26

3. MATERIALS AND METHODS

3.1 Materials

Human fibrinogen, human thrombin, bovine fibrinogen, bovine thrombin,

human plasmin, α-casein, tyrosine, urokinase, streptokinase, bacterial proteinase, 2,2-

bypirimidine, leupeptin, soybean trypsin inhibitor (SBTI), phenylmethanesulfonyl

fluoride (PMSF), tosyl phenylalanyl chloromethyl ketone (TPCK), tosyllysine

chloromethyl ketone (TLCK), E64, pepstatin-A, ethylene glycolbis (beta-aminoethyl

ether)-N,N,N,N’-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA),

synthetic chromogenic substrates N-Succinyl-Ala-Ala-Pro-Phe-pNA, D-Val-Leu-Lys-

pNA, D-Val-Leu-Arg-pNA, D-Ile-pro-Arg-pNA and NA-Benzoyl-L-Arg-pNA, p-

nitroanilide were obtained from Sigma-Aldrich. DEAE-Sepharose FF columns, Q-

Sepharose FF column, SP-Sepharose FF column, Sephacryl-S100, Sephacryl-S200

different size column were obtained from GE healthcare. 2D gel electrophoresis kit and

related reagents were obtained from Biorad. All molecular biology reagents for

polymerase chain reaction (PCR), restriction digestion, gene cloning, agarose gel

electrophoresis, polyacrylaimde gel electrophoresis, buffer salts, DNA and protein

ladders were obtained from Promega, GeNei and Sigma-Aldrich. Primers used for this

study were synthesized from Sigma. All other chemicals used were of analytical reagent

grade.

27

3.2 Collection of fermented food samples

Samples of traditional fermented foods of Northeast India (Fig. 3.1) were collected from

different markets of 7 states of North East India namely Agartala of Tripura state;

Silchar, Johrat and Guwahati of Assam state; Dimahpur of Nagaland; Aizawl of

Mizoram state; Shillong of Meghalaya state; Itanagar and Pashighat of Arunachal

Pradesh state and Imphal, Bishnupur, Kakching of Manipur state. These samples were

aseptically collected in sample containers and transported to the laboratory using cooler

boxes within 2 days of collection from the markets. The samples were immediately

processed for subsequent analyses. Ten gram of each sample was transferred to a sterile

stomacher bag containing 90 mL of physiological saline (0.1% w/v bacteriological

peptone, 0.85% w/v NaCl) and homogenized (Stomacher 400- circulator, Seward, UK)

at 250 ×g for 3 min. The homogenate was divided into two portions, one for screening

fibrinolytic activity and other for microbial isolation.

28

Fig. 3.1 Traditional fermented soybean and fish product of Northeast India. A. Peru of

Arunachal Pradesh, B. Akhoni of Nagaland, C. Hawaijar of Manipur, D. Bekang of

Mizoram, E. Tumgrymbai of Meghalaya, F. Perumb of Arunachal Pradesh are

traditional fermented soybean products. G. Shedal of Assam, H. Ngawum of Mizoram,

I. Shidal, J. Khoka illish and K. Lona illish of Tripura; L. Ango-ango from Arunachal

Pradesh are different traditional fermented fish products.

29

Ta

ble

3.1

Lis

t o

f tr

adit

ion

al f

erm

ente

d f

ood

s o

f N

ort

hea

st I

nd

ia s

cree

ned

fo

r fi

bri

no

lyti

c ac

tiv

ity i

n t

he

stu

dy

Typ

e

Food

cod

e

Loca

l n

am

e

Ind

igen

ou

s fe

rmen

tati

on

pro

cess

P

lace

of

coll

ecti

on

N

um

ber

of

sam

ple

s

Aver

age

pH

(mea

n±

SD

) M

ark

et

Pla

ce

Sta

te

Fer

men

ted

soyb

ean

Gly

cine

max

(L.)

Mer

r

1R

T

ung

rym

bai

Soak

ed

in

wat

er

for

6-7

h,

seed

coat

of

soyb

ean

was

re

mo

ved

,

cooked

fo

r 1

-2 h,

dew

ater

ed an

d

ferm

enta

tion

was

ca

rrie

d

out

at

room

te

mp

erat

ure

in

a

bam

boo

bas

ket

li

ned

w

ith

leav

es

of

Cli

nogyn

e dic

hoto

ma

(R

oxb

.)

Sal

isb

. fo

r 3

-5 day

s (T

aman

g et

al.

, 2009).

Bar

a baz

aar

Lab

an b

azaa

r

Shil

long

Meg

hal

aya

5

8.1

0 ±

0.1

0

A

Haw

aij

ar

Over

nig

ht

soak

ed

soyb

ean

was

boil

ed f

or

1-2

h,

was

hed

wit

h h

ot

Nao

rem

thong

Imp

hal

M

anip

ur

5

8.1

7 ±

0.1

3

30

wat

er a

nd t

hen

dra

ined

. It

was

then

pac

ked

in

b

amb

oo

bas

ket

li

ned

wit

h F

icus

his

pid

a L

.f.

leav

es a

nd

incu

bat

ed

for

3-5

day

s in

ro

om

tem

per

ature

. U

sual

ly t

his

bam

boo

bas

ket

w

as

cover

ed

wit

h

gunny

bag

s or

pad

dy

stra

ws

whil

e

incu

bat

ing (

Jeyar

am e

t al.

, 2008).

AK

P

eru

Boil

ed

soyb

ean

was

dra

ined

an

d

deh

usk

ed,

mad

e in

to

pas

te

and

wra

pp

ed

wit

h

ban

ana

leav

es.

Nat

ura

l fe

rmen

tati

on i

n 4

-5 d

ays.

Nah

arla

gun

baz

ar

Itan

agar

A

runac

hal

Pra

des

h

5

7.1

7 ±

0.0

9

AL

P

erum

b

Sim

ilar

to

P

eru

but

pas

te

was

mad

e af

ter

ferm

enta

tion.

Mai

n c

ity

mar

ket

and

Pas

hig

at b

azar

Itan

agar

Pas

hig

hat

Aru

nac

hal

Pra

des

h

5

7.1

4 ±

0.1

3

31

AB

A

khoni

Soak

ed s

oyb

ean w

as b

oil

ed f

or

2-3

h, co

ole

d a

nd p

acked

in a

conta

iner

lined

w

ith

ban

ana

leav

es.

It

was

incu

bat

ed

for

at

leas

t 6–

8

day

s

(Tam

ang e

t al.

, 2009).

New

m

arket

D

imap

ur

Nag

alan

d

5

7.4

7 ±

0.9

7

I B

ekang

Sim

ilar

to

H

aw

aij

ar

pre

par

atio

n

exce

pt

the

linin

g

in

the

bam

boo

bas

ket

w

ere

eith

er

Call

icarp

a

aro

ria

R

oxb

. or

Phry

niu

m

sp.

(Tam

ang e

t al.

