abstract - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/38597/5/05_abstract.pdf · silk...
TRANSCRIPT
Abstract
1
ABSTRACT:
Protease has immense industrial application.
This study led to the exploration of the
marine coastal ecosystem (support ~3.67x
1030
microorganisms) to isolate protease
producing bacteria. Seven extracellular
protease producing pure bacterial isolates
were obtained from two sites of the marine
coast of West Bengal (Digha and
Mandarmani) and one site of Andhra
Pradesh (Vijag). They were characterized
morphologically, physiologically and at the
biochemical level. Molecular
characterization was based on 16S rDNA
sequence analysis. All isolates were
observed to be Gram positive bacilli. It was
further confirmed by Real time PCR
analysis. Presence of endospore in all
validated their survivability under stressful
condition. Except one of the bacterium, all
of them showed presence of catalase and
oxidase. The isolates were considered to be
non-pathogeneic, due to lack of lecithinase.
Beside the protease, two of the strains (SD2
& SD4) were also found to produce
extracellular lipase. Among all the strains,
three of them (SM1, SM2 & SV1) also
exhibited amylase activity.
Optimum pH and temperature range were
pH 6 -12 and 20°C-40°C respectively,
reflecting their range of adaptability.
Jaggery and tamarind were better carbon
source for their growth. All the strains
showed good biofilm forming ability when
checked in 24 well cell culture plate.
Isolates could tolerate higher concentration
of heavy metal salts namely Al, Fe, Ni, Pb,
leading to accumulation as indicated by
EDXRF (Energy Dispersive X-ray
Fluorescense Analysis). In order to find out
the exact location of the metals within the
cell, Transmission Electron Microscopy of
metal containing cell was done. The change
of cell surface morphology in response to
metal stress was determined by scanning
electron microscopy (SEM). The SEM
micrographs revealed distinct changes like
shortening and thickening in cell structure
and appearance of woolly coat around the
surface in metal treated cells as compared to
normal cells. Combination of all the isolates
under immobilized condition was efficient
in removing lead from sterile water, as well
as the naturally contaminated water bodies
like Bheri supplemented with 2 mM lead
nitrate solution, which would help them to
use as a bioremedial package.
Extracellular protease activity of isolates as
checked by azocasein assay was as follows:
SD1 (10.23+0.381 U), SM2 (3.425+0.106
Abstract
2
U), SD2 (3.275+0.318 U), SD4 (2.8+0.283
U), SD5 (2.65+0.24 U), SD3 (2.4+0.141 U),
SV1 (2.325+0.247 U), SM1 (1.83+0.24 U).
All the protease producing strains had
gelatinase activity, and they could be used
for silver recovery from used X-ray films
(except for SD1). The gelatinase activity
was reconfirmed by azocoll reaction.
Gelatinase activity of extracellular
supernatant from the seven isolates was
between 0.0855 to 0.1781 U, SM2 showed
maximum gelatinase activity, whereas SV1
had lowest gelatinase activity among all the
isolates. The isolates showed more or less
same efficiency in degumming process
during silk extraction from cocoon.
Extracellular protease from SM1 (closest to
Bacillus thuringensis) was able to degum
silk fabric within 4 hrs at RT with enzyme
dosage 0.8 unit/cm2 and the maximum
degumming loss of raw silk fabric was
21.72%. Post enzymatic degumming, a
shiny texture was observed under
Environmental scanning electron
microscope (ESEM) and the yarn volume
also increased. The quality of silk fabric was
either improved or remained unchanged.
Combined effect of mixing supernatants
from two isolates, SM2 (Bacillus cereus as
protease source) and SD2 (closest to
Bacillus pumilus, as protease and lipase
source) had shown the capability of
enhancing the cleaning efficiency of
detergent. The enhanced efficiency was
backed up by the market survey data.
When Bacillus cereus SM2 was grown in
presence of metal (PbNO3) salts, 36128.85
ppb of lead was found to accumulate, as
evident from EDXRF (Energy Dispersive X-
ray Fluorescense Analysis) data. It showed
equal efficiency of metal removal from solid
metal (gold and silver) strip at zero valent
state. SM2 also showed the efficient
reduction of two major parameter of dairy
effluent i.e., the high nitrate and protein
content, along with phosphate, chloride,
carbohydrate and calcium carbonate within
24 hrs interval at a pH of 3.92 – 4.2 of the
dairy effluent (without any added nutrient or
adjustment of pH).
Wide application of SM2 in various fields
prompted the purification of the enzyme. A
metalloprotease of 75.10 kDa from SM2
was purified through Phenyl sepharose CL-
4B column using 50-0% ammonium
sulphate gradient, followed by dialysis
against 20 mM sodium phosphate buffer.
Enzyme activity was stable between wide
range of temperature (4 to 40oC) and pH 6 to
9.5 with optimum performance at 37oC and
Abstract
3
pH 7.5. Purified protein of SM2 was
inhibited by AgNO3 (61.79%) and
CuSO4.5H2O (50.02%), whereas the activity
was increased in presence of CoCl2.6H2O,
Cr2O3, NiCl2.6H2O and Pb(NO3)2. Purified
protein quenched in presence of CuSO4
solution, as well as phosphoramidon. CD
analysis revealed the unfolding of purified
protein in presence of CuSO4 solution, as
well as phosphoramidon, which might be
due to strong interaction between purified
protein and phosphoramidon.
As SM2 was isolated from the coastal region
of Mandarmani, the exposure to radiation
was examined in laboratory scale. DNA
damage was observed in control cell, as well
as metal treated cells. The damage in
presence of metal was higher, a
phenomenon known as radiosensitization.
Homologous recombination was the
underlying repair mechanism as it got
inhibited in presence a polymerase inhibitor
(Arabinose CTP). But this damage did not
affect the protease activity of the isolates, as
the damage did not immediately affect the
enzyme production.
The dissertation was an attempt to develop a
bioremedial package for environmental
decontamination of heavy metals along with
various other applications of extracellular
protease and their survivability assay upon
exposure to 60
Co-γ rays.
Prelude to work
4
PRELUDE TO WORK
Microbial life is widely distributed
(ubiquitous) in earth‘s ecosystem, the
presence of life anywhere indicates presence
of microbial life – microorganisms are found
in almost all niches. Microorganism--- the
term represents a diverse and extended
group of organism, which includes; bacteria,
viruses, protists and fungi which are widely
different in their characteristics. The
prokaryotic domain bacteria represent a
group which is characterized by their genetic
material rRNA and other biochemical
properties like characteristic enzymes and
membrane structure determinant
phospholipid profile (Woese, 1987; Das et
al., 2006).
Estimation of evolutionary history and
taxonomic assignment of individual
organisms are based on rRNA genes (Eigen
et al., 1985; Kuntzel et al., 1981; Woese,
1987 & 1998; Woese et al., 1990) which are
known to be highly conserved (Doolittle,
1999; Woese, 1987). The ribosome consists
of greater than 50 proteins and three classes
of RNA molecules including 16S rRNA
genes which are essential for assembly of
functional ribosomes, and hence prevents
any drastic change to the structure (Clayton
et al., 1995; Doolittle, 1999). In bacteria, the
three rRNA genes are organized into a gene
cluster which is expressed as single operon,
and is present in multiple copies in the
genome which over the time gets
homogenized through homologous
recombination (Hashimoto et al., 2003) .
Most environmental surveys including the
recently initiated Human Microbiome
Project (HMP) (Peterson et al., 2009) use
cultivation-independent techniques to
examine microbiomes that contain mixed
species.
Bacteria have been isolated and cultivated
from all possible region of the earth, on the
basis of their habitat, diversity, ecological
function, degree of pathogenicity and
biotechnological application. In the
beginning, bacteria were isolated from
common ecological niches, and variations
were sought in other extreme environments,
like acidic ponds, hot spring, saturated brine
and glaciers (Madigan and Marrs, 1997),
Bacteria has been found in earth‘s crust
(Kerr, 1997) and polar ice (Rothschild and
Mancinelli, 2001). Current investigations
predict there is great possibility in
extraterrestrial locations as well. But
principally marine ecosystem that covers
70% of the earth surface is now taken to be
the largest inhabitable space for the
Prelude to work
5
prokaryotes. Marine environments are also
microbial hotspots that support bacterial
abundance and activities. Marine microbes
are well distributed from surface water to
deep sea, they are also found in coastal
region shallow depth and coral reef.
Research on marine microbial diversity is
going on for couple of decades and recently
it has been reported that unknown groups
such as SAR11 (Brown et al., 2012) and
picoautotrophs such as Prochlorococcus are
significant contributors in marine microbial
diversity. With ~3.67x 1028
microorganisms
in marine environments, including
subsurface one exepects it to harbour a great
variety of biodiversity (Whitman et al.,
1998). Besides bacteria, archaea also play
significant role in microbial diversity of
marine environment (Table 1) (Das et al.,
2006).
Group Physiology Role in Marine environment Example
Archaebacteria
Sulphate-
reducing
bacteria
Chemoautotrophs, anaerobic,
thermophilic and mesophilic.
Contribute over 50% of the
carbon turnover of coastal
marine sediments; take part in
the cycling of sulphur
compounds in sea water.
Desulfomonas,
Desulfovibrio,
Desulfobulbus,
Desulfotomaculum
and Desulfococcus
Methanogenic
bacteria
Chemoautotrophs, strictest
anaerobes, utilize a limited
number of simple carbon
compounds (hydrogen,
carbon dioxide, formate,
acetate and methanol) as
their carbon and energy
sources for methanogenesis.
Utilize trimethylamine in the
marine environment as substrate
and produce methane as an end-
product of their energy-
generating metabolism.
