Production

of

a

recombinant

polyester-cleaving

hydrolase

from

Thermobifida

fusca

in

Escherichia

coli

The

hydrolase

-LRB-

Thermobifida

fusca

hydrolase

TfH

-RRB-

from

T.

fusca

was

produced

in

Escherichia

coli

as

fusion

protein

using

the

OmpA

leader

sequence

and

a

tag

Productivity

could

be

raised

more

than

Both

batch

and

fed-batch

cultivations

yield

comparable

cell

specific

productivities

whereas

volumetric

productivities

differ

largely

In

the

fed-batch

cultivations

final

rTfH

concentrations

of

g

L

could

be

achieved

In

batch

cultivations

the

generated

rTfH

is

translocated

to

the

periplasm

wherefrom

it

is

completely

released

into

the

extracellular

medium

In

fed-batch

runs

most

of

the

produced

rTfH

remains

as

soluble

protein

in

the

cytoplasm

and

only

a

fraction

of

about

is

translocated

to

the

periplasm

Migration

of

periplasmic

proteins

in

the

medium

is

obviously

coupled

with

growth

rate

and

this

final

transport

step

possibly

plays

an

important

role

in

product

localization

and

efficacy

of

the

Sec

translocation

process

Introduction

Since

more

than

years

the

interest

in

biodegradable

plastics

-LRB-

polymeric

materials

which

can

be

cleaved

by

enzymes

and

assimilated

by

microorganisms

respectively

-RRB-

is

steadily

raising

and

nowadays

a

number

of

commercial

products

are

on

the

market

Polymers

containing

ester

groups

especially

polyesters

play

a

central

role

as

they

are

easily

subject

to

microbial

attack

the

degradation

rate

being

largely

dependent

on

their

physical

structure

and

chemical

composition

First

attempts

to

produce

biodegradable

plastics

were

based

on

natural

polymers

like

poly

-LRB-

β-hydroxybutyrate

-RRB-

-LRB-

PHB

-RRB-

However

their

material

properties

proved

to

be

insufficient

and

could

not

compete

with

those

of

synthetic

polyesters

Tokiwa

and

Suzuki

-LSB-

-RSB-

already

showed

that

also

synthetic

aliphatic

polyesters

are

accessible

to

a

biological

degradation

However

aliphatic

polyesters

exhibit

similar

disadvantages

concerning

their

material

properties

-LRB-

e.g.

minor

tensile

strength

low

melting

points

-RRB-

and

therefore

are

hardly

applicable

for

every

day

life

products

On

the

other

hand

polyesters

such

as

poly

-LRB-

ethylene-terephthalate

-RRB-

-LRB-

PET

-RRB-

and

poly

-LRB-

butylene-terephthalate

-RRB-

-LRB-

PBT

-RRB-

exhibit

excellent

material

properties

but

were

classified

as

non-biodegradable

Therefore

aliphatic

aromatic

copolyesters

which

combine

both

acceptable

use

properties

and

sufficient

biodegradability

-LSB-

-RSB-

were

developed

The

material

properties

desired

for

specific

applications

can

be

easily

adjusted

by

the

ratio

of

the

aliphatic

to

the

aromatic

diacid

-LSB-

-RSB-

Commercial

products

are

available

now

under

the

trade

name

Ecoflex

-LRB-

BASF

AG

Germany

-RRB-

Recently

a

thermophilic

actinomycete

was

isolated

and

identified

as

Thermobifida

fusca

-LRB-

former

name

Thermomonospora

fusca

-RRB-

exhibiting

a

remarkable

degradation

capability

for

aliphatic

aromatic

copolyesters

-LSB-

-RSB-

The

extracellular

hydrolase

-LRB-

TfH

-RRB-

responsible

for

the

degradation

of

these

copolyesters

was

purified

from

the

culture

broth

and

classified

as

a

serine

hydrolase

with

the

highly

conserved

G-H-S-M-G

motif

-LSB-

-RSB-

The

mature

hydrolase

consists

of

amino

acids

corresponding

to

a

molecular

weight

of

kDa

Maximum

activity

was

found

at

°C

The

extracellular

hydrolase

gene

was

located

on

an

operon

containing

two

nearly

identical

genes

-LRB-

and

-RRB-

with

an

amino

acid

identity

of

-LSB-

-RSB-

The

enzyme

is

only

expressed

by

T.

fusca

if

a

polyester

such

as

poly

-LRB-

terephthalate

-RRB-

-LRB-

BTA

-RRB-

or

poly

-LRB-

ε-caprolactone

-RRB-

-LRB-

PCL

-RRB-

is

present

in

the

medium

Gouda

et

al.

-LSB-

-RSB-

observed

that

the

amount

of

enzyme

excreted

in

the

medium

correlates

with

the

amount

of

polyester

added

to

the

culture

This

induction

behaviour

as

well

as

adsorption

and

inhibition

phenomena

-LSB-

-RSB-

complicate

a

large-scale

production

of

this

enzyme

-LRB-

TfH

-RRB-

and

therefore

a

simpler

to

handle

recombinant

expression

system

for

producing

TfH

independent

of

the

presence

of

the

polymer

was

highly

desirable

particularly

as

recent

investigations

showed

that

TfH

is

capable

of

degrading

even

PET

-LSB-

-RSB-

In

this

paper

we

report

on

the

heterologous

expression

of

the

polyester-degrading

hydrolase

from

T.

fusca

in

E.

coli

To

this

end

the

TfH-encoding

gene

-LRB-

-RRB-

was

cloned

together

with

a

C-terminal

tag

and

the

ompA-secretion

signal

under

the

control

of

a

thermo-inducible

promoter

This

strategy

should

enable

translocation

of

the

gene

product

by

the

Sec-transport

system

from

the

cytoplasm

into

the

periplasmic

space

where

the

properly

folded

TfH

should

accumulate

after

cleavage

of

the

OmpA

sequence

Several

cultivation

strategies

and

purification

routes

were

considered

The

aim

of

the

investigations

was

to

provide

sufficient

amounts

of

TfH

for

larger-scale

polyester

degradation

studies

-LRB-

low

purification

grade

-RRB-

and

a

highly

purified

TfH

for

mechanistic

studies

and

structural

analysis

Materials

and

methods

Cloning

of

the

recombinant

TfH

Escherichia

coli

-LRB-

DSM

-RRB-

was

used

for

the

production

of

the

recombinant

hydrolase

from

Thermobifida

fusca

-LRB-

rTfH

-RRB-

is

characterized

by

the

following

genetic

markers

supE

thiΔ

-LRB-

lac-proAB

-RRB-

F

-LSB-

proAB

lacIq

-RSB-

-LSB-

-RSB-

The

vector

-LSB-

-RSB-

was

digested

with

NdeI

and

EcoRI

to

obtain

a

DNA

fragment

consisting

of

bp

This

DNA

fragment

was

used

as

a

template

to

clone

a

tag

at

the

C-terminal

end

using

the

primer

set

-LRB-

TAC

TTA

GGA

TCC

TTA

TCA

GTG

GTG

GTG

GTG

GTG

GTG

CTC

GAG

GAA

CGG

GCA

GGT

GGA

-RRB-

and

-LRB-

GTC

ATG

GGC

CAC

TCC

ATG

-RRB-

The

DNA

fragment

was

amplified

using

VENT

DNA

polymerase

-LRB-

New

England

Biolabs

USA

-RRB-

the

cycle

programme

was

run

at

°C

for

min

cycles

of

s

at

°C

s

at

°C

and

min

at

°C

followed

by

an

extension

reaction

for

min

at

°C

The

reaction

mixture

was

designed

according

to

the

description

of

the

manufacturer

The

resulting

PCR-fragment

was

purified

using

the

JETQUICK

PCR

Fragment

purification

kit

-LRB-

GENOMED

GmbH

Germany

-RRB-

was

ligated

to

a

DNA

fragment

obtained

from

the

digestion

of

the

origin

vector

with

NcoI

and

BamHI

The

resulting

plasmid

with

the

secretion

signal

of

OmpA

the

hydrolase

from

T.

fusca

-LRB-

TfH

-RRB-

and

the

affinity

tag

was

used

for

the

expression

of

rTfH

Transformation

of

into

E.

coli

was

done

by

electroporation

Expression

of

the

recombinant

TfH

-LRB-

rTfH

-RRB-

was

controlled

by

the

strong

tandem

promoter

λpRpL

which

is

expressed

by

the

thermolabile

repressor

Media

Louria-Bertani

medium

-LRB-

LB

medium

-RRB-

g

tryptone

g

L

NaCl

g

L

yeast

extract

and

g

L

glucose

was

used

for

cultivations

of

E.

coli

in

the

complex

medium

-LSB-

-RSB-

The

medium

was

autoclaved

at

°C

for

min

Before

cultivation

ampicillin

-LRB-

μg

mL

-RRB-

was

added

A

defined

medium

as

described

by

Korz

et

al.