, 2009).

Bad

a b

azar

A

izaw

l M

izora

m

5

7.3

9 ±

0.0

9

Fer

men

ted

fish

, H

ilsa

(Ten

ualo

sa

ilis

ha,

(F.

Ham

ilto

n,

AA

L

ona I

lish

Des

cale

d,

beh

eaded

fre

sh h

ilsa

fis

h

wer

e cu

t dia

gonal

ly

into

p

iece

s

wit

hout

rem

ovin

g t

he

gut.

Sal

t w

as

added

at

a

rati

o

of

4:1

w

ith

the

fish

, la

yer

ed in

a

bam

boo b

asket

and k

ept

for

48 h

in d

ark a

t ro

om

Bel

tola

mar

ket

G

uw

ahat

i A

ssam

3

5.3

5 ±

0.1

4

32

1822)

tem

per

ature

. A

gai

n,

this

w

as

pac

ked

in

a

tin

conta

iner

fi

lled

wit

h b

rine.

It

was

kep

t in

dar

k f

or

4-6

m

onth

s fo

r nat

ura

l

ferm

enta

tion (

Maj

um

dar

and B

asu,

2010).

F

Lona I

lish

S

ame

as a

bove.

L

ake

choum

uni

and B

arta

la

Agar

tala

T

rip

ura

5

5.4

4 ±

0.1

9

Y

Ilis

ha n

gari

W

hole

fi

sh

was

use

d

for

this

pre

par

atio

n.

The

fish

es

wer

e

rubb

ed

wit

h

turm

eric

(C

urc

um

a

long

a

L.)

p

ow

der

an

d

suff

icie

nt

salt

was

als

o a

dded

. It

was

pre

ssed

in

a ti

n

conta

iner

. F

erm

enta

tion

norm

ally

req

uir

ed 4

-6 m

onth

s.

Pal

tan b

azaa

r

and B

elto

la

mar

ket

Guw

ahat

i A

ssam

3

5.2

8 ±

0.1

2

33

Fer

men

ted

fish

(P

unti

us

sop

hore

,

Ham

.)

B

Ng

ari

D

ried

fis

h w

as w

ashed

wit

h w

ater

and

dra

ined

; it

w

as

spre

ad

over

poly

then

e sh

eet

and p

ress

ed u

sing

hea

vy w

eights

or

by l

egs

cover

ed

wit

h gunny b

ags.

S

pec

ial

eart

hen

pots

(4

5-5

0

kg

cap

acit

y)

whic

h

inner

su

rfac

e w

as

coat

ed

wit

h

must

ard

oil

w

ere

tightl

y

pac

ked

wit

h

the

pre

ssed

dri

ed

fish

. T

he

pots

w

ere

seal

ed t

ightl

y an

d kep

t

for

incu

bat

ion

for

6–

12

month

s

(Jey

aram

et

al.

, 2009).

Khw

aira

mb

and

mar

ket

Imp

hal

M

anip

ur

5

6.0

9 ±

0.2

0

AS

Shed

al

Sim

ilar

to

N

gari

pre

par

atio

n

but

not

tightl

y p

acked

. T

he

ferm

enti

ng

pots

are

als

o s

mal

l in

siz

e (5

–10 k

g

cap

acit

y).

Der

gao

n b

azar

Jo

rhat

A

ssam

5

6.8

2 ±

0.7

9

34

X

Shed

al

Sim

ilar

to

N

gari

pre

par

atio

n

but

not

tightl

y p

acked

. T

he

ferm

enti

ng

pots

are

als

o s

mal

l in

siz

e (5

–10 k

g

cap

acit

y).

Bel

tola

mar

ket

G

uw

ahat

i A

ssam

5

6.4

8 ±

0.3

5

Ns

Shed

al

Sim

ilar

to

N

gari

pre

par

atio

n

but

not

tightl

y p

acked

. T

he

ferm

enti

ng

pots

are

als

o s

mal

l in

siz

e (5

–10 k

g

cap

acit

y).

Bir

bal

mar

ket

and F

atak

baz

ar

Sil

char

A

ssam

5

6.7

5 ±

0.3

0

G

Shed

al/

ber

ma

Sim

ilar

to

N

gari

p

roduct

ion

but

ferm

enta

tion t

ime

is 1

–2 m

onth

s.

Lak

e ch

oum

uni

and B

arta

la

Agar

tala

A

ssam

5

6.4

6 ±

0.2

2

Js

Ng

aw

um

S

imil

ar

to

ngari

p

roduct

ion

but

usu

ally

p

roduce

d

in

ver

y

low

quan

tity

(5

- 6 k

g/b

atch

).

5

6.3

1 ±

0.3

0

35

P

Tung

tap

Was

hed

, dri

ed f

ishes

wer

e p

acked

tightl

y

in

a ti

n

conta

iner

. A

fter

addin

g

salt

it

w

as

ferm

ente

d

for

10–15

day

s (R

apsa

ng

and

Josh

i,

2012).

Bar

a b

azaa

r an

d

Lab

an b

azar

Shil

long

Meg

hal

aya

5

6.4

0 ±

0.2

2

Fer

men

ted

fish

, (n

ot

yet

iden

tifi

ed)

Nb

Shed

al

Sim

ilar

to n

gari

pro

duct

ion b

ut

the

fish

sp

ecie

s use

d a

s ra

w m

ate

rial

is

dif

fere

nt

and n

ot

yet

iden

tifi

ed.

Fat

ak b

azaa

r an

d

Bir

bal

baz

ar

Sil

char

A

ssam

5

7.6

3 ±

0.4

0

Jb

Ng

aw

um

S

imil

ar t

o n

gari

pro

duct

ion b

ut

the

fish

sp

ecie

s use

d a

s ra

w m

ate

rial

is

dif

fere

nt

and n

ot

yet

iden

tifi

ed.

Bad

a b

azaa

r an

d

Tre

asury

squar

e

Aiz

awl

Miz

ora

m

5

8.1

8 ±

0.0

9

Fer

men

ted

smal

l

fish

/shri

mp

pas

te

D

Hen

tak

Equal

am

ount

of

dri

ed s

mal

l fi

sh,

Eso

mus

danri

cus

(F.

Ham

ilto

n,

1822)

and

pet

iole

s of

Alo

casi

a

macr

orr

hiz

a

(L.)

G

.Don

wer

e

crush

ed

and

mak

e a

pas

te,

smal

l

Khw

aira

mb

and

mar

ket

Imp

hal

M

anip

ur

5

6.1

4 ±

0.3

1

36

bal

ls

wer

e pre

par

ed

and

kep

t in

eart

hen

pots

and s

eale

d f

or

nat

ura

l

ferm

enta

tion f

or

a w

eek (

Jeyar

am

et a

l., 2009).

L

Dang

-pui-

thu

Shri

mp

an

d

dif

fere

nt

typ

es

of

smal

l fi

sh

wer

e cr

ush

ed

and

pac

ked

in

b

amb

oo

culm

s ti

ghtl

y

and f

erm

ente

d i

n f

or

15

-20 d

ays.