Methanococcus,
Methanosarcina,
Methanomicrobium,
Methanogenium,
Methanoplanus,
Methanococcoides
and
Methanobolus
Halophilic
bacteria
Require at least 12–15%
NaCl to survive and grow
well even at concentrations
up to saturation.
Red colonies formed due to high
carotenoid content and dominate
in high salt environments, such
as salterns and salt lakes;
regulate the osmotic pressure
there by resisting the denaturing
effects of salt in their
environment.
Haloarcula,
Halobacterium,
Haloferax and
Halococcus
Prelude to work
6
Eubacteria
Luminous
bacteria
Produce light by a simple
proteinlike substance called
luciferin in contact with the
oxygen molecule; Gram-
negative and motile
heterotrophic rods.
Bioluminescence in the deep
ocean helps the organisms
defensively to startle and divert
predators (defence), to attract
prey (offence) and to
camouflage. Luminous bacteria
help in cycling of nutrients in
the sea and contribute in the
nutrition of marine organisms as
gut microflora.
Photobacterium
leiognathi,
Photobacterium
phosphoreum,
Vibrio fischeri and
Vibrio harveyi
Nitrifying
bacteria
Oxidize either ammonia to
nitrite (Nitrosococcus) or
nitrite to nitrate
(Nitrococcus) and convert
nitrogen to a form readily
available for other biological
processes.
Extremely important process,
since positively charged
ammonium ions bind to acidic
sediment particles, where they
become available for biological
processes; more abundant in
nearshore waters than in
offshore regions.
Nitrosococcus,
Nitrococcus, etc.
Table 1. Representing the different physiological groups of marine bacteria
Marine environment differs from other
water bodies in terms of their high pressure,
salinity, low temperature and absence of
light etc. Along the depth of oceans as well
as different temperature zones, there exists a
diverse microbial population in the marine
environment. They can belong to aerobic,
anoxygenic, and phototrophic groups and
are quite capable of using light and organic
matter simultaneously. To survive in this
environment heterotrophic bacteria have
adapted themselves in such a way that they
can maintain Na+ concentration of the cell to
overcome the osmotic shock caused due to
salt water. (Das et al., 2006). Marine
microbes also exhibit oligotrophicity, which
is adaptability due to limitation in the
amount of nutrient.
Marine organisms represent a promising
source for natural products of the future due
to the incredible diversity of chemical
compounds that have been isolated.
Burkholder and his co-workers isolated the
first marine metabolite from the bacterium
Pseudomonas bromoutilis, which is the
highly brominated pyrrole antibiotic
pentabromopseudiline which was active
against Gram positive bacteria (Burkholder
et al., 1966).
Prelude to work
7
Fig. 1. Diagrammatic representation of marine ecosystem and bio geo-chemical recycling of different organic and
inorganic materials. Where: DOM - Dissolved organic matter, DMS- Dimethylsulphide, POM - Particulate organic
matter. Taken from articles Microbial structuring of marine ecosystems by Farooq and Malfatti, 2007
Secretion of secondary metabolite by
marine bacteria:
Researchers are trying to find out new drugs
from marine bacteria for more than two
decades (Anand et al., 2006). In 1996 first
antibiotics were isolated from marine
bacteria (Burkholder et al., 1966), biofilm
forming bacteria were well known to
produce more amount of antibiotic than
other marine bacteria (Anand et al., 2006).
A number of surface associated marine
bacteria have also been found to produce
antibiotics (Holmstrom and Kjelleberg,
1999; Hans-Peter et al., 2004). An antibiotic
from marine bacterium Alteromonas rava
was found to produce thiomarinol
(Shiozawa et al., 1993). Other antibiotics
which were reported to have been isolated
from different bacteria are; loloatins from
Bacillus. Agrochelin and sesbanimides from
Agrobacterium (Acebal et al., 1999),
pelagiomicins from Pelagiobacter variabilis
(Imamura, 1997), pyrones from
Pseudomonas (Singh et al., 2003).
Marine microorganism as a source of
enzymes:
Marine microorganisms represent a novel
source for various enzymes. As compared to
the terrestrial counterparts, marine micro-
organisms possess specific physiological
characteristics, metabolic patterns and
Prelude to work
8
nutrient utilization; for their different
habitats (Sana et al., 2006). Therefore the
enzymes recovered from marine
microorganisms are expected to possess
unique properties (Jackson and Young,
2001). Some of the marine microorganisms
have enzymes which hydrolyzes the
polysaccharide; lignin, alginate, agar,
cellulose, carrageenan and xylan
(Andykovich and Marx, 1998; Marrs et al.,
1999). They are used in the
biodegradation,e.g: nylon 6 and nylon 66 are
hydrolyzed by marine micro-organisms;
Bacillus cereus, Bacillus sphericus, Vibrio
furnisii, Brevundimonas vesicularis
(Sudhakar et al., 2007). Two γ-
proteobacteria: Alcanivorax and
Cycloclasticus play an important role in
petroleum hydrocarbon degradation; they
can be used as candidate for bioremediation
of oil spillage (Harayama et al., 2004).
Lipases are ubiquitous enzymes found in
animals, plants, and microorganisms,
including fungi and bacteria. Lipases find
use in food industry, organic chemistry
(Gunstone, 1999; Pandey et al.,1999; Reetz,
2002), laundry industry (Cordon et al.,
1958), paper industry (Pandey et al., 1999;
Guiterrez et al., 2001).
Among all the enzymes, protease plays an
important role in the enzymatic world; it is a
single class of enzymes which occupy a
pivotal position with respect to its
application in both physiological and
commercial fields. In various industrial
sectors extracellular proteases have multiple
applications (Gupta et al., 2002), it accounts
for 60% of total worldwide sale of enzymes
(Rao et al., 1998), among which 40% are
originated from microbes (Godfrey et al.,
1996).
Objective of work
9
OBJECTIVE OF WORK
The marine bacteria were isolated from
different coastal areas of the Indian
peninsula. Out of eight, seven were isolated
from the coastal region of West Bangal from
two different regions namely Digha and
Mandarmani. Remaining one was isolated
from the Rishikonda beach near
Vishakhapatnam in Andhra Pradesh.
Digha is West Bengal's most popular sea
beach. It was originally known as Beerkul, it
is mentioned as the "Brighton of the East" in
one of Warren Hasting's letters (1780 AD)
to his wife. It is situated in East Midnapore,
185 km south-west from Kolkata/Howrah.
Its geographical coordinates are 21.68°
North, 87.55° East. Isolates SD1, SD2, SD3
and SD4 were isolated from Digha.
Mandarmani is another popular tourist
destination (13 km) in East Midnapore district
of South Bengal, one of the southern districts
of West Bengal. It is almost 180 km from
Kolkata Airport on the Kolkata- Digha route.
Its geographical coordinates are 21°39‘58‖
North and 87°42‘18‖ East. SM1 and SM2
were isolated from Mandarmani.
Rishikonda Beach is a beach 8 km from
Vishakhapatnam in Andhra Pradesh. Isolate
SV1 was isolated from the Rishikonda
beach.
The marine bacteria were isolated with an
aim to
Discover extracellular enzyme
producing bacteria which can have
industrial application.
Purification and characterization of
the enzyme from one of the isolates.
Discover the heavy metal
bioremediation potential of these
bacteria.
Testing the effect of metal as a
radiosensitizer during ionizing
radiation induced DNA damage.
Introduction
10
INTRODUCTION:
Coastal ecosystem:
The area where sea/ocean touches the
terrestrial land is known as coast. The
―Coastal Zone‖ describes the geographical
region where interaction of the sea and land
processes occurs. Sediment nature of the
coastline is often determined by the river
and tidal nature; river deposits minerals and
soils at the junction of sea/ocean, which are
carried out along with journey. Tide breaks
on the shore with high energy and moves the
sediment. The coastal area covers the
extensive areas of estuaries, brackish water
lagoons, mangroves, coral reefs and
seaweed beds, which are rich in specific
diversities, have different ecological,
economical and social significance. Human
activity has changed the coastal ecosystem
over time, pollution caused by
anthropogenic activities decrease the normal
flora and fauna of coastal region; the coral
reefs are now in threat. (McQuatters-Gollop,
2012)
Coastal region of Bay of Bengal :
The Bay of Bengal covers the eastern coast
of India, extending from international border
of India-Bangladesh in northeastern side to
Kannyakumari in south. It is 2545 km long,
and covers West Bengal, Orissa, Andha
Pradesh and Tamilnadu.
West Bengal Orissa Andhapradesh Tamilnadu
Kak
Dwip
Coast
Contai-
Digha
Coast
Salinity (ppt) 15-27 20-30 18-35 18-33 31
Temperature° C 25-35 22-37 10-43 20-30 27-30
Relative
humidity (%)
80-92 Upto 70 61-81 60-75 -----
Total rain fall
(mm/year)
1722 2000 995-1914 1000-1500 900
Wind velocity
(Km/hr)
---- 3.0-16.6 7.7-17.7 (70-120
in stormy
weather)
----- 5-10 (100-200 in
stormy weather)
Table 2. Physical parameters of the coastal states of the Bay of Bengal.
Introduction
11
The average salinity of Bay of Bengal is
between 30 to 34 ppt (parts per thousand),
the low salinity is mainly due to the dilution
by river water. Table 2 represents the
physical parameters of Bay of Bengal of
different states
(ftp://ftp.fao.org/docrep/fao/007/ad894e/AD
894E06.pdf). Due to discharge of the river
water into marine environment the microbial
profile in coastal area are sometimes similar
to fresh water bodies. Eight protease
producing microbes have been isolated from
the soil sample of east coast of Andhra
Pradesh (Singh et al, 2012). Different
bacterial species such as Vibrio,
Pseudomonas, Streptococcus, Esherichia,
Shigella, Salmonella, Proteus and Klebsiella
have been isolated from the coastal
environment of Little Andaman Island
(Swarnakumar et al., 2008).