-LSB-

-RSB-

was

used

for

batch

and

fed-batch

cultivation

of

E.

coli

The

medium

contained

-LRB-

per

litre

-RRB-

-LRB-

-RRB-

g

g

citric

acid

g

glucose

g

g

trace

elements

-LRB-

Fe

-LRB-

III

-RRB-

citrate

mg

mg

mg

mg

mg

mg

Zn

-LRB-

-RRB-

mg

EDTA

mg

-RRB-

and

μL

antifoam

-LRB-

Sigma

Antifoam

Sigma-Aldrich

USA

-RRB-

and

glucose

solution

were

autoclaved

separately

from

the

phosphate

buffer

and

trace

elements

at

°C

for

min

pH

was

adjusted

to

with

M

NaOH

before

Ampicillin

mg

L

and

thiamine

hydrochloride

mg

L

were

sterilized

by

filtration

-LRB-

Sterivex

cut

off

of

μm

Millipore

USA

-RRB-

and

added

before

starting

the

cultivation

The

feed

solution

for

fed-batch

cultivations

contained

the

following

components

-LRB-

in

L

demineralized

water

-RRB-

glucose

g

g

trace

elements

-LRB-

Fe

-LRB-

III

-RRB-

citrate

mg

mg

mg

mg

mg

mg

Zn

-LRB-

-RRB-

mg

EDTA

mg

-RRB-

thiamine

hydrochloride

mg

and

ampicillin

mg

The

feed

solution

was

sterilized

by

filtration

Cultivation

A

bioreactor

-LRB-

Biostat

B.

Braun

Melsungen

Germany

-RRB-

with

L

working

volume

connected

to

an

exhaust

gas

analysis

unit

-LRB-

Sick

Maihak

Germany

-RRB-

was

used

for

the

cultivations

Bioreactor

control

was

done

by

a

computer

operating

the

MFCS

software

-LRB-

B.

Braun

-RRB-

Feed

and

base

reservoirs

were

stored

on

scales

connected

to

the

computer

Starting

conditions

of

the

reactor

were

set

to

°C

rpm

pH

and

L

min

airflow

pH

was

controlled

by

adding

either

-LRB-

in

case

of

the

complex

medium

M

NaOH

-RRB-

or

M

The

minimum

level

of

dissolved

oxygen

-LRB-

-RRB-

was

set

to

and

was

adjusted

automatically

by

the

stirrer

speed

and

aeration

rate

If

the

stirrer

speed

and

aeration

rate

run

at

maximum

pure

had

to

be

enriched

to

the

inlet

airflow

Antifoam

was

added

manually

to

the

cultivation

under

sterile

conditions

if

necessary

Pre-cultures

were

carried

out

in

the

respective

medium

of

the

main

culture

by

inoculation

with

a

glycerol

stock

of

E.

coli

containing

the

plasmid

at

°C

and

rpm

for

h.

Batch

and

fed-batch

cultivation

in

the

bioreactor

was

started

by

adding

mL

of

preculture

In

batch

cultivation

induction

of

rTfH

production

was

started

at

an

OD

of

by

raising

the

cultivation

temperature

from

to

°C

or

°C

respectively

within

about

min

In

the

fed-batch

cultivation

feed

was

started

after

the

initial

glucose

was

consumed

as

indicated

by

a

sharp

increase

in

An

exponential

feed

profile

was

used

For

a

desired

specific

growth

rate

μset

-LRB-

h

-RRB-

the

volumetric

feed

rate

Fglc

-LRB-

L

h

-RRB-

was

calculated

from

-LRB-

Eq

-RRB-

-LSB-

-RSB-

where

-LRB-

XV

-RRB-

is

the

biomass

-LRB-

g

-RRB-

at

feed

start

tf

the

time

-LRB-

h

-RRB-

after

feed

start

YX/S

the

biomass

yield

coefficient

-LRB-

g

g

-RRB-

and

S

the

glucose

concentration

in

the

feed

-LRB-

g

L

-RRB-

Substrate

consumption

by

maintenance

is

not

considered

in

Eq

Therefore

the

actual

growth

rate

may

be

less

than

the

set

value

-LRB-

μset

h

-RRB-

Feed

rate

and

volume

variations

were

corrected

automatically

by

the

MFCS

software

The

expression

of

rTfH

was

initiated

at

a

biomass

level

of

about

g

L

by

shifting

the

temperature

to

°C

which

was

found

optimal

in

the

preliminary

runs

After

induction

the

feed-rate

profiles

were

kept

constant

at

the

value

of

the

induction

time

or

it

was

continously

increased

as

given

by

Eq

for

μset

h

Analytical

methods

Cell-mass

concentration

was

followed

by

OD

at

nm

-LRB-

Ultrospec

pro

Amersham

Biosciences

Sweden

-RRB-

For

the

determination

of

the

dry

cell

weight

-LRB-

DCW

-RRB-

mL

of

the

culture

was

added

in

triplicate

to

pre-weighed

tubes

and

centrifuged

for

min

at

g

and

°C

After

a

subsequent

washing

step

the

pellet

was

dried

until

constant

mass

at

°C

under

vacuum

Extracellular

glucose

and

metabolites

such

as

acetate

formate

succinate

propionate

and

isobutyrate

were

determined

in

the

supernatant

To

this

end

the

supernatant

was

heated

up

to

°C

for

min

and

centrifuged

-LRB-

g

min

°C

-RRB-

to

eliminate

the

precipitated

proteins

The

supernatant

was

additionally

filtered

-LRB-

pore

size

μm

-RRB-

before

applied

into

the

HPLC

-LRB-

Shimadzu

Japan

-RRB-

using

an

Aminex

column

-LRB-

Biorad

USA

-RRB-

mM

with

a

flow

rate

of

mL

min

was

used

as

mobile

phase

at

a

temperature

of

°C

Electrophoresis

SDS-PAGE

was

performed

with

a

Mini

Protean

apparatus

-LRB-

Bio-Rad

USA

-RRB-

according

to

the

manufacturer

's

recommendations

The

samples

were

run

on

polyacrylamide

gel

Molecular

mass

marker

was

purchased

from

Bio-Rad

Proteins

were

stained

with

Comassie-Blue

-LSB-

-RSB-

or

silver

-LSB-

-RSB-

Proteins

were

transferred

onto

a

PVDF

membrane

-LRB-

Immobilon

P

Transfer

Membrane

USA

-RRB-

using

a

Semi-Dry

Transfer

Unit

-LRB-

Semiphor

Hoefer

TE

Series

Amersham

Bioscience

Sweden

-RRB-

rTfH

was

detected

by

Comassie-Blue

staining

Hydrolase

activity

Hydrolase

activity

was

measured

by

monitoring

the

cleavage

of

p-nitrophenylpalmitate

-LRB-

pNPP

-RRB-

as

described

by

Schmidt-Dannert

et

al.

-LSB-

-RSB-

The

released

p-nitrophenol

was

determined

spectrophotometrically

-LRB-

Ultrospec

pro

Amersham

Biosciences

Sweden

-RRB-

pNPP

solution

was

freshly

prepared

by

mixing

mL

pNPP

dissolved

in

at

a

concentration

of

g

L

with

mL

mM

phosphate

buffer

-LRB-

pH

sodium

taurocholate

g

L

gum

arabic

g

L

-RRB-

Immediately

after

addition

of

the

enzyme

sample

the

absorption

was

measured

at

nm

and

°C

against

a

sample

without

enzyme

for

min

One

unit

was

defined

as

the

amount

of

enzyme

that

caused

the

release

of

μmol

p-nitrophenol

per

minute

under

test

conditions

The

extinction

coefficient

of

p-nitrophenol

is

μmol

To

compare

the

photometrically

determined

pNPP

activity

units

with

a

previously

used

activity

test

-LSB-

-RSB-

the

cleavage

of

the

polyester

bonds

of

PCL

-LRB-

poly

-LRB-

ε-caprolactone

-RRB-

-RRB-

films

in

a

pHstat

titration

system

was

additionally

measured

as

described

by

Gouda

et

al.