Bad

a b

azaa

r an

d

Tre

asury

squar

e

Aiz

awl

Meg

hal

aya

5

7.3

5 ±

0.0

6

1L

N

gap

i S

hri

mp

an

d

dif

fere

nt

typ

es

of

smal

l fi

shes

w

ere

use

d

as

raw

mat

eria

l.

Not

pro

per

ly

docu

men

ted.

Ima

mar

ket

Im

phal

M

anip

ur

5

6.7

0 ±

0.1

3

U

Uto

ng

a

kupsu

Sim

ilar

to

hen

tak

pre

par

atio

n,

t

he

pre

par

ed

pas

te

was

p

acked

tightl

y

layer

b

y

layer

in

a

dri

ed

Man

ipuri

mar

ket

Sil

char

A

ssam

2

8.0

2 ±

0.0

8

37

bam

boo

(Bam

busa

tu

lda

Roxb

.

or

Bam

busa

ca

chare

nsi

s

R.B

.

Maj

um

dar

) cu

lm.

Lea

ves

of

Cit

rus

sp.

wer

e la

yer

ed i

nsi

de

the

bam

boo

culm

p

rior

to pac

kin

g an

d se

aled

wit

h m

ud.

It w

as i

ncu

bat

ed f

or

at

leas

t one

month

to

get

good

pro

duct

.

Fer

men

ted

veg

etab

les

AQ

K

ahudi

Must

ard s

eeds

wer

e gro

und a

long

wit

h l

emon j

uic

e an

d s

alt

to m

ake

it a

pas

te o

r m

ore

lik

ely d

ough.

It

was

th

en

wra

pp

ed

wit

h

pla

nta

in

leav

es

and

put

into

a

conta

iner

wit

h

lid.

Thre

e to

fi

ve

day

s

ferm

enta

tion n

orm

ally

req

uir

ed f

or

the

pre

par

atio

n.

Jorh

at m

ain

mar

ket

Jorh

at

Ass

am

5

4.9

5 ±

1.1

4

38

AP

K

hara

li

Sim

ilar

to

ka

hudi

pre

par

atio

n

exce

pt

that

the

addit

ion o

f le

mon

juic

e w

as n

ot

requir

ed h

ere.

Jorh

at m

ain

mar

ket

Jorh

at

Ass

am

5

7.3

6 ±

0.5

3

AF

In

ziang

sang

Wil

ted

must

ard

leav

es

wer

e

crush

ed i

n a

n i

ndig

enous

wooden

crush

er.

Exce

ss

wat

er

was

rem

oved

and p

ress

into

a c

onta

iner

wit

h ai

r ti

ght

seal

ing.

Aft

er 7

-10

day

s fe

rmen

tati

on

in

room

tem

per

ature

, th

e fe

rmen

ted

juic

e

was

extr

acte

d a

nd c

once

ntr

ated

by

boil

ing

to

form

p

aste

(D

as

and

Dek

a, 2

012).

New

mar

ket

D

imah

pur

Nag

alan

d

5

5.3

0 ±

0.2

1

SB

Soib

um

S

ucc

ule

nt

bam

boo

shoots

(Den

dro

cala

mus

ham

ilto

nii

N

ees

and

A

rn.

ex

Munro

) w

ere

Kak

chin

g

mar

ket

Kak

chin

g

Man

ipur

5

4.5

±

0.2

0

39

def

oli

ated

an

d

slic

ed,

pac

ked

tightl

y i

nto

a b

amb

oo c

ham

ber

and

allo

wed

to

fe

rmen

t fo

r 6

–12

month

s (J

eyar

am e

t al.

, 2009).

SD

Soid

on

Ap

ical

m

eris

tem

of

a p

arti

cula

r

bam

boo,

Tei

nost

ach

yum

w

ighti

i

(Munro

) B

edd.

loca

lly c

alle

d n

aat

is

excl

usi

vel

y

use

fo

r so

idon

pre

par

atio

n.

The

apic

al m

eris

tem

s

wer

e def

oli

ated

an

d

des

ired

port

ion w

ere

tran

sver

sely

cut

into

pie

ces.

A

fter

w

ashin

g,

this

cu

t

pie

ces

wer

e su

bm

erged

in a

mil

ky

star

ter

cult

ure

fro

m p

revio

us

bat

ch.

Usu

ally

, G

arc

inia

p

enducu

lata

Roxb

. ex

B

uch

. -H

am

. fr

uit

sl

ices

Bis

hen

pur

mar

ket

Bis

hen

pur

Man

ipur

5

4.1

6 ±

0.1

7

40

and r

ice

was

hed

wat

er w

ere

use

d

for

star

ter

pre

par

atio

n.

Fer

men

tati

on

tim

e is

3

-5

day

s

(Jey

aram

et

al.

, 2009).

Fer

men

ted

mea

t

pro

duct

K

Sa’um

T

he

pork

fa

t w

ere

was

hed

an

d

boil

ed in

sm

all

quan

tity

of

wat

er

for

1-2

h

unti

l it

b

ecom

es

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2 d

ays.

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tan

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and

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mto

la

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ahat

i

and S

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am

5

3.9

4 ±

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7

41

1M

M

isti

doi

Cow

m

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, co

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mal

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. S

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6

42

3.3 Screening for fibrinolytic and caseinolytic activities

The homogenate was centrifuged at 10,000 ×g for 10 min at 4 °C (5810 R,

Eppendorf) and the supernatant was used for studying fibrinolytic activity. Fibrin plate

assay was carried out by Astrup and Mullertz (1952) method with modifications as

described by Kim et al (1997). Briefly, 6 mL of 0.6% fibrinogen (F3879, Sigma-

Aldrich) in 0.1M phosphate buffer, pH 7.4 was mixed with 6 mL of 1.5% low melting

agarose (V2111, Promega) in the same buffer containing 10 NIH unit of human

thrombin (T6884, Sigma-Aldrich). It was mixed gently and poured in a 90 mm diameter

Petri plate. It was allowed to stand for 1h in room temperature before use. Holes of 2

mm diameter were made using cork borer on the fibrin agar plates in which 4 µL of

crude enzymes were loaded and incubated for 2 h at 37 °C. Plasmin (P1867, Sigma-

Aldrich) in different concentrations were used as standard. The diameter of the halo of

clearing zone was measured for fibrinolytic activity. Five replications of each plasmin

concentration were used for preparing a standard graph to measure the fibrinolytic

activity by using fibrin plate assay. Spectrophotometric assay of the samples were also

carried out with slight modification of the method described by Kim et al. (1997). In 0.2

mL centrifuge tubes 45 µL of 0.6% fibrinogen and 5 µL of 0.01 NIH units/µL of

thrombin were mixed and allowed to stand for 1 h to form fibrin clogs. Further, 40 µL

of 0.1 M phosphate buffer at pH 7.4 and 10 µL of crude enzyme were added to the

fibrin clog and incubated at 37 °C for 30 min in water bath. The reaction was stopped

by addition of 10% trichloroacetic acid (T9159, Sigma-Aldrich). Then the mixture was

kept at room temperature for 20 min before being centrifuged at 16,000 xg for 15 min.

