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2. Literature review
2.1 Biosphere
The biosphere is the aggregate of the earth’s self- regulating ecosystems
that continuously interact with the lithosphere, hydrosphere and
stratosphere governed by the natural forces (Vernadsky, 1998). This
biosphere is postulated to have been evolving for more than 3.5 billion
years, through a process of biogenesis or biopoiesis (Campbell et al., 2006).
This biogenesis has started with the evolution of unicellular
microorganisms and then expanded to multicellular microorganisms fungi,
and the process of evolution had been in place on this earth for nearly 4
billion years.
These microbial organisms including both unicellular and multicellular
organisms are capable of exploiting a vast range of energy sources for their
growth and survival in every habitat including the habitats of extremes of
heat, cold, radiation, pressure, salt, acidity and darkness. So, the
microorganisms had been working in these habitats for over 2 billion years
preparing the ground for the evolution of higher order life forms like
insects, animals, plants and ultimately the humans. Although there are
diverse life forms existing in this biosphere, the microbial populations are
estimated to constitute 50% of the living protoplasm thriving on this earth’s
biosphere (Whitman et al., 1998). So the microbial populations in the
biosphere represents the richest repertoire of molecular and chemical
diversity in the nature and hence they serves as the basis for the sustenance
of life in this biosphere, through their interactions and associations with the
higher order life forms like, plants, animals and human beings. The
richness of the biosphere is seen through diverse life forms and the dynamic
natural forces resulting in the ever changing face of the biosphere.
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2.2 Role of microbes in the sustenance of life
The earth has the reservoirs of life forms at various locations in the
ecological domains for the sustenance of biosphere. One of those important
life reservoirs comes as the diverse microbial flora that can be revived and
sustained by the soil along with other natural forces in the biosphere. These
microbial flora has the capability to recycle the nutrients, produce and
consume gases that affect global climate, destroy pollutants, treat our
wastes and they can be used even for biological control of plant and animal
pests (Kalia et al., 2007a). Because of these peculiar properties, the
microorganisms from different explored and unexplored ecological niches
were isolated repeatedly all over the world for the novel microbial strains
with special characteristics like secondary metabolites for e.g., antibiotics,
hydrogen, methane, bioplastic, enzymes, etc. Along with these characters
other properties can be established that may be beneficial for improving the
products and services to the mankind and the environment (Kalia et al.,
2000; Kalia et al., 2003a; Kalia et al., 2003b; Kalia et al., 2007a; Kalia et
al., 2007b). So, the ecological studies on microbes were made continuously
to understand the diversity of microorganisms and how micro-organisms
interact with each other and with their environment to generate and
maintain such diversities (Konstantinidis et al., 2006) and subsequently use
those studies as key concepts for the creation of novel products and services
to serve the mankind and the society.
2.3 Microbial diversity
Microbial organisms had been evolving every day since the biogenesis
depending on the ecological variations subjected to the climatic conditions,
habitat conditions, radiation, alkalinity, acidity, availability of nutrients, air
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and its constituents, geographic locations, and so on. In other words, these
ecological factors influence microbial activities and play very important
roles in determining the dynamics of micro-organisms in natural
environments (Fenchel, 2005). So the scientists have been studying the
microbial organisms isolated from various habitats with the existing
permutations and combinations of the various environmental parameters
discussed above, at that given point of time, for their identification and
characterization. In this way there had been the huge database of isolated
microbial flora all over the world by various scientists at different point of
times. In spite of this huge data accumulated, there are still unknown
microbial flora existing in the reserve of biosphere, cultivated and exposed
by the nature in the soil, water bodies and the air as per the needs,
sustenance and maintenance of life on the planet earth. Considering this
universal fact, the research had been pursued to isolate the microbial flora
from various habitats and various experiments were conducted to
understand the diversity of microorganisms in terms of variations in
microbial metabolism, structural components, morphological and genetic
characters.
2.4 Habitats of microbial populations
Microbial life forms can thrive in every possible place on this earth where
the essential elements of the life, like water, carbon source and nitrogen
source along with the trace elements are available. Depending on the
amount of nutrients present, the survival and growth of microbial cultures
of diverse species can thrive to the extent that is possible at any given point
of time. So the habitats of microbial organisms are explored by scientists
all over the biosphere (Fenchel 2005) as discussed below.
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2.4.1 Soil
Soil is defined as the layers of minerals drawn from the small rocky particles
and sediments, over a period of time by various factors like, wind, water
etc., having their characteristic texture, structure, color and chemistry. In
other words, the soil is the unconsolidated or loosely bound layer of fine
rock particles that cover the surface of the earth as part of the lithosphere
(Birkeland 1999). So the soil forms a structure filled with pore spaces and
can be considered as a mixture of solids, water, and air (gas) (Taylor and
Ashcroft 1972). Accordingly, soils are often treated as a three-state system
(McCarty and David 2006) and most of the soils have a density between 1
and 2 g/cm³ (http://www.pedosphere.com/ ).
On the basis of volume, a good quality soil contain sand slit and clay as
minerals of 45% and 5% organic material including both live and dead life
forms along with 25% water and 25% air. The content of mineral remain
mostly constant in the soil while the organic contents, water and air are the
variable parameters where the increase in one of these components is
balanced by the reduction in the other component(s). So the organic matter
of the soil sprouts the microbial growth in association with the other
elements of the soil depending on the climatic and favorable conditions as
directed by the nature (Frank et al., 2011). If unfavorable conditions arise
in the form of change in the composition and physiochemical conditions of
the soil the microbial organisms will cease their metabolic activities and
slip in to the dormant stages like spore formations, and frozen conditions.
This cyclic phenomena of microbial life forms in the soil along with the
other higher order life forms makes the soil as one of the best choices for
the isolation of novel microbial organisms depending on the geological
position and the climatic conditions ( Ajay et al., 2007; Eichorst et al., 2007;
Stephanie et al., 2012; Bahig et al., 2008; Srivasthava et al., 2009).
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2.4.2 Water bodies
Water is the major constituent of the life systems in this biosphere and there
are various type of water bodies like, oceans, rivers, lakes, ponds, aquifers,
hot springs where the life forms can be found. Most of these water bodies
contain various minerals and nutrients that have been brought by various
forces of the nature and these nutrients and minerals support the life forms
in these water bodies along with other sources of energy like radiation from
the sun. Since the time of biogenesis these larger water bodies have been
cultivating diverse species of microbial flora (French et al., 1987; Morris et
al., 2002) compared to the other species of higher life forms. In addition to
the geographical locations, the changes in the climatic conditions and the
dynamic nature of the earth, the microbial organisms are revived and
cultivated by these water bodies (Saleem et al., 2011; Hussin et al., 2011;
Velankar 1957) and at the same time they also preserve these microbial life
forms in the sediments till the favorable conditions.
In addition to these natural water bodies, there are many man made water
bodies like artificial ponds, lakes, dams, canals, drainages, water treatment
plants etc. (Kumar et al., 2012; Biswapriya et al., 2012; Vessoni et al., 2002;
Kehinde et al., 2008; Selvi et al., 2012). All these man made artificial water
bodies and drainages from various industries and cities also has vast
collection of microbial organisms deposited by the nature forces like, wind,
rains, volcanic eruptions etc., either by a sole or in combination. In addition
the availability of diverse nutrients in association of water revives diverse
microbial organisms depending on the ambient physiological conditions.
All these water bodies with their unique characters towards the microbial
flora lures the scientists for the search of novel microbial organisms
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24
(Fenchel 2005; Campbell et al., 2006; Purohit et al., 2007; Tiquia et al
2008).
2.4.3 Air
Microbial organisms being the lighter forms of biomass, have been carried
away by the wind or air depending on the dynamic conditions of the earth.
Air of the atmosphere also got water in the form of water vapor as one of
the main constituents and this water content along with the available gases
and organic aerosols in the presence of energy sources like sun’s radiation
can sustain the life forms (Bovallius et al., 1978). Further the climatic
changes on the earth drives the air from one location to the other depending
on the variation in energy levels (Fulton and Mitchell 1966). In this process
the air also erodes the nutrients and minerals from the lithosphere and use
them as the nutrition to cultivate microbial flora. In addition, the volcanic
eruptions also throw various nutrients and gases in to the atmosphere along
with the hidden microbial flora either in live or spore forms. So all these
dynamics of air combined with the other physiological and climatic
conditions along with the human activities revive the microbial organisms
and transport these life forms from one geographical location to the other
(Heidelberg et al., 1997; Rahkio and Korkeala 1997; Jericho et al., 2000;
Amy et al., 2005; Kang and Frank 1989; Chasseignaux et al., 2002). Hence
the air also considered as one of the choices for isolating some air born
microbial species from time to time.
2.4.4 Host organisms
In the biosphere there are various diverse life forms starting from the
unicellular microorganism to multicellular organisms of plant and animal
kingdom to the most complex organisms such as the humans (Desissa et al.,
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25
2009; Feng et al., 2011; Denise et al., 2002; Ishimaru et al., 2001).
Microbial organisms being the smallest life forms can grow in the higher
order life forms either in the symbiotic way or in the parasitic way or may
be as pathogens invading the other life forms (Julien et al., 2003; David
1991; Casper 1934). In such host systems the microbial organisms can grow
themselves as the nutrients can be derived easily from the host organisms.
Although there are diverse species of higher order life forms as per the
changes in the geographical locations, and the climatic conditions, the
microbial organisms make themselves adapted to the diverse situations and
the climatic conditions, and thrives to and make the largest diversity of life
forms on the earth. In this process some of the microbial species have
become part of the higher order life forms and keep surviving in the host
organism throughout its life time; in some cases the microbial organisms
have become the pathogenic organisms causing diseases to the host
organisms (Toth et al., 2006; Midekssa 2011; Drancourt et al., 2000;). In
other cases some of the microbial species do the recycling work of the
nutrients from the dead life forms in to the nutrient pool of the biosphere.
In all these situations there lies the opportunities for the researchers to
isolate the novel microbial organisms that can be used for various purposes
in the industries, institutions and the academia. One of the best practiced
microbial isolations were seen as the clinical isolates of pathogenic
microbial organisms from the infected humans or animals in health centers
of the society (Drancourt et al., 2000; Sujatha et al., 2011).
2.4.5 Caves
During the formation of landscapes on the earth since the last 4 billion
years, the dynamics of the earth’s natural forces made the formation of
caves in the lithosphere. These caves have been under exploration every
day all over the world and has brought the new insights of the ancient
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26
microbial samples that got preserved in the rocky layers of the soil
(Filomena et al., 2012; Barton et al., 2004; Laiz et al., 1999). Caves are
considered to be the most undisturbed areas from the natural physiological
changes and free from the erosion process. These caves are not the every
body’s choice of microbial isolation habitats, but they are being explored
by a very few scientists who are adventurous and driven by passion to
identify the novel microbial strains. In this direction, though such kind of
adventurous and exploration projects are risky in nature, especially the
young scientists and engineers have been willing to take up such projects
and the findings have been excellent as they have added some new species
and genera the existing microbial world. So there are some caves explored
specifically to find the appropriate strains that could produce the antibiotics
and the required medicine for the known and unknown diseases for humans
and animals (Bhullar et al., 2012; Barton et al., 2001; Northup et al., 2003).
