Separation Recovery and Purification in Biotechnology 1986 Recent Advances and Mathematical Modeling

8/10/2019 Separation Recovery and Purification in Biotechnology 1986 Recent Advances and Mathematical Modeling

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Separation,Recovery,

and

Purification

inBiotechnology

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In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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CS SYMPOSIUM SERIES 314

Separation,

Recovery,

and

Purification

inBiotechnology

RecentAdvancesand

Mathematical

Modeling

JuanA

Asenjo,

EDITOR

olumbia

University

Juan Hong,

EDITOR

Illinois

nstituteof

Technology

Developedfroma symposiumsponsoredby

theDivisionof

Microbial

andBiochemical

Technology

at the

190th

Meeting

of theAmerican ChemicalSociety,

Chicago,

Illinois,

September

8-13, 1985

Amer i can Chemica l Soc ie ty Wash ington D 1986


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Library

of Congress Cataloging-in-Publication Data

Separation, recovery, and purification in

biotechnology.

(ACS symposiumseries,ISSN0097-6156;314)

Includes bibliographies and index.

1 BiotechnologyTechniqueCongresses.

2. BiomoleculesPurificationCongresses.

3. Biological chemistryTechniqueCongresses.

I. Asenjo,

Juan

., 1949- . II.Hong, Juan.

III.

American

Chemical Society. Meeting

(190th:

1985:

Chicago,

Ill.) IV. Series.

TP248.24.S47 1986 660 .6 028 86-10833

ISBN0 8412 0978 2

Copyright

1986

American

Chemical Society

Rights Reserved. The appearance of the code at the bottom of the first page of each

chapter in this volume indicates the copyright owner s

consent

that reprographic

copies

of the

chapter may be made for personal or internal use or for the personal or internal use of specific

clients.

This

consent is given on the condition, however, that the copier pay the statedper

copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem,

MA01970,

for

copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law.

This

consentdoesnot extend to copying or transmission by any meansgraphic or electronicfor

any other purpose, such as for general distribution, for advertising or promotional purposes,

for creating a new collective work, for resale, or for informationstorageand retrievalsystems.

The

copying fee for each chapter is indicated in the code at the bottom of the firstpageof the

chapter.

The citation of trade names and/or names of manufacturers in this publication is not to be

construed as an endorsement or as approval by ACS of the commercial products orservices

referenced herein; nor should the mere reference herein to any drawing, specification, chemical

process, or other data be regarded as alicenseor as a conveyance of any right or permission,

to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or

sell any patented invention or copyrighted work that may in any way be related thereto.

Registered names, trademarks, etc., used in this publication,evenwithout specific indication

thereof, are not to be considered unprotected by law.

PRINTED

IN

THE UNITED STATES OF AMERICA

American

Chemical

Society

Library

1155 16th St . N W

Washington D C 20036


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ACSSymposiumSeries

M Joan Comstock,SeriesEditor

dvisory

Board

Harvey

W Bl an ch

University

of

CaliforniaBerkeley

A l a n

Elzerman

Clemson University

John W Fin l ey

Nabisco Brands,

Inc.

M a r ye Ann e Fox

The University

of

TexasAustin

M a r t i n L Gorb at y

Exxon Research and Engineering Co.

Rol an d F Hir sc h

U.S. Department

of

Energy

Rudol ph J Ma rc us

Consultant, Computers

Chemistry Research

Vincent D M cGi nn i ss

BattelleColumbus Laboratories

Donal d E M ore l and

USDA,Agricultural Research Service

W H No r t on

J

T. Baker Chemical Company

James C Ran da l l

Exxon Chemical Company

W D Shul t s

Oak

Ridge National Laboratory

Geoff rey K Smi t h

Rohm

Haas Co.

Charles S Tuesday

General Motors Research Laboratory

Dougl as B Walt ers

NationalInstitute of

Environmental Health

C Gra nt Wi l l son

IBM Research Department


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F O R E W O R D

The ACS

S Y M P O S I U M S E RI ES

was

foun e

in 1974 to

provide

a

medium for publishing symposia quickly in book

form.

