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STUDY OF PROTEASES FROM BIOLOGICAL
SOURCES


S
ynopsis submitted in fulfilment of the requirements for the degree of


DOCTOR OF PHILOSOPHY


By


SMRITI GAUR






Department of B
iotechnology


JAYPEE INSTITUE OF INFORMATION TECHNOLOGY UNIVERSITY

A
-
10, SECTOR
-
62, NOIDA, INDIA



October 2009



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Biotechnology is gaining ground rapidly due to the various advantages that it offers over
conventional chemical processes especially regarding environment and cost involvement.
Industrial enzymes represent the heart
of biotechnology processes. According to a recent
report from Business Communications Company, (BCC 2008)

the global market for industrial
enzymes increased from $2.2 billion in 2006 to $2.3 billion by the end of
2007,

$2.7 billion
by 2012 and $4.9 billion

by

2013
[1]
. New and emerging applications have
helped drive
demand for enzymes

and the industry is responding with a continuous stream of innovative
products. The industrial enzyme market is divided into three appl
ication segments: technical
enzymes, food enzymes and animal feed enzymes. Tech
nical enzymes for detergent,
pulp and
paper manufacturing,
have a

largest segment with
approximately
52%
market
share
and

the
largest share of the enzyme market has been held by

alkaline proteases.


Proteases cover the 60% of total enzyme market and amongst the most valuable commercial
enzyme.

Alkaline proteases hold a great potential for application in the de
tergent and leather
industries

and there is an ever

increasing

trend to

develop environment

friendly
technologies
[2]
,
[3]
. Plants, animals and microbes are the main sources for protease production.

The
preferred sources of proteases are microbe
s

because of their rapid growth
and the ease with
which they can be genetically manipulated to generate new e
nzymes with altered properties
and are

currently being
utilized

by
the detergent

industry
eg.
Serine

proteases

produced by
Bacillus
strains
[4]
,
[5]
.

Proteases from several bacteria have been purified a
nd characterized
[6]
,
[7]
,
[8]
,
[9]
. Genus
Pseudomonas

a gram
-
negative bacterium that predominantl
y produces
alkaline proteolytic
enzymes

and the proteases
has

been purified
[10]
,
[11]
.
F
ungal alkaline
proteases

are advantageous

because of the ease of downstream processing to prepare a
microbe
-
free

enzyme at low cost production
.
[12]
,
[13]
.


The bulk of the plant proteases eg Papain,

Bromelain and
Ficin have major application in the
food industry where they are added at different stages of production.

T
hermostable serine
proteases named ‘wrightin’ from the l
atex of the plant
Wrightia tinctoria
, ‘Carnein’ from the
latex of the weed
Ipomoea carnea
spp. fistulosa (Morning glory)

and ‘Milin’ from the latex of

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3
-

Euphorbia milii
,
too
have found
applications
in food and other biotechnology
industries

[14]
,
[15]
,
[16]
.




During leaf senescence protein degradation is enhanced
,

e
ndoproteases
have been
isolated
from alfalfa, oat and barley senesced

leav
es which are involved in the process of protein
degrad
ation during foliar senescence

[17]
,
[18]
,
[19]
,
[20]
.



A 70
-
kDa serine protease was
identified
from artificially

senescing parsley leaves

t
his
protease activity is low in young leaves, was found to increase considerably in parallel to the
advance of senescence and the reduction in the protein
content of the
leaves [
21
]
.
Senescing
leaves show dominance of proteolytic enzymes belonging to four major classes, which are
common in mammals
, and

microbes. Senescence induced protein breakdown has been well

reported in many plant systems,

it results in a
vailabi
lity of transportable nitrogen

[22]
,
[23]
,
[24]

and degradation of protein
[25]
,
[26]
,
[27]
.

However
, till date
there are no reports
of utilization
of protease in industry from

this abundant senesced leaf waste.


The innovative aspect of the present work

was to

identify
and
isolate

alkaline
proteases from
various biological
sources such as
senesced
leaves of
regional plants that are currently not
used in agriculture
and from

soil microbes
with a purpose to have positive effect for

solid
waste management
.

T
he

purified enzyme would be
checked for

their

potential industrial
application.

The

first objective
of the present
work was to identify
new
sources of
alkaline proteases

eg:

i.

F
rom
senesceing

leaves
as it
show
s

dominance of proteolytic enzymes.

ii.