The released tyrosine due to fibrinolytic activity was measured at 280 nm absorbance

43

(A280). A strandard graph for plasmin with five replications of different plasmin

concentration against tyrosine released due to fibrinolytic activity was prepared.

Fibrinolytic activity was calculated by comparing with the plasmin standard.

Fibrinolytic activity equivalent to one plasmin unit (PU) was defined as the A280

equivalent of perchloric acid soluble products released from fibrinogen in a reaction

volume of 100 µL by 1 NIH unit of plasmin in 30 min at pH 7.4 at 37 °C. One A280 was

equivalent to 167 plasmin unit (PU) in the above defined conditions. Similarly, using

0.6% of casein (C7078, Sigma-Aldrich) solution (in 0.1 M phosphate buffer, pH 7.4)

was used for caseinolytic activity. The ratio of fibrinolytic to caseinolytic activity (F/C)

was calculated from the above data.

3.4 Fibrin zymogram based cluster analysis

With some minor modification, the method of Kim and Choi (2000) was

followed for fibrin zymogram analysis. A native polyacrylamide gel of 4% stacking gel

and 12% separating gel copolymerized with 0.12% fibrinogen and 10 µL of thrombin 5

NIH units/mL was used. Four microlitre of the crude enzyme was mixed with equal

volume of sample loading buffer without β-mercaptoethanol and loaded in the

acrylamide gel without heating. Electrophoresis (Mini protean, BioRad) was carried out

initially for 15 min at 60 V followed by 100V for 90 min. The gel was washed with

sterile 2.5% triton-X solution (Sigma-Aldrich, T8787) for 1 h in a platform rocker. It

was followed by 3 washing steps of 15 min each by 25 mL of distilled water to remove

traces of the detergent. It was followed by incubation of the gel at 37 °C for 3 h in the

incubating buffer (50 mM phosphate buffer, pH 7.4 and 0.2% NaN3). The gel was

44

stained with coomassie brilliant blue (Merck) solution (40% methanol, 10% acetic acid

and 0.5% coomassie brilliant blue-R250) for 2 h and de-stained. The colourless clearing

band indicated the enzyme and its isozymes. In this study, we compared the zymograms

of different fermented foods. The zymogram profile was scored manually and cluster

analysis was done based on simple matching similarity co-efficient and unweighted pair

group method with arithmetic mean (UPGMA) using NTSYSpc version 2.20f software.

3.5 Microbial cultures

The homogenate was serially diluted using physiological saline and spread

plated in triplicates over plate count agar (PCA) for mesophilic aerobes, de Man Rogosa

and Sharpe (MRS) agar supplemented with 1% CaCO3 for facultative anaerobes and

yeast extract peptone dextrose (YEPD) agar supplemented with 100 µg/mL each of

filter-sterilized ampicillin and tetracycline for yeast. PCA and YEPD plates were

incubated for a period of 24-48 h. The MRS plates were incubated in anaerobic Jars

(Merck) with anaerobic gas packs (Anaerocult C, Merck) for maintaining a micro-

aerobic environment for 48-120 h. From each plate 10-15 colonies were subcultured

after selecting on the basis of colony and cell morphology and their relative abundance.

These microbial isolates were maintained as agar slants on respective agar media at 4

°C up to two weeks for further studies. Reference strains namely B. subtilis MTCC

2451, B. amyloliquefaciens MTCC 1270, B. licheniformis MTCC 429, B. cereus MTCC

430, B. circulans MTCC 490 and Bacillus sp. MTCC 1747 (isolated from Indian

Kinema) were also included for comparison. The cultures were revived and purified in

Luria Bertani (LB) agar with the following composition (g/l): casein enzymatic

45

hydrolysate 10; Yeast extract 5; NaCl 10; Agar 15; pH 7.5 and maintained at 4°C during

this study.

3.6 Screening of microbial isolates for fibrinolytic activity

For rapid and economic screening, a direct colony spotting method was

employed. In this method, the previously maintained sub-cultured single colonies were

spotted on the fibrin plates prepared as described elsewhere by using sterile toothpicks.

The inoculated fibrin plates were incubated at 37 °C for up to 16 h with regular

observation for every 1 h of incubation. The colonies showing clearing zones on the

fibrin plates were selected as fibrinolytic isolates. The selected fibrinolytic bacterial

isolates were grown in 5 mL Luria Bertani (LB) broth and yeast isolate in YEPD broth

at 30 °C using shaking incubator at 250 × g for 24-72 h depending on its growth rate.

The culture broth was divided into three portions; one portion (1 mL) was stored as 25%

glycerol stock in -80 °C, the second portion (2 mL) was utilized for DNA extraction and

the third portion (2 mL) was used for fibrinolytic assay. For this assay the broth culture

was centrifuged at 10,000 ×g for 8 min at 4 °C. One mL of the culture supernatant was

mixed with three volumes of acetone and kept overnight in 4 °C to allow the enzyme to

precipitate. It was then centrifuged at 10,000 ×g for 10 min and the precipitate was

lyophilized until it became dry. The dried protein was dissolved in 100 µL of 0.1 M

phosphate buffer, pH 7.4 to prepare crude enzyme solution. The spectrophotomertic

assay for fibrinolytic activity was carried out as mentioned earlier.

46

3.7 Amplified ribosomal DNA restriction analysis

DNA from the bacterial isolates was extracted by lysozyme (62971, Sigma-

Aldrich) and mutanolysin (M9901, Sigma-Aldrich) treatment was followed by heat lysis

method. The quantity and purity of the extracted DNA was checked in

spectrophotometer (ND-1000; NanoDrop Technologies, USA). The 16S rRNA gene

was amplified using universal primer pair by the following PCR program. In short, PCR

mixture of 25 µL contains 30 ng of cell-free DNA lysate, 1 X PCR Buffer [10 mM

Tris.Cl (pH 8.3 at 25 °C), 50 mM KCl, 0.001 % gelatin], 2.5 mM MgCl2, 0.8 µM of

each forward primer 5¢-AGAGTTTGATCCTGGCTCAG-3¢ and reverse primer 5¢-

AAGGAGGTGATCCAGCCGCA-3¢, 200 µM of each dNTP, 1.5 U of Taq DNA

polymerase and final volume make-up with DNase-free sterile deionized MilliQ water.