-LSB-

-RSB-

It

was

found

that

one

pNPP

enzyme

unit

corresponds

to

three

PCL

units

Localization

of

rTfH

In

this

paper

it

is

discriminated

among

the

various

rTfH

fractions

located

in

the

extracellular

medium

the

periplasmic

space

and

the

cytoplasm

as

soluble

or

insoluble

protein

The

extracellular

fraction

-LRB-

EF

-RRB-

was

obtained

from

the

culture

supernatant

after

centrifugation

-LRB-

g

min

°C

-RRB-

A

variation

of

the

protocol

of

Neu

et

al.

-LSB-

-RSB-

was

used

to

obtain

the

periplasmic

fraction

-LRB-

PF

-RRB-

Biomass

-LRB-

about

mg

-RRB-

corresponding

to

ten

OD

was

resuspended

in

mL

ice-cold

mM

Tris

HCl

-LRB-

pH

-RRB-

containing

sucrose

and

mM

EDTA

and

after

incubation

for

min

at

room

temperature

centrifuged

at

g

and

°C

for

min

The

supernatant

was

separated

from

the

cell

pellet

and

stored

at

an

ice

bath

The

cell

pellet

was

again

resuspended

in

mL

ice-cold

mM

solution

and

was

slightly

rotated

on

ice

for

min

After

centrifugation

the

supernatant

was

pooled

with

the

previously

obtained

supernatant

giving

the

total

periplasmic

proteins

-LRB-

PF

-RRB-

Each

fraction

-LRB-

EF

and

PF

-RRB-

was

stored

at

°C

until

further

use

Soluble

proteins

harboured

in

the

cytoplasm

were

obtained

by

resuspending

the

cells

in

μL

PBS

buffer

-LSB-

-RSB-

-LSB-

pH

-LRB-

v/v

-RRB-

BugBuster

-LRB-

v/v

-RRB-

benzonase

-LRB-

both

Novagen

USA

-RRB-

U

lysozyme

-RSB-

slightly

rotated

at

room

temperature

for

min

and

centrifuged

at

g

and

°C

for

min

The

supernatant

formed

the

soluble

cytoplasmic

fraction

-LRB-

sCF

-RRB-

To

gain

the

insoluble

cytoplasmic

protein

fraction

-LRB-

iCF

-RRB-

cells

were

again

resuspended

in

PBS

buffer

-LRB-

-LRB-

v/v

-RRB-

BugBuster

U

lysozyme

-RRB-

and

after

incubation

for

min

diluted

sixfold

with

PBS

buffer

-LRB-

-LRB-

v/v

-RRB-

BugBuster

-RRB-

After

centrifugation

at

g

and

°C

for

min

cells

were

washed

twice

with

μL

PBS

-LRB-

-LRB-

v/v

-RRB-

BugBuster

-RRB-

Final

centrifugation

was

done

at

g

and

°C

for

min

The

pellet

was

then

resuspended

in

μL

of

-LRB-

w/v

-RRB-

SDS

Purification

of

rTfH

To

gain

a

rTfH

solution

of

low

purification

grade

mg

biomass

was

resuspended

in

mL

ice-cold

PBS

buffer

pH

-LSB-

-RSB-

Cells

were

disrupted

by

ultrasonification

-LRB-

Sonopuls

HD

Sonotrode

UW

Bandelin

electronics

Germany

-RRB-

in

an

ice

bath

using

three

intervals

each

of

s

pulse

and

min

break

Subsequently

disrupted

cell

were

denaturated

by

heat

at

°C

for

min

and

centrifuged

-LRB-

g

min

°C

-RRB-

The

supernatant

was

stored

at

°C

Highly

purified

rTfH

was

recovered

from

the

extracellular

and

the

PF

-LRB-

see

Localization

of

rTfH

section

-RRB-

Chelating

Sepharose

Fast

Flow

Gel

-LRB-

Amersham

Bioscience

-RRB-

was

activated

as

described

by

the

manufacturer

and

added

to

the

solution

harbouring

rTfH

-LRB-

mL

chromatographic

material

was

used

for

U

-RRB-

The

suspension

was

stirred

for

h

and

filled

then

into

a

XK

-LRB-

Amersham-Bioscience

-RRB-

and

after

the

chromatographic

material

has

settled

the

column

was

connected

to

the

FPLC

system

-LRB-

Plus

Amersham

Bioscience

Sweden

-RRB-

Column

was

washed

with

column

volumes

-LRB-

CV

-RRB-

binding

buffer

containing

mM

Tris

-LRB-

pH

-RRB-

mM

NaCl

and

mM

imidazole

at

a

flow

rate

of

mL

min

Elution

of

the

bound

rTfH

was

done

by

a

linear

increase

of

the

imidazole

concentration

to

mM

within

CV

Fractions

with

activity

were

pooled

and

concentrated

by

ultrafiltration

-LRB-

Amicon

Stirred

Cell

or

Amicon

USA

-RRB-

using

a

regenerated

cellulose

membrane

-LRB-

YM

Ultrafiltration

Membranes

cut

off

kDa

Amicon

USA

-RRB-

mL

of

the

concentrated

enzyme

was

applied

manually

to

the

gel

filtration

column

-LRB-

HiLoad

Superdex

prep

grade

Amersham

Bioscience

-RRB-

equilibrated

with

NaCl

The

flow

rate

was

set

to

mL

min

to

elute

the

proteins

Fractions

with

activity

were

pooled

and

stored

at

°C

until

further

use

Whenever

necessary

rTfH

solution

was

concentrated

by

ultrafiltration

Protein

determination

Protein

was

determined

by

Roti

Nanoquant

-LRB-

Roth

Germany

-RRB-

as

described

by

the

manufacturer

Results

Batch

cultivations

E.

coli

harbouring

the

plasmid

was

firstly

cultivated

batchwise

with

complex

medium

in

L

scale

monitoring

OD

DCW

glucose

and

metabolites

in

the

supernatant

At

an

OD

of

about

cultivation

temperature

was

raised

to

either

or

°C

to

induce

rTfH

production

After

starting

induction

generation

of

rTfH

was

pursued

by

the

spectrophotometric

assay

in

the

extracellular

and

PFs

Results

are

shown

in

Fig.

Fig.

of

rTfH

in

E.

coli

during

batch

cultivation

with

LB

medium

Cells

grew

at

°C

to

an

OD

of

before

starting

induction

by

a

temperature

shift

to

or

°C

a

growth

and

generation

b

rTfH

activity

in

different

locations

extracellular

fraction

-LRB-

EF

open

square

-RRB-

periplasmic

fraction

-LRB-

PF

open

circle

-RRB-

sum

of

all

fractions

-LRB-

continuous

line

-RRB-

c

glucose

and

acetate

in

broth

Glucose

present

in

the

complex

medium

is

quickly

consumed

at

about

h

after

induction

-LRB-

Fig.

-RRB-

Consumption

of

glucose

is

accompanied

by

formation

of

acetate

its

extracellular

concentration

peaks

at

°C

at

h

-LRB-

h

at

°C

respectively

-RRB-

which

is

remetabolized

after

total

glucose

consumption

The

consumption

pattern

is

reflected

by

biomass

development

and

generation

-LRB-

Fig.

-RRB-

which

show

a

two

and

three-phase

behaviour

respectively

Firstly

growth

rate

is

highest

as

long

as

glucose

is

available

-LRB-

induction

at

°C

-RRB-

The

following

consumption

of

the

intermediate

acetate

leads

to

reduced

growth

rate

After

acetate

consumption

the

growth

rate

further

declines

and

finally

growth

ceases

however

evolution

is

yet

observed

in

the

stationary

phase

From

the

biomass

and

evolution

one

has

to

conclude

that

in

all

cultivation

phases

considerable

amounts

of

constituents

of

the

complex

fractions

of

the

medium

are

also

consumed

For

instance

the

g

L

biomass

-LRB-

h

after

induction

at

°C

-RRB-

can

not

be

generated

alone

by

glucose

consumption

Unfortunately

the

complex

constituents

are

not

known

Therefore

a

carbon

balance

is

not

possible

The

production

of

the

hydrolase

rTfH

is

shown

in

Fig.