The

formatof the Series parallels that of the continuing

A D V A N C E S

IN C H E M I S T RY S E RIE S

exceptthat, in order to

save

time, the

papers are not

typeset

but are reproduced as they are submitted

by the authors in camera-readyform. Papers are reviewed under

the supervision of the Editors with the assistanceof the Series

Advisory

Boardand areselectedto maintain the integrity of the

symposia; however, verbatim reproductions of previously pub

lished papers are not accepted. Both reviews and reports of

research are acceptable, because symposia may embrace both

typesof presentation.


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P R E F C E

ONE

O F T H E M O S T D I FFI CU L T

and challenging problems facing large-scale

biotechnology today is to

find

and develop appropriate recovery, separation,

an d

purification processes. The area of large-scale bioseparations is one to

which biologists, physical biochemists, and particularly biochemical engi

neers have important contributions to make. Some of the most recent

advances and developments that have already started to

find

practical

applications are

membrane separations, including the use of membrane bioreactors

and

liquidemulsion membranes;

continuous or semicontinuous chromatographic separations,

includ

ing

the use of

a

number of affinity methods and monoclonal antibodies;

two-phase extraction

processes

such as aqueous

systems

and the use

of

reverse micelles;

precipitation techniques;

electrically driven separation processes;

methods of product secretion, cell permeation, disruption, and

selective

enzymatic lysis of

microbialcells

for

intracellular

product release;

product solubilization and renaturation of proteins or polysaccharides

present in inclusion bodies or granules.

This book covers several of the emerging areas of separations in

biotechnology and is not intended to be a comprehensive handbook. It

includes recent advances and

latest

developments in techniques and

operations used for bioproduct recovery in biotechnology and applied to

fermentation

systems

as well as mathematical analysis and modeling of such

operations. The topics have been arranged in three

sections

beginning with

product

release

from

the cell and recovery

from

the bioreactor.Thissection

is followed by one on broader separation and concentration processes, and

the

final

section is on purification operations. The operations covered in

these last two

sections

can be used at a number of differentst gesin the

downstream process.

crucial

question remaining is how to design aflowsheet or product

recovery operation sequence. Three main points to keep in

mind

are

1) integrating recovery with the fermentation system, 2) integrating the

different separation and purification

st ges

to design the optimum sequence,

an d

3)

assessing

the possibility of a continuous operation.

Revised versions of papers presented in the symposium upon which this

book

is based as well as papers presented in other

sessions

that were relevant

IX


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to the topic have been included in this volume. In addition we have included

a few keynote chapters on areas we felthad not been well covered at the

meeting.

We

gratefully acknowledge the

assistance

of many reviewers who helped

us with

critical

and constructive comments on the original manuscripts. We

wouldalso

like to acknowledge the support and well-organized help of the

staff at the C S Books Department.

J U N A. S E N J O

ColumbiaUniversity

NewYork NY10027

J U N H O N G

Illinois

Institute

of Technology

Chicago

IL

2 742


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1

ProteinReleasefromChemicallyPermeabilized

Escherichiacoli

David

J

Hettwer

and

Henry

Y Wang

Department

of

ChemicalEngineering,The University

of

Michigan,A nn Arbor,

M I48109-2136

n important factor complicating the recovery of

recombinant proteins from Escherichia

coli

is their

intracellular location. An alternative to the commonly

used method of releasing theseproteins by mechanical

disruption is to chemically permeabilize the cells. The

objective of this research was to characterize the pro

tein release

kinetics and mechanism of a permeabiliza-

tion

process

using guanidine-HCl and Triton-X100. The

protein release

kinetics were determined as a function

o

the guanidine

Triton

and

cell

concentrations.

Some

o

theadvantages over mechanical disruption include

avoidance of

extensive

fragmentation of the cells and

retention of the nucleic

acids

inside the

cell

structure.

