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.