The coastal areas are getting increasingly
polluted by domestic, commercial,
agricultural and industrial pollutants. The
metal contamination of sea water is mainly
due to discharge of the chemical load from
various industries into the rivers, and from
the rivers to the sea. Some of the metals like
cadmium, arsenic, lead and mercury are
toxic in nature. According to literature,
heavy metals like zinc, copper, nickel,
chromium, mercury, cadmium, cobalt, lead
and arsenic have been found in the coastal
regions of the Bay of Bengal (Das et al.,
2012). Marine mercury resistant bacterium
has been isolated and used in bioremedial
purpose for detoxification (De et al., 2004).
53 different bacterial organisms were found
to be resistant against 350 ppm of mercury
(11.53%), 250 ppm of cadmium (3.77%),
700 ppm of chromate (50.94%) and 250
ppm zinc (13.20%) from Krishna-Godavari
basin of Bay of Bengal (Gunaseelan and
Ruban, 2011). Chromium (VI and III)
resistant Streptomyces spp. VITSVK5 has
been isolated from Marakkanam, which is
also resistant to arsenic. However it was
sensitive to lead and nickel nitrate (Kumar
and Kannabiran, 2009). Pathogenic bacteria
such as Vibrio, Pseudomonas, Coliforms,
Salmonella and Shigella were isolated from
the Chennai coastal area and it was found
that isolates were resistant to heavy metals
at the concentration of 50 mM of Ni, Cr, Cu,
Co, Pb and Hg (Santhiya et al., 2011).The
resistance could have been due to the
selective pressure exerted on the organisms
by pollution of the marine atmosphere by
heavy metals. These all isolates from
different sites of eastern coastal region of
India could be used as a potent individual
Introduction
12
candidate for bioremediation of heavy metal
or they could be used as consortium,
cleaning up the environment off heavy metal
pollutants.
Proteases are enzymes of class 3,
hydrolases, and subclass 3.4, the peptide
hydrolases or peptiodases. In aqueous
environment it hydrolyzes the peptide bond,
where as in non-aqueous environment it
synthesizes the peptide bond (Sana et al.,
2006). Proteases are found in a wide variety
of sources such as plants, animals and
microorganisms. But microbial proteases are
preferred over other sources as they are fast
growing and can easily meet the current
world demand. Also, microorganisms have
broad biochemical diversity and are easy to
genetically manipulate.
They are subdivided into two major groups,
i.e., exopeptidases and endopeptidases,
depending on their site of action. The
exopeptidase acting on carboxy terminal is
called carboxypeptidase and that which
acts on amino terminal is called
aminopeptidase. Trypsin, chymotrypsin,
pepsin, papain and elastase are the examples
of endopeptidase. Based on the functional
group present at the active site; proteases are
further classified into four prominent
groups, i.e.,
a. Serine proteases
b. Aspartic proteases
c. Cysteine proteases
d. Metalloproteases
e. Threonine proteases
f. Glutamic acid proteases.
Characteristic features of the four types of proteases
Properties
EC
No.
Mo
lar
mass
ran
ge/
kD
a
pH
op
tim
um
Tem
per
atu
re
op
tim
um
/ºC
Met
al
ion
req
uir
emen
t(s)
Act
ive
site
am
ino
aci
d(s
) Major
inhibitor(s)
Major source(s)
Aspartic
or
carboxyl
proteases
3.4.23 30–45 3–5 40–55 Ca2+
Aspartate
or
cysteine
Pepstatin Aspergillus, Mucor,
Endothia, Rhizopus,
Penicillium,
Neurospora,
animal tissue
(stomach)
Cysteine
or thiol
3.4.22 34–35 2–3 40–55 - Aspartate
or
Indoacetamide, Aspergillus, stem of
pineapple (Ananas
Introduction
13
proteases cysteine p-CMB comorus), latex of fig
tree (Ficus sp.),
papaya
(Carica papaya),
Streptococcus,
Clostridium
Metallo
proteases
3.4.24 19–37 5–7 65-85 Zn2+,
Ca2+
Phenylal
anine
or
leucine
Chelating agents
such as EDTA,
EGTA
Bacillus, Aspergillus,
Penicillium,
Pseudomonas,
Streptomyces
Serine
proteases
3.4.21 18–35 6–11 50-70 Ca2+
Serine,
histidine
and
aspartate
PMSF, DIFP,
EDTA, soybean
trypsin inhibitor,
phosphate buffers,
indole, phenol,
triamino acetic acid
Bacillus, Aspergillus,
animal tissue (gut),
Tritirachium album
(thermostable)
Table 3. Representing the different features of four major proteases and their isolation sources, as well as common
inhibitors of these proteases (Sumantha et al., 2006).
Chymotrypsin, subtilisin are few examples
of serine proteases. Pepsin, rennin are
examples of aspartic proteases. Cystine
proteases are also known as thiol protease,
mainly found in fruit like papaya, pine apple
and kiwifruit, the amount being higher in
unripe condition. Papein, actinidain are
examples of cystein protease, these
proteases are used in meat tenderization.
Metalloproteases are characterized by the
requirement for a divalent metal ion for their
activity; they are subdivided into two
groups: metalloexoprotease and
metalloendoprotease. Most of the
metalloproteases are zinc-dependent but
some use cobalt. Coordination between
metal and protein requires histidine,
glutamate, aspartate, lysine and arginine.
Thermolysin, collagenase, elastase are
examples of metalloprotease. Threonine and
glutamic acid proteases act on threonine and
glutamic acid respectively present at the
active site of protein.
Depending on the pH at which they are
active, proteases are also classified into acid,
alkaline, and neutral proteases. Acid
proteases are mainly rennin like proteases
secreted by fungi; they act within the pH
range of 2 – 4. They are used in medicine, in
digestion of soy protein and to break down
wheat gluten in the baking industry. Neutral
proteases are secreted by both fungi and
Introduction
14
bacteria; pH range of their activity is narrow
and they become unstable with increasing
temperature. Neutral proteases require
different ions such as Ca++
, Na+ and Cl
- for
stable activity. They are used in leather and
food industry for the production of crackers,
bread and idli. Alkaline proteases are those,
in which optimum pH is greater than 9 and
working range is between 9-12. They are
stable at higher temperature around 60ºC,
also have broad substrate specificity, which
make them suitable in detergent industry.
They are also stable in association with
chelating agents. B.licheniformis and
B.coagulans have been found to produce
alkaline protease (Kumar and Takagi, 1999).
Extracellular alkaline protease finds
numerous applications in industrial
processes like in detergents, leather tanning,
dairy, meat tenderization, baking, brewery,
photographic industry etc. (Moses and Cape,
1991). Most commercial proteases are
neutral or alkaline by nature, and mainly
produced by the genus Bacillus. Neutral
proteases generate less bitterness in
hydrolyzed food proteins than the animal
proteases and hence are valuable for use in
the food industry. Table representing the
application of different type of protease in
industry is presented below (Table 4).
Industry Application Enzyme
Baking and milling Bread baking Neutral Protease
Beer Chill proofing Neutral Protease
Cereals Condiments Neutral Protease
Dairy Milk prevention of oxidation
flavor, Milk protein
hydrolysates,
Acid Protease
Dry cleaning, Laundry Spot removal Alkaline Protease, Lipase
Leather Bating, Dehairing Neutral Protease
Meat, Fish Meat tenderizing, Condensed Several Protease
Introduction
15
fish soluble
Pharmaceutical and
clinical
Digestive aids Several Protease, Lipase
Table 4. Application of different proteases in various industry.
Microbial Protease:
The improvement of industrial processes
with microbial enzymes is one of the most
important fields of research because
enzyme-catalyzed reactions are highly
efficient and selective, are less polluting,
and usually require mild conditions and less
energy, which leads to the lowering of costs
(Cherry and Fidanstef, 2003). Thus, there is
an increasing interest for isolating new
enzymes and new enzyme-producing strains
for their use in industrial conversions
(Cherry and Fidanstef, 2003). Among these
enzymes, lipases, esterases, cellulases,
xylanases, pectinases, amylases and
proteases are some of the most important.
Protease from microbial resources are used
in food, pharmaceutical, detergent and
leather industry, they are also used for basic
research purpose (Tunga et al., 2003;
Manachini and Fortina, 1998). In detergent
industry protease acts as an additive to
increase the wash performance. Protease
recently isolated from various environment
includes those from a new gamma-
Proteobacterium isolated from the marine
environment of the Sundarbans (Sana et al.,
2006), a thermostable alkaline protease from
Bacillus subtilis PE-11 (Adinarayana et al.,
2003) and extracellular alkaline protease
from Teredinobacter turnirae‘s (Nogueira et
al, 2006), extracellular protease of
Microbacterium luteolum isolated from East
Calcutta Wetland (Malathu et al., 2008) as
detergent additive. Protease is also used in
leather industry in soaking, dehairing and
bating (Cordon et al., 1958; Underkofler et
al., 1958 Rao et al., 1998). It also helps to
overcome the pollution occurred in
conventional method. In food industry
protease helps to defatting the meat and fish
(Esakkiraj et al., 2009).
Commercial application of protease:
Dr. Jokichi Takamine (1894) introduced the
possibility of cultivation of enzymes and to
introduce them in industry. He mainly tried
with fungal enzymes, whereas Boidin and
Introduction
16
Effront (1917) in France extracted the
bacterial enzymes about 20 years later.
Recent developments in industrial
biotechnology have resulted in the
exploitation of new and undiscovered
microorganisms and the devising of
improved methods for enzyme production,
which have led to increased yields of the
enzyme, thus making a viable industrial
process feasible.