Both

induction

profiles

shifting

the

temperature

to

or

°C

revealed

similar

rTfH

production

kinetics

Shortly

after

induction

rTfH

is

only

found

in

the

periplasm

However

in

the

course

of

the

cultivation

rTfH

did

not

accumulate

in

the

PF

as

expected

due

to

the

leader

signal

OmpA

Surprisingly

rTFH

was

entirely

found

in

the

extracellular

medium

at

the

end

of

the

cultivation

At

an

induction

of

°C

the

promoter

λpRpL

in

front

of

the

gene

is

stronger

than

at

°C

but

expression

of

rTfH

is

nearly

finished

about

h

after

induction

resulting

in

a

lower

volumetric

activity

-LRB-

only

-RRB-

About

h

after

induction

a

remarkable

increase

of

extracellular

rTfH

activity

was

observed

which

obviously

correlates

with

a

high

metabolic

activity

as

indicated

by

the

second

strong

increase

in

evolution

The

rise

in

rTfH

activity

can

not

solely

originate

from

translocation

but

suggest

additional

secretion

from

cytoplasm

Apparently

expression

of

rTfH

is

faster

than

its

transport

through

the

cytoplasmic

membrane

in

the

early

phase

shortly

after

induction

In

shake-flask

cultivation

the

rTfH

production

was

monitored

in

the

cytoplasm

as

soluble

and

insoluble

cytoplasmic

fractions

-LRB-

iCFs

data

not

shown

-RRB-

Maximum

hydrolase

activity

was

achieved

after

about

h

summing

up

all

compartments

Subsequently

the

total

hydrolase

activity

was

slightly

decreased

and

finally

rTfH

was

only

detected

in

the

supernatant

To

study

the

influence

of

medium

composition

on

recombinant

TfH

production

and

to

follow

the

transport

of

the

enzyme

into

the

medium

batch

cultivations

with

the

defined

medium

containing

g

L

glucose

were

carried

out

-LRB-

Fig.

-RRB-

rTfH

was

again

induced

at

low

OD

The

course

of

the

cultivation

with

the

defined

medium

is

remarkably

different

from

that

using

the

complex

medium

Raising

the

temperature

to

either

°C

or

°C

caused

firstly

a

low

growth

rate

The

consumption

of

glucose

was

coupled

with

acetate

formation

h

after

induction

at

°C

growth

rate

rised

to

about

h

-LRB-

h

at

an

induction

at

°C

-RRB-

and

both

glucose

and

acetate

were

consumed

while

a

low

amount

of

succinate

was

generated

which

was

remetabolized

after

exhaustion

of

other

C-substrates

As

expected

growth

was

directly

coupled

with

evolution

The

respiration

coefficient

RQ

was

around

the

yield

coefficient

-LRB-

biomass

on

glucose

-RRB-

was

determined

as

g

g

for

the

batch

cultivation

at

°C

Over

the

entire

induction

period

-LRB-

about

h

-RRB-

carbon

balances

of

the

cultivation

with

the

defined

medium

were

close

to

Fig.

of

rTfH

in

E.

coli

during

batch

cultivation

with

the

defined

medium

Cells

grew

at

°C

up

to

an

OD

of

before

starting

induction

by

a

temperature

shift

to

°C

or

°C

a

growth

and

generation

b

rTfH

activity

in

different

locations

EF

-LRB-

open

square

-RRB-

PF

-LRB-

open

circle

-RRB-

sum

of

all

fractions

-LRB-

continuous

line

-RRB-

c

glucose

and

metabolites

in

broth

In

contrast

to

the

cultivation

in

LB

medium

succinate

was

detected

in

the

extracellular

medium

under

both

the

induction

profiles

Succinate

is

a

typical

product

of

the

anaerobic

metabolism

of

E.

coli

It

is

produced

during

the

mixed

acid

fermentation

and

the

fumarate

reduction

Both

pathways

are

strictly

regulated

by

Fnr

the

global

regulator

of

the

anaerobic

metabolism

-LSB-

-RSB-

With

the

applied

experimental

approach

anaerobiosis

can

be

excluded

The

formation

of

succinate

was

coupled

with

the

evolution

and

hence

with

a

high

metabolic

activity

This

may

lead

to

an

imbalance

in

the

citric

cycle

or

reduction

equivalents

Unfortunately

other

metabolites

of

the

citric

cycle

were

not

determined

in

the

supernatant

supporting

this

assumption

Similar

observations

were

made

by

Korz

in

batch

cultivations

using

the

same

defined

medium

He

correlated

the

succinate

formation

with

the

amount

of

raising

in

the

exhaust

gas

-LSB-

-RSB-

While

secretion

of

rTfH

in

the

periplasm

started

immediately

after

induction

the

rate

of

rTfH

release

in

the

cultivation

medium

correlates

with

the

growth

rate

i.e.

growth

and

product

transport

from

the

cytosol

via

the

periplasm

into

the

extracellular

space

are

coupled

At

the

end

of

the

cultivation

almost

the

entire

product

was

located

outside

the

cells

The

final

results

of

the

batch

cultivations

at

controlled

pH

with

the

complex

and

the

defined

medium

are

summarized

in

Table

For

induction

in

both

media

by

shifting

the

temperature

to

°C

gives

better

results

than

the

increase

to

°C

While

the

volumetric

activities

achieved

for

the

complex

and

the

defined

media

at

the

same

induction

temperature

are

comparable

the

specific

activities

differ

significantly

due

to

the

higher

biomass

obtained

with

the

defined

medium

As

the

induction

time

is

considerably

longer

for

the

cultivation

in

the

defined

medium

the

specific

productivities

differ

even

more

These

results

clearly

indicate

that

production

of

the

rTfH

in

batch

cultivation

becomes

more

attractive

when

the

complex

medium

is

employed

Table

of

results

on

rTfH

production

in

batch

cultivationsComplex

mediaDefined

mediaInduction

temperature

-LRB-

°C

-RRB-

-LRB-

gDCW

L

-RRB-

Induction

time

-LRB-

h

-RRB-

Volumetric

activity

-LRB-

U

L

-RRB-

Specific

activity

-LRB-

U

gDCW

-RRB-

Volumetric

productivity

-LSB-

U

-LRB-

L

h

-RRB-

-RSB-

Specific

productivity

-LSB-

U

-LRB-

gDCW

h

-RRB-

-RSB-

During

the

induction

phase

the

protein

content

in

the

culture

supernatant

was

analysed

by

SDS-PAGE

-LRB-

Fig.

-RRB-

About

h

after

the

temperature

upshift

the

first

protein

bands

can

be

recognized

particularly

for

the

cultivation

with

LB

medium

where

rTfH

and

other

protein

bands

appear

simultaneously

In

cultivations

with

the

defined

medium

the

pattern

looks

differently

In

the

first

phase

up

to

almost

h

after

induction

a

single

protein

of

about

kDa

is

found

By

N-terminal

sequencing

this

band

could

be

identified

as

the

periplasmic

located

dipeptide

binding

protein

-LRB-

DBP

-RRB-

which

is

involved

in

the

transport

of

dipeptides

-LSB-

-RSB-

In

the

cultivation

with

complex

medium

leakage

of

the

outer

membrane

increased

significantly

h

after

induction

when

C-starvation

began

growth

was

slow

but

generation

was

still

high

With

the

defined

medium

protein

release

in

the

extracellular

medium

was

drastically

rising

at

about

h

after

induction

when

all

glucose

was

consumed

The

findings

shown

in

Fig.

are

in

accordance

with

the

results

reported

by

Rinas

and

Hoffmann

-LSB-

-RSB-

who

studied

in

detail

the

leakage

of

host-cell

proteins

of

recombinant

and

non-recombinant

E.

coli

These

authors

concluded

that

irrespective

of

the

cell

line

proteins

of

periplasmic

origin

are

predominantly

released

in

the

medium

specifically

under

stress

conditions

like

temperature

increase

nutrient

starvation

and

high

cell

densities

Fig.

under

denaturating

conditions

EFs

of

different

cultivations

are

shown

after

increasing

the

temperature

to

°C

to

induce

rTfH

production

a

batch

cultivation

with

complex

medium

b

batch

cultivation

with

the

defined

medium

dashed

line

marks

the

beginning

of

glucose

starvation

The

SDS-PAGE

shown

in

Fig.

indicates

that

the

further

protein

bands

observed

for

each

media

differ

only

in

their

intensity

but

not

in

their

composition

This

suggests

that

protein

release

in

the

medium

can

not

be

attributed

to

cell

lysis

but

is

mainly

due

to

an

increasing

leakage

of

proteins

located

and

transported

to

the

periplasm

from

the

cytoplasm

by

the

Sec-transport

system

This

conclusion

is

also

confirmed

by

analysis

of

the

N-terminal

sequence

of

rTfH

which

clearly

revealed

that

the

OmpA

leader

sequence

has

been

cleaved

However

cleavage

was

not

uniform

Only

of

the

secreted

rTfH

found

in

the

extracellular

medium

was

cleaved

at

the

expected

cleavage

site

-LRB-

Fig.