T he r e c e n t d e v e l o p m e n t of r e c o mb i n a n t DNA t e c h n o l og y has made i t

f e a s i b l e

to

p r o d u c e

i n t e r f e r o n ,human g r o w t h hormone,

i n s u l i n ,

and

o th e r p r o t e i n s in the b a c t e r i u m E s c h e r i c h i a c o l i . An i m p o r t a n t

f a c t o r c o m pl i c a t i n g the r e c o v e r y p r oc e s s is the r e t e n t i o n of the

p r o t e i n

p r o d u c t i n s i d ethem i c r o b i a l c e l l . T h i shasn e c e s s i t a t e dthe

d e v e l o p m e n t of p r o c e s s e s c a p ab l e of r e l e a s i n g p r o t e i n f r o m E. c o l i .

P r o t e i n r e le a s e on an i n d u s t r i a l s c a le iscommonly a c h i e v e d by

m e c h a n i c a l l y b r e a k i n g the c e l l in a h i g h p r e s s u r e h o m o g e n i z e r or a

b a l l m i l l .

D i s r u p t i o n

in a

h i g h p r e s s u r e

h o m o g e n i z e r

is

c a u s e d

by

p r e s s u r e g r ad ie n t s e s t a b l i s h e dwhen a pr e s s u r i z e d c e l l s u s p e n s i o nis

f o r c e d t h r o u g hanarrow

o r i f i c e

whereas

w i t ha b a l l

m i l l

d i s r u p t i o n

i s c a u s e d by

s h e a r

f o r c e s

g e n e r a t e d

by g r i n d i n g the c e l l s w i t h

a b r a s i ve p a r t i c l e s (1.).

T h e s e

m e c h a n i c a l ly

b a s e d

p r o t e i n r e l e a s e

methods have

s e v e r a l

u n d e s i r a b le p r o p e r t i e s . One

p r o b l e m

ist h a t e x t e n s i v e

f r a g m e n t a t i o n

of the c e l l s

makes

the

s u b s e q u e n t

c e n t r i f u g a t i o n

d i f f i c u l t

( 2 , 3 ) .

A d d i n g

to the

p r o b l e m

of

c e l l f r a g m e n t r e m o va l

is theh i gh v i s c o s i t y

i m p a r t e d

to the s o l u t i o n by the r e l e a s e d n u c l e i c a c i d s (4). A

n u c l e i c

a c i d

r e m o v a l

s t e p is

n e c e s s a r y

to

d e c r e a s e

the s o l u t i o n

v i s c o s i t y and

a v o id p o t e n t i a l i n t e r f e r e n c e

w i t h f r a c t i o n a l

p r e c i p i t a t i o n and c h r o m a t o g r a p h y

(5.).

A n o t h e r

u n d e s i r a b l e p r o p e r t y

i s

t h a t

theh a r s h

a c t i o n

ofme c h a n i c a l

d i s r u p t i o n

c a u s e sthe

r e l e a s e

0097-6156/

86/

0314-0002 06.00/

0

1986American ChemicalSociety


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1. H E T T W E R A N D

W A N G

ProteinReleasefrom hemically Permeabilized E

coli

3

o f n e a r l y a l lthes o l u b l e c e l l u l a r p r o t e i n . E x t e n s i v e p u r i f i c a t i o n

schemes are r e q u i r e d to i s o l a t e the

p r o d u c t f r om t h e s e e x t r a n e o u s

c e l l u l a r p r o t e i n s .

On e

a lt e r n a t i v e

tom e c h a n i c a l d i s r u p t i o n is to

t r e a t

the

c e l l s

w i t h

membrane

a c t i v e compounds

t h a t

can p e r m e a b i l i ze the c e l l to

p r o t e i n

wi t h o u t c a u s i n g e x t e n s i v e b r e a k ag e of the c e l l . The

o b j e c t i v eoft h i s

r e s e a r c h

was to

s t u dy

thep r o t e i n r e l e a s e k i n e t i c s

an d mechanismof a p e r m e a b i l i z a t i o n p r o c e s s u s i n g g u a n i di n e - H C land