The different industrial applications of
microbial proteases are:
Detergent additive:
Proteases are used in commercial industry as
additive to remove the stain of the cloth; it
breaks down the proteinaceous material. The
acceptability of this enzyme in industry is
due to their lower wash temperature, shorter
agitation after soaking and their non-
phosphate nature. Proteases are usually used
in formulation having high activity and
stability in broader range of temperature and
pH. They need to fulfill some criteria to
become a good additive to detergent:
effective at low levels (0.4 – 0.8%), should
also be compatible with various detergent
components along with oxidizing and
sequestering agents and have a long shelf
life (Ward, 1985).
A thermostable extracellular serine alkaline
protease from Vibrio fluvialis having
molecular weight of 33.5 kDa was reported
to be used successfully as additive to
laundry detergent. Co2+
, Ca2+
and Fe3+
were
found to enhance the activity of the enzyme
(Venugopal and Saramma, 2006). An
alkaline protease from Bacillus clausii was
found to be highly compatible and stable
with the commercial detergents (Joomand
Chang, 2006). An alkaline subtilisin like
protease from Bacillus clausii KSM-K16,
was successfully used in laundry detergents
(Saeki et al., 2007). In 2009, two alkaline-
serine proteases BM1 and BM2 were
isolated from Bacillus mojavensis A21; they
were used in detergent industry. Both of
them showed stability in presence of non-
ionic detergent, and also showed
compatibility with a wide range of
commercial liquid and solid detergents
(Haddar et al., 2009). TC4, a detergent-
stable alkaline protease isolated from B.
Alcalophilus TCCC11004 was purified and
characterized for detergent formulation
(Cheng et al., 2010). Extracellular alkaline
protease from Bacillus licheniformis
KBDL4 of Lonar Lake was found to be
compatible with various detergents (Pathak
and Deshmukh, 2012). In combination with
Introduction
17
protease, lipase improves the efficiency and
quality of detergent by degrading the soluble
lipid. Sometime amylase is also used in
detergent which helps to break down
polysaccharide material (Simpson and
Russel, 1998).
Besides washing of clothes, alkaline
proteases are in demand for dish cleaning,
cleaning of ultrafiltration (UF) and reverse
osmosis (RO) membranes. It forms one of
the most important aspects of modern dairy
and food industries (Glover, 1985; Cheryan,
1986). Alkaline protease isolated from
marine shipworm bacterium is used to clean
contact lenses. Novozymes, Denmark
brought Clear-Lens Pro® to market, which
is used in cleaning of contact lenses by
removing protein-based deposits and protein
films from contact lenses (Sumantha et al.,
2006). In India, M/s Bausch and Lomb
(India) Ltd. has formulated an enzyme based
optical cleaner, containing Subtilopeptidase
A (Kumar and Takagi, 1999).
Tannary industry:
Elastolytic and keratinolytic activity of
alkaline protease help them in biotreatment
such as dehairing and bating of skins and
hides (Taylor et al., 1987). Alkaline
condition swells the hair root and protease
gradually decomposes the keratin so that it
comes out easily from the skin. Bating after
dehairing degrades the elastin and keratin
and de-swells the collagen and produces a
good soft leather, ready for commercial
purpose. In conventional processes harsh
chemicals such as lime, sodium sulphide,
salts, solvents are used, which subsequently
pollutes the environment (Saravanabhavan
et al., 2003). Enzymatic dehairing process
reduces the use of sodium sulphide and
creates an eco-friendly atmosphere for the
workers.
Protease from Pseudomonas aeruginosa PD
100 was used for dehairing of cow skin
(Najafi et al., 2005). Alkaline protease
isolated from Bacillus subtilis AKRS3 is
effectively used in removing of hair from
goat and sheep skin, which indicates its
application in leather industry (Ravishankar
et al., 2012). Keratin degrading protease
have been found to be secreted by three
Bacillus sp showing 99% identity with B.
Subtilis, B. Amyloliquefaciens and B.
Velesensis which is able to dehair the bovine
skin (Giongo et al., 2007). An alkaline
protease from Bacillus circulans having size
of 39.5 kDa was able to dehair goat skin
using purified enzyme (Subba Rao et al.,
2009). Proteases from B. pucilum and S.
Introduction
18
auricularis were efficient in dehairing and
depilating of raw leather (Bholay et al.,
2012). It has been reported (Verma et al.,
2011) that the protease from
Thermoactinomyces sp. RM4 can dehair
goat hides.
Food Industry:
Protease plays an important role in food
industry such as cheese making, fruit juice
and soya protein preparation. Protease has
also applications in baking, milling and
brewing industries. Hydrolysate produced
by the action of protease used as food
additive, improves the nutritional value of
food. Acidic protease coagulates the milk
protein, and helps in cheese formation
(Neelakantan et al., 1999). Novozymes from
Denmark formulated different commercial
bacterial proteases such as Alcalase®,
Neutrase®, Esperase®, Protamex™, and
Novozym® FM to improve functional,
nutritional and flavour properties of
proteins. Neutrase® is used in brewing and
baking industry. Neutral protease is used in
the extraction of rice starch (Sumantha et
al., 2006). The bitter taste of meat
hydrolysate was overcome by commercial
Novozymes‘s Flavourzyme®, which
degrades bitter peptide groups and makes it
possible to obtain 20% of hydrolysis (DOH)
without bitterness. Commercially available
proteases SEB and Tender 70 are used in
meat tenderization by breaking down
collagens in meat to make it easily digestible
(Singhal et al., 2012). Commercial alkaline
protease alcalase, hydrolyze the terminal
hydrophobic amino acid. This enzyme was
used in the production of a less bitter
hydrolysate (Adler-Nissen, 1986) and a
debittered enzymatic whey protein
hydrolysate (Nakamura et al., 1993).
Alkaline protease from B. Amyloliquefaciens
has been used to produce infant food from
the hydrolysate of casein, whey protein and
soya protein. Sardine muscle hydrolysate
produced by the action of protease derived
from B. Licheniformis was used in
formulation of food, which have role in
blood pressure regulation (Kumar and
Takagi, 1999).
Chemical industry:
Stability of the protease in presence of
organic solvent makes them suitable
candidate as biocatalyst in non-aqueous
medium for protein synthesis. A drawback
of this approach is that the enzyme activity
is reduced under anhydrous conditions.
Bacillus pseudofirmus SVB1, Pseudomonas
Introduction
19
aeruginosa psea have proved themselves as
potential candidate for peptide synthesis
(Yadav et al., 2011; Sen et al., 2011; Gupta
and Khare, 2007). Alkaline proteases from
B. pumilus strain CBS and Streptomyces sp.
strain AB1 are also used in peptide synthesis
in low water system (Jaouadi et al., 2011).
Besides peptide synthesis alkaline protease
also have role in synthesis of chemical
components. In 2011 Wang et al, reported
that alkaline protease from Bacillus
licheniformis has been used in synthesis of
2H-1-benzopyran-2-one derivative (Wang et
al., 2011). Commercial alkaline protease
Proleather isolated from Bacillus sp. form an
intermediate component sucrose-polyester
which is used in production of
biodegradable plastic (Patil et al., 1991).
Medical use:
Protease have role in medicinal field as
therapeutic agents. Soft gel-based medicinal
formulas, ointment compositions, gauze,
non-woven tissues and new bandage
materials has been prepared for therapeutic
purpose by using the immobilized alkaline
protease of Bacillus subtilis (Davidenko,
1999). A serine protease elastoterase,
isolated from Bacillus subtilis 316M strain
having high elastolytic activity was found to
have therapeutic application in the treatment
of burns and purulent wounds, carbuncles
and deep abscesses in immobilized
condition on a bandage (Kudrya and
Simonenko, 1994). Alkaline fibrinolytic
protease is suggested for future application
in thrombolytic therapy and anticancer drugs
(Mukherjee and Rai, 2011; Simkhada et al.,
2010).
Silk degumming:
Degumming is a process where sericin is
totally removed from the fibroin wall to
obtain shine, smoothness and other
properties in commercial silk (Freddi, et al.,
2003). A series of steps are involved in the
silk processing: reeling, weaving,
degumming, dyeing/printing and finishing
(Zahn, 1993). In conventional process silk
fiber is boiled in an aqueous solution
containing soap, alkali, synthetic detergent
and organic acids (Svilokos Binachi and
Colonna, 1992; Freddi, 1996). Enzymatic
reatment of silk fiber as an alternative of
conventional process is now in focus.
Alkaline proteases perform better than other
proteases (acid and neutral) with respect to
uniform sericin removal and improvement
of silk quality. In comparison with
conventional process there are certain
Introduction
20
drawbacks which are found in enzymatically
degummed silk fiber quality: higher shear
and bending rigidity, lower fullness and
softness to handle, remnant of the sericin at
cross over points between wrap and weft
(Chopra et al., 1996). Inspite of lower
performance and higher cost of enzyme
compared to chemical, enzymatic treatment
attract the attention of scientists and
technologists for the eco friendliness of the
process (Duran and Duran, 2000; Gubitz and
Cavaco-Paulo, 2001). Alkaline protease
from Bacillus sp. RGR-14 was reported for
removal of sericin during degumming
(Gupta et al., 2002). Three different types of
proteases were studied in silk industry for
the degumming of silk (Freddi et al., 2003).
Waste management:
Protease plays a role in cleaning up of the
environment by the degradation of deposited
waste material from food, leather, poultry
industry and house hold activities. Chemical
and mechanical processes of degrading
waste is successful, but they have some
disadvantages like energy intensive,
polluting and leading to loss of essential
amino acids. Keratinase isolated from
Bacillus is used to degrade feather (Ni et al.,
2011; Kojima et al., 2006; Cortezi et al.,
2008). There are other keratinase producing
bacterial strains of which Pseudomonas sp.