-RRB-

The

remaining

rTfH

still

carried

parts

of

the

OmpA

leader

peptide

The

incomplete

processing

of

signal

sequences

has

been

reported

before

-LSB-

-RSB-

The

cleavage

motif

of

a

signal

petidase

is

the

A-X-A

box

-LSB-

-RSB-

and

fission

occurs

after

the

X

The

N-terminal

analysis

of

the

secreted

rTfH

revealed

that

the

cleavage

motif

is

kept

These

observations

may

allow

the

conclusion

that

the

Sec-transportation

pathway

is

overloaded

which

might

explain

the

improper

cleavage

of

the

signal

peptidase

Fig.

acid

sequence

of

the

signal

peptide

OmpA

-LRB-

underlined

-RRB-

and

the

beginning

of

rTfH

arrows

mark

the

cleavage

site

and

their

relative

frequency

Fed-batch

cultivation

To

enlarge

volumetric

rTfH

activities

fed-batch

cultivations

were

carried

out

The

basic

assumption

is

that

the

volumetric

productivity

Qp

-LSB-

U

-LRB-

L

h

-RRB-

-RSB-

is

given

by

equation

with

the

specific

productivity

qp

-LSB-

U

-LRB-

gDCW

h

-RRB-

-RSB-

which

is

anticipated

to

be

approximately

constant

when

increasing

the

biomass

concentration

As

observed

in

the

controlled

batch

cultivation

it

was

also

thought

that

the

target

enzyme

may

be

released

into

the

cultivations

medium

from

the

periplasmic

space

under

high

cell

density

conditions

thus

facilitating

down-stream

processing

-LSB-

-RSB-

High

cell

density

cultivation

-LRB-

HCDC

-RRB-

for

E.

coli

can

be

effectively

performed

by

using

an

appropriate

defined

medium

After

the

batch

phase

with

unlimited

growth

and

consumption

of

glucose

the

cultivation

is

continued

by

feeding

glucose

at

a

feed

rate

resulting

in

a

reduced

growth

rate

-LRB-

μset

μmax

Eq

-RRB-

Thus

oxygen

starvation

can

be

prevented

and

the

culture

grows

under

glucose

limitation

in

the

feed

phase

The

oxygen

partial

pressure

was

kept

constant

at

as

described

in

the

Cultivation

section

Two

typical

HCDC

runs

are

shown

in

Fig.

Fig.

of

rTfH

in

E.

coli

during

HCDC

Cells

grew

at

°C

before

induction

by

shifting

the

temperature

to

°C

-LRB-

dashed

line

-RRB-

a

After

h

the

batch

phase

ended

and

exponential

feed

was

started

and

set

to

a

growth

rate

h

At

the

time

point

of

h

feeding

was

set

to

a

constant

rate

-LRB-

gglc

h

-RRB-

b

Fed

batch

with

an

exponential

feeding

profile

with

μset

h

over

the

entire

feed

phase

including

induction

Starting

with

an

initial

glucose

concentrations

of

g

L

the

biomass

concentration

is

about

gDCW

L

at

the

end

of

the

batch

phase

-LRB-

Fig.

b

-RRB-

Due

to

the

metabolic

overflow

acetate

was

generated

in

the

unlimited

growth

phase

-LRB-

μmax

h

-RRB-

which

was

remetabolized

at

the

end

of

the

batch

phase

-LRB-

Fig.

-RRB-

Then

the

glucose

feed

was

started

The

feed

rate

was

adjusted

to

give

a

growth

rate

of

about

h

according

to

Eq

by

assuming

a

yield

coefficient

YX/S

of

g

g

The

actually

measured

growth

rates

were

-LRB-

Fig.

b

-RRB-

In

Run

A

after

a

feed

phase

of

h

a

biomass

concentration

of

gDCW

L

was

obtained

and

at

this

point

the

expression

of

rTfH

was

induced

by

shifting

the

temperature

from

to

°C

within

min

The

glucose

feed

rate

was

not

further

increased

-LRB-

gglc

h

-RRB-

but

kept

constant

to

the

value

when

the

induction

was

started

Therefore

the

growth

rate

steadily

decreased

and

after

about

h

the

induction

growth

ceased

In

the

first

phase

after

induction

-LRB-

h

-RRB-

an

average

growth

rate

of

roughly

h

can

be

estimated

from

the

data

However

over

the

entire

period

of

h

with

the

constant

feed

rate

rate

was

also

constant

Therefore

the

fed

glucose

is

used

only

for

maintenance

in

the

second

part

of

the

induction

phase

when

there

was

zero

growth

In

the

fed-batch

phase

of

Run

B

the

same

exponentially

increasing

glucose-feed

rate

as

for

Run

A

was

applied

i.e.

Fglc

was

calculated

from

Eq

for

μset

h

and

YX/S

g

g

After

starting

the

induction

at

g

L

DCW

exponential

glucose

feeding

was

continued

for

the

same

μset

-LRB-

h

-RRB-

In

spite

of

the

exponential

glucose

feeding

growth

decreased

and

ceased

about

h

after

induction

An

average

growth

of

h

can

be

estimated

from

the

experimental

biomass

data

at

the

first

h

of

the

induction

phase

generation

increased

exponentially

even

when

the

growth

was

negligible

in

the

second

half

of

the

induction

phase

After

h

induction

a

final

biomass

concentration

of

gDCW

L

was

obtained

Fig.

and

metabolites

formed

during

HCDC

-LRB-

Run

A

-RRB-

cf.

Fig.

In

the

course

of

both

runs

the

respiratory

quotient

was

always

about

-LRB-

-RRB-

This

indicates

that

consumed

glucose

was

exclusively

used

for

ATP

production

Indeed

an

intermediate

formation

of

fermentative

by-products

such

as

acetate

succinate

and

formate

was

only

observed

at

low

level

at

the

beginning

and

end

of

the

induction

period

-LRB-

Fig.

-RRB-

Both

during

the

fed-batch

and

the

induction

phase

the

glucose

concentration

was

always

zero

Carbon

balances

were

checked

by

the

installed

computer

program

with

consideration

of

volume

changes

by

feeding

and

sampling

and

found

to

be

close

to

The

kinetics

of

the

appearance

of

the

heterologous

gene

product

-LRB-

rTfH

-RRB-

in

the

periplasm

and

cultivation

broth

is

shown

in

Fig.

for

Run

A

with

a

long

induction

phase

-LRB-

h

-RRB-

The

activity

in

the

periplasm

steadily

increased

up

to

a

value

above

U

mL

while

in

the

first

h

no

rTfH

was

released

in

the

supernatant

The

final

activity

in

the

medium

was

rather

low

-LRB-

U

mL

-RRB-

A

similar

release

kinetics

as

shown

in

Fig.

was

also

observed

for

Run

B

which

differs

largely

from

the

behaviour

found

for

batch

cultures

-LRB-

Figs.

-RRB-

In

contrast

to

the

batch

cultivations

the

periplasmic

activity

does

not

pass

a

maximum

and

particularly

little

product

-LRB-

say

less

than

-RRB-

is

found

in

the

medium

Fig.

kinetic

of

rTfH

in

the

fed-batch

cultivation

-LRB-

Run

A

-RRB-

At

a

biomass

of

gDCW

L

rTfH

expression

was

induced

by

raising

the

temperature

to

°C

However

despite

the

low

leakage

of

rTfH

into

the

medium

a

SDS-PAGE

-LRB-

Fig.

