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Stimulation of novel thermostable extracellular lipolytic
enzyme in cultures of Thermus sp.
Alberto Domnguez
a
, Pablo Fuci nos
b
, M. Luisa R ua
b
, Lorenzo Pastrana
b
,
Mara A. Longo
a
, M. Angeles Sanrom an
a,
a
Department of Chemical Engineering, University of Vigo, Lagoas-Marcosende, 36310 Vigo, Spain
b
Department of Biochemistry, Genetics and Immunology, University of Vigo, Spain
Received 29 September 2005; received in revised form 5 September 2006; accepted 12 September 2006
Abstract
Selected organisms from the genus Thermus (T. aquaticus YT1, T. thermophilus HB8 and HB27) offer new opportunities for biocatalysis and
biotransformations as a result of the extreme stability of their enzymes. In order to favour the secretion of extracellular lipolytic enzymes the effect
of temperature and carbon source has been studied. All strains were able to grow within a wide temperature range (from 60 to 80
C) with an
optimum value of 70
Corresponding author. Tel.: +34 986 812383; fax: +34 986 812380.
E-mail address: sanroman@uvigo.es (M.A. Sanrom an).
in a complex medium has been demonstrated in our laboratory.
All enzymes were stable at 80
C). The
cultures were stopped after 30 h of incubation, since previous
experiments indicated that, no signicant increases in lipolytic
enzyme activity were attained later in ask cultures. Besides,
cell lysis was mostly negligible at this time. Moreover, the
authors indicated that no kinetic typication of the enzymes as
primary metabolites was possible for any of the Thermus strains,
because of the lack of a good tting of the experimental lipolytic
activity production rates to the Luedecking & Piret model
[68].
Time-course of cultures and nal biomass, intra- and
extracellular lipolytic activity are shown in Fig. 1 and Table 1,
respectively. In the three studied strains, the increase in temper-
ature seemed to have a negative effect on biomass production:
nal cell growth at 60
C. Similar nal
cell growth levels are attained at 60 and 70
C, although a
longer lag phase is generally observed at 60
C, as it was
foreseeable.
More signicant differences between strains were observed
for lipolytic enzyme production. Maximum intracellular
activity was very similar in all T. aquaticus YT1 cultures, with
an average value of 66 Udm
3
, and production proles were
fairly close. On the other hand, the increase in temperature had
an acute effect on T. thermophilus HB8 and HB27 intracellular
enzyme levels. Final activities of 125 and 93 Udm
3
were
obtained, respectively, operating at 80
C, () 70
C, and () 80
C.
intracellular chaperonins from late log phase T. thermophilus
HB8 cultures performed at 75
C spontaneous
refolding fails but if the native protein is sufciently stable, the
chaperonin induces productive refolding in an ATP-dependent
manner. At temperatures below 60
C (i.e. 50
C) spontaneous
refolding of the proteins occurs, and the chaperonin arrests this
spontaneous refolding in the absence of ATP.
It could be hypothesised that the occurrence of chaperonins
within T. thermophilus cells might contribute to the phenomena
described in this work, by favouring lipolytic enzyme folding
and therefore stability at high temperature. Since the mechanism
of chaperonin action seems to be dependent of temperature, this
could account to a certain extent for the differences observed in
intracellular lipolytic enzyme levels for T. thermophilus HB27
and HB8.
As for extracellular lipolytic activities, they were rather low
in comparison with the intracellular enzymes. In all cases, the
highest nal values were obtained when operating at 70
C. A
parabolic dependence of temperature was postulated for extra-
cellular lipolytic activity. Experimental results for nal activity
were tted to polynomic equations, and subsequently derivated
to obtain the theoretical optimum. According to this procedure,
optimal temperatures were found at 66.9, 70.6 and 70.5
C for
T. thermophilus HB8 and HB27 and T. aquaticus YT1, respec-
tively.
The lower extracellular enzyme activity at high tempera-
ture could in part be due to the secretion of specic proteases
during the last phase of the cultures. This fact was described
by Matsuzawa et al. [14] in previous studies carried out with
other Thermus strains. Also, temperature-dependent differences
in extracellular enzyme activity could be related to the occur-
rence of special enzyme secretion mechanisms in the studied
microorganisms, a hypothesis that has already been proposed
by the authors in a previous work [8] to explain the seemingly
independent dynamics of extracellular lipolytic activity and
intracellular enzyme and biomass production. Several species
of Thermus genus are able to form, under certain environmental
conditions, clusters of cells surrounded by a membrane formed
by the fusion of the individual external membranes of each cell.