MS21, Microbacterium sp.,
Chryseobacterium sp. and streptomyces sp.
have been reported (Tork et al., 2010; Thys
and Brandelli, 2006; Brandelli and Riffel,
2005; Tapia and Simoes, 2008). Hydrolysate
component of feather by keratinases is used
for various purposes: as additives for
feedstuffs, fertilizers, glues and films or
used for the production of the rare amino
acids: serine, cysteine, and proline (Gupta
and Ramnani, 2006).
Silver recovery:
One of the noble metals, silver is used in
various applications such as photography.
Silver is impinged within gelatin layer of X-
ray film. It contains 1.5–2.0% silver by
weight, which can be recovered and used for
a variety of purposes (Gupta et al., 2002).
Recovery of this precious metal by
traditional process include burning of
photographic plate, oxidation of the metallic
silver following electrolysis, stripping the
gelatin-silver layer using different chemical
solutions, which causes environmental
pollution. Enzymatic decomposition of
gelatin layer minimizes all these impacts
(Nakiboglu et al., 2003). Alkaline protease
Introduction
21
produced by Bacillus subtilis, Conidiobolus
coronatus, Streptomyces avermectinus are
reported to decompose gelatin layer of X-ray
film (Nakiboglu et al., 2001; Shankar et al.,
2010; Ahmed et al., 2008).
Production of protease:
Owing to its potential applications and
desirable properties, plenty of research is
being done on proteases. In industry large
scale production can only suffice the
ongoing demand. Fermentation and the
immobilization of the bacterial cell or
enzyme would be able to provide continuous
supply of enzyme in industry. Alkaline
protease can be produced by solid state or
submerged fermentation in industrial scale.
Submerged fermentation:
In submerged process free flowing liquid
like molasses, fruit and vegetable juice,
liquid broth and waste water are used as
substrate. In this process substrate is used up
very rapidly and continuous supplement of
the substrate is essential. This process is
suitable for organism like bacteria, they
release bioactive components within the
broth or liquid substrate, and the purification
of the bioactive component is easier.
Solid-state fermentation:
In this process bacterium is grown on a solid
matrix, in a moist environment with little or
no free water. Solid substrates, like bran,
bagasse and paper pulp are used can be used
for a sustained period of time as matrix. In
certain cases it is better than submerged
fermentation, because product can be
recovered in highly concentrated manner.
Three methods of fermentation – drum, pot
and tray method are use in the production of
enzyme. But this fermentation process is not
suitable for organisms that require high aw
(water activity), such as bacteria. (Babu and
Satyanarayana, 1996). Extracellular alkaline
protease production from Bacillus subtilis
RSKK96 was studied using solid state
fermentation (SSF), using different substrate
such as Wheat bran (WB), rice husk (RH),
lentil husk (LH), cotton stalk (CS), crushed
maize (CM) and millet cereal (MC). Highest
enzyme production (5759.2 U/mg) found
using lentil husk (1000 ml of fermentation
media) (Akcan and Uyar, 2011). Beef
extract as nitrogen source, and arabinose
followed by lactose, galactose, and fructose
as carbon sources was found to be the best
inducer of alkaline protease. In presence of
metal salts FeSO4.7H2O and MgSO4.7H2O
Introduction
22
protease production was increased (Akcan
and Uyar, 2011).
Recent study showed that solid state
fermentation is more efficient than
submerged fermentation for the bacterial
enzyme production (Subramaniyam and
Vimala, 2012). In submerged fermentation
metabolic intermediate was found to
accumulate within the substrate along with
desired products, which lowered the enzyme
activity and product efficiency.
Immobilization:
Enzyme immobilization is a technique
where enzyme is entrapped within inert,
insoluble gel like material for
immobilization. Calcium alginate, produced
by reaction of sodium alginate solution with
calcium chloride is one such material which
is well known for enzyme immobilization.
Immobilized enzyme have greater
operational stability (they are more resistant
to change in pH, temperature) than the
soluble form of the enzyme and can be
reused and easily separated from the
products (Barabino et al., 1978).
Adsorption, entrapment and cross-linkage
are three different processes by which
enzyme can be immobilized. Synthetic
polymers like polyacrylamide, polyethylene
glycol, Polyvinyl alcohol (PVA), cellulose
triacetate, Poly (Tetrafluoro-ethylene)
membranes, polyurethane have also been
used for immobilization studies (Hsu et al.,
2010; Lozinsky and Plieva, 1998; Hyde et
al., 1991), though they are toxic as
compared to natural polymer.
Fig.2. Representing the different method of immobilization of enzyme. Taken from:
(loschmidt.chemi.muni.cz/peg/lecture/biocat_lecture10.pdf) , Accesed on 20th
December 2012, at 12.30 p.m.
Introduction
23
Silva reported the immobilization of a
commercial protease, Esperase. This
protease was covalently linked to Eudragit
S-100, a reversible soluble–insoluble
polymer, and showed higher thermal
stability, good storage stability and
reusability. The immobilized protease has
shrink-resist finishing in wool industry
(Silva et al., 2006). Extracellular protease of
Pseudomonas aeruginosa PD100 with
application for amino acid production,
clearing of juice was entrapped within
polyacrylamide gel retaining 90% of its total
activity compared with the soluble enzyme,
and the pH, temperature optima of the
enzyme remain unaltered. The reuse of
immobilized enzyme retained 83% of its
initial activity after six cycles (Najafi et al.,
2005; Mansson et al., 1983). There are
several immobilized enzymes used in
various purposes in industrial scale, table 5
presents examples of some commercial
immobilized enzyme, with their product and
immobilization techniques.
(loschmidt.chemi.muni.cz/peg/lecture/biocat
_lecture10.pdf) , Accesed on 20th
December
2012, at 12.30 p.m.
Table 5. Commercial product formulation using enzyme immobilization techniques.
(loschmidt.chemi.muni.cz/peg/lecture/biocat_lecture10.pdf) , Accesed on 20th
December 2012, at 12.30 p.m
Introduction
24
Whole cell immobilization is an alternative
process of enzyme immobilization. When
extraction and recovery of the enzyme (e.g:
intracellular enzyme) is difficult and
expensive, or a series of enzyme is required
for a particular reaction from the same cell
and so the target cell immobilization is used
for convenience (Burrill et al., 1983). This
process reduces the cost of multiple enzyme
immobilizations, whereas the undesired
enzyme or by-product of the cell reduces the
yield of desired product.
Table 6 represents some of the commercially
reported organic product produced by whole
cell immobilization techniques
(loschmidt.chemi.muni.cz/peg/lecture/biocat
_lecture10.pdf) , Accesed on 20th
December
2012, at 12.30 p.m
Table 6. Commercial product using whole cell immobilization techniques.
(loschmidt.chemi.muni.cz/peg/lecture/biocat_lecture10.pdf) , Accesed on 20 December 2012, at 12.30 p.m
Immobilization of the microorganisms is
done for large scale industrial use.
Immobilization can be carried out in
bioreactors.
Bioreactor:
Bioreactor is a device, where chemical
reaction occurs with help of biological
organisms or biologically active substrate
derived from such organisms. According to
mode of operation, bioreactors are classified
into: batch, fedbatch and continuous.
In batch mode the reaction is allowed to
continue for certain time after which the
product and byproduct is taken out. A fresh
process is then started.
Introduction
25
In fed batch process where nutrient is added
in controlled manner to avoid dilution, as
well as to maintain the growth rate and
oxygen limitation of the culture.
In continuous mode the continuous supply
of the raw material is maintained with
continuous collection of the product and
byproduct.
The microorganisms can be immobilized in
inert matrix and the different modes can be
used for operation. Upon immobilization in
a reactor the following criteria may be taken
into account
a. Uniform hydrodynamics at the solid
support surface
b. High experimental surface area
c. Lower adhesion of the bacteria to the
reactor surface and low adsorption of the
toxic material to the reactor surface.
d. Maintenance of sterile environment in
the reactor (Hsieh et al., 1985).
Parameters which influence protease
production:
There are different parameters which
influence the production of enzyme: media
composition (Varela et al., 1996),
particularly carbon and nitrogen source
(Kole et al., 1988) and process parameters
such as temperature, pH, agitation speed
(Hameed et al., 1999). All of these
parameters vary from one to another
organism. Effect of various culture
conditions on the production of an
extracellular protease by Bacillus sp. was
studied by Sepahy and Jabalameli (Sepahy
and Jabalameli, 2011) and they reported that
sucrose and corn steep liquor are the best
substrate for enzyme production. Several
workers reported that use of different sugars
such as lactose (Malathi and Chakraborty,
1991) maltose (Tsuchiya et al., 1991),
sucrose (Phadatare et al., 1993) and fructose
(Sen and satyanarayana, 1993) as carbon
source increased the yield of alkaline
protease. Various organic acids, such as
acetic acid (Ikeda et al., 1974), methyl
acetate (Kitada and Horikoshi, 1976) and
citric acid or sodium citrate (Takii et al.,
1990; Kumar et al., 1997) also have been
reported to enhance production of proteases
at alkaline pH. To overcome the expense of
fermentation, different agro industrial
wastes (green gram husk, chick pea, wheat
bran, rice husk, lentil husk, cotton stalk,
crushed maize, millet cereal), tannery
wastes, shrimp wastes, date wastes etc. have
been used (Nadeem et al., 2008; Prakasham
et al., 2005; Mukherjee et al., 2008;
Ravindran et al., 2011). In presence of
Introduction
26
certain amino acids alkaline protease
production by Bacillus sp increased (Ikura
and Horikoshi, 1987), whereas in presence
of glycine, casamino acid protease
production decreases (Ong and Gaucher,
1976). In presence of Feso4.7H2O and
MgSO4.7H2O protease production by
Bacillus subtilis RSKK96 was enhanced
(Akcan and Uyar, 2011). Beside the medium
source and supplement, pH, temperature and
agitation rate also varied the production of
protease. Traditionally scientists considered
―one variable at a time‖ strategy, where they
only varied one parameters by keeping other
factors constant, which is time consuming
(Bhunia et al., 2010; Jayasree et al., 2009).
