bleaching [185] and to the purification of waste gases
expelled from factories [203]. The fascinating role of A.
oryzae lipase in the degradation of waste hair for the
production of cystine was another milestone in the bio-
remedial application of lipase [202]. The production of
single-cell protein from industrial effluents with the use of
lipophilic micro-organisms, especially yeast, could provide
yet another prospect for the application of lipases to waste
management.
Lipases have also been used for the degradation of
wastewater contaminants such as olive oil from oil mills. The
treatment process generally consists of the cultivation of
lipase-producing microbial strains in the effluents [86,205].
Wakelin and Forster [9] investigated the microbial treatment
of waste from fast-food restaurants for the removal of fats,
oils and greases. They cultivated pure and mixed microbial
flora (known to produce lipases and other enzymes).
Acinetobacter was the most effective of the pure cultures,
typically degrading 60–65 % of the fatty material.
Lipases in the cosmetics and perfume industry
The overwhelming interest of technocrats in screening
lipases for use in the cosmetic and perfume industry has
mainly been due to its activity in surfactants and in aroma
production, which are the main ingredients in cosmetics and
perfumes [206]. Monoacylglycerols and diacylglycerols, pre-
pared by the lipase-catalysed esterification of glycerol, are
useful as surfactants in cosmetics [207,208]. Mixed-acid-type
polyester [209] and acylglycerol ester fatty acids [210] were
the main components in lipase-mediated cosmetics. Izumi et
al. [45] performed the transesterification of 3,7-dimethyl-
4,7-octadien-1-ol with lipases from various microbial
sources to prepare rose oxide, which is an important
fragrance ingredient in the perfume industry.
Two patents were obtained by Asahi-Electrochem for
the production of maltose-like and lactose-like sugar fatty
acid esters [16,17]. These esters have numerous ap-
plications, for example in cosmetics, food and medicines.

Miyamoto et al. [211] obtained a patent for the utilization of
lipases in the production of glycerine mixtures. This was to
be used as an external preparation or by application directly
to skin. Another patent involved lipases as the main catalyst
for cosmetic production from plant ash. This was claimed to
be an efficient skin-conditioning agent [212]. Nippon Oil &
Fats also obtained a patent for the preparation of propyl-
eneglycerol mono-fatty acid ester in the presence of lipase ;
this ester has uses as, for example, an emulsifier and a
pearling agent in cosmetics and foods [21].
Conclusions
The exponential increase in the use of lipases in bio-
technology in comparison with other hydrolytic enzymes
indicates the growing demand for and application of lipases,
especially in making fine chemicals. The concept of ‘ weight-
less fatty acid ’, which could be obtained from outer space, is
ideally suited to the manufacture of improved food additives :
a diet control foodstuff of the twenty-first century. There is
a gradual emergence of a fascinating aspect of meat
technology, the processing of sausages with microbial lipases,
which shows tremendous potential, but is not yet fully
developed. The widening applications of biotechnology and
the necessity for continued research and development on
fats and oils in relation to the food industry suggest that
microbial lipases will have an increasingly important role in
this area. A host of new fields are still to be exploited for the
commercial application of lipases, for instance the pro-
duction of single-cell protein\probiotics. Resource recovery
by single-cell protein with the use of yeasts (Candida sp.,
Saccharomyces sp.) and bacteria (Lactobacillus sp., Alcaligens
sp.) offer much potential. The use of lipases in fields such as
cosmetics, bleaching, pulping, plastics, lubricants and fil-
tration are under-exploited. As a whole, lipase bio-
technology has just reached the end of the lag phase and the
beginning of the exponential phase : it demands extension in
terms of both quality and quantity. Qualitative improvements
can be achieved by employing already established recom-
binant DNA technology and protein engineering to restruc-
ture the lipase gene and its protein. Quantitative enhance-
ment needs strain improvement, especially through site-
directed mutagenesis and standardizing the nutrient medium
for the overproduction of lipids\lipases. Finally, we hope
that this review will yield insight into the lipase-mediated
biotechnology with a view to broadening its scope.
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Received 7 December 1998 ; accepted 13 January 1999
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