2.4.6 Polar ice caps
The polar ice caps of the earth, has the extreme weather conditions with
two type of seasonal changes, one being the winter and the other being the
summer. However there had been the life forms both at micro and macro
levels adopted to these extreme physiological and climatic conditions
(Bowman et al., 1997). There would be a complete freezing of the
vegetation, sea and the other life forms subjected to extreme low
temperature breezes triggering complete ice covering of the land and
mountain ranges. But then some of the life forms still thrive in these
extreme cold conditions especially like seal fish-tiger, polar bear and on
microscopic scale there would be the growth of psychrophilic
microorganisms both on the land and underwater sea in association with the
macroscopic life forms (Boyd 1967; Olson et al., 1998). In the next season
there would be the sunny atmosphere melting the ice caps to certain extent
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27
making the way for vegetation to start growing, and reviving the other
frozen life forms. In addition there would be the visiting life forms like
penguins, seal, whales, birds etc. through the sea waters from one continent
to the other. These visiting life forms also bring diversified microbial flora
along with them across the continents. So all these nature driven activities
at the polar ice caps of Arctic and Antarctic continents cultivate
extremophiles of microbial organisms from time to time as desired by the
nature (Dancer et al., 1997; Junge et al., 1998; Thompson et al., 1998).
Hence these polar caps also make one of the best choices for the scientists
to look for the novel microbial organisms and species that may produce
new products and services for the mankind.
Apart from these above discussed habitats, the microbial organisms can
also be found in many other natural and manmade habitats that are
discovered and made at different points of time.
2.5 Lipase producing microbial organisms
All the life forms in the biosphere possesses lipase as per the needs of the
biochemical reactions being carried out on routine basis and for the special
purposes of lipid metabolism (Rubin and Dennis 1997a; Rubin and Dennis
1997b). Since the lipases produced in these biosystems are part of the
sequential biochemical reactions there would be a limited life span and
activity of these special biomolecules. So as per the design and purpose
every biomolecule in the biosphere has the life cycle and then it would
eventually be degraded. But the microbial systems among the life forms of
the biosphere has the capability of producing excess quantities of
biomolecules like lipases and be released in to the ambient environment to
aid in the consumption of bountiful complex organic molecules like lipids,
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28
oils etc.(Kazlauskas and Bornscheuer 1998; Thomson et al., 1999; Hasan et
al., 2008). This particular property of the microorganisms were exploited to
produce the lipase in excess quantities by stimulating or mitigating suitable
culture and production conditions. In this direction, research has been
carried out on diverse microbial species as shown in the Table-6 by some
of scientists from time to time.
Table 6. Bacterial species explored for the production of lipases by
scientists (Sharma et al., 2001)
Source Genus Species Reference(s)
Bacteria
(Gram-
positive)
Bacillus
B. megaterium Godtfredsen,
1990
B.cereus El-Shafei and
Rezkallah, 1997
B.stearothermophilus
Gowland et al.,
1987;
Kim et al., 1998
B.subtilis Kennedy and
Rennarz,1979
Recombinant B.
subtilis 168
Lesuisse et al.,
1979
B. brevis Hou, 1994
B. thermocatenulatus Rua et al., 1998
Bacillus sp. IHI-91 Becker et
al.,1997
Bacillus strain WAI
28A5
Janssen et
al.,1994
Bacillus sp. Helisto and
Korpela, 1998
B. coagulans EI-Shafei and
Rezkallah, 1997
B. acidocaldarius Manco et al.,
1998
Bacillus sp. RS-12 Sindhu et al.,
1998
B. thermoleovorans
ID-I Lee et al., 1999
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29
Source Genus Species Reference(s)
Bacillus sp. J 33 Nawani and
kaur, 2000
B. subtilis Jaeger et al.
1999
B. alcalophilus Ghanem et al.,
2000
B. pumilus Jaeger et al.
1999
Staphylococcus
S. canosus Tahoun et al.,
1985
S. aureus Lee and
Yandolo, 1986
S. hyicus
Van Oort et al.,
Meens et al.,
1997;
van Kampen et
al.,1998
S. epidermidis
Farrell et
al.,1993;
Simons et al.,
1998
S. haemolyticus Oh et al. 1999
S. xylosus
Pandey et al.,
1999;
Van Kampen et
al.2001
S. warneri Talon et al.,
1995
Lactobacillus
Lactobacillus
delbruckii sub sp.
Bulgaricus
EI-Sawah et al.,
1995
Lactobacillus sp. Meyers et al.,
1996
L. plantarum Lopes Mde et
al., 2002
Streptococcus Streptococcus lactis
Sztajer et
al.,1988
Micrococcus
Micrococcus
freudenreichii Hou, 1994
M. luteus Hou, 1994
Propionibacterium
Propionibacterium
acne
Sztajer et
al.,1988
Pr. granulosum Sztajer et
al.,1988
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30
Source Genus Species Reference(s)
Pr. avidium Brune and Gotz
1992
Burkholderia
Burkholderia sp. Yeo et al., 1998
Bu. glumae EI Khattabi et
al., 2000
Bacteria
(Gram-
negative)
Pseudomonas
P. aeruginosa
Aoyama et al.,
1988;
Hou, 1994
Ito et al., 2001
P. fragi Mencher and
Alford, 1967
P. mendocina Jaeger and
Reetz, 1998
P. putida 3SK Lee and Rhee,
1993
P. glumae
Farrell et
al.,1993;
Noble et al.,
1993;
P. cepacia
Penereach and
Baratti, 1996;
Lang et al.,
1998;
Hsu et al., 2000
P. fluorescens
Maragoni,
1994;
Lacointe et al.,
1996
P. aeruginosa KKA-
5
Sharon et al.,
1996
P. pseudoalcaligenes
F-111
L in et al.,
1995, 1996
Pseudomonas sp.
Sin et al., 1998;
Miyazawa et
al., 1998;
Reetz and
Jaeger, 1998;
Dong et al.,
1999
P. fluorescens MF0 Guillou et al.,
1995
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31
Source Genus Species Reference(s)
P. luteola
Arpigny and
Jaeger 1999;
Litthauer et al.
2000
P. nitroreducens var.
thermotolerans
Ghanem et al.,
2000
Pseudomonas sp.
KW156
Yang et al.,
2000
Chromobacterium
Ch. viscosum
Rees and
Robinson,
1995;
Helisto and
Korpela, 1998;
Jaeger and
Reetz, 1998
Diogo et al.,
1999
Ch. violaceum Koritala et al.
1987
Acinetobacter
Aci.
pseudoalcaligenes
Sztajer et al.,
1988;
Aci. radioresistens Chen et al.,
1999
Acinetobacter sp.
Wakelin and
Forster 1997;
Barbaro et al.,
2001
Aeromonas
Ae. hydrophila Anguita et al.,
1993
Ae. sorbia LP004
Lotrakul and
Dharmsthiti,
1997
Fungi Rhizopus
Rhizop.delemar
Salleh et al.,
1993
Coenen et al.,
1997;
Beer et al.,
1998;
Essamri et al.,
1998;
Hiol et al., 2000
Rhizop. arrhizus
Sztajer and
Maliszewska,
1989;
Page 14
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32
Source Genus Species Reference(s)
Elibol and
Ozer, 2001
Rhizop. nigricans Ghosh et al.,
1996
Rhizop. nodosus Nakashima et
al., 1988
Rhizop.
microsporous
Ghosh et al.,
1996
Rhizop. chinensis Ghosh et al.,
1996
Rhizop. japonicus Nakashima et
al., 1988
Rhizop. niveus Kohno et al.,
1994, 1999
Aspergillus
A. flavus Long et al.,
1996, 1998
A. niger Chen et al.,
1995
A. japonicus Satynarayana
and Johri, 1981
A. awamori Satynarayana
and Johri, 1981
A. fumigatus Satynarayana
and Johri, 1981
A. oryzae Ohnishi et al.,
1994
A. carneus Helisto and
Korpela, 1998
A. repens Kaminishi et
al., 1999
A. nidulans Mayordomo et
al., 2000
Penicillium
Pe. cyclopium Chahinian et
al., 2000
Pe. citrinum
Sztajer and
Maliszewska,
1989;
Pe. roqueforti Petrovic et al.,
1990
Pe. fumiculosum Hou, 1994
Penicillium sp. Helisto and
Korpela, 1998
Page 15
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33
Source Genus Species Reference(s)
Pe. camambertii Ghosh et al.,
1996
Pe. wortmanii Costan and
Peralta, 1999
Mucor
Mucor miehei
Rantakyla et al.,
1996
Lacointe et al.,
1996
Plou et al., 1998
Mu. javanicus Ishihara et al.,
1975
Mu. circinelloides Balcao et al.,
1998
Mu. hiemalis Ghosh et al.,
1996
Mu. racemosus Ghosh et al.,
1996
Ashbya Ashbya gossypii
Stahmann et al.,
1997
Geotrichum
G. candidum
Sugihara et al.,
1991
Ghosh et al.,
1996
Geotrichum sp. Macedo et al.,
1997
Beauveria
Beauveria bassiana
Hegedus and
Khachatourians,
1998
Humicola H. lanuginosa
Ghosh et al.,
1996
Takahashi et
al., 1998
Plou et al., 1998
Zhu et al., 2001
Rhizomucor R. miehei
Merek and
Bednasski,
1996
Weber et al.,
1999
Jaeger and
Reetz, 1998
Dellamora-
Ortiz et al.,
1997
Page 16
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34
Source Genus Species Reference(s)
Fusarium
Fusarium oxysporum Rapp, 1995
F. heterosporum Takahashi et
al., 1998
Acremonium Ac. strictum
Okeke and
Okolo, 1990
Alternaria
Alternaria
brassicicola
Berto et al.,
1997
Eurotrium Eu. herbanorium
Kaminishi et
al., 1999
Ophiostoma O. piliferum
Brush et al.,
1999
Yeast
Candida
C. rugosa
Wang et al.,
1995
Frense et al.,
1996
Yee et al., 1995
Brocca et al.,
1998
Xie et al., 1998
C. tropicalis Takahashi et
al., 1998
C. antarctica
Weber et al.,
1999
Jaeger and
Reetz, 1998
Arroyo et al.,
1999
C. cylindracea
Kamiya and
Gotto, 1998
Helisto and
Korpela, 1998
C. parapsilosis Lacointe et al.,
1996
C. deformans Lacointe et al.,
1996
C. curvata Ghosh et al.,
1996
C. valida Ghosh et al.,
1996
Yarrowia Y. lipolytica
Merek and
Bednasski,
1996
Pignede et al.,
2000
Page 17
CHAPTER-2
35
Source Genus Species Reference(s)
Rhodotorula Rho. Glutinis
Papaparaskevas
et al., 1992
Tahoun et al.,
1985
Pichia
Pi. bispora Hou, 1994
Pi. maxicana Hou, 1994
Pi. sivicola Sugihara et al.,
1995
Pi. xylosa Sugihara et al.,
1995
Pi. burtonii Sugihara et al.,
1995
Saccharomyces Sa. lipolytica
Tahoun et al.,
1985
Sa. crataegenesis Hou, 1994
Torulospora Torulospora globora Hou, 1994
Trichosporon
Trichosporon
asteroides
Dharmsthiti and
Ammaranond,
1997
Actinomycetes Steptomyces
Streptomyces fradiae
NCIB 8233
Sztajer et al.,
1988
Strptomyces sp.
PCB27
Sztajer et al.,
1988
Strptomyces sp.