The reaction mixture was subjected to 10 min initial denaturation at 94 °C, followed by

36 cycles of 94 °C for 1 min denaturation, 60 °C for 1 min annealing and 72 °C for 30

sec for elongation with final extension of 7 min at 72 °C to complete the PCR

amplification. The amplification was confirmed by horizontal gel electrophoresis in

0.8% agarose by comparing its size with 1Kb DNA ladder (G5711, Promega). The

amplified products were digested by restriction enzymes in the order HinfI (R620,

Promega), RsaI (R637, Promega) and CfoI (R624, Promega) (Jeyaram et al., 2010)

separately for each enzyme following manufacturer’s instructions. The digested

products were subjected to electrophoresis using 0.5 µg/mL ethidium bromide

containing 1.5% agarose gel at constant voltage of 80V for 2 h in 0.5X TBE buffer.

Gels were documented and analysed using gel documentation system (GelDoc EQ,

BioRad, USA). ARDRA groups were formed based on similar digestion profile.

47

3.8 Internal transcribed spacer restriction (ITS-RFLP) analysis

DNA from the isolates was extracted by lysozyme (62971, Sigma-Aldrich)

treatment followed heat lysis method (Jeyaram et al., 2010). The quantity and purity of

the extracted DNA was checked in spectrophotometer (ND-1000; NanoDrop

Technologies, USA). The 16S rRNA-23S RNA gene internal transcribed spacer (ITS)

was amplified using universal primer pair 16-1A (5’-GAATCGCTAGTAATCG-3’, 23-

1B 5’-GGGTTCCCCCATTCGGA-3’) by following PCR program. In short, PCR

mixture of 25 µl contains 30 ng of cell-free DNA lysate, 1 X PCR Buffer [10 mM

Tris.Cl (pH 8.3 at 25 °C), 50 mM KCl, 0.001% gelatin], 2.5 mM MgCl2, 0.8 µM of each

forward primer 16F-R2 and reverse primer 23SR-R10, 200 µM of each dNTP, 1.5 U of

Taq DNA polymerase and final volume make-up with DNase-free sterile deionized

MilliQ water. The reaction mixture was subjected to 10 min initial denaturation at 94

°C, followed by 36 cycles of 94 °C for 1 min denaturation, 55 °C for 1 min annealing

and 72 °C for 30 sec for elongation with final extension of 7 min at 72 °C to complete

the PCR amplification. The amplification was confirmed by horizontal gel

electrophoresis using 0.5 µg/mL ethidium bromide containing 0.8% agarose by

comparing its size with 1 kb DNA ladder (G5711, Promega). The amplified products

were double digested by restriction enzymes HaeIII and HindIII (Promega) by

following manufacturer’s instructions. The digested products were subjected to

electrophoresis in 1.5% agarose gel at constant voltage of 80V for 2 h in 0.5X TBE

buffer. Gels were documented and analysed using gel documentation system (GelDoc

EQ, BioRad, USA). ITS-RFLP groups were formed based on the similar digestion

profile.

48

3.9 Ribosomal DNA sequencing for identification of the isolates

Representative strains of each ARDRA groups were selected randomly for PCR

amplification of 16S rRNA gene as mentioned elsewhere. Representative strains of each

ITS-RFLP groups were selected randomly for PCR amplification of 16S rRNA gene by

using primers fD1 and rD1 ( Forward fD1: 5’-AGAGTTTGATCCTGGCTCAG-3’,

rD1: 5’-AAGGAGGTGATCCAGCCGCA-3’)) with PCR conditions as described in

earlier section with annealing temperature of 65 °C. Three isolates each from major

ARDRA groups with more than 10 isolates and two isolates each from minor ARDRA

groups were identified by ribosomal DNA sequencing. The amplified products were

purified using NucleoSpin® Extract II gel extraction kit (Macherey-Nagel, Germany)

following manufacturer’s instructions. Sequencing of the purified DNA was carried out

in both the directions with the same primers used for amplification in automated ABI

3100 genetic analyzer (Applied Biosystems, USA) by service provider (GeNei,

Bengaluru, India). For yeast isolate, DNA was extracted as cell-free DNA lysate using

lyticase-heat lysis method. Briefly, yeast cells from 1 mL of 24 h old yeast extract

peptone dextrose (YEPD) broth culture were harvested at 5000 ×g for 5 min. The cell

pellet was washed twice with 1 mL sterile 0.5 M NaCl and sterile deionized water (Milli

Q, Millipore, Molsheim, France). The cells pellet was resuspended in 500 µL of 1X TE

buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) with 10 μL of lyticase (5U/µL) (Sigma-

Aldrich) and incubated at 37°C for 1 h. The cell suspension was incubated at 95 °C for

20 min and centrifuged at 10,000 ×g for 10 min at 4°C. The DNA present in the

supernatant of cell-free lysate was quantified spectrophotometrically (Nanodrop ND-

1000, NanoDrop Technologies Inc, Rockland, USA). The 26S rDNA D1/D2 region was

49

amplified using primers NL1 (5¢-GCATATCAATAAGCGGAGGAAAAG-3¢) and NL4

(5¢-GGTCCGTGTTTCAAGACGG-3¢) with PCR conditions as described in section 3.6

with annealing temperature of 52 °C. The electropherogram data of sequences were

validated using Chromas LITE 2.01 software (www.technelysium.com.au) and the

sequence similarity search was carried out in NCBI and Ribosomal Database project

(RDP) databases and the closest known relatives were identified. A neighbour joining

phylogenetic tree was constructed using Mega software (Tamura et al., 2007). The

evolutionary distance of closely related type strain was computed by Kimura-2

parameter.

3.10. Optimisation of growth conditions for fibrinolytic enzyme production

3.10.1 Bacillus cultures

Casein broth containing 0.5% of casein, 0.5% NaCl, 0.25% K2HPO4, 0.25%

dextrose, 0.1% beef extract and 0.2% peptone with pH of 8.5 was used for fibrinolytic

enzyme production. A seed culture of 2 mL casein broth was inoculated with loopful

culture of freshly grown single colony of B. subtilis MH12B1 (MCC 2516), B.

amyloliquefaciens MH18B1 (MCC 2517), B. subtilis MH12B3 (MCC 2511) and B.

subtilis MH10B5 (MTCC 5481) separately and incubated at 37°C at 200 rpm shaking

speed for 16 h and further inoculated to 100 mL of casein broth with similar condition.

The seed culture was transferred to 5 L flask containing 2.5 L of casein broth in

fermenter (Bioflo, New Brunswick) at 37°C with agitation speed of 200 rpm and

airflow rate of half volume/ min for 18 h.

50

3.10.2 Enterococcus faecalis ALL10 and Vagococcus carniphilus ALL3

Single colonies of E. faecalis and V. carniphilus were inoculated in the fish

waste broth at different incubation temperature of 25 °C, 30 °C, 35 °C and 40 °C were

allowed to grow up to 120 h. The growth was monitored by measuring OD at different

time interval up to 120 h. The fibrinolytic activity was measured by spectrophotometric

method as described in the following section on daily basis after 24 h, 48 h, 72h, 96 h

and 120 h. Similarly, the microbial cultures were also inoculated at 35 °C in different

starting pH with pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0 and pH 9.0 for checking the

optimum pH for fibrinolytic enzyme production. The fibrinolytic activity was also

measured by spectrophometric method as described earlier.