Soil sample
of the poultry waste site which is rich in organic waste was selected
for
Screen
ing of

microbial isolate that can
produce alkaline protease.

Secondly
,

check

for application of

isolated
enzymes
in industry

processes.



Since
there
are

no reports
available
on the use of plant proteases in detergent
industry w
e

were interes
ted in exploiting the use
of plant

proteases for
its
commercial
a
pplication

in
detergent industry.

W
e chose
senesced
leaves of
Lantana
camara

after initial screening

for
protease from vario
us plants
. Lantana camara is

most

commonly occurring weed

in the
world

and

the proteases extracted from this weed would be cost effective
.


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W
e
describe an
easy protocol of production and potential application of caseinolytic,
thermostable alkaline protease

by utilizing the senesced leaves of common
weed
L
. camara.

W
e also report
the purification of a novel extracellular
alkaline
protease which was produced
by bacterium,
Pseudomonas
therma
e
r
um

GW1
isolated from
soil.

Protease from senesced
leaves of the weed

Lantana camara
was purified

by

a
two
-
step procedure involving
ammonium sulfate precipitation and Sephadex G
-
250 gel permeati
on chromatography. The
Sephadex
G
-
250 fraction of senesced leaves of
Lantana camara
showed 28.31 fold with a
yield of 6.19%. The en
zyme was shown to have a low molecular weight of 43 kda by SDS
-
PAGE. It was strongly activated by metal ions such as Cu
2+
, Zn
2+
, Mg
2
,
Co
2+

and Mn
2+
. It
remained active at 60
°
C, pH 10.5 even after 1 h
our

of incubation when casein was used as
substrate. The
compatibility of the enzyme was studied with commercial and local
detergents, 60% activity of the enzyme was retained even after 1 h of

incubation at pH 10.0.
The easy availability of the senesced leaves of this common weed makes it a cheaper enzyme
source

and po
tential additive in detergents

[28]
,
[29]



Secondly an

extracellular protease

was purified and characterized from
Pseudomonas
therma
e
rum
GW1
a new strain identified
isolated f
rom soil of Poultry waste
site. Based on
biochemical characteristics,
nucleotides homology and phylogenetic analysis the
m
icrobe was

detected to
Pseudomonas thermaerum

and its

nearest

homolog was

found to be

Pseudomonas
aeruginosa
.

The strain produces extr
a
cellular protease in the culture media that was
maintained at 37
°
C and at 140 rpm.
The media was harvested for protease after 48 hrs of
incubation at 37
°
C in basal media supplemented with 1% casein. Enzyme

was purified by

ammonium sulphate precipitation a
nd DEAE
-
cellulose chromatography. The molecular
weight of the enzyme was estimated to be 43
,
000 daltons as shown by casein zymography
studies. The optimum pH for the proteolytic activity was pH 8.0 and enzyme remained stable
between pH 5
-
11 at 60
°
C.
Inter
estingly
Mn
2+

(5mM)
strongly activated enzyme activity by 5
fold, while
Cu
2+
, Mg
2+
and
Ca
2+

moderately activated enzyme activity,

where as Zn
2+
, Fe
2+
and Hg
2+
inhibited enzyme activity.


The protease produced was stable in presence of 50 %
(v/v) ethylacet
ate and acetone

and however showed 50% reduction on enzyme activity in the
presence of
glycerol.
Isopropanol, methanol and benzene increased protease activity by 2.7,

1.3 and 1.1 fold respectively



suggesting

its
potential
industrial
application

[30]
,
[31]
.




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-

Future studies regarding upgrading

the protease production technology from laboratory to a
large
-
scale process, allowing for a new green industrial process to be developed especially
where enzymatic tr
eatment of protein fibers, like hair
,

wool and silk is involved so that there
is significant reduction in the chemical use and cost. Development of new formulations for
industrial and domestic wool carpet cleaning, garment washing, dyeing processes for pr
otein
fibers by pre
-
tr
eatment with the proteases can

be performed.

The amino acid sequence determination of purified alkaline protease from senesced leaves of
Lantana camara

and
Pseudomonas thermaerum

would be performed and checked for
innovative applica
tion in other biotechnology industries.


