T r i t o n - X l O O . G u a n i d i n e - H C l , a c h a o t r o p i c a g e n t , has been

d e m o n s t r a t e d to be c a p a b l e of

s o l u b i li z i n g

p r o t e i n f r o m E. c o l i

membrane

f r a g me n t s (6). P r e s u m a b l y ,

t h i s

o c c u r s v ia g u a n i d in e s

i n t e r a c t i o n

w i t h

w a t e r wh i c h

a l l o w s h y d r o p h o b i c g r o u ps tobecome

t h e r m o d y n a m i c a l l y more

s t a b l e

in anaqueousphase ( 7 ) . T r i t o n - X l O O ,

a n o n i o n i c de t e r g e n t

t h a thas a

h i g h b i n d i n g

a f f i n i t y

f o r h y d r o p h o b ic

s p e c i e s ,

i s v e r y e f f e c t i v e i n b i n d i n g to and s o l u b i l i z i n g

p h o s p h o l i p i d s

f r o m E. c o l i i n n e rmembraneand o u t e r w a l l f r a g m e n t s

( 8 ) .

Methods

C e l l

p r e p a r a t i o n .

E s c h e r i c h i a

c o l iK12,

s t r a i nW3110,

was

grown

i n a

14 l i t e r f e r m e n t e r at

3 7 C ,

pH 7.0 u s i n g d e f i n e d

media. A d d i t i o n a l

n i t r o g e nwas

s u p p l i e d

by NH OH

w h i c h

was a u t o m a t i c a l l y f e dtoc o n t r o l

t h epH. Thef e r m e n t a t i o n b r o t hwash a r v e s t e din thel a t e e x p o n e n t i a l

phase

andc o o l e dto4C . The

c e l l s

were i m m e d i a t e l y

c e n t r i f u g e d

at

4 C andwashed w i t h

b u f f e r

(.1M

T r i s

pH 7 . 0 ) . F o l l o w i n ga s e c o n d

c e n t r i f u g a t i o n , the

c e l l s

were r e s u s p e n d e d in

b u f f e r

tog i v ea d e n s e

c e l l

s u s p e n s i on

(^50 g

p r o t e i n / 1 ) .

C e l l p e r m e a b i l iz a t i o n .

The

p e r m e a b i l i za t i o n

p r o c e s swas s t a r t e dby

a d d i n g 30 ml of the c e l l s u s p e n s i onto 70 ml of a b u f f e r e d

s o l u t i o n

c o n t a i n i n g

g u a n i d i n e - H C l a n d / o r

T r i t o n

X100. The r e p o r t e d

c o n c e n t r a t i o n s

of

T r i t o n ,

g u a n i d i n e , and

c e l l s

a lw ay s c o r r e s p o n dto

t h e c o n c e n t r a t i o n s a f t e r m i x i n g t h e s e s o l u t i o n s . The m i x t u r e was

s h a k e n

at 200 rpm in a 4C i n c u b a t o r . Samples were w i t h d r a w nat

v a r i o u s

t i m e sandwere i m m e d i a t e l y

c e n t r i f u g e d .

The s u p e r n a t a n twas

a s s a y e d to de t e r m i n e d the

r e l e a s e

of thev a r i o u s c e l l components.

A n a l y s i sof thep e l l e twasdonetop e r f o r mamass b a l a n c e .

A n a l y s i s of c e l l components. P r o t e i n was d e t e r m i n e d w i t h the

B r a d f o r d dye b i n d i n g as s a y u s i n g b ov i n e serum a l b u m i n as s t a n d a r d

( 9 ) . I n t e r f e r e n c eby T r i t o nX100 was a c c o u n t e d f o rby e n s u r i n g

t h a t

e v e r y

sample

had .2

T r i t o n .

In o r d e r to d e t e r m i n e theamountof

u n r e l e a s e d p r o t e i n

f r o m

thesample p e l l e t s ,

a l l

s a m p l e swere t r e a t e d

f o r 5m i n ut e s w i t hIN NaOH at1 0 0 C .

DNA was d e t e r m i n e d by thed i ph e n y l a mi n e

r e a c t i o n

( 1 0 ) . Two 45

m i n u t e

e x t r a c t i o n s

at7 C w i t h ,5NHCIO were u s e d to

r e l e a s e

DNA

f r o m the

sample p e l l e t s .

I n t e r f e r e n c e f r om g u an i d in ewas a c c o u n t e d

f o r bymaking

e a c h

sample.4M

g u a n i d i n e .