CCM33
Sztajer et al.,
1988
Str. coelicolor Hou, 1994
Str. Cinnamomeus Sommer et al.,
1997
The above data includes the microbial organisms from bacteria, fungi, yeast
and actinomycetes, and shows the dominance of bacterial and fungal
organisms for the production of lipase from the explored microbial
organisms compared to the other reported species. Further the genus
Bacillus covers more than 50% of the gram positive bacterial domain
followed by 25% of the genus Staphylococcus and the remaining 25% is
covered by the rest of genera. On the other hand the genus Pseudomonas
dominates more than 80% of the species in gram negative bacterial domain
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36
and the rest 20 % is shared by the genera Chromobacterium, Acinetobacter
and Aeromonas. Similarly, the fungal domain of lipase producing
organisms distributed among the genera Rhizopus (20%), Aspergillus
(20%), Penicillium (18%), Mucor (15%), Geotricum (5%), Fusarium (5%)
and the rest of the genera constitute 17% of the total explored fungal
species. The other important lipase producing microbial domain, yeast is
dominated by the genus Candida (50%), followed by Pichia (20%) and
Saccharomyces (10%) and the rest of the genera constitute 30% of the
distribution in yeast. On the tail end the domain of Actinomycetes is
dominated by the genus Streptomyces completely. However the lipase
producing microbial organisms are not limited to these organisms and there
can be many more microbial species that need to be explored from the
possible habitats of life in the biosphere.
2.6 Lipases
Lipase is a biomolecule categorized as an enzyme of functional protein and
it is further categorized under the enzymes as hydrolases that catalyzes the
reactions of hydrolytic breakdown of complex molecules, with a bond
specificity of ester linkages between the glycerol and hydrocarbon chains
that may be of fatty acids. So these lipase molecules are named by the
enzyme classification committee of IUBMB in the systematic classification
as triacylglycerol lipases with the code EC 3.1.1.3. The important
distinctive characteristic feature of lipase catalysis of esterification,
transesterification, aminolysis and acidolysis reactions (Joseph et al., 2008)
at the interface of the aqueous and non-aqueous (organic) fluid molecules
on the target substrate molecules makes it as a special enzyme compared to
the other enzyme in the class of esterases. However, esterases catalyzes
water soluble short chain triglycerides, whereas the lipases act on the long
chain water insoluble aggregate lipid molecules (triglycerides) emulsified
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37
in aqueous environment, to produce diglycerides, monoglycerides and fatty
acids (Giham and Lehner 2005; Angkawidjaja and Kanaya 2006).
2.6.1 Genesis and exploration of lipases
Lipases were first noticed by the Claude Bernard in the year 1848, as the
pancreatic secreted juices emulsified and saponified the fatty substances,
and these observations were later attributed to the enzymes named
pancreatic lipases. Traditionally the animal pancreatic juices and extracts
were used as the source of lipases for commercial applications. As the use
of these lipases were increasing the search for other sources of lipase led to
the exploration of lipases from all the life forms including plants and
microbial organisms. In that endeavor by 1901 the bacterial lipase
production was first reported in the strains of Serratia marescens and
Pseudomonas aeruginosa (Hasan et al., 2006) and then onwards the
bacterial and fungal exploration had been on the first priority for the
production of lipases (Table-6), though other species from the yeast, and
actinomycetes were also explored for lipase production However the
bacterial lipases were relatively more in demand due to ease in production
and processing of the bacterial lipases and their versatile characters. Some
of the commercial lipases of bacterial origin are shown in Table-7 along
with their commercial names and applications.
Table-7. Commercial bacterial lipases used in various applications
(Balakrishnan et al., 2011)
Commercial
lipase Source Supplier
Applicati
on Reference
Lumafast Pseudomonas
menodocina
Genencor
International,
USA
Detergent Jaeger et al.
1994;
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38
Commercial
lipase Source Supplier
Applicati
on Reference
Jaeger and
Reetz 1998
Lipomax P. alcaligenes
Gist-Brocades,
The
Netherlands
Detergent Jaeger et al.
1994;
Genencor
International,
USA
Detergent Jaeger and
Reetz 1998
n.s P. glumae Unilever, The
Netherlands Detergent
Jaeger et al.
1994;
n.s Bacillus
pumilus
Solvay,
Belgium Detergent
Jaeger et al.
1994;
Chiro CLEC-
PC,
Chirazyme L-
1
P. cepacia
Altus
Biologics,
Manheim
Organic
synthesis
Jaeger and
Reetz 1998
Amano P, P-
30, PS, LPL-
80, LPL-200S
P. cepacia
Amano
Pharmaceutical
s, Japan
Organic
synthesis
Jaeger and
Reetz 1998
Lipase AH P. cepacia
Amano
Pharmaceutical
s, Japan
Organic
synthesis
Jaeger and
Reetz 1998
Lipase AK,
YS P. fluorescens
Amano
Pharmaceutical
s, Japan
Organic
synthesis
Jaeger and
Reetz 1998
Lipase 56P P. fluorescens Biocatalysts,
UK
Biotransfer
mations,
chemicals
Godfrey and
west 1996
Lipase K-10 Pseudomonas
sp.
Amano
Pharmaceutical
s, Japan
Organic
synthesis
Jaeger and
Reetz 1998
Chromobacter
ium viscosum
lipase
C. viscosum
Asahi
Chemical
Biocatalysts
Organic
synthesis
Godfrey and
west 1996
Lipase 50P C. viscosum Biocatalysts,
UK
Biotransfer
mations,
chemicals
Godfrey and
west 1996
Lipase QL Alcaligenes
sp.
Meito Sankyo
Co., Japan
Organic
synthesis
Jaeger et al.
1994;
Lipoprotein
lipase
Alcaligenes
sp.
Meito Sankyo
Co., Japan Research
Godfrey and
west 1996
Lipase PL,
QL/QLL,
PLC/PLG,
QLC/QLG
Alcaligenes
sp.
Meito Sankyo
Co., Japan
Technical
grade
Godfrey and
west 1996
Alkaline lipase Achromobacte
r sp.
Meito Sankyo
Co., Japan Research
Godfrey and
west 1996
Page 21
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39
Commercial
lipase Source Supplier
Applicati
on Reference
Lipase AL,
ALC/ALG
Achromobacte
r sp.
Meito Sankyo
Co., Japan
Technical
grade
Godfrey and
west 1996
Combizyme
23P
(proteinase/lip
ase mix)
n.s Biocatalysts,
UK
Waste
treatment
Godfrey and
west 1996
Combizyme
61P
(proteinase/lip
ase mix)
n.s Biocatalysts,
UK
Waste
treatment
Godfrey and
west 1996
Combizyme
209P
(/Amylaseprot
einase/lipase
mix)
n.s Biocatalysts,
UK
Waste
treatment,
grease
disposal
Godfrey and
west 1996
Greasex
(lipase) n.s Novo Nordisk Leather
Godfrey and
west 1996
n.s : Not specified
2.6.2 Bacterial lipases and classification
Bacterial lipolytic enzymes are produced in various forms such as maybe
classified in to different families including carboxylesterases (EC 3.1.1.1),
which hydrolyze the ester bonds in smaller lipid molecules dissolved in
aqueous solutions, and true lipases (EC 3.1.1.3), that catalyzes water
insoluble long-chain triglycerides. These lipases were analyzed with
respect to amino acid sequence and found them as part of the serine
hydrolases whose activity is dependent on catalytic triad consisting of
serine, histidine and aspartate along with α or β hydrolase folding (De-
Pascale et al., 2008). Since there is a large collection of bacterial lipase
data accumulated, these bacterial lipases may be classified in to different
families and sub families as proposed by Arpigny and Jaeger (1999) based
on the conserved amino acid sequence motifs and the biochemical
properties of lipases. So the bacterial lipases were classified in to 8 families
and the first family being the largest family was subdivided in to 6 sub
families (Arpigny and Jaeger 1999) as shown in the Table-8.
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40
Table 8. Classification of bacterial produced lipolytic enzymes (Arpigny
and Jaeger 1999)
Family Sub
family Enzyme - producing strain Properties
I
1
Pseudomonas aeruginosa
True lipases
Pseudomonas fluorescens
C9
Vibrio cholerae
Acinetobacter calcoaceticus
Pseudomonas fragi
Pseudomonas
wisconsinensis
Proteus vulgaris
2
Burkholderia glumae
Chromobacterium viscosum
Burkholderia cepacia
Pseudomonas luteola
3
Pseudomonas fluorescens
SIK W1
Serratia marcesens
4 Bacillus subtilis
Bacillus pumilus
5
Bacillus stearothermophilus
Bacillus thermocatenulalus
Staphylococcus hyicus
Phospholipase
Staphylococcus aureus
Staphylococcus epidermidis
6 Propionibacterium acnes
Streptomyces cinnamoneus
II (GDSL)
Aeromonas hydrophila Secreted
acyltransferase
Streptomyces scabies Secreted esterase
Pseudomonas aeruginosa OM-bound
esterase
Salmonella typhimurium OM-bound
esterase
Photorhabdus luminescens Secreted esterase
Page 23
CHAPTER-2
41
Family Sub
family Enzyme - producing strain Properties
III
Streptomyces exfoliatus Extracellular
lipase
Streptomyces albus Extracellular
lipase
Moraxella sp. Extracellular
estarase 1
IV (HSL)
Alicyclobacillus
acidocaldarius Esterase
Pseudomonas sp. B11-1 Lipase
Archaeoglobus fulgidus Carboxylesterase
Alcaligenes eutrophus Putative lipase
Escheria coli Carboxylesterase
Moraxella sp. Extracellular
estarase 2
V
Pseudomonas olevorans PHA-
depolymerase
Haemophilus influenzae Putative esterase
Psychrobacter immobilis Extracellular
estarase
Moraxella sp. Extracellular
estarase 3
Sulfolobus acidocaldanus Esterase
Acetobacter pasteurianus Esterase
VI
Synechocystis sp.
Carboxylesterase
Spirdina platensis
Pseudomonas fluroescens
Rickettsia prowazekii
Chalamydia trachomatis
VII
Arthobacter oxydans Carbamate
hydrolase
Bacillus subtilis p-Nitrobenzyl
esterase
Streptomyces coelicolor Putative
carboxylesterase
VIII
Arthobacter globiforms Stereoselective
esterase
Streptomyces chrysomallus Cell-bound
esterase
Pseudomonas fluorescens
SIK W1 Esterase 3
Page 24
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42
In this lipase classification, true lipases are classified under Family I, which
is dominated mostly by Pseudomonas, Bacillus and Staphylococcus lipases
where the catalytic center possess pentapeptide, Gly-Xaa-Ser-Xaa-Gly.
Conversely the lipases of Family II possess Gly-Asp-Ser-(Leu), [GDS(L)],
peptide sequence as the catalytic motif, where the catalytic serine residue
lies much closer to the N-terminus compared to other lipases. However this
Family II is one of the shorter families dominated by the secreted and
membrane bound esterases of Streptomyces, Aeromonas, Pseudomonas,
Photorabdus and Salmonella. Family III lipases consists of Streptomyces
and Moraxella lipases that are extra cellular in nature having the typical
catalytic triad, Ser-His-Asp, as the catalytic center. Further, the bacterial
enzymes showing the peptide sequence similarity to that of the mammalian
hormone sensitive lipases, were grouped under Family IV, consisting
mostly the esterases of Pseudomonas, Archaeoglobus, Alcaligenes,
Escherichia and Moraxella, with the similar catalytic peptide motif Gly-
Asp-Ser-Ala-Gly-Gly-Xaa-Leu-Ala-Xaa. Similarly the enzymes of Family
V are constituted from the esterases of mesophilic bacteria like
Pseudomonas oleovorans, Heamophilus influenza and Acetobacter
pasteurianus; psychrophilic bacteria like Moraxella sp. and Psychrobacter
immobilis and even thermophilic bacteria like Sulfolobus acidocaldarius.