These formations are usually known as rotund bodies [15]. It
might be assumed that proteins could be rstly secreted by indi-
vidual cells to this internal cavity and, from there poured into
the culture medium. Therefore, the integrity and permeability
of the external membrane of the rotund bodies might be impor-
tant to control the liberation of enzymes to the culture medium,
and these properties could be inuenced by the temperature.
Thus, the combined and opposed effects of increased release of
190 A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194
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enzymes to the culture medium and thermal or proteolytic deac-
tivation at high temperatures could account for the results found
in the present work.
Although maximum intracellular lipolytic activity was
obtained at 80
C as culture temperature.
3.2. Effect of the type and concentration of carbon source
A high degree of nutritional diversity has been detected in
Thermus strains (i.e. T. thermophilus HB8, T. aquaticus YT1) by
Degryse et al. [16], Alfredsson et al. [17] and Santos et al. [18].
These strains grewwell on different carbon sources, although the
presence of glucose, galactose, fructose, sucrose or maltose in
the culture mediumseemed to have a particularly positive effect.
However, the above-mentioned studies were carried out by
means of agar plate cultures, and only provided qualitative
results. Besides, they focused on the investigation of cell
growth, and no information was given on the production of
potentially interesting enzymes. In the present work T. ther-
mophilus HB8, HB27 and T. aquaticus YT1 have been grown
in submerged cultures, using a complex medium supplemented
with several concentrations (from 0.5 to 14 g dm
3
) of mono-
and disaccharides (glucose, fructose, maltose and sucrose). The
end-culture values of biomass, intra- and extracellular lipolytic
activity (after 30 h of incubation) for the three strains are
compared in Figs. 24, respectively. The data are expressed as
percentages referred to the results obtained in the basal medium
(with no additional carbon source) at 70
C, shown in Table 1.
The effect of carbon source appeared to be somewhat
strain-dependent and therefore difcult to generalise. However,
some common patterns could be established for the tested
strains. As it can be observed in Fig. 2, supplementation of
the basal medium with low concentrations (up to 1.5 g dm
3
)
of mono- or disaccharides led, in most cases, to a moderate
increase (2050%) in biomass production. Higher carbon
source concentrations did not result in further improvements in
cell growth, with the exception of T. thermophilus HB8 cultures,
in which biomass was increased two-fold and 2.5-fold when
the basal medium was supplemented with sucrose (7 g dm
3
)
or maltose (14 g dm
3
). Also, it is noteworthy that cell growth
was signicantly diminished when high levels of glucose were
added to the medium, in all the studied strains.
When intracellular lipolytic activity production was con-
sidered (Fig. 3), the general behaviour of the microorganisms
was similar to that described for cell growth. However, the
comparison of both sets of data indicated that intracellular
enzyme and biomass productions were not fully associated. The
addition of low concentrations (0.51.5 g dm
3
) of mono- and
disaccharides generally resulted in a noticeable amelioration
in enzyme production, the most promising results having been
A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194 191
Fig. 2. Inuence of carbon source on biomass production by Thermus strains,
after 30 h of culture time. Carbon source: () glucose, () fructose, () maltose,
and () sucrose (100%biomass production: T. thermophilus HB8, 0.75 g dm
3
;
T. thermophilus HB27, 1.04 g dm
3
; T. aquaticus YT1, 1.18 g dm
3
).
obtained with disaccharides, and for T. thermophilus HB8.
Carbon source supplementation above this level does not
seem to be advisable, since intracellular lipolytic activities
remained mostly unaltered, or decreased. Only T. thermophilus
HB8 and HB27 showed any amelioration in intracellular
enzyme levels at high carbon source concentrations, with
three-fold and 1.6-fold rises in activity (referred to control) in
the presence of sucrose (14 g dm
3
) and maltose (7 g dm
3
),
respectively.
Finally, extracellular lipolytic enzyme concentrations were
assessed. No signicant levels of extracellular activity were
detected for T. thermophilus HB8, in any case. As for the other
two strains (Fig. 4), the general effect of carbon source supple-
mentation was similar to that previously described for biomass
Fig. 3. Inuence of carbon source on intracellular lipolytic activity production
by Thermus strains, after 30 h of culture time. Carbon source: () glucose, ()
fructose, () maltose, and () sucrose (100% lipolytic activity: T. thermophilus
HB8, 72.80 Udm
3
; T. thermophilus HB27, 81.79 Udm
3
; T. aquaticus YT1,
68.70 Udm
3
).
and intracellular enzyme. A slight enhancement in extracellular
enzyme production was obtained when low concentrations of
the different carbon sources were added. The best results were
obtained with sucrose for T. thermophilus HB27 and fructose
for T. aquaticus YT1 (both sugars at 0.5 g dm
3
).