-RRB-

made

from

the

supernatant

after

the

enzyme

induction

showed

that

the

cells

were

already

leaky

at

the

beginning

of

the

induction

which

is

proven

by

the

appearance

of

the

periplasmic

DBP

During

the

induction

phase

an

increase

both

of

DBP

and

rTFH

is

observed

Fig.

EFs

of

different

cultivations

are

shown

after

increasing

the

temperature

to

°C

to

induce

rTfH

production

HCDC

-LRB-

Run

A

-RRB-

The

hydrolase

activity

-LRB-

pNPP

test

system

-RRB-

was

also

measured

in

the

cytoplasm

before

the

induction

and

at

the

end

of

the

HCDC

While

no

activity

was

found

prior

to

induction

a

surprisingly

high

intracellular

activity

was

detected

at

the

end

of

the

cultivation

process

Indeed

about

two-thirds

of

the

entire

rTfH

activity

was

located

in

the

cytoplasm

apparently

in

soluble

and

active

form

Any

significant

inclusion-body

formation

was

not

observed

The

operational

parameters

and

the

results

received

in

the

two

HCDC

runs

shown

in

Fig.

are

summarized

in

Table

In

spite

of

the

different

feed

rates

and

the

total

induction

times

the

rTfH

activities

found

in

the

different

compartments

are

in

reasonable

agreement

The

constant

feed

rate

after

induction

in

Run

A

yields

higher

specific

activities

obviously

due

to

lower

biomass

formation

However

as

a

result

of

the

long

induction

time

the

productivity

is

lower

for

Run

A

than

for

Run

B

In

comparison

to

the

batch

runs

-LRB-

Table

-RRB-

the

accumulation

of

product

in

the

periplasm

and

cytoplasm

is

most

noticeable

Thus

the

total

volumetric

activity

is

about

larger

than

that

achieved

in

the

batch

runs

These

higher

overall

product

concentrations

are

advantageously

for

rTfH

recovery

and

purification

Table

operation

parameters

and

achieved

rTfH

activities

in

fed-batch

runs

-LRB-

starting

glucose

concentration

g

L

pH

T

°C

Tind

°C

-RRB-

Cultivation

Run

ARun

BDCW

at

induction

-LRB-

g

L

-RRB-

Feed

rateConstant

-LRB-

gglc

h

-RRB-

Exponential

-LRB-

μ

h

-RRB-

Estimated

mean

growth

rate

after

induction

-LRB-

h

-RRB-

Total

induction

time

-LRB-

h

-RRB-

Final

DCW

-LRB-

g

L

-RRB-

SupernatantVolumetric

activity

-LRB-

U

L

-RRB-

Specific

activity

-LRB-

U

gDCW

-RRB-

PeriplasmVolumetric

activity

-LRB-

U

L

-RRB-

Specific

activity

-LRB-

U

gDCW

-RRB-

Cytoplasm

-LRB-

soluble

-RRB-

Volumetric

activity

-LRB-

U

L

-RRB-

Specific

activity

-LRB-

U

gDCW

-RRB-

TotalVolumetric

activity

-LRB-

U

L

-RRB-

Specific

activity

-LRB-

U

gDCW

-RRB-

Productivity

-LSB-

U

-LRB-

gDCW

h

-RRB-

-RSB-

aTotal

induction

time

Purification

Two

different

strategies

were

applied

to

purify

rTfH

with

regard

to

different

uses

To

gain

rTfH

with

a

high

purification

grade

the

tag

cloned

at

the

C-terminal

end

of

the

gene

allowed

a

simple

and

effective

chromatographic

procedure

rTfH

was

purified

from

the

culture

broth

or

the

periplasm

which

was

obtained

by

an

osmotic

shock

treatment

-LRB-

Localization

of

rTfH

section

-RRB-

In

the

following

the

purification

of

rTfH

is

described

which

originated

from

the

periplasmic

space

About

g

of

wet

biomass

-LRB-

corresponding

to

ca.

gDCW

-RRB-

was

subjected

to

osmotic

shock

treatment

-LRB-

L

scale

-RRB-

After

separating

the

cells

by

centrifugation

mL

activated

sepharose

was

directly

added

to

the

supernatant

harbouring

the

periplasmic

protein

After

h

incubation

only

low

amounts

of

rTfH

activity

-LRB-

-RRB-

could

be

detected

in

the

supernatant

The

suspension

was

then

filled

into

a

glass

column

Particle

settling

could

be

improved

by

connecting

a

peristaltic

pump

to

the

column

outlet

After

connecting

the

column

to

a

FPLC

system

rTfH

was

washed

with

bindung

buffer

rTfH

was

eluted

by

applying

a

linear

gradient

of

imidazole

-LRB-

Fig.

-RRB-

rTfH

elution

was

observed

at

an

imidazole

concentration

of

about

mM

In

the

pooled

fractions

a

rTfH

activity

of

U

mL

could

be

detected

The

step

resulted

in

a

purification

and

a

loss

of

about

rTfH

activity

Fig.

of

rTfH

from

the

PF

E.

coli

cells

derived

from

a

fed-batch

cultivation

-LRB-

Run

A

-RRB-

g

biomass

was

used

for

osmotic

shock

treatment

a

affinity

chromatography

with

Chelating

Sepharose

Fast

Flow

b

gelfiltration

with

Superdex

prep

grade

pooled

fraction

are

marked

by

a

line

To

further

eliminate

impurities

and

to

remove

high

concentrations

of

NaCl

and

imidazole

from

the

protein

sample

gelfiltration

was

done

using

a

Hi

Load

Superdex

prep

grade

column

with

a

NaCl

buffer

-LRB-

Fig.

-RRB-

Before

applying

the

sample

to

the

column

pooled

fractions

with

rTfH

from

the

affinity

chromatography

were

concentrated

by

ultrafiltration

-LRB-

concentration

factor

cut-off

kDa

-RRB-

Gelfiltration

assured

purification

to

homogeneity

as

shown

by

SDS-PAGE

-LRB-

Fig.

-RRB-

although

only

a

minor

increase

of

the

specific

activity

was

observed

-LRB-

Table

-RRB-

Gel

filtration

resulted

in

a

further

loss

of

rTfH

activity

of

about

Totally

rTfH

could

be

purified

with

a

recovery

in

activity

by

the

applied

purification

route

Table

gives

an

overview

of

the

procedure

to

obtain

the

highly

purified

rTfH

which

will

be

used

for

mechanistic

studies

-LRB-

adsorption

kinetic

-RRB-

and

structural

analysis

Fig.

rTfH

at

different

purification

steps

-LRB-

silver

staining

-RRB-

g

biomass

was

used

for

the

osmotic

shock

treatment

cells

derived

from

HCDC

-LRB-

Run

A

-RRB-

standard

molecular

mass

protein

standard

supernatant

from

the

periplasm

affinity

chromatography

pooled

fraction

after

Superdex

gel

chromatographyTable

of

two

routes

used

to

recover

and

purify

rTfH

from

HCDC

the

highly

purified

product

was

obtained

from

the

periplasmic

fraction

-LRB-

Run

A

-RRB-

the

low

grade

product

was

recovered

from

the

cytoplasm

and

periplasm

by

ultrasonification

-LRB-

Run

B

-RRB-

Purification

stepTotal

units

-LRB-

U

L

-RRB-

Total

protein

-LRB-

mg

L

-RRB-

Specific

activity

-LRB-

U

mg

-RRB-

Purification-factorYield

-LRB-

-RRB-

High

grade

rTfH

Supernatant

with

periplasmic

affinity

Gel

Low

grade

rTfH

Crude

Heat

Ultrasonification

heat

aaCrude

extract

-LRB-

ultrasonification

plus

centrifugation

-RRB-

Contrary

to

the

purification

profile

of

the

TfH

from

the

culture

broth

of

T.

fusca

cultivations

the

overall

yield

could

be

increased

from

to

-LSB-

-RSB-

Prior

to

performing

the

first

purification

step

the

supernatant

containing

TfH

had

to

be

concentrated

by

ammonium

sulphate

precipitation

By

this

method

a

final

recovery

of

only

could

be

achieved

because

of

the

limited

amounts

of

the

precipitated

proteins

that

could

be

redissolved

-LSB-

-RSB-

By

inserting

the

tag

at

the

C-terminal

end

of

rTfH

precipitation

could

be

avoided

and

the

recombinant

enzyme

could

be

easily

obtained

from

E.