The conserved catalytic peptide motif for the esterases of Family V was
noted as Gly-Xaa-Ser-Xaa-Gly-Gly. On the contrary the lipases of Family
VI are the smallest (23-26kD) known esterases constituted of two subunits,
having α/β hydrolase fold and a classical Ser-Asp-His catalytic triad, in the
active site of the enzyme. These carboxyl esterases hydrolyses simple
substrate molecules with broad spectrum of specificity, however they do
not catalyze the long chain triglycerides. On the other hand the lipases of
Family VII consists of large esterases having their peptide sequence
homologous to that of the eukaryotic acetyl choline esterases and they are
mostly derived from Arthrobacter oxydans, Bacillus subtilis and
Streptomyces coelicolor. The lipases of Family VIII are constituted from
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43
the esterases derived from Arthrobacter globiformis, Streptomyces
chrysomallus and Pseudomonas fluorescens SIK W1, having the peptide
length of approximately 380 residues long, showing the sharp similarity to
several class C, β-lactamases.
Other lipase enzymes that could not be placed in any of the 8 super families
described by Arpigny and Jaeger, are placed in new families 9 and 10 in an
arbitrary manner. Similarly the lipases that could not be categorized in to
any of these discussed families are grouped under novel lipolytic family.
Microbial Esterases and Lipases Data Base, MELDB, is a similar
comprehensive protein database of microbial esterases and lipases (Kang et
al., 2009), where the enzymes in MELDB are clustered into groups
according to their peptide sequence similarities analyzed on a local pairwise
alignment algorithm and a graph clustering algorithm (TribeMCL), which
differs from traditional approaches involving global pairwise alignment and
joining methods. So, the more elaborated and comprehensive data on lipase
and its related properties along with classification may also be found from
MELOB.
2.6.3 Processing of Lipases in cellular system
Processing of lipases can be better understood with simplest cellular
structures like bacteria. Like many other functional biomolecules, bacterial
lipases are found as either intracellular or extracellular or even like
membrane bound enzymes. So the cells that grow on simpler molecules
like glycerol and short chained lipids can channelize these nutrient
molecules to the cellular cytoplasm where the intracellular lipases can break
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44
them down further to lower molecules that can be metabolized and
absorbed easily in to the cellular biochemical reactions. One of such kind
of intra cellular lipase catabolized systems was reported in Bacillus clausii
SKAL-16 (Lee and Park 2008), isolated from the soil contaminated with
vegetable oil.
In other case, the cells can grow on the larger complex molecules like long
chain triglycerides as well as on short chain lipids and glycerol, where the
cell can produce both intra cellular and extra cellular lipase so as to
catabolize the simpler as well as complex lipid molecules. This kind of
intra cellular and extra cellular lipase systems were observed in the bacteria
isolated from the oil mill waste water such as Bacillus sp. (Ertugrul et al.,
2007) where the intracellular lipase activity was reported more than to be
11 times to that of the extra cellular lipase.
The extra cellular lipase can also be induced in the cellular system in higher
quantities depending on the kind of substrate or nutrients available in the
ambient. The lipase inducers like Tween 80, hexadecane, olive oil etc. in
the ambience of cells can trigger the lipase gene expressions leading to the
excess production of lipase that can be released in to the ambience and
catabolize the substrates to the easily assimilated molecules. Such kind of
extra cellular lipase secretions by membrane bound chaperons are observed
in the cellular systems where the growth media has multiple and complex
substrate molecules like the growth of Burkholderia glumae strains
(Boekerma et al., 2007), observed in the presence of glucose and
hexadecane rich culture medium.
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45
Apart from the intra cellular and extracellular lipase catabolized cellular
systems, there were the cases where the membrane bound lipases were
produced by the cells may be for the optimal utilization of the lipase
catalytic activity and prevent the loss of lipase activity and optimal
utilization of the simple broken molecules in the ambient. Such kind of
membrane bound lipase catalyzed cellular systems are rare and may be they
are part of the evolving cellular systems. However there had been the
cellular systems engineered through molecular biology tools to construct
the membrane bound lipase producing cellular systems such as recombinant
yeast (Matsumoto et al., 2002) reported to produce the membrane bond
lipases.
In all these cellular lipase systems, the lipase molecules in the cells are
synthesized in the ribosomes with the expression of lipase genes in the
DNA upon the signals received by the membrane bound receptors and then
in case of intra cellular lipases, these raw lipase molecules would be
processed to the correct folding through the Golgi apparatus and be released
in to the cytosol ready for the transport to the targeted biochemical reactions
and places. In case of extra cellular secretion, the lipase peptide molecules
are synthesized in ribosomes outside of the Endoplasmic reticulum and then
be docked in to the rough endoplasmic reticulum lumen where the raw
protein molecules are glycosylated and molecular chaperones shape the
folding to the correct functioning. These properly folded proteins would
enter the Golgi complex through the vesicles generated from the rough
endoplasmic reticulum, and there the post translational modifications of the
lipase are made and functionality of the lipase are attained. These functional
lipases are ready for secretion to the extra cellular ambient.
Page 28
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46
In general bacteria secrete the lipases to the extracellular medium through
various type secretion systems (T1SS TO T6SS). However the most
significant secretory system are Type I Secretory System (T1SS), consisting
of energy driven three component exporter protein complex; and the Type
II Secretory System that has two secretion pathways as Sec dependent
(general secretion) path way and Tat (Twin-arginine translocation) path
way. Bacterial lipases are secreted in the unfolded state through the Sec-
dependent path way in to the periplasmic space where the lipase specific
foldase (Lif) chaperons fold the protein to the functional lipase and then it
is transported to the external medium through the transporter complex
(Angkawidjaja and Kanaya 2006; Buist et al., 2006). The better
understanding of such complex lipase processing with in the bacterial
cytoplasm would help in design and production of novel experimental
procedures and possible solutions for the existing and prospective
difficulties in lipase catalyzed processes.
2.6.4 Production of lipases
Production of lipase by bacterial microorganisms was dealt in detail by
various scientists and engineers by using different strategies with direct and
indirect methods of detection, screening and measurement of lipase activity
as discussed below.
2.6.4.1 Screening of lipase production
During the century long research and development in the area of lipases,
the literature has recorded several methods and procedures for the
production of lipase by microbial organisms. These methods and
procedures were developed based on either direct use of microorganism
under study (Nair and Kumar 2007) or the indirect method involving
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47
measurement of lipolytic activity of crude or purified lipase preparations
(Singh et al., 2010).
Among those methods, the preliminary detection methods like plate
detection method was developed using agar plates containing lipid
molecules and the subsequent was detected either by direct lipolysis or
indirect use of lipase, as clear halos or opaque zones around the well
containing culture or lipase enzyme preparation (Nair and Kumar 2007;
Hun et al., 2003). Alternatively the lipase detection can also be observed by
preparing the agar plates with lipids and chromophoric substances having
sensitivity towards the pH, like phenol red and victoria blue (Singh et al.,
2006; Rahman et al., 2005), where the lipolytic activity of lipases releases
fatty acids decreasing the pH, which in turn can change the color of the
chromophoric dyes used in the plate. In another method, the plates prepared
by the agar medium containing lipids and the Rhodamine B, a fluorescent
dye, indicates the lipolysis by formation of fluorescent orange halos under
the UV illumination (Kim et al., 2001).
On the other hand, colorimetric methods are developed based on the
measurement of chromophoric complexes formed by the reaction of fatty
acids released by the lipolysis and a divalent metal ion like copper ions
(Rahman et al., 2005). In the similar lines, spectrophotometric methods
were developed to measure the lipolytic activity of the lipases in an indirect
way of detection and measurement of lipase activity. One of such popular
method uses the designed lipid like substrate, usually p-nitro-phenol esters
of fatty acids (simplest molecule being p-nitro-phenol acetate) in the
reaction mixture that would be incubated with the lipase at the optimal
conditions and the resultant lipolytic activity was measured as the
Page 30
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48
absorbance of light at 415nm, which is directly proportional to the amount
of p-nitro-phenol released after the lipolysis (Wang et al., 2009).
Another earliest known direct measurement of lipase activity was
developed by mixing the lipase enzyme preparation with the reaction
mixture containing pH sensitive chromophoric indicator and substrate lipid
molecules such as olive oil; and incubation of the reaction mixture added
with known lipase quantity, at the optimal enzyme conditions, leading to
the release of free fatty acids that create an acidic environment in the
reaction mixture. These released free fatty acids are neutralized by titrating
the reaction mixture with known basic solution such as 50mM NaOH
solution, to the desired pH level where all the released free fatty acids are
consumed by the titrant basic solution and the pH indicator produces the
characteristic color, indicating the end of titration process. In the latest
technological developments, the auto-titrators are developed that can
perform the titration process to the greatest precision as per the
programmed end point of titration. The resultant titrant, basic solution,
consumed in the titration process can be measured and equated to get the
equivalent moles of free fatty acids released by the lipolytic activity of the
lipase enzyme (Shukla et al., 2007). This method is popularly known as
titrimetric method or pH stat method of lipase assay, and this method was
followed in most of the lipase assay procedures as it is the direct method of
measuring the lipase activity and is more accurate provided the errors in
this tedious assay are handled and eliminated properly.
There are other developed methods like, chromatographic, turbidometric,
fluorimetric, immunological and radioactive assays which are mostly based
on the indirect methods of detection and measurement of lipolytic activity.
Due to the errors and procedural problems involved in these methods the
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49
researchers consider them as the least preferred choices for the lipase assay
(Sumitra et al., 2012).
2.6.4.2 Lipase production media
Media plays an important role in the culturing of microorganisms and
production of microbial derived products like enzymes. Media can be either
submerged or solid state media containing the essential and necessary
nutrients for the growth and multiplication of microbial organisms, during
which the products of either intra cellular or extra cellular are produced.
Media composition is also selective to the type of microorganism and
determines the production and yield of the products produced by the
fermentation process. Optimal media composition also helps in the isolation
and purification of the lipase produced by minimizing the byproduct release
in to the fermentation broth. However, the bacterial lipases are produced by
both submerged (Chakraorty et al., 2008) as well as solid state fermentation
process (Alkan et al., 2007) as shown in the Table-9 with their respective
limitations in the production parameters of temperature, pH, carbon source
and nitrogen source etc. depending on the causative bacteria under the
fermentation process.
Table-9. Comparison of submerged and solid state fermentation for the
bacterial lipase production
Solid state Fermentation Submerged Fermentation
Requires much space for trays
Requires much hand labor
Uses low pressure air blower
Little power requirement
Uses compact closed fermenters
Requires minimum of labor
Requires high pressure air
Needs considerable power for
Page 32
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50
Solid state Fermentation Submerged Fermentation
Minimum control necessary
Little contamination problem
Recovery involves extraction
with aqueous solution, filtration and
or centrifugation, perhaps
evaporation and/or
precipitation
air compressors and agitators
Requires careful control
Contamination is frequent and a
serious problem
Recovery involves filtration or
centrifugation, and perhaps
evaporation and/or precipitation
Although most of the fermentation studies use simple sugars as the carbon
source in the media, the lipase production media utilizes lipids as the sole
carbon source and substrate (Zhang et al., 2009a; Zhang et al., 2009). It was
also found that lipase production was rarely constitutive under general
fermentation conditions, and the extra cellular lipase production is meagre
(Lee et al., 2001). But then there are also the fermentation studies where
sugars like glucose (Anbu et al., 2011) are used as carbon source whereas
lipids in the media are used as inducers for lipase production (Hun et al.,
2003). So the lipase inducers like vegetable oils (Kumar et al., 2005),
Tween 20 or Tween 80 (Li et al., 2004), hexadecane (Boekema et al., 2007)
and synthetic triglycerides like tributyrin and tripalmitin (Rahman et al.,
2006) are used in the fermentation media either in combination with
nutrients or as sole carbon source for the production of microbial lipases.