Now-a-days different statistical methods
have been developed such as Astaguchi
methodology, Plackett–Burman design and
response surface methodology (RSM) for
optimization of the production of enzyme.
These methods give a better understanding
of interaction of different parameters using
minimum experiments (Hajji et al., 2008).
Maintenance of each parameter condition
for optimum production is essential; a small
deviation from the specified parameter can
lead to the production of undesirable
products (Subramaniyam and Vimala,
2012).
Protease purification:
Isolation and identification of promising
strains, characterization of enzymes and
optimization of products leads to improve
their application. Advances in microbiology
and biotechnology have created a favorable
condition for the development of proteases.
Different protease producing microbes were
isolated from the marine environment,
which were subsequently purified and
characterized. Green mussels (Perna viridis)
were collected from Kanyakumari coast and
protease producing Bacillus sp. was isolated
from mussel‘s cell. A 37 kDa alkaline serine
protease was isolated and purified, which
was active at pH 7 and 70°C that can be
used in detergent industry as additive
(Padmapriya et al., 2012). Two alkaline
serine proteases (Pro 1 and Pro 2) were
purified from marine Bacillus sp. by using
cation exchange chromatography on CM-
Sepharose CL-6B followed by Sephadex G-
75 superfine. These proteases were stable
over pH range from 7.0-11 and temperatures
of 50ºC and 55
ºC, and were partially
inhibited by Ag+
and Hg2+
and stable in the
presence of the surfactants and bleaching
agent (H2O2) (Gouda, 2006). Two novel
halotolerant extracellular proteases were
derived from Bacillus subtilis strain FP-133,
Introduction
27
isolated from a fermented fish paste by
Setyorini. One of these two enzymes was
non-alkaline serine protease with a
molecular mass of 29 kDa while the other
was a metalloprotease with a molecular
mass of 34 kDa (Setyorini et al., 2006).
Tang recently reported an organic solvent
tolerant, alkaline metalloprotease from
Pseudomonas aeruginosa PT121. The
protease was purified in a single step by
hydrophobic interaction chromatography on
a phenyl sepharose matrix (Tang et al.,
2010). The purified protease had molecular
mass of 33 kDa. The activity of protease
was inhibited by EDTA and 1,10-
phenanthroline and it was found to have
broad specificity for carboxylic acid residue
(Tang et al., 2010). Bacillus subtilis ICTF-1
was isolated from western sea coast of
Maharastra (India). Fibrinolytic enzyme
(28kDa) isolated from marine Bacillus
subtilis ICTF-1 was stable at pH 5.0-11.0
and temperature of 25-37°C. The enzyme
activity of purified protease was activated by
Ca2+
and inhibited by Zn2+
, Fe3+
, Hg2+
and
PMSF and the enzyme have found as an
applicant in laundry detergent (Mahajan et
al., 2012) Bacillus cereus having alkaline
protease activity was isolated from the
Marsa-Matrouh sea shores (North-west of
Egypt). 31 kDa protease was purified from
the strain by ammonium sulfate precipitation
and Sephadex G-200 chromatography,
which showed maximum activity at pH 10,
50°C and in presence of 5 mM Cu2+
ions the
relative enzyme activity enhanced up to
112%. This protease has some application in
removing the stain of blood from clothes
(Abou-Elela et al., 2011).With an optimum
temperature and pH for activity being 40oC
and 7.0 respectively a marine protease
producing bacterium was isolated from
Indian Ocean (Fulzele et al., 2011). Bacillus
halodurans CAS6, a protease producing
bacterial strain was isolated from marine
sediments of Parangipettai coast, Tamilnadu,
India. Protease was purified using DEAE-
Cellulose and Sephadex G-50; when
purified protease was treated with ionic,
non-ionic and commercial detergents and
organic solvents, it retained its activity 72–
94% , 76–88 %, and 88–126 % respectively
(Annamalai et al., 2012). A salt tolerant
thermostable 66 kDa protease was purified
using ultrafiltration, ethanol precipitation,
hydrophobic interaction column
chromatography and gel permeation
chromatography. This protease was purified
from the bacterium Chromohalobacter sp.
Strain TVSP101, which was isolated from
Introduction
28
solar saltern samples of Tuticorin,
Tamilnadu, India. The purified protease
activity was completely inhibited by ZnCl2
(2 mM), 0.1% SDS and PMSF (1 mM),
whereas it was able to retain its activity in
presence of 1 mM of pepstatin, EDTA and
PCMB. It also retained 100% of it activity in
presence of 10% (v/v) DMSO, DMF,
ethanol and acetone (Vidyasagar et al.,
2009).
Besides marine bacteria, protease was also
extracted and purified from different sources
of microbial origin. P. aeruginosa ATCC
15442 (KCCM 11321) strain was reported to
secrete an alkaline protease that can cleave
transferrins with the production of
siderophores, and thus helps itself to
overcome iron deficiency during human
infection (Kim et al., 2006). An intracellular
protease from Pseudomonas aeruginosa was
characterized and purified by Shahanara and
co workers. The molecular weight of the
protease was about 48-49 kDa. This was
reported to be a glycoprotein and
monomeric in nature. The Km value of the
protease was found to be 0.48% against
casein as substrate (Shahanara et al., 2007).
Ghosh characterized an extracellular serine
protease from a feather degrading bacterium,
Bacillus cereus DCUW. The structural
analysis of the protease by SMART domain
analysis revealed that N-terminal end of the
protease had a signal sequence for secretion,
a catalytic S_8 peptidase domain and a long
C-terminal protease associated region
containing nine intrinsically disordered sub-
domains (Ghosh et al., 2009). A
thermotolerant 58 kDa alkaline protease was
purified from Serratia marcescens Subsp.
sakuensis TKU019 from northern Taiwan
soil (Liang et al., 2010). An extracellular
protease (43 kDa) was purified from
Pseudomonas thermaerum GW1, by using
ammonium sulphate precipitation and
DEAE-cellulose chromatography achieving
a 6.08 fold purification. The optimum
proteolytic activity of purified enzyme was
found at pH 8. Enzyme activity was
increased 5 fold in presence of 5mM Mn2+
,
while Cu2+
, Mg2+
and Ca2+
moderately
activated enzyme activity, but Zn2+
, Fe2+
and
Hg2+
inhibited enzyme activity. This
protease was stable in presence of different
organic solvent like ethylacetate, acetone,
isopropanol, methanol and benzene; which
pointed that it could be used as biocatalyst
for enzymatic peptide synthesis (Gaur et al.,
2010). A metalloprotease producing
Pseudomonas aeruginosa was isolated from
CharakDanga Bheri, East Calcutta wetland.
Introduction
29
Using Phenyl Sepharose CL-4B column,
36.18 kDa protease was purified, resulting in
1.2 fold increase in specific activity and
28% recovery. Enzyme activity was
optimum at 40°C, and was stable within the
range of pH 5 to 8 (Yadav et al., 2010).
Environmental pollution:
The industrial development and
anthropogenic sources (metalliferous
mining, fossil fuel combustion, waste
disposal, fertilizers in agriculture) has led to
an increase in the release of pollutant in
environment which is an important cause of
the natural climate change. Pollution can be
divided in four general categories: i) air, ii)
noise, iii) soil and iv) water. Urbanization,
deforesting, use of chemical pesticide in
agricultural, release of waste product from
chemical industry all contribute as point
sources leading to environmental pollution.
These waste products are either deposited in
soil, ground water or along with runoff
water it comes to river and thus to
sea/ocean. Some common soil contaminants
are dichlorodiphenyltrichloroethane(DDT),
chlorinated hydrocarbons (CFH), methyl
tertiary butyl ether (MTBE), arsenic,
benzene and heavy metals (such as lead,
chromium, cadmium, zinc, mercury etc)
(Jarup, 2003).
Biomagnification and bioaccumulation are
the major problems associated with heavy
metals, which is a threat to human health
and also for other animals and plants, as
well as for microorganisms. Skin irritations
and rashes can occur due to oil spill. Due to
exposure to heavy metals like lead and
mercury neurological problems are
generated. Due to ozone pollution humans
are suffering from various respiratory,
cardiovascular diseases, throat
inflammation, chest pain, and congestion
(Spengler and Sexton, 1983).
Here we are interested in heavy metal
pollution and its removal by bioremediation
process.
Heavy metals:
The metals with density 5 or higher than that
are considered as heavy metals. Mercury
(Hg), Cadmium (Cd), Arsenic (As) and Lead
(Pb) are few examples of heavy metals.
Sometimes, the metals having toxic effect to
human health or negative impact to
environment are also defined as heavy
metals, like cobalt, chromium, lithium and
even iron. They are natural components of
earth crust. The toxicity of metal depends on
Introduction
30
the allotrope or oxidation state of the metal.
For example, chromium with hexavalent
state is deadly; whereas the trivalent state of
chromium is nutritionally significant in
many organisms, including humans (Jarup,
2003). Some heavy metals like Cu and Zn
serve as trace elements and are essential to
maintain metabolism at a lower
concentration, but in higher concentration
they are toxic. Even over-used cookware
made of iron (Fe) and aluminum (Al) may
produce toxic side-effects by repeated
ingestion of metal (elemental state) into
human food chain.