Total carbohydrates and protein consumption were evalu-
ated in all the cultures. The assessed nutrients were not totally
depleted in any case, and most consumption occurred during
the early stages of the cultures. Low nutrients consumption
in Thermus strains has already been mentioned in previous
works [6,19,20]. No relevant information could be inferred from
these data, concerning the dynamics of cell growth and lipolytic
enzyme production on different carbon sources.
192 A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194
Fig. 4. Inuence of carbon source on extracellular lipolytic activity production
by T. thermophilus HB27 and T. aquaticus YT1, after 30 h of culture time.
Carbon source: () glucose, () fructose, () maltose, and () sucrose (100%
lipolytic activity: T. thermophilus HB8, 4.50 Udm
3
; T. thermophilus HB27,
34.40 Udm
3
; T. aquaticus YT1, 27.25 Udm
3
).
To our knowledge there are few reports about the produc-
tion of thermostable lipases or esterases from thermophilic
microorganisms, and none on how it can be affected by the
choice and concentration of carbon source. However, the inu-
ence of this factor on lipolytic enzyme secretion by mesophilic
microorganisms has been investigated. The results are very dif-
ferent depending on the microorganism and the experimental
conditions employed. Generally, monosaccharides have been
reported to favour lipase production by mesophilic microor-
ganisms. So, Benjamin and Pandey [21] found that glucose
and fructose enhanced lipase activity in submerged cultures of
Candida rugosa. Dalmau et al. [22] proposed a mixture of com-
pounds as optimum carbon source for lipase production by the
same microorganism. Polysaccharides (i.e. starch) and glycerol
have been described as poor carbon sources for lipase production
by C. rugosa and Yarrowia lipolytica [2123], although they led
to good results in the case of Penicillium citrinum and Rhizopus
delemar [24,25]. Costa et al. [26] concluded that the ability of the
yeast Issatchenkia orientalis to secrete lipolytic activity in sub-
merged culture was improved by using monosaccharides (glu-
cose, fructose), although di- and polysaccharides (sucrose, lac-
tose, maltose, starch) as well as glycerol were not recommended.
The results found in the present work seemto indicate that the
presence of certain carbon sources in the culture medium might
have aninuence oncell growthandlipolytic enzyme production
by Thermus strains. However, the observed effects appeared to
be somehowless dramatic than those reported in some instances
for mesophilic microorganisms. This could be related to the
extreme environments in which thermophilic microorganisms
are usually found, generally characterised by lownutrients avail-
ability. Therefore, the strains are naturally conditioned to survive
in oligotrophic environments, and inhibition by excess of sub-
strates is often encountered [27].
Supplementation of the medium with low concentrations of
mono- anddisaccharides generallyresultedinimprovedbiomass
and lipolytic enzyme production. However, when high carbohy-
drate concentrations were used, no further improvements were
observed, and in some cases (i.e. glucose) strong decreases
in biomass and enzymatic activity were detected. This could
be attributed to the occurrence of Maillard reactions between
amino compounds and reducing sugars, promoted by the high
culture temperatures utilised and favoured by values of pH in
the medium between 7.5 (initial) and 8.5 (at the end of culture).
This kind of reaction is often found in cultures of thermophilic
strains and may result in products that can be inhibitory to
the microorganisms [27]. In the present case, this potentially
inhibitory effect appears to be most remarkable with monosac-
charides, and more precisely glucose. This could be due both
to the higher reactivity of this sugar towards Maillard reaction,
and a possible stronger toxic effect of the formed products. Sus-
ceptibility of disaccharides to undergo the reaction is smaller
(i.e. maltose) or nonexistent (i.e. sucrose, non-reducing sugar),
which agrees with the lesser adverse effects associated to their
presence in the culture medium.
With respect to the mechanismby which disaccharides might
be metabolised by the studied strains, the results seem to indi-
cate that they are not massively hydrolysed in the mediumbefore
being incorporated to the cells. Extracellular hydrolysis of both
disaccharides would imply the generation of glucose, which was
shown to exert a strong inhibitory effect on both biomass and
enzyme production, yet no drastic negative repercussions were
detected when high concentrations of maltose or sucrose were
considered. Therefore, either the disaccharides are hydrolysed
at approximately the uptake rate by the cells, thus maintain-
ing a concentration below the critical one for inhibition, or
they are directly up taken by the cells. The latter hypothesis
is supported by the recently ascertained genome sequence of T.
thermophilus, in which maltose/trehalose permease and ABC
transporter encoding genes were found [28]. Besides, the mal-
tose/trehalose ABC transporter was recently reported to also
recognize sucrose [29]. The genes encoding two -glucosidades,
a -glucosidase and a maltodextrin glucosidase were also found,
but there are no indications that these enzymes could be secreted
by the microorganisms to the culture medium.