In this context it is also noteworthy that high concentration of carbon source
like vegetable oils, or the free fatty acids repress the lipase synthesis may
be by the feedback repression mechanism. Considering all these aspects,
use of either a single carbon source like tween 80 and the moderately
sustained release of free fatty acids from the lipids (Li et al., 2004) or the
appropriate combination of carbon source and lipids in the media may not
Page 33
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51
cause the repression of lipase during the fermentation process, and further
would enhance the production of lipase.
In other cases, nitrogen sources in the media composition were found to
have varied effects on the growth of microbial organisms and the
production of lipase. Organic nitrogen sources are generally provided as
yeast extract, peptone, polypeptone, meat extract, beef extract, corn steep
liquor, soybean meal etc. whereas ammonium nitrate, ammonium chloride,
ammonium hydrogen phosphate, ammonium sulphate, sodium nitrate and
urea are used as inorganic nitrogen sources in the media composition for
lipase production. However the use of either organic nitrogen or inorganic
nitrogen source in the lipase production media varies in selection as well as
concentration depending on the bacterial organism used for the
fermentation process. In some cases the use of mixed nitrogen sources
along with the carbon source gave the best results for the lipase production
(Kumar et al., 2005). The organic nitrogen sources like peptone and corn
steep liquor were reported to promote the increased lipase productions in
many of the bacterial fermentation processes (Gunasekaran et al., 2006;
Rathi et al., 2011) and similarly, the inorganic nitrogen source like
ammonium nitrate was also reported to produce the mixed results of lipase
production. However there were no reports of lipase repression by the
nitrogen content of the fermentation media by bacterial microorganisms.
Another important aspect in the lipase related lipid metabolism and
assimilation is the presence of surfactants that lowers the surface tension at
the interfacial surface of immiscible aqueous and organic media. In this
context the use of vegetable oils such as olive oil in the aqueous based
fermentation media brings out two different aqueous and organic phases of
liquids as the components of the media, and the use of surfactants enhances
Page 34
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52
the formation of emulsion solution of these two otherwise immiscible
liquids. This emulsified media preparations with the help of surfactants
increases interface between the two liquids in the form of micelles and
hence the greater accessibility of substrate enhancing the catalytic activity
of lipases to have better enzyme productivity. In these regard, the most
commonly used surfactants are Tween and Triton-X 100 (Pogaku et al.,
2010).
2.6.5 Biochemical characteristics of bacterial lipases
The globalized markets in this modern era has brought all the products to
work or provide services at their best performance with the competitive
price dictated by the market forces. This increasing trend made the research
and development teams across the world to extract and find all governing
factors influencing the performance of the products being manufactured
round the clock. Such concepts applied on to the biotechnologically derived
products like enzymes, brought the biochemical characters in terms of
optimal pH and temperature of enzyme, the influence of the presence of
cofactors, inhibitors and enhancers on the catalytic activity, the enzyme
acceptance to the non-aqueous solvents and the presence of co-produced
enzymes in the media, to the limelight in order to derive the best
performance of the produced products.
2.6.5.1 Alkaline and acidic lipases
Over the past century long research and developments on lipases the
reported bacterial lipases were mostly of alkaline in nature and such
alkaline lipases have been used to catalyze many reactions in industrial
processes (Nawani et al., 2007; Ahmed et al., 2010); on the other hand there
are a few reports on acidic lipases in the bacterial domain by genus
Page 35
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53
Pseudomonas whereas those from the fungal domain are mostly from the
genus Aspergillus (Ramani et al., 2010).
2.6.5.2 Thermophilic and psychrophilic lipases
Temperature is one of the most important factor influencing the catalytic
activity to the greatest extent for enzymes. Among the bacterial lipases
produced, the optimal catalytic temperature had been varying from 0oC to
around 60oC and these lipases may be broadly classified as psychrophilic
and thermophilic lipases with optimal temperature ranges of (0-30)oC and
(31-70)oC respectively. There had been the great demand for thermo stable
lipases in the industry as many of the reactions were carried out around
60oC and such thermo stable lipases isolated from the genera Pseudomonas
and Bacillus were studied for their optimal utilization in industries (Kumar
et al., 2005; Ahmed et al., 2010; Nawani et al., 2000; Dutta and Ray 2009).
Similarly the cold adapted or psychrophilic lipases were produced and
studied extensively from the psychrophilic microorganisms that survive at
temperatures around 5oC (Joseph et al., 2008). There were also the cases
where the cold adapted lipases were produced from the non-psychrophilic
microorganisms like Geotrichum sp. which is a mesophilic bacterial
species. However the spectrum of the industrial potential of spychrophilic
lipases sweeps a wide range of biotechnology products like dairy products,
detergents, food preparations, beverages etc.
2.6.5.3 Effect of surfactants
Surfactants are known for their ability to decrease the surface tension of the
immiscible liquids at their interfaces, which in turn provide the better
access for the lipases to catalyze the lipolytic reactions. However there are
limitations for the use of surfactants depending on the type of surfactant
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54
and their concentrations in the lipase production media. In this regard, it
was reported that about 1% concentration of the surfactant like Tween 80
in the broth media inhibited lipase production by Bacillus pumilus and on
the other hand, 0.5% concentration of Tween 80 enhanced the lipase
production (Zhang et al., 2009a; Zhang et al., 2009). In another case, SDS
exhibited the inhibitory activity whereas Triton X100 and Tween 20/80
have enhanced the lipase activity (Quyen et al., 2003; Lianghua and Liming
2005); conversely it was also reported that SDS exhibited the stimulatory
effect enhancing the lipase activity whereas the Triton X100 and Tween
have inhibited lipase activity (Dutta and Ray 2009). So in general it was
found that the surfactants can act as both inhibitors as well as stimulators
depending on the concentration and the other constituents of the lipase
production media.
2.6.4.4 Effect of proteases
In many of the industrial applications of enzymes such as detergents and
food preparations, lipases have been used in combination with other
industrial enzymes like, proteases, amylases etc. But the proteases are
hydrolytic enzymes capable of auto-digestion and simultaneous
degradation of other enzymes produced by the biological systems (Aguilar
et al., 2002) whereas lipases and proteases were reported as inter related
and produced simultaneously (Rajmohan et al., 2002) with variation in their
concentrations subjected to the alterations in the production parameters
such as increased aeration, and or due to the genetic changes in the
microbial genome (Lopes et al., 2008). In another finding the susceptibility
of Bacillus subtilis produced lipases to the extra cytoplasmic proteases
located either in the cell wall or the broth were presented whereas a few
reports in the production of bacterial lipases from Streptomyces fradiae and
Bacillus cereus were reported as resistant lipases to commercial neutral and
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55
alkaline proteases (Dutta et al., 2009; Zhang et al., 2008). Similarly the
lipases produced by Pseudomonas aeuruginosa, Bacillus pumilus and
Bacillus licheniformis were reported as the lipases that were resistant to the
native and simultaneously produced proteases (Ruchi et al., 2008;
Sangeetha et al., 2010; Sangeetha et al., 2010).
2.7 Recombinant Bacterial lipases
Recombinant DNA technology had been thriving as the latest modern
technology developed using the principles of molecular biology and genetic
engineering in the developing and expanding area of biotechnology. In any
biological product development it has become a norm to perform the
recombinant studies may be for the commercial production, after the lab
scale and pilot scale production studies using the wild strain and further the
process continues with the recombinant organism(s) in a cyclic manner to
enhance the productivity and minimize the downstream processing. This
recombinant technology helped the scientists and engineers to understand
the enzymes like lipases in terms of substrate binding, the catalytic site, and
over expression of the enzymes in suitable host organisms to meet the
commercial demands. This continuous exercise had been helping to
engineer the enzymes and the corresponding host organisms to enhance the
productivity and analyze the probable anomalies to predict any unstable
conditions and subsequent by correct them for the smooth and efficient
functioning of the commercial production.
In case of recombinant lipases, it has been reported that they overcome the
limitations of crude lipases from the culture supernatants such as the non-
reproducibility of the results, undesirable side effects and the tedious
downstream processing requirements and moreover they ease the
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56
commercial production of pure lipases (Schmidit-Dannert 1999). Though
the success of this recombinant technology depends on the selection of the
expression system to be employed and the genetic engineering of the host
for the over expression and production of lipases, many researchers had
been working in this area with wide range of host organisms like
Escherichia coli (Anet al., 2003; Long et al., 2007) Pichia pastoris and
Saccharomyces cerevisiae (Ramchuran et al., 2006; Mormeneo et al., 2008)
and successfully cloned, sequenced and expressed the lipase genes.
During the development of recombinant lipases, there were some hurdles
observed, as the formation of insoluble and inactive inclusion bodies due to
the over expression of lipase in the cytoplasm or periplasm of the host
organisms like Escherichia coli (Akbari et al., 2010), and the inefficient
processing of lipase proteins by the post translational machinery of the host
leading to protein mis-folding (Goldberg 2003). But then these mis-folding
problems were also addressed for recombinant lipases from bacteria like
Pseudomonas sp. by the response surface optimization studies (Akbari et
al., 2010) with the addition of low molecular osmolytes such as polyols,
sugars, polysaccharides, amino acids and natural polymers that can enhance
the refolding of inactive lipases. In another attempt to solve the mis-folding
of recombinant lipases, in addition to the conventional methods using
chemical additives to help lipase refolding, the lipase specific foldase (Lif),
chaperones, genes were cloned along with the lipase genes in the same
plasmid of the host organism, as these chaperones were found to assist
lipases both in vivo and in vitro. However the analysis of the expression
results predicted the lipase production as a complex process involving both
gene regulation and secretion mechanism; suggesting an alternate
expression system involving cloning and expression of lipase genes and Lif
genes in separate vectors in different hosts. But then these alternate
expression studies resulted in the enhanced inclusion bodies of mis-folded
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lipases rather than active recombinant lipases (An et al., 2003); so in a
mixed attempt, the isolated lipase specific foldases were used along with
osmolytes to refold the recombinant lipases of Pseudomonas expressed as
inclusion bodies in host Escherichia coli, and subsequently found several
fold increase in the refolding yield of recombinant lipase (Akbari et al.,
2010). There were also the reports on the successful expression of
recombinant lipases from Pseudomonas fragi IFO 34584, Pseudomonas
fluorescens C9 and Pseudomonas fluorescens JCM 5963 in Escherichia
coli (Zhang et al., 2009a; Zhang et al., 2009) as the host organism.
2.8 Applications of bacterial lipases
The potential of lipases were reported by many researchers and eventually
their potential was harvested and realized by the popular world enzyme
producers like Novozyme (Denmark), Amano Enzyme Inc (Japan),
Biocatalysts (UK), Unilever (Netherlands) and Genencor (USA) by
commercializing many microbial lipases. Though fungal lipases were
considered initially as the better lipases for commercial production
compared to the animal derived lipases, the bacterial lipases later
dominated the commercial lipase production and to name a few, Amano
Enzyme Inc had been producing lipase (named as Lipase PS) from
Burkholderia cepacia and Pseudomonas fluorescens (Lipase named as
Lipase AK); similarly Meito Sangyo (Japan) had been producing lipases
named as Lipase SL isolated from Bacillus cepacia and Lipase TL isolated
from Pseudomonas stutzeri. Inspite of the availability of commercial
lipases, they share about 5%of the global industrial enzyme market,
however gained the attention as biotechnologically valuable enzymes due
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to their diverse applications especially in the areas of food, detergent and
pharmaceutical industries.