3.11 Production and purification of fibrinolytic enzymes

3.11.1 Fibrinolytic enzymes from five Bacillus strains

The culture broth was centrifuged at 6000 ×g for 10 minutes. The culture

supernatant was mixed with 3 volumes of acetone and kept overnight at 4 °C. The

fibrinolytic mucilage complex floated as clump along with the protein mass settled at

the bottom of the container was recovered without centrifugation and lyophilised to

remove residual acetone. This precipitate was added with 20 mL of 0.1M phosphate

buffer, pH 7.4 and kept in 4 °C for 3 to 5 days which allowed dissolving the mucin

complex. The crude enzyme extract was dialysed with 10 kDa cut off membrane in 10

51

mM Tris buffer, pH 7.8 at 4 °C overnight. The buffer was changed twice during

dialysis. This crude enzyme extract was filtered through 0.2 µm membrane filter and

injected to the low pressure liquid chromatography (AKTA prime Plus, GE healthcare)

with anion exchange DEAE Sepharose-FF column. The column was preconditioned

with buffer A (20 mM Tris-Cl buffer, pH 7.8) with a flow rate of 2.0 mL/min. The

bound protein was eluted with a 0% to 50% gradient of buffer B (buffer A + 0.5 M

NaCl) for a run volume of 200 mL and 100% buffer B was used with additional volume

of 40 mL. The active fractions were precipitated using 3 volumes of acetone,

centrifuged and lyophilized. The dried precipitate of active fraction was dissolved in

buffer A and subjected to gel permeation chromatography with sephacryl-200S matrix

with elution buffer of 20 mM Tris-Cl buffer with 0.25 M NaCl, pH 7.8, flow rate of 1

mL/min. Collected active fractions were checked for activity on fibrin plate. The active

fractions were pooled and concentrated by lyophilisation and stored at 4 ºC for further

characterisation.

3.11.2 Fibrinolytic enzymes from E. faecalis ALL10 and V. carniphilus ALL3

The above two strains were grown in 5 mL of fish waste broth at 35 °C as seed

culture in a shaking incubator at 250 rpm for 24 h, which was further inoculated to 100

mL of the same broth and grown for another 24 h in similar conditions. This serve as

the seed culture for the 5 L fermenter (Bioflo, New Brunswick) containing 2.5 L of the

fish waste broth set at 35°C with agitation speed of 200 rpm and airflow rate of half

volume/ min for 48h. The cells from culture broth were separated by centrifugation at

6000×g for 10 min. The culture supernatant was fractionated by gradually adding

52

ammonium sulphate upto 80% saturation and incubating overnight at 4°C. The protein

precipitate was obtained by centrifugation (14000xg, 15 min) at 4°C, and the precipitate

was dissolved in 20 mL of 0.1M phosphate buffer, pH 7.4. This crude enzyme extract

was dialysed, filtered and injected to the low pressure liquid chromatography (AKTA

prime Plus, GE healthcare) with anion exchange Q Sepharose- FF column as described

earlier. The column was preconditioned with buffer A (20 mM Tris-Cl buffer, pH 8.5)

with a flow rate of 2.0 mL/min. The bound protein was eluted with a gradient of buffer

B (buffer A + 0.5 M NaCl) for a run volume of 200 mL and 100% buffer B was used

with additional volume of 40 mL. The active fractions were pooled and concentrated

using Amicon Ultra centrifugal filtration (Millipore) with 10 kDa cut-off and

lyophilized and subjected to gel permeation chromatography with sephacryl-100S

matrix, flow rate of 1 mL/min. Collected active fractions were checked for activity on

fibrin plate and stored at 4 ºC.

3.11.3 Fibrinolytic enzymes from Streptomyces sp.

The strains Streptomyces sp. LY9 was grown in LB broth at 37 °C as described

elsewhere for 72h. The culture broth was carefully separated by decanting through

muslin cloth in a clean beaker. Enzyme was extracted by acetone precipitation method

as described and dialysed with 10 kDa cut off membrane in 10 mM Tris buffer, pH 8.5

at 4°C overnight. This crude enzyme extract was filtered through 0.2 µm membrane

filter and injected to the anion exchange Q Sepharose-FF column. The column was

preconditioned with buffer A (20 mM Tris-Cl buffer, pH 8.5) with a flow rate of 1.5

mL/min. The bound protein was eluted with a gradient of buffer B (buffer A + 0.5 M

53

NaCl) for a run volume of 200 ml and 100% buffer B was used with additional volume

of 40 mL. The active fractions were concentrated using Amicon Ultra centrifugal

filtration (Millipore) with 10 kDa cut-off and subjected to gel permeation

chromatography with sephacryl-100S matrix in the condition described elsewhere. The

active fractions were concentrated and lyophilised and stored at 4 ºC for further

characterisation.

3.12 SDS-PAGE and fibrin zymogram

Purity of the enzyme was checked by running sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE) and its activity was related with fibrin

zymography. The molecular mass of the purified enzyme was determined by SDS-

PAGE using 12% polyacrylamide gel (Laemmli, 1970). The medium range protein

markers and broad range protein markers (Promega and Bangalore Genei) were used for

standard graph preparation. For fibrin zymography, the method of Kim and Choi (2000)

with minor modifications was followed. SDS-PAGE with 4% stacking gel and 12%

separating gel was copolymerized with 0.12% (w/v) fibrinogen and 10 µl of thrombin 5

NIH units/ml was used. The purified enzyme was mixed with equal volume of sample

loading buffer without β-mercaptoethanol and loaded in the acrylamide gel without

heating. Electrophoresis (Mini protean, BioRad) was carried out initially for 15 min at

60 V followed by 100 V for 90 min. The gel was washed with sterile 2.5% Triton-X

solution (Sigma-Aldrich, T8787) for 1 h in a platform rocker. It was followed by 3

washing steps of 15 min each by 25 ml of distilled water to remove traces of the

detergent. It was followed by incubation of the gel at 37 °C for 3 h in the incubating

54

buffer (50 mM phosphate buffer, pH 7.4 and 0.2% NaN3). The gel was stained with

coomasie brilliant blue (Merck) solution (40% methanol, 10% acetic acid and 0.5%

coomassie brilliant blue-R250) for 2 h and de-stained. The colourless clearing band

developed during fibrin zymography indicated the fibrinolytic activity of purified

enzyme.