Finally, the effect of the addition of lipidic compounds to the
culture mediumin biomass and lipolytic enzyme production has
been assessed in T. thermophilus HB27 cultures. This strain was
selected for this preliminary study, due to the higher enzyme
secretion levels detected in previous cultures, and the more
abundant information available on its lipolytic enzymes [7]. The
presence of lipids has been reported to be crucial for lipases and
esterases production by a number of microorganisms [30]. Thus,
A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194 193
Fig. 5. Intra- and extracellular lipolytic activity production by T. thermophilus
HB27 cultures in presence of several lipidic compounds with different con-
centrations (1 and 10 g dm
3
): B, control; OO, olive oil; CO, coconut oil; TB,
tributyrin.
the basal medium was supplemented with different concentra-
tions (1 and 10 g dm
3
) of triglycerides (olive oil, coconut oil
and tributyrin). Intra- and extracellular enzyme activities after
30 h culture time are presented in Fig. 5. Tributyrin appeared
to have a strong inhibitory effect in the microorganism, while
cell growth did not undergo dramatic changes with the addition
of olive or coconut oil. On the other hand, the presence of olive
oil helped to increase lipolytic enzyme production levels, both
intra and extracellular up to 33% and 70%, respectively. As it
occurred with carbohydrates, the best results were obtained with
low concentrations of added carbon source. Therefore, it would
appear that the presence of lipids could trigger to some extent the
production of lipolytic enzymes. The occurrence of a biphasic
medium could lead to hydrophobic interactions that might alter
the permeability of the membranes surrounding the already
mentioned rotund bodies, therefore accounting for the observed
variations in enzyme secretion levels. However, further inves-
tigation is required on this topic before attempting any denite
conclusion.
4. Conclusions
According to the results obtained, it can be concluded that
the progressive increase in incubation temperature appeared to
have a negative effect on cell growth, and 70
C was selected
as an optimum for this variable, a compromise in terms of
biomass, intra- and extracellular enzyme production, and cost
optimisation.
Disaccharides (i.e. sucrose and maltose) have been estab-
lishedas goodcarbonsources allowingtoimprove growthand/or
favour enzyme secretion by these microorganisms, although the
most adequate concentration seems to depend on the strain.
On the other hand, the addition of high levels of glucose or
fructose to submerged cultures of Thermus sp. did not bring
about signicant ameliorations in lipolytic activity. Moreover,
the addition of some lipidic compounds (olive and coconut oils)
to the culture medium increased total lipolytic activity levels up
to two-fold with respect to the control culture. These promising
results encourage to realise more studies in which surfactants
or other lipidic compounds would be added to culture medium.
These compounds could help to increase the secretion of lipoly-
tic enzymes due to changes in cell wall permeability or surfactant
effects on cell bound enzyme.
Acknowledgements
This work was nanced by the Spanish Ministry of Science
and Technology and European FEDER(Project PPQ2001-3361)
and XUNTA de Galicia (Project PGIDT03PXIB30103PR).
References
[1] Hough DW, Danson MJ. Extremozymes. Biocatal Biotransfor
1999;3:3946.
[2] Schiraldi C, De Rosa M. The production of biocatalysts and biomolecules
from extremophiles. Trends Biotechnol 2002;20:51521.
[3] Pantazaki AA, Pritsa AA, Kyriadakidis DA. Biotechnologically rele-
vant enzymes from Thermus thermophilus. Appl Microbiol Biotechnol
2000;58:112.
[4] Berger JL, Lee BH, Lacroix C. Identication of new enzyme activi-
ties of several strains of Thermus species. Appl Microbiol Biotechnol
1995;44:817.
[5] Lagarde D, Nguyen HK, Ravot G, Wahler D, Reymond JL, Hills G, et al.
High-throughput screening of thermostable esterases for industrial biocon-
versions. Org Process Res Dev 2002;6:4415.
[6] Dominguez A, Sanroman A, Fuci nos P, Rua ML, Pastrana L, Longo MA.
Quantication of intra- and extra-cellular thermophilic lipase/esterase pro-
duction by Thermus sp. Biotechnol Lett 2004;26:7058.
[7] Fuci nos P, Abadin CM, Sanroman A, Longo MA, Pastrana L, Rua ML.