2.8.1 Lipases in food industry
The use of lipases is majorly found in the food industry to catalyze the
breakdown of various lipid based complex molecules to simpler molecules
as well as the transesterification and other biochemical reactions to meet
the industry needs as discussed below.
2.8.1.1 Hydrolysis of oils
Fatty acids are produced mostly by the hydrolysis of oils and such fatty
acids are fats, and used for the production of high value products like
adhesives, cosmetics and other personal care products, lubricants and
coatings. So, industries exploit vegetable oils and fats as the cost effective
raw materials for the commercial production of fatty acids, using lipase
catalyzed hydrolysis (Murty et al., 2002) and one such popular fatty acids
production was practiced as the hydrolysis of olive oil with immobilized
acidic lipase from Pseudomonas gessardii (Ramani et al., 2010).
The Polyunsaturated Fatty acids (PUFAs) having two or more double bonds
in their hydrocarbon chain, categorized as ω3 fatty acids such as α-
Linolenic Acid (ALA), Eicosatrienoic Acid (ETA), Eicosapentanoic Acid
(EPA), docosahexaenoic aicd (DHA) and ω6 fatty acids, like Linoleic acid
and arachidonic acid are extensively used in the formulations of
nutraceuticals and pharmaceutical products due to their physiological
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importance in the growth and development of human beings. The
increasing demand for these PUFAs are supported by increasing the content
of PUFAs in the alga, fish oil, fish by-products and the edible oils
(Chakraborty et al., 2010) through the hydrolysis of lipids by bacterial
lipases (Chakraborty and Paulraj 2008; Kumar et al., 2005).
Commercial lipases such as AK-lipase and HU-lipase produced from
Pseudomonas fluorescens (Kojima et al., 2006) were used successfully to
enhance the concentration of EPA and DHA selectively in fish oils, instead
of the costly and cumbersome traditional methods involving distillation,
chromatography and solvent extraction. Hence, the AK-lipase catalyzed
lipid hydrolysis, increased the DHA concentration from 16.3% to 44.6% in
cuttle fish oil, whereas the catalyzis of HU-lipase increased the EPA content
in the cod-oil from 12 % to 43% (Kojima et al., 2006).
In another finding, Pseudomonas lipase was used to hydrolyze the sardine
oil in the presence of emulsifiers where the overall content of saturated fatty
acids got decreased and led to the increase in the mono and poly unsaturated
fatty acids in the sardine oil (Byun et al., 2007). Further a three-step PUFAs
purification was noted where arachidonic acid content was enhanced and
purified from Mortierella alpine single cell oil using non-selective
Alcaligenes lipase hydrolysis followed by selective elimination of saturated
fatty acids using urea adduct fractionation and finally the selective
esterification enrichment of the oils by using the lipases of Bacillus cepacia
(Yamauchi et al., 2005).
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2.8.1.2 Interesterification of fats and oils
The health benefits of ingested fats and oils are essentially dependent on
the ratio (ω6/ω3) of fatty acids from various sources especially for humans.
So the ratio (ω6/ω3) of fatty acids from PUFAs for the ingested fats and
oils must be maintained between 1 and 4 to reap the health benefits for
better growth and maintenance of the body (Griffin 2008). It is also known
that many vegetable oils like sunflower oil, coconut oil, olive oil, corn oil
and rice bran oil are rich in ω6 fatty acids, whereas fish oil, linseed oil,
walnuts and milk are rich in ω3 fatty acids. Hence the vegetable oils are
tailor made to meet the health requirements in terms of the structured lipids
with appropriate fatty acid composition.
To meet the fatty acid requirements of the ratio (ω6/ω3) in the structured
lipids, the use of lipase catalyzed interesterification of lipids had been one
of the best strategies reported (Mitra et al., 2010) and one of such structured
lipid, dioleyl palmitoyl glycerol, was produced by acidolysis of tripalmitin
with oleic acid. However such structured lipid synthesis require high
temperatures for the better homogenization of lipids and which in turn
necessitates the presences of organic solvent to resist the high temperature
denaturation problems of lipases; but then some times the organic solvents
may initiate the undesired product isomeric forms. Even in such kind of
process difficulties, the Bacillus stearothemophilus MC7 derived lipases
produced the dioleyl palmitoyl glycerol in non-solvent high temperature
reaction systems avoiding the probable side reaction of acidolysis
(Guncheva et al., 2008).
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2.8.1.3 Glycerolysis
The lipase catalyzed glycerolysis (Guo and Xu 2005; Kristensen et al.,
2005) of fats and oils were used to produce diacy glycerols and monoacyl
glycerols, that are widely used as emulsifiers in food, pharmaceutical and
personal care products. Otherwise these mono and diacyl glycerols were
produced traditionally by chemical glycerolysis, where there may be the
occurrence of negative side effects in terms of undesirable color and flavor
to the products (Cheirsilp et al., 2009). However the use of lipase catalyzed
glycerolysis avoids all the negative side effects and produces the more
efficient mono and diacyl glycerides with better surface activity as
emulsifiers and adds value to health unlike triglycerides (Cheirsilp et al.,
2009; Kahveci et al., 2009).
2.8.1.4 Synthesis of flavor esters
Flavor or fragrance materials like aliphatic and aromatic compounds share
the major market of food additives around the globe. But the flavor
compounds extracted from the plants are very expensive, so the low
molecular flavor esters are synthesized by the microbial lipase catalyzed
esterification of fatty acids and some of these flavor esters were reported as
ethyl acetate, ethyl butyrate, ethyl methyl butyrate, ethyl valerate and ethyl
caprylate (Talon et al., 1996; Dandavate et al., 2009; Ahmed et al., 2010).
However these synthesized flavor esters carry the natural tag in spite of
their synthesis by the lipase esterification process, and hence this area of
research had been one of the most intensively researched domains (Gillies
et al., 1987).
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2.8.1.5 Tea seed oil
Cocoa butter had been the primary ingredient for many dark chocolate
formulations throughout the world, but the increase in the demand of dark
chocolate had increased the cocoa butter price by several folds, forcing for
a cheaper alternative. In such condition, interesterification of tea seed oil
was identified to possess the similar properties of cocoa butter and hence
used as a best replacement in partial terms to meet the demand and pricing
requirements of confectionary products (Zarringhalami et al., 2010).
2.8.1.6 Lipolysed Milk Fat (LMF)
The LMF is prepared from condensed aggregates of milk or the butter oil
with added lipase catalyzed lipolysis to produce free fatty acids that impart
the cheesy aroma to the product. Such type of LMF is used for chocolate
coatings, artificial flavor additives, margarine etc. So, bacterial lipases used
for these products may be derived from the microbial genera of
Achromobacter and Pseudomonas (Sangeetha et al., 2011).
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2.8.1.7 Cheese
Cheese has been produced traditionally in most of the homes in the villages
if not in the cities. However the commercial cheese like Parmesan and
Grana Padano cheese, and Amul cheese, have been thriving in the market
with their added flavors and enhanced ripening derived from the lipolysis
or esterification of lipids or fatty acids. Though Lactobacillus lipases had
been in the cheese industry for centuries, the recent investigations found
that recombinant lipases were more efficient than the native lipases for
imparting flavor and to enhance cheese ripening (Sangeetha et al., 2011).
An important cheese derived product is the Enzyme Modified Cheese
(EMC), a concentrated cheese flavor produced by treating the curd with
lipolytic enzymes and used as food additive for many FMCG products like,
cheese powders in soups, salads, sauces and coatings (Guinee and
Kilcawley 2004). EMC products have more intense flavor than the naturally
ripened cheese, where the short chained fatty acids (C2-C6) are the major
contributors for the indication of ripened cheese. The product variations of
the EMC were seen in the market due to the esterification of fatty acids
releasing the characteristic cheese flavor molecules like ethyl butanoate and
ethyl hexanoate (Fenster et al., 2003). Some of those EMC products may
be seen as Amul processed cheese, Amul Gouda cheese and pizza
Mozzarella cheese from Amul dairy, India.
2.8.1.8 Bread making
To improve the bread quality, emulsifiers are added and these can increase
the bread volume, improve the texture and provides the dough stability, but
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the problem with these added emulsifiers had been their protruding
appearance on the final baked and marketed loaf, forcing the manufacturer
to put all the additives on the labels. On the other hand the use of enzymes
like lipases from Bacillus subtilis (Sanchez et al., 2002) as bread improvers
can enhance the bread quality and in addition, they get denatured during the
baking process leaving the fine texture on the bread loaf, and this
technology can replace the emulsifying additives either completely or
partially (Moavyedallaie et al., 2010) with the better quality and efficiency
of bread making process.
2.8.2 Lipases in detergents
Lipases were developed as part of the detergents, after the successful
introduction of proteases in detergents both in powder and liquid forms,
where lipases were investigated to meet the detergent requirements of
stability at alkaline pH, solubility in water, resistance to detergent proteases
and surfactants and minimum substrate specificity (Rahman et al., 2006;
Quax et al 2006). The industrial bodies like Genencor International
presented Lipomax and Lumafast as the commercial bacterial lipases
derived from Pseudomonas alcaligenes and Pseudomonas mendocina
respectively to the detergent market in the year 1995 (Rahman et al 2006)
as an alternative to the existing acidophilic fungal lipases that were
suffering from the incompatible alkaline wash conditions (Quax 2006).
Such bacterial lipases get adsorbed on to the fabric during the laundering,
to form a fabric–lipase complex that is tolerant to harsh wash condition and
be stable through the laundering period and deliver the better washing
performance by hydrolyzing the oil stains (Hasa et al., 2006).
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The continued research on the detergent lipases, brought better detergent
stable lipase isolated from bacteria like Bacillus cepacia (Rathi et al.,
2001), with properties desired for detergent additives and gave a better
stability and performance than the existing detergent stable lipases like
Lipolase, from Novo Nordisk, Denmark. Conversely, a lipase derived from
Bacillus licheniformis, aspart of commercial detergents was found to lose
its stability and hence the loss of lipolytic activity; and further remedial
studies revealed that addition of calcium chloride could restore the stability
and activity of the lipase (Bayoumi et al., 2007) in the presence of the
detergent but then such lipases are prone to lose the activity in the presence
of chelating agents if any in the detergent formulations. Similarly, detergent
compatible lipases were also isolated from the bacterial species such as
Bacillus cepacia (Wang et al., 2009) and Pseudomonas fluorescens (Zhang
et al., 2009a; Zhang et al., 2009). In another development, a low
temperature, detergent stable active lipase was produced from
Pseudomonas with better wash performance (Suzuki 2001) and this
alkaline lipase was patented along with its detergent composition.