coli-culture

broths

independently

of

its

composition

For

industrial

processes

purification

with

affinity

chromatography

is

too

costly

of

course

Therefore

an

alternative

purification

route

was

developed

which

aimed

at

a

rTfH

preparation

of

a

low

purification

grade

but

sufficient

activity

and

stability

for

larger-scale

investigations

on

polyester

degradation

Activity

test

of

the

soluble

protein

in

the

cytoplasm

obtained

during

HCDC

showed

that

most

of

the

expressed

rTfH

was

not

secreted

into

the

periplasm

but

remained

in

the

cytoplasm

Hence

the

cells

were

disrupted

by

ultrasonification

to

get

rTfH

both

from

the

cytoplasm

and

the

periplasm

In

preliminary

experiments

different

ultrasonication

conditions

were

tested

Three-fold

pulses

of

s

each

with

min

break

were

found

as

the

best

treatment

for

the

test

conditions

of

mg

biomass

resuspended

in

mL

PBS

buffer

Here

biomass

from

the

HCDC

Run

B

was

used

After

the

separation

of

cell

debris

by

centrifugation

an

activity

of

U

mL

was

detected

corresponding

to

a

specific

activity

of

U

gDCW

Chemical

disruption

of

the

cells

by

a

detergent

formulation

-LRB-

BugBuster

see

Localization

of

rTfH

section

-RRB-

resulted

only

in

a

specific

activity

of

U

gDCW

implying

that

cell

disruption

is

more

effective

than

the

chemical

treatment

Protein

concentration

of

the

crude

extract

was

mg

mL

Taking

the

specific

activity

of

the

high-grade

rTfH

into

account

-LRB-

U

mgProtein

-RRB-

mg

protein

harboured

heterologous

produced

rTfH

Because

rTfH

is

very

stable

against

higher

temperatures

-LSB-

-RSB-

heat

treatment

was

carried

out

at

°C

for

min

Two

routes

were

tested

namely

either

heat

treatment

of

the

disrupted

cells

immediately

after

the

ultrasonication

or

heat

treatment

after

the

separation

of

the

cell

debris

Activity

tests

revealed

that

a

heat

treatment

directly

after

the

cell

disruption

yielded

the

best

results

Thus

the

purification

factor

could

be

increased

from

to

and

a

higher

yield

was

observed

Table

summarized

the

results

in

detail

Discussion

The

recombinant

strain

E.

coli

harbouring

the

plasmid

proved

to

be

an

appropriate

system

for

expression

secretion

and

purification

of

the

hydrolase

from

T.

fusca

In

comparison

to

cultivations

with

T.

fusca

and

induction

of

TfH

by

adding

polyesters

a

higher

volumetric

activity

could

be

obtained

in

batch

cultivations

of

the

recombinant

E.

coli

strain

The

volumetric

activity

could

be

further

improved

in

fed-batch

cultivations

with

glucose

limitation

Considering

only

the

extracellular

and

PFs

the

increase

is

If

one

also

accounts

for

the

activity

found

in

the

cytoplasm

the

total

volumetric

activity

is

over

higher

than

that

obtained

from

T.

fusca

The

overall

volumetric

productivity

-LSB-

U

-LRB-

L

h

-RRB-

-RSB-

increased

by

a

factor

more

than

Hence

the

production

of

rTfH

in

recombinant

E.

coli

is

an

attractive

and

promising

approach

for

providing

hydrolytic

activity

for

polyester

cleavage

in

degradation

and

recycling

processes

This

view

is

supported

by

the

simple

recovery

route

to

give

a

crude

and

stable

rTfH

preparation

which

did

not

exhibit

activity

losses

after

storage

at

°C

for

several

weeks

A

further

improvement

of

rTfH

production

in

E.

coli

is

the

insertion

of

the

tag

at

the

C-terminal

end

of

the

enzyme

Purification

of

the

wild-type

TfH

proved

to

be

quite

ineffective

A

three-step

purification

strategy

-LRB-

precipitation

ion-exchange

chromatography

and

hydrophobic

interaction

chromatography

-RRB-

resulted

in

only

yield

in

activity

-LSB-

-RSB-

With

the

tag

activity

yield

of

rTfH

could

be

obtained

in

a

two-step

purification

route

starting

from

the

supernatant

and

PFs

A

purification

was

achieved

yielding

a

specific

activity

of

U

mg

From

this

value

the

overall

concentration

of

the

produced

rTfH

can

be

estimated

as

about

g

L

In

the

batch

runs

with

LB

and

the

defined

medium

better

results

were

found

by

induction

to

only

°C

Therefore

fed-batch

cultivations

were

carried

out

with

this

induction

temperature

While

both

the

media

yielded

comparable

volumetric

activities

the

specific

activities

differ

significantly

-LRB-

about

fivefold

in

LB

-RRB-

LB

medium

obviously

supplies

E.

coli

with

all

nutrients

to

easily

synthesize

the

recombinant

protein

at

high

specific

productivity

and

to

secrete

it

across

the

cell

envelope

Increased

growth

rates

and

facilitated

protein

production

in

LB

medium

are

frequently

reported

in

the

literature

-LSB-

-RSB-

In

batch

cultivations

with

both

media

rTfH

expression

as

well

as

its

transport

across

the

cell

membrane

in

the

periplasm

and

the

subsequent

release

into

the

cultivation

media

are

seemingly

coupled

with

growth

and

generation

-LRB-

Figs.

-RRB-

In

the

fed-batch

cultivations

with

the

defined

medium

roughly

tenfold

higher

activities

could

be

achieved

in

the

supernatant

and

periplasm

than

in

batch

operation

Surprisingly

the

specific

activities

in

the

glucose

limited

fed-batch

process

are

even

twofold

higher

If

one

accounts

for

the

soluble

cytosolic

fraction

the

activity

yields

further

improve

remarkably

The

total

specific

activity

of

the

HCDCs

is

in

the

same

range

as

those

obtained

for

the

batch

process

with

LB

medium

An

influence

of

the

tag

and

the

full

or

truncated

OmpA

leader

sequence

on

TfH

activity

can

hardly

be

estimated

from

the

results

of

this

study

However

the

high

activities

of

rTfH

obtained

suggest

that

small

sequences

at

the

C

and

N

terminals

might

have

little

effect

on

TfH

activity

The

fraction

of

soluble

active

protein

located

in

the

cytoplasm

is

remarkably

high

but

its

amount

can

be

estimated

to

be

less

than

of

the

total

protein

content

of

the

cells

-LRB-

assuming

protein

of

DCW

-RRB-

Therefore

it

is

understood

that

inclusion

body

formation

could

not

be

detected

Among

the

different

mechanisms

to

transport

protein

from

the

cytoplasm

across

the

cell

membrane

to

the

periplasm

the

Sec

pathway

is

the

most

prominent

for

prokaryotic

organisms

-LSB-

-RSB-

The

Sec-translocation

process

is

thought

to

be

generally

responsible

for

the

transport

of

newly

synthesized

but

unfolded

proteins

out

of

the

cytosol

to

the

periplasm

This

process

is

accompanied

by

the

cleavage

of

the

leader

sequence

-LRB-

OmpA

-RRB-

from

the

pre-protein

and

proper

folding

in

the

periplasm

If

we

assume

that

the

Sec

pathway

is

also

active

for

the

transport

of

the

heterologous

rTfH

out

of

the

cytoplasm

it

is

obvious

that

due

to

its

accumulation

in

the

cytoplasm

during

the

fed-batch

runs

the

translocation

rate

is

less

than

the

expression

rate

Hence

transport

into

the

periplasm

apparently

presents

a

limiting

step

It

might

be

possible

that

the

entire

translocation

process

is

subjected

to

an

equilibrium

phenomenon

in

as

far

as

high

protein

concentrations

in

the

periplasm

reduces

the

transport

rate

This

is

evident

if

the

rTfH

masses

-LRB-

or

units

-RRB-

in

periplasm

and

cytoplasm

are

not

referred

to

cultivation

volume

as

given

in

Table

but

to

their

respective

actual

volumes

If

it

is

assumed

that

the

volume

of

periplasm

is

about

of

the

cell

volume

then

the

rTfH

concentration

in

the

periplasm

is

more

than

tenfold

higher

than

in

the

cytoplasm

This

might

explain

that

no

cytoplasmic

rTfH

activity

was

found

in

the

batch

runs

where

the

periplasmic

protein

was

entirely

released

in

the

extracellular

space

In

contrast

relatively

little

protein

was

transported

across

the

outer

cell

wall

into

the

medium

in

the

fed-batch

runs

with

high

cell

densities

and

higher

rTfH

activities

were

found

in

both

the

cytoplasm

and

periplasm

As

to

the

Sec

pathway

the

rTfH

in

the

cytoplasm

should

be

unfolded

and

hence

is

not

expected

to

be

in

the

active

form

However

when

gaining

the

cytoplasmic

fraction

-LRB-

see

Localization

of

rTfH

section

-RRB-

immediate

activity

was

observed

This

suggests

that

either

folding

takes

place

spontaneously

in

the

PBS

buffer

or

the

cytosol

already

harbours

the

active

folded

rTfH

and

its

translocation

follows

another

mechanism

Unfortunately

our

study

does

not

allow

to

distinguish

between

the

two

possibilities

Though

it

is

often

assumed

that

E.