Identication of extracellular lipases/esterases produced by Thermus ther-
mophilus HB27: partial purication and preliminary biochemical charac-
terisation. J Biotechnol 2005;117:23341.
[8] Fuci nos P, Dominguez A, Sanroman MA, Longo MA, Rua ML, Pastrana L.
Production of thermostable lipolytic activity by Thermus species. Biotech-
nol Prog 2005;21:1198205.
[9] Dominguez A, Pastrana L, Longo MA, Rua ML, Sanroman MA. Lipoly-
tic enzyme production by Thermus thermophilus HB27 in a stirred tank
bioreactor. Biochem Eng J 2005;26:959.
[10] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement
with the Folin phenol reagent. J Biol Chem 1951;193:26575.
[11] Dubois M, Giles UA, Hamilton JK, Rebers PA, Smith F. Colorimetric
method for determination of sugars and related substances. Anal Chem
1956;28:3506.
[12] Oshima T, Imahori K. Description of Thermus thermophilus combination
nova, a non-sporulation thermophilic bacterium from a Japanese thermal
spa. Int J Syst Bacteriol 1974;24(1):10212.
[13] Taguchi H, Konishi J, Ishii N, Yoshida M. Achaperoninfroma thermophilic
bacterium, Thermus thermophilus, that controls refoldings of several ther-
mophilic enzyme. J Biol Chem 1991;266(33):224118.
194 A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194
[14] Matsuzawa H, Hamaoki M, Ohta T. Production of thermophilic extracellu-
lar proteases (aqualysins I and II) by Thermus aquaticus YT-1, an extreme
thermophile. Agric Biol Chem 1983;47(1):258.
[15] Becker RJ, StarzykMJ. MorphologyandrotundbodyformationinThermus
aquaticus. Microbios 1984;41:11529.
[16] Degryse E, Glansdorff N, Pierard A. A comparative analysis of extreme
thermophilic bacteria belonging to the genus Thermus. Arch Microbiol
1978;117(2):18996.
[17] Alfredsson GA, Baldursson S, Kristjansson K. Nutritional diversity among
Thermus spp. isolated from Icelandic hot springs. Syst Appl Microbiol
1985;6(3):30811.
[18] Santos MA, Williams RAD, Da Costa MS. Numerical taxonomy of
Thermus isolates from hot springs in Portugal. Syst Appl Microbiol
1989;12:3105.
[19] Sonnleitner B, Cometta S, Fiechter A. Growth kinetics of Thermus ther-
mophilus. Eur J Appl Microbiol Biotechnol 1982;15:7582.
[20] Cometta S, Sonnleitner B, Fiechter A. The growth behaviour of Ther-
mus aquaticus in continuous cultivation. Eur J Appl Microbiol Biotechnol
1982;15:6974.
[21] Benjamin S, Pandey A. Optimisation of liquid media for lipase production
by Candida rugosa. Bioresour Technol 1996;55:16770.
[22] Dalmau E, Montesinos JL, Lotti M, Casas C. Effect of different carbon
sources on lipase production by Candida rugosa. Enzyme Microb Technol
2000;26:65763.
[23] Corzo G, Revah S. Production and characteristics of the lipase
from Yarrowia lipolytica 681. Bioresour Technol 1999;70(2):
17380.
[24] Haas MJ, Bailey DG. Glycerol as carbon source for lipase pro-
duction by the fungus Rhizopus delemar. Food Biotechnol 1993;7:
4953.
[25] Sztajer H, Maliszewska I. The effect of culture conditions on lipoly-
tic productivity of Penicillium citrinum. Biotechnol Lett 1989;11:
8958.
[26] Costa M, Deive FJ, Longo MA. Lipolytic activity in submerged
cultures of Issatchenkia orientalis. Process Biochem 2004;39(12):
210914.
[27] Holst O, Manelius A, Krahe M, M arkl H, Raven N, Sharp R. Thermophiles
and fermentation technology. Comp Biochem Physiol 1997;118A:
41522.
[28] Henne A, Br uggemann H, Raasch C, Wiezer A, Hartsch T, Liesegang H,
et al. The genome sequence of the extreme thermophile Thermus ther-
mophilus. Nature Biotechnol 2004;22:54753.
[29] Silva Z, Sampaio MM, Henne A, Boehm A, Gutzat R, Boos W, et al.
The high-afnity maltose/trehalose ABC transporter in the extremely ther-
mophilic bacterium Thermus thermophilus HB27 also recognizes sucrose
and palatinose. J Bacteriol 2005;187:12108.
[30] Sharma R, Chisti Y, Banerjee U. Production, purication, characterisation,
and applications of lipases. Biotechnol Adv 2001;19:62762.