2.8.3 Lipases in Tannery
Many chemicals and enzymes are used in the three stage leather processing
where the first stage called pre-tanning cleans the hides, and tanning, the
second stage, where the hides are stabilized and in the third stage called
post-tanning, the esthetic values are imparted to the processed leather
(Thanikaivelan et al., 2004). Though the leather processing include curing,
soaking, liming, dehairing, bating, pickling, degreasing and tanning; only
at certain processes like soaking, bating and degreasing lipases are
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employed (Choudary et al 2004). The conventional soaking processes for
the rehydration of hides use the soda ash or the sodium tetrasulphide in the
presence of surfactants whereas the enzymatic processes use lipases and
proteases (Choudary et al 2004; Tancous et al., 1994), and the mixture of
these lipases and proteases are commercially available from bacterial
sources in the market as Forezym SK, marketed by La Forestal Tanica,
Spain. Similarly the dehairing process of the hides was performed
conventionally by using lime and sulphide, whereas the enzymatic
processes use proteases for loosening of the hair from the hides and the
commercial enzymes are available from La Forestal Tanica, for dehairing
as Dorezym LM, which is also a mixture of proteases and lipases. Another
important degreasing process is performed to remove the natural fat present
in the skin, especially sheep skin, by using proteases to breakdown the
protein sacs of lipids and subsequent removal of fat and its emulsification
in water or solvent by lipolysis of lipases (Padmapriya et al., 2011), in
addition to the removal of residual grease left out after liming process. So
the combination of bacterial proteases and lipases (both acid and neutral
lipases) from the marketed products like Forezym WG-L and Forezym DG
of La Forestal Tanica, together with enzyme compatible surfactants
(Thanikaivelan et al., 2004) are necessarily used in the degreasing
formulations.
2.8.4 Lipases in textile industry
Garment processing to have the refined and polished look in the finished
fabric, has brought the denim culture to daily life style in the world
including India. One of the important step in the garment processing that
results in much sort after finished fabrics in denim fashion is desizing,
where the size material from the fabric is removed, traditionally by acid or
base or oxidizing agents that can damage the fabric. Alternatively, enzyme
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desizing has been developed averting the probable damages of traditional
methods and added multiple advantages by using enzymes like cellulases,
amylases, proteases and lipases depending on the sizing material used
(Sangeetha et al., 2011). In this concern, there had been the reports on the
use of bacterial lipases from Pseudomonas cepacia, Pseudomonas
fluorescens, Pseudomonas fragi and Pseudomonas stutzeri for desizing
where the synthetic sizing material like polyester (www.wipo.int) was used.
In another development, lipases from Pseudomonas cepacia and
Pseudomonas fluorescens were used to improve the fashionability of
polyethylene Terephthalate (PET) fabrics by increasing the wettability of
the PET fabrics and better dyeing (Kim and Song 2006) compared to the
alkaline dyeing methods; otherwise the PET fabrics were known for their
hydrophobic nature, high strength and wrinkle resistance.
2.8.5 Lipases for synthesis of polymers
Lipases are used to modify the surface of polylactate (PLA) fibers (Wang
et al., 2002), synthesized from lactic acid produced by the fermentation of
natural sugars, so as to make the surface wettable which in turn can
facilitate dyeing, otherwise the PLA fibers are hydrophobic in nature having
high biodegradability ad low in energy consumption. These PLA fibers are
used in textile fabrics, packaging material, biomedicine and bio-plastics
(Drumright et al., 2000) due to their similar mechanical properties to that
of polystyrene and polyethylene tetraphthalate and they can be synthesized
from different isomers of lactic acid (Garcia et al., 2009). Further there had
been the reports on the enzymatic synthesis (Matsumura et al., 1997) of
Poly-L-Lactic acid (PLLA) using Bacillus cepacia lipase, with more
potential than the amorphous PLA (Priya and Chadha 2003).
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2.8.6 Lipases in Pharmaceutical applications
Pseudomonas cepacia lipases were used for the synthesis of
hydrocinnamate esters (Priya and Chadha 2003) that act as precursors for
the synthesis of 1,3,4,9-tetrahydropyrano (3,4-b)indole-1-acetic acid, the
medicine in analgesics, antipyretics and anti-inflammatory agents and
additionally these esters can also act as the inhibitors of HIV-protease.
Similarly, an enatio-selective Acinetobacter lipase had been reported to
catalyze the hydrolysis of cis-(±)-2-(bromomethyl)-2-(2,4-dichlorophenyl)-
1,3-dioxolane-4-methyl acetate, the intermediates in the synthesis of an
antifungal agent, Itraconazole (BALDESSARI AND Iqlesias 2012). In
other findings, Serratia marescens lipases were used to resolve the racemic
mixtures of Ketoprofen (Long et al., 2007) which act as non-steroidal anti-
inflammatory drug and (±) trans-methoxylphenyl glycidic acid methyl ester
where (-) trans-methoxylphenyl glycidic acid methyl ester (Baldessari and
Iqlesias 2012; Hu et al., 2009) in its pure form is used for the synthesis of
Diltiazem hycrochloride that acts as calcium channel blocker to treat angina
pectoris, hypertension and other vascular disorders.
Lipases derived from Pseudomonas were reported to catalyze the synthesis
of lipophilic phenols (Choo and Birch 2009) like cinnamoyl esters with
higher free radical scavenging activity (Buisman et al., 1998) than the
conventional antioxidant, cinnamic acid that suffer from the disadvantage
of being hydrophilic leading to deactivation as they get involved in the
stabilization of lipids in water, whereas these antioxidants are desired to be
active in the lipid region.
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In another case it was found that animal derived pancreatic lipases used as
digestive aid in lipid malabsorption disorders, lose their catalytic activity at
low pH and in the presence of proteases but the bacterial lipases of low pH
activity and protease resistance were proposed in place of these pancreatic
lipases to treat disorders of cystic fibrosis and pancreatitis (Sani 2006).
Similarly in the recent developments, a treatment method was developed
and patented with the composition of proteases, lipases and a metallic salt
for the skin and scalp disease and the hair loss (Miyazaki and Fujikawa
2009). The lipase in the developed composition was preferred to be of
Pseudomonas although the lipase can be used from both fungal and
bacterial origin.
2.8.7 Lipases for Biosensors
There had been the reports on immobilization of bacterial lipases using
micro-emulsion based gel and mesoporous silica matrix separately to
fabricate the glass based electrode (Huang et al., 2001) and potentiometric
(Setzu et al., 2007) lipase biosensors respectively. Such biosensors
developed can be used for the detection of lipids and the lipid binding
proteins like triglycerides in food and clinical samples, lipid based
pollutants and contaminants in pollution samples and pharmaceutical
samples (Pandey et al., 1999).
2.8.8 Lipases in Agrochemical Industry
Pseudomonas lipases were reported to produce insecticides, fungicides
(Pandey et al., 1999) and secondary alcohols like (R, S)-4-hydroxy-3-
methyl-2-(2’-propenyl)-2-cyclopenten-1-one (Xu et al 2005) by
trnsesterification and resolution of the enantiomeric alcohols used in the
formulations of pesticides. So these lipases were found to be an ecofriendly
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alternative to the production of precursor molecules required for the
synthesis of pesticides, insecticides and other agro chemically useful
compounds.
2.8.9 Lipases in Cosmetics and personal care products
Lipase catalyzed flavor production by transesterification and resolution of
racemic intermediates boosted the cosmetic and perfume industry and one
of such process was reported from the lipases of Pseudomonas cepacia to
resolve the racemic rose oxides produced by the bromomethoxylation of
citronellol (Tanej et al., 2005). Similarly transesterification by lipases were
used to produce the flavor materials like esters of aliphatic and aromatic
acids, and alcohols including terpene alcohols, aldehydes and phenols for
perfumes and other personal care products (Franssen et al., 2005).
In other investigation, Pseudomonas cepacia lipases were used for the
synthesis of hydrocinnamic acid esters (Priya and Chadha 2003) that forms
the composition of the perfumes, sunscreen lotions etc. Similarly, lipases
from Pseudomonas fluorescens and Pseudomonas cepacia (Chaplin et al.,
2006) were used to produce the menthol esters and similar compounds for
the formulations of mouth washes and shaving creams known for their
peppermint flavor and cooling sensation respectively.
2.8.10 Lipases in biodiesel production
Biodiesel has been produced as an alternative to petroleum based diesel, all
over the world and the research had been intensified to increase the yield
and productivity of biodiesel to meet the energy needs of industry and the
transport facilities. Biodiesel is defined as the monoalkyl esters of long
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chain fatty acids like methyl ester of fatty acid with remarkable properties
like renewability, biodegradability, non-inflammatory and non-toxic nature
and such biodiesel is denoted as FAME, Fatty Acid Methyl Ester
(Sangeetha et al., 2011).
Though biodiesel is synthesized by chemo catalytic and thermo catalytic
approaches, the bio-catalytic approach is more appreciated as it uses the
biodegradable and ecofriendly lipases from Pseudomonas cepacia (Li and
Yan 2008) to catalyze the trans esterification of lipids and short chain
alcohols (Chen et al., 2009; Dizge et al., 2009; Raita et al., 2010) from the
sources like soybean oil, rice bran oil, sunflower oil, palm oil, cotton seed
oil, jatropha oil, animal fat, algae, waste edible oil and industrial acid oil to
produce esters and glycerol, the byproduct . Since the aqueous environment
favors the hydrolysis reaction by lipids, the micro-aqueous environment
(Xu et al., 2009) like that of immobilized enzyme systems, is preferred for
the production of biodiesel from oils and fats. Further, the reports on the
efforts to improve the methanolysis for biodiesel production prefers solute
like ectoine that decreases the affinity of the lipase for methanol and
increases the affinity of lipase for triglycerides leading to the improved
yield of biodiesel (Wang and Zhang., 2009).
As the biodiesel production leads to the production of glycerol as a
byproduct, it has been utilized by the industry, to convert it to 1,3
propanediol, a monomer for the synthesis of novel polymers like
polymethylene terephthalate. In this concern, the bacterial species like
Klebsiella, Citrobacter, Clostridium, Enterobacter and Lactobacillus were
reported to convert glycerol to 1, 3 Propanediol (Xu et al., 2009).
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2.8.11 Lipases in Environment Management
Waste-water generated from the metropolitan cities, industrial area and
other health centers, contain high amount of lipidic content in addition to
other organic matter, but the disposal of such contaminated and polluted
water pose threat to water bodies and ground water by consuming the
dissolved oxygen as well as decreasing the oxygen transfer rate
necessitating the human intervention may be in the form of ecofriendly
bioremediation (Mongkolthanaruk and Dharmsthiti 2002) that involves the
use of enzymes such as lipases, proteases etc. Another important area where
the lipases are used is the oil spills that contaminate both soil and water
during the rigging and refining processes (Pandey et al., 1999) in the oil
fields. Similarly, the effluents emanating from food processing, tanner,
automobile industries, restaurant and fast-food outlets can be treated by
inoculating the effluents with lipase producing bacteria (Pandey et al.,
1999; Nelson and Rawson 2010) and cultivating microbial organisms for
longer periods of time. Such waste water containing the lipidic and organic
matter the treatment process was performed by culturing the bacterial
organisms from the genera such as Pseudomonas, Bacillus and
Acinetobacter (Mongkolthanaruk and Dharmsthiti 2002; Padmapriya et al.,
2011).
2.8.12 Lipases in Environmental studies
Environmental studies are emerging to understand the interdependence of
the geological forces and biological forces and one such studies is the
analysis of the relationship between Dissolved Organic Matter (DOM) and
bacterial dynamics in terms of bacterial response to environmental changes
and the human intervention if any. These studies utilize various parameters
like the soil composition, moisture levels, air and water percolation,
biological systems etc. for analysis and among such complex situations
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bacteria use the readily available small molecules from the pool of DOM
for their growth and subsequently breakdown the complex molecules by
amino peptide hydrolysis and polysaccharide degradation in situ as
indicators of DOM dynamics. Similarly in the recent past, there was also a
report on the relationship between the levels of lipids and in situ lipase
activities during different environmental conditions like Spring and
Summer (Bourguet et al., 2009).