coli

laboratory

strains

do

not

secrete

proteins

across

the

cell

envelope

in

the

extracellular

medium

at

normal

growth

conditions

-LSB-

-RSB-

leakage

of

the

outer

cell

membrane

is

a

common

phenomenon

observed

for

a

variety

of

cultivation

conditions

as

reported

in

recent

papers

-LSB-

-RSB-

and

review

articles

-LSB-

-RSB-

Permeability

of

the

outer

cell

wall

may

depend

on

medium

composition

growth

rates

cell

concentration

changes

in

cultivation

conditions

etc.

For

instance

and

increase

leakage

of

the

outer

membrane

-LSB-

-RSB-

Rise

of

temperature

causes

changes

in

membrane

permeability

and

fluidity

due

to

alterations

in

the

lipid

composition

of

the

cell

wall

This

aspect

was

particularly

studied

in

continuous

cultures

of

E.

coli

-LSB-

-RSB-

and

an

increased

permeability

was

found

at

higher

growth

rates

Rinas

and

Hoffmann

-LSB-

-RSB-

studied

in

detail

the

leakage

of

host-cell

proteins

during

HCDCs

of

recombinant

and

non-recombinant

E.

coli

These

authors

reported

that

the

proteins

found

extracellularly

are

of

periplasmic

origin

except

the

heat-shock

protein

DnaK

Various

and

interrelated

stress

factors

in

the

temperature-induced

HCDC

are

suggested

to

be

responsible

for

the

migration

of

periplasmic

proteins

in

the

extracellular

medium

In

all

cultivations

performed

in

this

study

rTfH

could

be

detected

in

the

medium

without

applying

any

additional

measures

to

permeabilize

the

outer

cell

membrane

Cell

lysis

as

a

possible

mechanism

of

rTfH

release

can

be

excluded

as

the

cleavage

of

the

OmpA

sequence

in

the

extracellularly

found

hydrolase

indicates

transport

across

the

cytoplasmic

membrane

by

the

Sec-translocation

mechanism

Any

significant

cell

lysis

would

also

release

cytoplasmic

rTfH

still

containing

the

complete

signal

sequence

In

batch

cultivations

-LRB-

Figs.

-RRB-

with

LB

and

the

defined

medium

induction

of

rTfH

expression

was

started

at

low

cell

densities

Immediately

after

the

temperature

upshift

rTfH

appeared

in

the

periplasm

but

its

periplasmic

activity

passes

a

maximal

value

just

in

the

first

part

of

the

exponential

growth

phase

In

accordance

with

the

findings

of

Shokri

et

al.

-LSB-

-RSB-

the

migration

of

the

periplasmic

rTfH

in

the

medium

is

obviously

growth

coupled

This

effect

may

be

attributed

to

changes

in

cell-wall

composition

which

facilitate

transport

into

the

extracellular

medium

On

the

other

hand

during

cell

growth

and

cell-wall

biosynthesis

transient

openings

of

the

outer

membrane

and

the

murein

layer

are

unavoidable

-LSB-

-RSB-

thus

the

protein

of

periplasmic

origin

can

migrate

into

the

extracellular

medium

Furthermore

vesiculation

and

fragmentation

of

the

outer

membrane

may

occur

as

a

natural

phenomenon

during

the

exponential

growth

phase

-LSB-

-RSB-

which

is

thought

to

be

caused

by

an

imbalance

of

the

outer-membrane

biogenesis

and

peptidoglucan

synthesis

Therefore

several

growth-coupled

phenomena

may

contribute

to

the

migration

of

periplasmic

proteins

in

the

medium

At

the

end

of

the

batch

cultivations

the

produced

rTfH

has

entirely

accumulated

in

the

medium

As

can

be

discerned

from

the

SDS-PAGE

shown

in

Fig.

the

transport

of

rTfH

across

the

cell

envelope

is

accompanied

simultaneously

by

release

of

other

periplasmic

proteins

-LSB-

-RSB-

The

kinetics

of

rTfH

accumulation

in

the

extracellular

medium

-LRB-

Figs.

-RRB-

agrees

qualitatively

with

the

results

shown

in

Fig.

particularly

no

significant

migration

is

observed

at

low

growth

rates

after

the

induction

when

the

culture

adapts

to

the

higher

temperature

Rinas

and

Hoffmann

-LSB-

-RSB-

found

DBP

as

the

most

prominent

periplasmic

protein

transported

to

the

medium

This

observation

and

even

the

release

kinetics

reported

by

these

authors

for

high

cell-density

conditions

are

in

striking

agreement

with

our

findings

for

batch

conditions

-LRB-

relatively

low

cell

densities

-RRB-

as

observed

in

the

defined

medium

-LRB-

Fig.

-RRB-

In

contrast

to

the

batch

runs

rTfH

accumulates

in

the

cytoplasm

and

periplasm

during

the

glucose-limited

fed-batch

cultivations

after

induction

at

higher

cell

densities

The

relative

fractions

of

rTfH

released

extracellularly

is

low

For

an

induction

period

of

h

only

and

of

the

PF

are

released

for

Run

A

and

B

respectively

As

in

Run

B

the

growth

rate

was

higher

as

a

result

of

exponential

glucose

feeding

this

finding

underpins

the

view

that

the

permeability

of

the

outer

membrane

is

related

to

growth

rate

As

shown

in

Fig.

the

outer

membrane

is

already

leaky

before

the

temperature

shift

from

to

°C

as

among

other

periplasmic

proteins

the

DBP

is

predominantly

found

in

the

extracellular

medium

as

reported

previously

-LSB-

-RSB-

Of

course

in

the

induction

phase

a

further

increase

of

DBP

is

observed

-LRB-

Fig.

-RRB-

Though

the

transport

of

rTfH

in

the

periplasm

as

well

as

its

migration

into

the

extracellular

medium

continued

-LRB-

Fig.

-RRB-

the

cultivation

was

terminated

to

prevent

cell

lysis

after

growth

ceased

However

in

the

non-growth

phase

glucose

does

not

accumulate

in

the

medium

but

is

consumed

as

is

also

exhibited

by

the

monotonous

increase

of

generation

This

points

to

the

increased

maintenance

and

ATP

demand

for

translocation

of

the

heterologous

gene

product

-LRB-

rTfH

-RRB-

across

the

inner

cell

membrane

These

mechanisms

which

govern

the

local

distribution

of

rTfH

are

not

yet

understood

Though

cell-specific

productivities

are

in

a

comparable

range

product

distributions

differ

largely

Our

results

suggest

that

the

outer

membrane

permeability

is

related

with

the

growth

rate

and

hence

all

rTfH

can

migrate

into

the

extracellular

medium

at

high

growth

rates

-LRB-

batch

runs

-RRB-

Therefore

final

product

release

across

the

cell

envelope

could

play

an

important

role

in

the

overall

translocation

process

as

product

accumulation

in

the

periplasm

seemingly

reduces

the

translocation

rate

from

the

cytoplasm

by

the

Sec

pathway

This

hypothesis

requires

further

investigations

which

are

presently

in

preparation

Therefrom

new

strategies

for

cultivation

and

downstream

processing

with

improved

product

qualities

are

expected

recombinant protein expression
batch culture
fed-batch culture