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REFERENCES


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Business Communications Company, Inc., (2008). In: Report
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E
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Y.
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S.
, Wang
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M.
, “
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stable protease producer

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Jellouli

K.
, Bougat
ef

A.
, Manni

L.
, Agrebi

R.
, Siala

R.
, Younes

I.,
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M.
,


Molecular and biochemical characterization of an extracellular serine
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protease
from Vibrio metschnikovii J1

,

J Ind Microbiol and Biotechnol, vol.14, no.7, pp. 939
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948, 2009.


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7
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[10]

Jellouli

K.
, Bayoudh

A.
, Manni

L.
, Agrebi
R.,
Nasri

M.
, “
Purification, biochemical
and molecular characterization of a metalloprotease from Pseudomonas aeruginosa
MN7 grown on shrimp wastes

,

Appl Microbiol Biotechnol
, vol. 79, pp. 989

99, 2008.

[11]

Rahman

R.N.Z.R.A.
, Geok

L.P.
,
Basri

M.,

A. B.,
Salleh, “
An organic solvent
-
stable
alkaline protease from Pseudomonas aeruginosa strain K: Enzyme purification and
characterization

,

Enzyme and Microbial Technology
,

vol. 39, pp. 1484

1491, 2006.

[12]

Tsomides

J.,
Goldberg

R.I.
, “
Controlled ev
aluation of oral chymotrypsin
-
trypsin
treatment of injuries to the head and face

,

Clin Med
,

vol. 76, no. 11, pp. 32
-

40,
1969.

[13]

Sharma

J.
, Singh

A.
, Kumar

R.
, Mittal

M.
, “
Partial purification of an alkaline
protease from a new strain of
Aspergillus oryzae
AWT 20 and its enhanced
stabilization in entrapped Ca
-
Alginate beads

,

The Internet Journal of M
icrobiology
,
vol. 2, no. 2, 2006.

[14]

Tomar

R.
, Kumar

R.,
Jagannadham

M.V.
, “
A Stable SerineProtease, Wrightin, from
the Latex of the Plant Wrightia tinctoria(Roxb
.) R. Br.: Purification and Biochemical
Properties
,”
J. Agric. Food Chem
, vol. 56, no. 4, pp. 1479
-
1487, 2008.

[15]

Patel

A.K.
, Singh

V.K.,

Jagannadham

M.V.
, “
Carnein, a Serine Protease from
Noxious Plant Weed
Ipomoea carnea
(Morning Glory)

,

J. Agric. Food Ch
em
,
vol. 55,
no. 14, pp. 5809
-
5818, 2007.

[16]

Yadav

S.C.
, Pande

M.
, Jagannadham M
.
V
.
, “
Highly stable glycosylated serine
protease from the medicinal plant Euphorbia milii
,”
Phytochemistry
,

Vol. 67, no. 14,
pp. 1414
-
1426, 2006.

[17]

Nieri

B.
, Canino

S.
, Versace

R.
,

Alpi

A.
, “
Purification and characterization of an
endoprotease from alfalfa senescent leaves
,”
Phytochemistry
, vol. 49, no. 3, pp. 643
-
649, 1998.

[18]

Miller

B.L.,

Huffaker

R.C.
, “
Partial Purification and Charac
-
terization of
Endoproteinases from Senescing Ba
rley Leaves
,”
Plant Physiol
, vol. 68, pp. 930
-
936,
1981.

[19]

Drivdahl

R.H.,
Thimann

K.V.,

Proteases of senescing oat leaves I. purification and
general properties
,”
Plant Physiol
, vol. 59, pp. 1059
-
1063, 1977.

[20]

Drivdahl R.H., Thimann K.V.,

“Proteases of senesc
ing oat leaves II. reaction to
substrates and inhibitors
,”
Plant Physiol
, vol. 61, pp. 501
-
505, 1978.


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-

[21]

Jiang W.B., Lers A., Lomaniec E., Aharoni N., “
Senescence
-
related serine protease
in parsley
”,
Phytochemistry,
vol. 50, no.3, pp.

377
-
382,

Feb.1999.


[22]

Sma
rt

C.M.
, Hosken

S.E.
, Thomas

H.
, Greaves

J.A.
, Blair

B.G.,

Schuch

W.
, “
The
timing of maize leaf senescence and characterization ofsenescence
-
related cDNAs
,”
Plant Physiol
., vol. 93, pp. 673
-
682, 1995.