Removal strategy of heavy metals:
Removal of toxic heavy metals from water
is essential from the environmental point of
view (Yuan et al., 2001). The conventional
methods adopted earlier for this purpose
included chemical precipitation, oxidation,
reduction, filtration, electrochemical
treatment, evaporation, adsorption and ion-
exchange resins. Conventional techniques
are cost-effective in terms of equipments,
chemicals. Intensive management and long-
term maintenance is also required (Brodie,
1993). These methods require high energy
inputs especially when it refers to dilute
solutions. All these techniques were able to
reduce the concentration of the contaminants
but they involved complex technologies and
were not very cost effective. Therefore now
a days bioremediation is replacing the
chemical treatment. It is the process of using
living organisms or its product for treatment
of waste. The contaminants are either
degraded completely or they are reduced to
a concentration much below the values
established by regulatory authorities (Vidali,
2001).
Metal-microbe interaction:
Metals play an integral role in the life
processes of microorganisms. Some metals,
such as calcium, cobalt, chromium, copper,
iron, potassium, magnesium, manganese,
sodium, nickel and zinc, are considered as
essential metals. These metals are used for
redox-processes; to stabilize molecules
through electrostatic interactions; as
cofactors of various enzymes and enzymatic
reactions; and for regulation of osmotic
pressure (Bruins et al., 2000). Many other
metals have no biological role (e.g. silver,
aluminium, cadmium, gold, lead and
mercury), and are nonessential (Bruins et
al., 2000) and potentially toxic to
microorganisms.
Introduction
31
Heavy-metal toxicity can cause poisoning
and inactivation of enzyme systems, and
also reduce microbial activity to a great
extent. Nonessential metals can displace the
essential metals from their original binding
sites or through ligand interactions (Nies,
1999; Bruins et al., 2000). For example,
Hg2+
, Cd2+
and Ag2+
can bind to SH groups,
and thus inhibit the activity of some
enzymes (Nies, 1999). Bong reported that
addition of zinc to growth medium decrease
the bacterial amino peptidase activity (Bong
et al., 2010). In 1993, Nair studied the effect
of Hg, Cd and Zn on Bacillus sp,
Flavobacterium sp., from Indian coastal
waters. Growth of both the species were
inhibited in presence of mentioned metals,
Hg>Zn>Cd and Hg>Cd>Zn are the order of
inhibition for Bacillus and Flavobacterium
sp. respectively (Nair et al., 1993). Due to
exposures to heavy metal contaminated
environment, microorganisms generate some
defense mechanisms among themselves to
protect themselves from this stress
condition. Zolgharnein reported the
occurrence of plasmid in bacteria isolated
from heavy metal contaminated water
source of Persian Gulf and surrounding
industrial area, highest percentage of
plasmid was detected from industrial waste
water (84.6%), followed by coastal
sediments (55.5%) and marine waters
(53.8%). His findings also stated that
frequencies of occurrence of plasmid in
heavy metal resistant microbes are much
higher than normal bacteria. These metal
resistant bacteria were able to remove 90%
lead and cadmium from the contaminated
source (Zolgharnein et al., 2007).
Interactions between microorganisms and
metals can be conveniently divided into
three distinct processes, all of which may be
important with respect to metal distribution
in natural waters: a) intracellular
interactions, b) cell-surface interactions, and
c) extracellular interactions.
Intracellular Interactions:
Assimilation of metals may be important to
the microbe in detoxification, enzyme
function, and physical characteristics of the
cell. Probably the most widely recognized
microbial interaction with toxic metals in the
aquatic environment is the microbial
methylation of mercury. Pure-culture
experiments have shown that many bacteria
and fungi have the capability to methylate
mercury (Gilmour and Henry, 1991).
Cell-Surface Interactions:
A number of authors have shown that metal
binding to cell surfaces is an important
Introduction
32
factor in the distribution of metals in natural
waters (Sigg, 1987; Xue et al., 1988). Heavy
metals may bind to the active groups of
chemical compounds of cell walls and
membranes. Gram-negative bacteria possess
lipopolysaccharides and phospholipids in
their cell walls, with phosphoryl groups as
the most abundant electronegative sites
available for metal binding (Coughlin et al.,
1983; Ferris, 1989). Gram-positive bacterial
cell walls possess teichoic acids and
peptidoglycan, providing carboxyl and
phosphoryl groups that are potential sites for
metal binding (Doyle, 1989). For both gram-
negative and positive bacteria, metal binding
to cell-surface functional groups is thought
to be an important step to intracellular
accumulation of trace metals required for
enzyme function.
Extracellular Interaction:
Extracellular interactions with toxic metals
range from the potential to leach metals
from sediments by production of acidic
metabolites to the formation of colloidal
sized extracellular polysaccharide (EPS)
metal complexes implicated in mobilization
and transport of toxic metals in soils (Black
et al., 1986; Chanmugathas and Bollag,
1988). Indirectly, toxic metals closely
associated with iron oxide (Cd and Zn) have
been shown to be solubilized by enzymatic
reduction of the ferric iron (Francis and
Dodge, 1990). Insoluble complexes may
also be formed by the activity of
microorganisms.
Microbes can obtain their energy by the
oxidation of iron, sulfur, manganese and
arsenic (Tebo et al., 1997). Through the
reduction of metals by dissimilatory
pathway, microorganisms utilize metals as a
terminal electron acceptor for anaerobic
respiration. For example, Microbes used
oxyanions of arsenic (Stolz and Oremland,
1999; Niggemyer et al., 2001), selenium
(Stolz and Oremland, 1999) and uranium
(Tebo and Obraztsova, 1998) through
anaerobic respiration as terminal electron
acceptors. Reduction of the metal not only
coupled with respiration process, but
microbes can used this property for metal
resistance. For example, aerobic and
anaerobic reduction of Cr(VI) to Cr(III)
(Nkhalambayausi-Chirwa and Wang, 2001);
reduction of Se(VI) to elemental selenium
(Lloyd et al., 2001); and reduction of Hg(II)
to Hg(0) (Brim et al., 2000; Wagner-Dobler
et al., 2003) are widespread detoxification
mechanisms among microorganisms.
Introduction
33
Bioremediation:
Bioremediation is the process where
microbial metabolism is used to remove the
pollutants from environment. Depending on
the site of action; it can be divided into in
situ and ex situ bioremediation. When
contaminated material is treated at
contaminated sites, it is known as in situ,
whereas the removal of the contaminated
material from their site of origin and their
treatment elsewhere is termed as ex situ
(Gaad, 2010). Bioremediation is widely
accepted due to the following advantages:
1. It is less expensive as compared to
other technologies and needs simpler
technology.
2. It is a natural process thus less energy is
required as compared to conventional
technique.
3. The end product is harmless and less
likely to affect human health.
4. The microorganisms may be indigenous to
the polluted environment and in situ
bioremediation can be carried out.
Role of the microbes in bioremediation:
Bacteria help in bioremediation by various
processes; in some cases they adsorb the
metal into cell wall, but do not accumulate
them inside the cell, this is known as
bioabsorption. Different process like
complexation, chelation, coordination, ion-
exchange, precipitation and reduction are
involved in bioabsorption process. Presence
of negatively charged particles on bacterial
cell membranes and polysaccharides attract
positively charged metal ions to get attached
(Ramasamy et al., 2007). The presence of
functional group like, hydroxyl, carbonyl,
carboxyl, sulfhydryl, thioether, sulfonate,
amine, imine, amide, imidazole,
phosphonate, and phosphodiester groups on
bacterial cell membrane, plasma membrane
and outer membrane facilitate metal binding
(Sannasi et al., 2009). The accumulation of
the metal within the bacterial cell leads to
the changes in bacterial cell morphology to
inhibit the entering of metal into the cell
(Chowdhury et al., 2008). At higher
concentrations metal can cause damage to
cell membranes, alter enzyme specificity;
disrupt cellular functions; and damage the
structure of DNA (Bruins et al., 2000).
Bacillus spp. Pseudomonas spp.,
Staphylococcus spp., and Aspergillus niger,
isolated from soil and sludge were used to
remove toxic metals from heavy metal (Cd,
Cr, Cu, Zn and Pb) contaminated industrial
waste and liquid waste water. Each of the
species were able to remove all the metals
Introduction
34
more than 45% by bio absorption process,
though it was reflected that the ability of
bioabsorption of Pseudomonas is much
higher than that of the others (Kumar et al.,
2010) bioremediate waste water by
bioabsorption of Cr (VI) in different
matrices (Srinath et al., 2003). Pseudomonas
isolated from the Uppanar estuarine water
was able to absorb 41% of Cd, 62.8% of Fe,
87.9% of Pd, 53% of Ni and 49.8% of from
estuarine water (Sri kumaran et al., 2011).
Precipitation of heavy metals to highly
insoluble form such as sulphides and
phosphates is another way of removing toxic
metals from environment. Citrobacter sp.
was reported to produce cell bound metal
phosphates of plutonium and neptunium
(Macaskie et al., 2006). An engineered E.
coli cell, containing acid phosphatase gene
phoN from Salmonella enterica sv. Typhi,
was reported to remove 21 mg cadmium /g
of dry weight from 1 mM solution within 3
hrs by precipitation. The precipitated
cadmium was recovered by 0.1N HCl wash,
after which the cells could be reused for
cadmium precipitation (Seetharam et al.,
2009).
Reduction of metals to a less toxic form is
another effective means of bioremediation.
Alcaligenes faecalis (seven isolates),
Bacillus pumilus (three isolates), Bacillus
sp. (one isolate), Pseudomonas aeruginosa
(one isolate), and Brevibacterium iodinium
(one isolate) were isolated from different
sites of Indian coastal region. These bacteria
were able to remove more than 70% of Cd
and 98% of Pb within 72 and 96 hrs,
respectively, from growth medium
supplemented with 100 ppm metal salts.