3.13 Effect of pH and temperature

The fibrin clog (as described elsewhere) was added with 40 µL of 0.5 M buffer

with different pH (sodium acetate buffer for pH 3.0 to 6.0, sodium phosphate buffer

for pH 7.0 to 8 and glycine NaOH buffer for pH 9 to 11) and 10 µL of purified enzyme

(0.5 mg), incubated at 37°C for 30 min. The reaction was stopped by adding 110 µL of

10% (v/v) TCA and allowed to stand for 30 min, centrifuged at 12,000 ×g for 15 min,

and the absorbance of supernatant was measured at 280 nm. The effect of temperature

was examined in 20 mM phosphate buffer with pH 7.2 and the reaction mixture was

incubated at temperature 30 to 65° C. The enzyme activity was expressed as % of

relative activity in comparison to control without enzyme addition.

3.14 Effect of metal ions and protease inhibitors

The fibrin clog prepared (as mentioned somewhere else) was added with 40 µl

0.1 M phosphate buffer pH 7.4, 5 µL of purified enzyme (0.5 mg/mL) and 5 µL of

different metal ions or protease inhibitors and incubated at 37°C for 30 min. The metal

ions studied were KCl, CaCl2, MgCl, ZnSO4, FeSO4, CuSO4, MnCl2 and Co(NO3)2 with

55

the final concentration of 2 mM . The protease inhibitors were 2,2-bypirimidine,

leupeptin, SBTI, PMSF, pepstatin-A and EGTA with final concentration of 1 mM and

EDTA with 5 mM concentration. The enzyme activity was expressed as relative activity

% in comparison of enzyme treated with metal ions or protease inhibitors to that of the

untreated control.

3.15 Amidolytic activity and enzyme kinetics

The amidolytic activity of the purified enzymes was measured using following

synthetic chromogenic substrates N-Succinyl-Ala-Ala-Pro-Phe-pNA, D-Val-Leu-Lys-

pNA, D-Val-Leu-Arg-pNA, D-Ile-pro-Arg-pNA and NA-Benzoyl-L-Arg-pNA. A

reaction mixture (100 µL) of 0.5 mM concentration of synthetic substrate and 0.5 µg of

purified enzyme was incubated for 5 min at 37°C and the liberated p-nitroanilide was

measured by spectrophotometer at 405 nm in comparison with a standard graph of p-

nitroanilide and the amidolytic activity was expressed as µM of p-nitroanilide released/

min/µg. The chromogenic substance N–Succinly-Ala-Ala-Pro-Phe-pNA (specific for

subtilisin) with a concentration range of 0.025 – 0.5 mM was used for enzyme kinetics

studies. The kinetic constants Km and Vmax of the fibrinolytic enzymes were determined

by the method of Lineweaver and Burk (1934).

3.16 Fibrinogen degradation assay

Fibrinogenolytic activity was determined by incubation of 40 µl of 1% (w/v)

human fibrinogen with 0.5 µg of purified enzymes and incubated at optimum

56

temperature of respective enzymes for 0, 2, 5, 10, 20, 30, 40, 60 min. The reaction was

stopped by addition of sample buffer with 10 mM PMSF, boiled for 3 min, analysed by

10% SDS-PAGE gel and stained with commassie brilliant blue.

3.17 Cloning and sequencing of fibrinolytic enzyme genes

Fifteen primer pairs were adapted from the earlier reported fibrinolytic enzyme

genes from the Bacillus strains for amplifying the open reading frame (ORF). In

addition to above primers set, five more primer pairs were designed from the available

NCBI GenBank data to amplify the fibrinolytic enzyme gene sequences. The primer

sequences, amplicon size and annealing temperature used are shown in Table 3.2. In

short, PCR mixture of 25 µL contains 30 ng of cell-free DNA lysate, 1X PCR Buffer

[10 mM Tris Cl (pH 8.3 at 25 °C), 50 mM KCl, 0.001% gelatin], 2.5 mM MgCl2, 0.8

µM of each primer, 200 µM of each dNTP, 1.5 U of Taq DNA polymerase and final

volume make-up with DNase-free sterile deionized MilliQ water. The reaction mixture

was subjected to 10 min initial denaturation at 95 °C, followed by 35 cycles of 94 °C for

1 min denaturation, respective annealing temperature for 1 min and 72 °C for 2 min

elongation with final extension of 15 min at 72 °C to complete the PCR amplification.

The amplification was confirmed by horizontal gel electrophoresis in 1% agarose gel by

comparing the product size with 100 bp and 1kb DNA ladders (G6951 and G5711,

Promega). The amplified products were purified using NucleoSpin® Extract II gel

extraction kit (Macherey-Nagel, Germany) following manufacturer’s instructions. The

amplified DNA fragments were ligated into pGEM-T Easy vector or TOPO-TA cloning

system (Promega) and introduced into Escherichia coli JM109 competent cells. The

57

cloned DNA fragments were released by EcoRI digestion and sequenced (ABI 3100

DNA sequencer, Applied Biosystems) as mentioned elsewhere. The sequence similarity

search was carried out in NCBI and a pair wise sequence alignment with closely related

fibrinolytic enzyme genes using Bioedit (version 5.0.9 and Clustal X (version 1.8). The

cluster analysis was performed using Mega software. The gene sequences were

translated into amino acid sequence by Generunner software (version 3.05) and the

deduced amino acid sequence similarities were compared with other reported closely

related fibrinolytic enzymes catalytic centre.

58

Tab

le 3

.2 P

rim

er s

equen

ces

des

igned

/ u

tili

zed f

or

PC

R a

mp

lifi

cati

on o

f fi

bri

noly

tic

gen

es f

rom

sel

ecte

d B

aci

llus

stra

ins

Pri

mer

set

Pri

mer

Nam

e

Seq

uen

ces(

5’-

3’)

P

rim

er

size

(bas

es)

Am

pli

con

size

(Kb

)

PC

R

annea

lin

g

tem

per

ature

(oC

)

Sp

ecif

ic

amp

lifi

cati

on

Ref

eren

ce

A

Sub

F

G

CG

CA

GT

CC

GT

GC

CT

TA

C

18

0.8

53

Yes

Pen

g e

t al.

, 2003

Sub

R

T

TA

CT

GA

GC

TG

CC

GC

CT

GT

AC

21

B

FE

ckF

A

TG

AT

GA

GG

AA

AA

AG

AG

TT

TT

TG

GC

- 25

0.8

52

No

Kim

et

al.

, 1996

FE

ckR

C

AT

CC

GA

CC

AT

AA

TG

GA

AC

GG

AT

TC

25

C

FE

dF

CT

GG

TA

CC

GT

GA

GA

AG

CA

AA

AA

AT

TG

TG

G

29

1.2

53

Yes

Wan

g e

t al.

, 2007

FE

dR

CG

AT

CA

GA

TT

AC

TA

TT

AT

TG

TG

CA

GC

T

27

D

ppB

H4

F

AC

GA

TC

CA

TG

GT

TA

AC

AC

GA

TG

AC

GC

TA

28

1.2

54

No

Dab

onne

et

al.