[23]

Buchanan
-
Wollaston

V.,
Ainsworth

C.
, “
Leaf senescence

in Brassica napus: cloning
of senescence related genes by subtractive hybridization
,”
Plant Mol. Biol
., vol. 33,
pp. 821
-
834, 1997.

[24]

Griffith

C.M.
, Hosken

S.E.
, Oliver

D.
, Chojecki

J.,

Thomas

H.
, “S
equencing,
expression pattern and RFLP mapping of a senesc
ence J
-
enhanced cDNA from
Zea
mays
with high homology to
Oryzain gamma
and
aleurain,

Plant Mol. Biol
., vol. 34,
pp. 815
-
821, 1997.

[25]

Dungey

N.O., Davies D.D.
, “
Protein turnover in isolated barley leaf segments and
the effect of stress
,” J
. Exp. Bot
, vol.
33, pp. 12
-
20, 1982.

[26]

Yoshida

T.,

Minamikawa

T.
, “
Successive amino
-
terminal proteolysis of the large
subunit of ribulose 1,5
-
bisphosphate carboxylase/ oxygenase by vacuolar enzymes
from French bean leaves
,”
Eur. J. Biochem
, vol. 238, pp. 317
-
324, 1996.

[27]

Ho
rtensteiner

S.,

Feller

U.
, “
Nitrogen metabolism and remobilization during
senescence
,”
J. Exp. Bot
., vol. 53, pp. 927
-
937, 2002.


[28]

Gaur S.,

Wadhwa N.,


Alkaline protease from senesced leaves of invasive weed
Lantana camara”
,
African Journal of Biotechnology
, vol. 7, no. 24, pp.4602

4608,
Dec.2008.

[29]

Gaur S.,

Wadhwa N.,


Thermostability and antimicrobial properties of protease
from senesced leaves of
Lantana camara
,” International Conference on Plant
Genomics and Biotechnology: Challenges and Opportunities, IGA
U Raipur, pp282
-
283, Oct. 2005.

[30]

Gaur S.,

Gupta S.,

Wadhwa N.,


Purification of protease from
Pseudomonas

thermaerum

GW1

isolated from poultry waste site

,

The Open Microbiology Journal,

2009 submitted for publication.

[31]

Gaur S.,

Gupta S.,

Wadhwa N.,


Isolati
on of Protease and Keratinase From
Microbes Isolated From

Ghazipur Poultry Waste Site, Ghaziabad, India
”, SIM Annual

-

9
-

Meeting and Exhibition Industrial Microbiology and Biotechnology
,

Toronto, Canada.
26

30, July, 2009.



AUTHOR’S

PUBLICATIONS


[1]

Gaur

S.
,

Gup
ta

S.,

Wadhwa

N.
,


Purification of protease from
Pseudomonas

thermaerum

GW1

isolated from poultry waste site

,

The Open Microbiology Journal,

2009 submitted for publication.

[2]

Gaur S.,

Wadhwa N.,


Alkaline protease from senesced leaves of invasive weed
Lanta
na camara”
,
African Journal of Biotechnology, vol. 7, no. 24, pp.4602

4608,
Dec.2008.

[3]

Gaur S.,

Gupta S.,

Wadhwa N.,


Isolation of Protease and Keratinase From Microbes
Isolated From

Ghazipur Poultry Waste Site, Ghaziabad, Indi
a

,

SIM Annual Meeting
and Exh
ibition Industrial Microbiology and Biotechnology
,

Toronto, Canada. 26

30,

July,

2009.

[4]

Gupta S.,

Gaur S.,

Wadhwa N.,


P
roduction of extracellularly secreted keratinase and
protease from bacteria of poultry waste site
”,

I
nternational Conference on Emerging
trends in Environmental Research, St. Al
bert’s College Kochi, Kerala"

14
-
16 Aug
.

2009
.

[5]

Gaur S.,

Wadhwa N.,


Thermostability and antimicrobial properties of protease from
senesced leaves of
Lantana camara
,”

International Conference on Plant Genomics and
Bio
technology: Challenges and Opportunities, IGAU Raipur,
pp282
-
283,
Oct.
2005.

[6]

Gaur

S.
,

Sabharwal

T.
, Gupta

P.,
Wadhwa

N.,


Increased activity of cysteine protease
in senesced leaves of Carica papaya and studies using bioinformatics tools


Cognizance,

IIT,

Roorkee, March 2004.










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