Bacteria convert toxic metals like Hg into
less toxic forms through volatilization, Cd
and Pb are detoxified by entrapment (De et
al., 2008). Heterotrophic bacteria isolated
from the water and the sandy sediment of
Sopot beach, Gdańsk Bay (Poland) are
resistant to 0.1 mM lead (Jankowska et al.,
2006). Bacteria such as Thiobacillus
ferrooxidans and iron bacteria of the genus
Gallionella are capable of oxidizing ferrous
(Fe2+
) iron into ferric (Fe3+
) iron.
Magnetotactic bacteria, exemplified by
Aquaspirillum magnetotacticum, can
transform iron into its magnetic salt
magnetite. These bacteria act as biological
magnets. Hexavalent chromium [Cr(VI)]
and hexavalent uranium [U(VI)] are highly
toxic and the soluble forms of these
elements are of great concern as pollutants
in the environment. When reduced to Cr(III)
or U(IV) these elements are much less
Introduction
35
soluble and hence less toxic. Therefore,
reduction of hexavalent Cr and U,
particularly by bacteria, is being explored as
a bioremediation strategy for these elements
(Lodish et al., 2004). Five bacterial samples
isolated from activated sludge sample of
Egypt were able to transform metal salts
such as Co, Cu, Cd, Fe, Hg, Ni, Mn, Pb and
Zn in individual or in mixed from into
insoluble precipitates (Essa et al., 2012).
A strain of Pseudomonas fluorescens
isolated from uranium mine was found to
uptake 1048 nmol Ni2+
/mg of
dry wt., 845
nmol Co2+
/mg of
dry wt., 828 nmol Cu
2+/mg
of dry wt. and 700 nmol Cd2+
/mg of dry wt
(Chaudhury and Sar, 2009) Bacillus,
Salmonella and Arthrobacter species
isolated from New Calabar River sediment
was able to tolerate 2 mM Zn2+
concentrations in a nutrient broth-glucose-
TTC medium. Salmonella sp. was able to
tolerate the highest Zinc concentration,
followed by Arthrobacter and Bacillus sp.
(Nweke et al., 2007). Four sewage isolated
Proteus vulgaris (BC1), Pseudomonas
aeruginosa (BC2), Acinetobacter
radioresistens (BC3) and Pseudomonas
aeruginosa (BC5) from Madurai district of
Tamilnadu showed variable degree of
resistance against different metals. They
were able to tolerate Cd (4-7 mM), Cr (0.7
mM), Ni (6.75-8.5 mM), Pb (6 mM), As
(6.5-15 mM) and Hg (0.75 mM) (Edward
Raja et al., 2009).
By all these methods (absorption of the
soluble and insoluble metal in the cell wall,
precipitation of the metal in the form of
insoluble sulphate or phosphate component,
transformation of toxic element to less toxic
form) bacteria help in decontamination of
the environment from heavy metals.
Application of metal resistant microbes:
Bacteria growing in the metal contaminated
environment often showed intracellular
accumulation. It would either be a whole
cell accumulation or a localized distribution.
In case of localized distribution, it could
either be as aggregates or as nanoparticles
(between 1 and 100 nanometers diameter).
Physiochemical properties of the metal are
being changed when they are converted into
nanoparticle, it may be due to the greater
surface area per weight than larger particles
which causes them to be more reactive to
some other molecules. Nanoparticles have
wide applications such as drug and gene
delivery, biodetection of pathogens,
detection of proteins, tissue engineering,
separation science, biosensors, enhancing
Introduction
36
reaction rates and magnetic resonance
imaging (MRI) etc (Li et al., 2011).
Synthesis of nanoparticles in laboratory is
cost effective and requires specialized
instrumentation and energy consumption. So
Researchers are interested in microbe
fabricated nanoparticles generation.
Here are some nanoparticle generating
microbes; a silver resistant Bacillus sp
isolated from atmosphere was reported to
grow in presence of 3.5 mM silver nitrate
solution and produce nanoparticles of size
range 5–15 nm at their periplasmic space
after 7 days incubation at room temperature
(Pugazhenthiran et al., 2009). A novel strain
of Marinobacter pelagius isolated from
water sample from solar saltern, Kakinada
was able to synthesize gold nanoparticles
from HAuCl4 solution. Gold nanoparticles
formed within the cell were less than 10 nm
size (~ 2 – 6 nm) with different shape
within a short period of time (Sharma et al.,
2012). Magnetotactic bacteria were reported
to generate crystals of magnetic iron
assembled in a chain within magnetosome
(Lang and Schuler, 2006). Magnetic
nanoparticles are of interest to researchers
for targeted cancer treatment (magnetic
hyperthermia), stem cell sorting and
manipulation, guided drug delivery, gene
therapy, DNA analysis, and magnetic
resonance imaging (MRI) (Fan et al., 2009).
Bacteria were able to synthesize silver
nanoparticle in different shape and form;
e.g. spherical (Fayaz et al., 2010), in form of
film by Aspergillus flavus (Jain et al., 2011).
Radiations:
Transmission of energetic particles or
energetic waves through space in various
form, is known as radiation. Depend on the
interaction with substrates radiations are
subdivided into ionizing and non-ionizing
radiation. The electromagnetic radiations
which have more than 10 eV energy are
known as ionizing radiation, which can
knock out electrons from molecule and
ionize them. When cells are exposed to
ionizing radiation they generate free
hydrogen radicals, hydroxyl radicals, and
some peroxides; which in turn cause various
intracellular damages. Non ionizing
radiations are more safer than ionizing
radiations. Ultraviolet radiations lies in non-
ionizing radiations though they having some
features of ionizing radiations. Small doses of
ultraviolet light is absorbed by different
cellular compounds, alter the chemical
bonds of biological molecule and damage
them. Gamma rays are high energy
radiations emitted from 60
Co, which
Introduction
37
penetrate into cell and directly damage the
DNA; which unless repaired results in death
of the cell.
Radiation and its effect on microbes:
Marine microbes are expected to be exposed
to higher doses of UV rays than terrestrial
fresh water counterparts and are
comparatively less susceptible to these non
ionizing radiations (Flint, 1987). In case of
coastal areas, due to shallow depth, the
penetration of UV rays would be even
greater thus influencing the population. It
was investigated that various part of the
coastal area of world are effected by
background radiation. Deposition of
monazite sand containing thorium (8-10%),
uranium (0.30%) and its radioactive decay
product within a part of Kerala beach (~55
km in length and 0.5-1.5 km in width) effect
human population in all stages of life. The
back ground radiation of beach area varies
(from <1-45 mGy/year) due to the non
uniform distribution of monazite. (Jaikrishan
et al., 2012) Gamma (γ) rays induce
different types of damage in organisms often
leading to cell death (unless repaired) and
permanent changes within daughter cells
(Legault et al., 1997; Harrison and
Malyarchuk, 2002). The most severe among
them is DNA double strand break (DSB)
which leads to chromosomal aberrations
including deletions. Damage of DNA
depends upon the quality of the radiation
and on the rate of energy deposition (i.e. the
dose-rate) (Harrison and Malyarchuk, 2002).
It has been reported that with increasing
dose rates the damage of DNA increases,
which is commonly known as dose-rate
effect (Bonura et al., 1975; Takahashi et al.,
2000, 2002).
DNA- DSB repair is predominant in higher
organism, though it was reported by
Hariharan and Hutchinson that very few
DSBs may be rejoined in Bacillus subtilis
(Bonura et al., 1975). Damage to DNA
alters the spatial configuration of the helix,
which is detected by the cell and thus repair
strategies are evolved to restore lost
information. There are three mechanisms
existing to repair double-strand breaks
(DSBs):non-homologous end joining
(NHEJ), microhomology-mediated end
joining (MMEJ), and homologous
recombination. Non-homologous end
joining (NHEJ) is a process where two ends
of DNA is ligated with the help of DNA
LigaseIV,
and
without the need of a
homologous template. Mutation can occur
due to inappropriate ligation which can lead
Introduction
38
to translocations and telomere fusion which
are hallmarks of tumor cells. NHEJ repair
protein was observed in Bacillus subtilis,
Mycobacterium tuberculosis and
Mycobacterium smegatis (Lodish et al.,
2004).
Microhomology-mediated end joining
(MMEJ) is a repair mechanism where 5-25
base pair microhomologous sequences are
aligned to the broken strands before joining.
It is an error-prone method, which causes
deletion mutations in the genetic code, and
which could lead to the development of
cancer. This repair mechanism only comes
to play a role when NHEJ method is
unavailable or unsuitable due to the
disadvantage posed by introducing deletions
into the genetic code (Okuno et al., 2004).
In homologous recombination repair, an
identical or nearly identical sequence is used
as a template for repair of the break. This
repair mechanism acts very accurately
against double-strand breaks. Homologous
recombination is observed across all three
domains. Homologous recombination occurs
in plants, animals, fungi, protists as well as
bacteria (Escherichia coli) and viruses
(herpes virus, retro virus), which help to
conclude it is a nearly universal biological
manner.
Radiosensitization:
Radiosensitization is an effect which
increase DNA damage and make them more
susceptible, as a result of which the applied
dose of irradiation becomes lethal. The
intracellular accumulation of metal within
the bacterial cell itself might lead to DNA
damage. When metal accumulated bacterial
cells are exposed to radiation, it is observed
that metal/metalloid (nickel, cadmium,
mercury, cobalt, lead and copper)
compounds inhibit DNA-DSB repair, a
phenomenon known as radiosensitization
(Takahashi et al., 2002). The inhibitory
effect of metal on DNA repair depends on
the nature of damage. Examples for the
above include the following: mercury
interfered with the repair of DNA damage
induced by X rays but not the repair of
damage induced by UV light; Arsenite has a
strong inhibitory effect on the repair of 60
Co
γ-radiation induced DSBs (Takahashi et al.,
2000). There are some chemicals which
inhibit DNA- DSB repair.