2.9 Bacterial identification
Identification of an unknown organism like bacteria in the interest of the
sustenance of life on the earth, is an important process to further
understand, characterize, preserve, organize and share the organism for the
benefit of the mankind and the biodiversity at large. There are two ways of
identifying an unknown bacterial organism like the series of phenotypic
methods, developed and practiced traditionally over the centuries of time,
and genotypic methods, developed and has been practiced over the last few
decades based on the molecular and genetic engineering principles.
2.9.1 Phenotypic methods
These are a series of methods or tests developed over two centuries to
identify and type the microbial organisms based upon their phenotypic
characteristics (Holt et al., 1994) as discussed below.
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2.9.1.1 Biotyping
This method is based on differentiation of the unknown organism using the
responses or properties like different biochemical reactions, morphological
characters and sustenance to the given stimulus in the form of media,
culturing methods, chemical treatment etc. in an in vitro laboratory
condition. So for every given stimulus the microbial organism respond in a
distinct way, such as the growth of the organism to the given differential
growth media displaying the ability of the organism to metabolize the given
nutrients in the media and the display of the morphological characters;
which would help in differentiation of the organism to a particular species,
and further display of different biochemical responses by the organism to
the given chemical treatment in various forms, will assist in the next level
of microbial identification and so on up to the strain level. All such
responses and characters of microbial organisms are recorded and
organized to suggest a series of tests to identify the unknown microbial
organisms at various laboratories and microbial repositories and they are
even programmed to automated systems to make the process more efficient
and time saving (Singh et al., 2006).
2.9.1.2 Antimicrobial susceptibility
Antimicrobial susceptibility testing or microbial diagnosis had been carried
out for any unknown suspected microorganism as common practice in
clinical microbiology laboratories. There are different antimicrobial test
methods but the satisfactory results were obtained by the methods like agar
dilution, micro-dilution, E-test and disk diffusion (Pankuch et al., 2006),
but then disk diffusion method is not being adopted as once it was due to
the difficulties in automation of the process, whereas micro-dilution testing
was preferred as it provides a quantitative measure of the MIC, the
minimum inhibitory concentration of the antimicrobial agent that inhibits
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the growth of the organism. However, disk diffusion as well as broth
dilution were standardized and hence are accepted as they are reproducible
within and among laboratories. Such antimicrobial tests on an unknown
microorganism yield a pattern of in vitro resistance to a panel of
antimicrobial agents or antibiotics, named antibiogram that indicates the
type of unknown microorganism, with some limitations as some of the
suspected microorganisms may have the same susceptibility pattern
although they are not genetically and epidemiologically related.
2.9.1.3 Serotyping
This method for the identification of an unknown organism, particularly
pathogens, involve a series of antibody stimulus on microbial organisms
like bacteria to which they respond in variable antigen antibody
characteristic binding (Babl et al., 2001). Such methods of pathogenic
bacterial identification are practiced for decades and continue to be the
preferred choice of identification especially for the genera of Salmonella,
Legionella, Shigella, and Streptococcus pneumonia; and in addition
serotyping has been used to differentiate strains within the species of
nosocomial pathogens such as Klebsiella and Pseudomonas. Further this
serotyping technology was used to find the relation of serotypic
distributions of strains causing invasive diseases, nasopharyngeal
colonization and antibiotic resistance, to age, geography and socio-
economic conditions of human populations (Aslan et al., 2007) and in turn
these studies be used for the distribution and evaluation of vaccines.
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2.9.1.4 Phage and bacteriocin typing
Bacteriophage and bacteriocin are the typing techniques developed as
epidemiological tools specifically to identify and characterize the bacteria
based on the pattern of resistance or susceptibility to a particular set of
phages (Hopkins et al., 2004). It is known that the bacteria having the phase
binding receptors in the cell wall are susceptible to the bacteriophages and
hence the phages can enter the cell, multiply and eventually lyse the cell;
such phage dependent phenomena varies across the bacterial species
corresponding to the variation and availability of phase binding receptors.
Though this phage typing method was applied to characterize the bacteria
associated with nosocomial infections, it suffers from the lack of
widespread availability of biologically active phages and the technical
difficulty in performing this technique for various bacterial species.
In the other bacteriocin typing technique, a series of heterogeneous
bacterial protein based molecules are used as inhibitors called bacteriocins,
to limit the growth of bacterial species, defining a particular inhibitory
pattern to identify and characterize the unknown and susceptible bacteria,
and one of such bacteria characterized by this technique was P. aureginosa
(Arbeit et al., 1995). However this bacteriocin typing, like bacteriophage
typing, had the limitations due to the non-availability of wide range of
bacteriocin molecules to characterize various species of bacteria.
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2.9.2 Genotyping techniques
Since the last three decades, the molecular techniques and genetic
engineering based technologies have been evolving and practiced to find
the characters and the interrelations between the species based on the
genetic or gene patterns conserved among different domains and genera of
species. Among those techniques, Polymerase Chain Reaction (PCR)
techniques including, real time PCR, multiplex PCR, nested PCR, and the
hybridization techniques like fluorescent in situ hybridization, are widely
used either in sole or in combination with other molecular and genetic
engineering methods of sequencing to characterize and identify the
organisms. However PCR had been the initial and primary technique
involved in most of the genotyping techniques developed.
PCR is a well-established biochemical in vitro reaction technique that
involves a DNA template from the organism of interest, two
complementary oligonucleotide primers designed to flank the sequence to
be amplified, in the template DNA, and a heat stable DNA polymerase,
usually Taq DNA Polymerase. Once these components of the PCR along
with the reaction cocktail were placed in the wells of the thermalcycler (the
PCR facilitating machine), the PCR process gets initiated by rapidly
increased temperature to 90oC, where double-stranded DNA gets separated
into single strands (denaturation) , then rapidly decreasing the temperature
to 40-65 oC, where the primers gets annealed (bind) on to the generated
complementary DNA templates specifically at the target sequences
(Annealing) and then the temperature gets raised to 72 oC, where the Taq
DNA polymerases binds to the target template DNAs at the primer and
starts to amplify the target sequence leading to the generation of two copies
of the target sequence (Extension) and there ends of the first cycle. In the
subsequent PCR cycles (denaturation, annealing and extension) of about
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20-30, the target DNA sequence gets amplified to sufficient quantity
required for further analysis as part of the characterization and
identification of the organism of interest (Grimm et al., 2004; Hussain et
al., 2004; Suzuki et al., 2004; Trad et al., 2004; Nakari et al., 2005; Shutt et
al., 2005).
For more advanced level of analysis, a set of molecular and genetic
engineering based typing techniques were developed to characterize and
find the pattern or profile of the community diversity (Meyer et al., 2007)
by rapid analysis methods such as denaturing-gradient gel electrophoresis
(DGGE), temperature-gradient gel electrophoresis (TGGE), and single-
stranded conformational polymorphism (SSCP) specifically for bacterial
domain. Further, there were also techniques developed based on
heteroduplex mobility analysis of 16S rDNA fragments for targeted
detection of sub-populations of bacteria even to the level of strains within
diverse microbial communities (Turner et al., 2002) and some of those
techniques include ribotyping, amplified ribosomal DNA restriction
analysis (ARDRA), pulsed-field gel electrophoresis (PFGE), random
amplification of polymorphic DNA (RAPD), repetitive element sequence-
based PCR (rep-PCR), and amplified fragment length polymorphism
(AFLP).
In spite of all these methods and the data built up on these methods, the
molecular and genetic engineering techniques have been evolving and
specially the gene sequencing technology has revolutionized the DNA
sequence based technologies and improved the accuracy and repetitiveness
of the results of bacterial identification among various laboratories. One of
such technology evolved as the most common housekeeping genetic marker
for identification of bacterial isolates is 16S rRNA gene sequencing
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technology, due to the following attributes of 16S rRNA genes (Janda and
Abbot 2007).
(i) 16S rRNA genes are present in almost all bacteria, often existing
as a multi-gene family or operons.
(ii) The function of 16S rRNA gene over the time has not changed,
suggesting that random sequence changes are a more accurate
measure of time (evolution), and
(iii) The 16S rRNA gene (1500 bp) is large enough for informatics
purposes.
2.9.2.1 Identification of microbial isolates by 16S rRNA gene
sequencing
The bacterial identification process in this technique, typically starts with
the isolation of genomic DNA samples form the bacterial isolates from the
chosen habitats, and the purified DNA sample of the isolate strain would be
used for the PCR amplification of 16S rRNA genes using appropriate
primers (Table-10). These amplified 16S rRNA gene sequences would be
read and compared to match with the existing database of the known 16S
rRNA gene sequences. If no close match to an existing 16S rRNA gene
sequence is found, then the test sequence represent a new bacterium and is
listed in GenBank as “uncultured bacterium”. However the sequence match
is not the ultimate or the absolute for the identification of the unknown
bacterial culture. So further characterization may be carried out by the
traditional phenotyping techniques to supplement the information of the
organism. The accumulating database of 16S rRNA sequences, over
2,578,902 (16S rRNA) sequences till date, provides additional benefits to
focus on microbial diversity of less explored extreme microbial flora whose
unidentified sequence similarities can be matched with these reported
sequences to find the close and divergent relatives of microbial ecosystem.
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Due to this vast availability of data and reliability of technique, the 16S
rRNA gene sequencing has emerged as one of the most used methods of
species identification for the bacterial genera.
Table 10. Primers used for amplification and sequencing of the 16S rRNA
genes (Claudio et al., 2002)
Primer,
Forward (F) /
Reverse (R) Sequence
Generic primers used for 16S rRNA amplification
8F 5´AGT TGA TCC TGG CTC AG 3´
1492R 5´ACC TTG TTA CGA CTT3´
Primers for amplification of the 16S rRNA gene
67F 5´TGA AAA CTG AAC GAA ACA AAC 3´
1671R 5´CTC TCA AAA CTG AAC AAA ACG AAA 3´
Inner primers used for nested PCR on clinical samples
23F 5´ACA AAC AAC GTG AAA CGT CAA 3´
136R 5´AAA CGA AAC ACG GAA ACT T 3´
Primers used for sequencing of the 16S rRNA gene
104F 5´GGA CGG GTG AGT AAC ACG TG 3´
104R 5´CAC GTG TTA CTC ACC CGT CC 3´
1230F 5´TAC ACA CGT GCT ACA ATG 3´
1390F 5´GGG CCT TGT ACA CAC CG 3´
1390R 5´CGG TGT GTA CAA GGC CC 3´
8F 5´AGT TGA TCC TGG CTC AG 3´
357F 5´TAC GGG AGG CAG CAG 3´
357R 5´CTG CTG CCT CCC GTA 3´
530F 5´CAG CAG CCG CGG TAA TAC 3´
530R 5´GTA TTA CCG CGG CTG CTG 3´
790F 5´ATT AGA TAC CCT GGT AG 3´
790R 5´CTA CCA GGG TAT CTA AT 3´
981F 5´CCC GCA ACG AGC GCA ACC C 3´
981R 5´GGG TTG CGC TCG TTG CGG G 3´
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The steps involved in the 16S rRNA gene sequencing technology may be
summarized as follows.
Isolation of genomic DNA from microbial strain
↓
Design of primers in the region of 16S RNA gene or usage of already
published set of suitable primers
↓
Amplification of 16S RNA gene using the primers and isolation of the
amplified DNA
↓
Gel purification of the amplified DNA fragments
↓
Sequencing of the amplified DNA fragments
↓
Searching for the match of similar sequences using NCBI search tool
(Ex.: BLAST)
↓
Analysis of the search results and construction of phylogenetic tree
↓
Identification of the most probable strain of the test microbial isolate
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