,

2005

ppB

H4R

T

AT

AC

TC

GG

TG

AA

TC

AA

TG

CT

TT

TG

GA

AG

29

E

F1 F

G

GA

TC

CT

CG

GT

CA

AG

AA

AG

CC

GA

AG

AC

27

1.7

53

60

Yes

P

res

ent

stu

dy

F1R

A

AG

CT

TG

AC

CG

CT

AC

GG

CA

TC

GA

GC

TG

27

F

F2F

G

GA

TC

CT

GG

CG

TT

CG

GC

AG

CA

CG

TC

TC

27

0.9

43

60

Yes

P

res

ent

stu

dy

59

F2R

A

AG

CT

TA

CG

TG

CG

GA

GA

TG

CC

AT

TG

AC

27

G

F3F

G

GA

TC

CT

CA

CG

GA

AG

CA

CA

CG

CA

GG

TC

27

1.6

17

61

Yes

P

res

ent

stu

dy

F3R

G

AA

TT

CG

TT

GA

TC

CT

CC

GG

TG

CT

TG

TG

27

H

F4F

G

CA

AC

AT

GT

CT

GC

GC

AG

GC

TG

21

0.9

87

58

Yes

P

res

ent

stu

dy

F4R

C

TT

GC

GC

GT

TT

GT

CC

AA

GT

CG

21

I F

5F

C

CG

TG

CC

TT

AC

GG

CG

TA

TC

AC

21

0.8

20

56

Yes

P

res

ent

stu

dy

F5R

T

AT

TG

AG

CT

GC

CG

CC

TG

TA

CG

21

K

P3F

7F

C

TG

AA

TC

CA

TG

GC

AG

GG

AA

AT

CA

AA

CG

G

28

1.5

58

Yes

Xia

o e

t al.

, 2004

P

4F

7R

G

TC

GG

AT

CC

TT

AC

TG

AG

CT

GC

CG

CC

TG

TA

C

30

L

R1F

8F

T

CA

CA

GC

TT

TT

CT

CG

GT

C

18

1.8

42

No

Pen

g e

t al.

, 2003

R2F

8R

T

GA

TC

CG

AT

TA

CG

AA

TG

C

18

M

F9F

G

TG

AG

AR

GC

AA

AA

AR

KT

RT

GG

AT

CA

G

26

1.1

4

57

Yes

Kim

et

al.

, 2

006

F9R

A

WT

GT

GC

WG

CT

GC

TT

GT

AC

GT

TG

AT

Y

26

N

F10F

C

GC

GG

AT

CC

TT

GA

AA

AA

GG

GG

AT

CA

TT

CG

C

TT

TC

34

1.8

64

Yes

Kho et

al.

, 2005

F10R

A

CG

CG

TC

GA

CG

TC

TT

CA

AC

AG

TG

AA

AG

GT

T38

60

CT

TC

GG

AC

O

F11F

A

GG

AT

CC

CA

AG

AG

AG

CG

AT

TG

CG

GC

TG

TG

T

AC

32

1.5

64

Yes

Kim

et

al.

, 2

009

F11R

A

GA

AT

TC

TT

CA

GA

GG

GA

GC

CA

CC

CG

TC

GA

T

CA

32

P

F12F

C

AG

AA

TA

GT

CT

TT

TA

AG

TA

AG

TC

TA

C

26

1.2

37

58

Yes

Agre

bi

et

al.

, 2009

F12R

A

AG

CA

GG

TA

TG

GA

GG

AA

CC

TG

C

22

Q

R

F13F

C

TG

AA

TC

CA

TG

GC

AG

GG

AA

AT

CA

AA

CG

G

28

0.8

5

58

Yes

Zhan

g

et a

l., 2005

F13F

1

CG

CC

AT

GG

CG

CA

GT

CC

GT

GC

CT

TA

C

25

58

F13R

G

TC

GG

AT

CC

TT

AC

TG

AG

CT

GC

CG

CC

TG

TA

C

30

0.8

5

Y

es

61

3.18 Nucleotide sequence accession numbers

The 16S rRNA gene sequences and the cloned fibrinolytic gene sequences

obtained during this study were submitted to NCBI GenBank nucleotide sequence

database using the BankIt web-based submission tool (http://www.ncbi.nlm.nih.gov)

under the accession numbers JQ266337 to JQ266358, JN255700, JN255701, JQ255702,

JN558839, GQ268319, KJ470768, KJ470769, KJ470770, KJ470771, KJ470772 and

KJ470773.

3.19 2D gel electrophoresis and MALDI-TOF-MS peptide mass fingerprinting

for identification of the fibrinolytic enzyme

The purified enzyme was processed for 2D gel electrophesis by using the

ReadyPrep 2D Cleanup Kit (Biorad, USA) following the company’s instructions. The

cleaned protein was dissolved in ReadyPrep Rehydration buffer (Biorad). Immobilized

pH gradient (IPG) strips (7 cm, pH 3 to 6; Biorad, USA) were rehydrated overnight with

approximately 25 μg (130 μL) protein and then subjected to isoelectric focusing (IEF).

IEF was performed with a PROTEAN i12 IEF Cell system (Biorad, USA) at 20°C with

a 3-phase gradient program: 500 V for 0.25 kVh, 4000 V for 5.25 kV h, and 4000 V for

28 kVh. Following IEF, each strip was equilibrated for 12 min in 2 mL of equilibration

buffer-I [50 mMTris-HCl, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002%

bromophenol blue] containing 1% (w/v) dithiothreitol. This step was followed by a

second equilibration step with equilibration buffer-II containing 2.5% (w/v)

Iodoacetamide in the SDS equilibration buffer-I. The strips were then loaded onto 12%

polyacrylamide gel. The second dimension of the separation was performed using the

Mini PROTEAN cell system (BIORAD) and with the following 2-phase gradient

62

program: 60 V for 15 min and 90 V for 1 h. After electrophoresis, the gels were stained

with Coomassie Brilliant Blue (CBB) R250. The 2D gels were documented in the

ChemiDoc (Biorad) and the protein spots were subjevted to MALDI-TOF-MS analysis.

The spot in the gel was excise carefully and processed as per the company manuals.

Matrix-assisted laser desorption ionization (MALDI)–mass spectrometry (MS) was

performed using an UltrafleX III MALDI-TOF/TOF (Bruker Daltonik) analyzer. The

following parameters were used for database search: (1) taxonomy group: Bacteria

(Eubacteria) (2) mass tolerance of 150 ppm, (3) one missed tryptic cleavage allowed,

(4) carboamidomethylation of cysteine (as a fixed modification) and (5) oxidation of

methionine (as a variable modification). The spectra were acquired in reflector mode in

the mass range of 700 to 3,500 Da. and peak lists were generated by Bruker Daltonics

flexAnalysis (Bruker Daltonik) and searched against bacterial proteins in the NCBI

nonredundant database (NCBInr20140323) and NCBInr using the Mascot search

engine (Matrix Sciences, London, United Kingdom). Protein identifications were

assigned where the MOESE scores were significant (P < 0.05).