RNA was de t e r m i n e d by theo r c i n o l pr o c e d u r e ( 1 1 ) . Two 15

m i n u t e

e x t r a c t i o n s at7 C in ,5N

HC10

4

were

u s e d

tor e l e a s eRNA

f r o m

the

sample p e l le t s . I n t e r f e r e n c e f rom T r i t o nX100 was a c c o u n t e d f o rby

making e a c h sample1

T r i t o n .


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4

SEPARATION

R EC O V ER Y A N D PURIFICATION IN B I O T E C H N O L O G Y

R e s u l t s and D i s c u s s i o n

F i g u r e 1

shows

t h e p r o t e i n , DNA, a nd RNA r e l e a s e

p r o f i l e s

o b t a i n e d

when

E .

c o l i

c e l l s a r e m e c h a n i c a l l y d i s r u p t e d

w i t h

.1 mm g l a s s

b e a d s .

The

c e l l

c o n c e n t r a t i o n

p r o f i l e

n o r m a l i z e d

t o t h e

i n i t i a l

c e l l

c o n c e n t r a t i o n , was o b t a i n e d w i t h a b a c t e r i a l c o u n t i n g

chamber.

The

d e c r e a s e

i n t h e c e l l c o n c e n t r a t i o n i n d i c a t e s t h a t e x t e n s i v e

f r a g m e n t a t i o n o f t h e

c e l l s

i s o c c u r r i n g . A n e a r l y m i r r o r

image

r e l e a s e o f DNA, RNA, a nd p r o t e i n r e s u l t s a s c e l l u l a r

components s p i l l

o u t i n t o t he e x t r a c e l l u l a r f l u i d . T h e maximum p r o t e i n r e l e a s e ,

7 0 , i s p r o b a b l y i n d i c a t i v e o f a s i g n i f i c a n t

amount

o f c e l l u l a r

p r o t e i n b e i n g a s s o c i a t e d w i t h t h e membrane a nd w a l l f r a g m e n t s .

A s i m i l a r c h a r a c t e r i z a t i o n f o r c e l l s t r e a t e d

w i t h

2M

g u a n i d i n e

an d 2 T r i t o n i s shown i n F i g u r e 2. T he p r o t e i n r e l e a s e ,

b a s e d

o n

t o t a l c e l l u l a r p r o t e i n , l e v e l s o f f a t 3 5 . RNA i s r e l e a s e d t o a

l e s s e r e x t e n t (^15 ) a nd v e r y l i t t l e D NA (^5 ) i s r e l e a s e d f r o m t h e

c e l l s .

T he c o n s t a n t

c e l l

c o n c e n t r a t i o n i n d i c a t e s t h a t t he r e l e a s e i s

n o t t he r e s u l t o f

c e l l f r a g m e n t a t i o n .

From t h e s e

r e s u l t s , t h r e e

m a j o r

d i f f e r e n c e s

between c h e m i c a l

p e r m e a b i l i z a t i o n a nd

m e c h a n i c a l

d i s r u p t i o n c a n be

i d e n t i f i e d .

F i r s t

t he r e l e a s e o c c u r s b y

f u n d a m e n t a l l y

d i f f e r e n t

mechanisms.

W i t h

m e c h a n i c a l d i s r u p t i o n t he c e l l s a re e s s e n t i a l l y t o r n a p a r t ,

whereas

w i t h c h e m i c a l t r e a t m e n t

t h e

c e l l

s t r u c t u r e i s

s t i l l

p r e s e n t b u t h a s

been a l t e r e d t o a l l o w r e l e a s e o f

i n t r a c e l l u l a r

components. Se c o n d ,

t h e r e i s a n e a r l y

c o m p l e t e

p r e f e r e n t i a l r e l e a s e o f p r o t e i n

o v e r

DNA.

T h i r d ,

t h e r e i s a p a r t i a l s e l e c t i v e r e l e a s e o f p r o t e i n

o v e r

RNA.

T h i s

s e l e c t i v i t y

may r e s u l t

f r o m

a

m o l e c u l a r

s i e v i n g

mechanism.

The

a v e r a g e

p r o t e i n

m o l e c u l a r

w e i g h t

i s

40,000

whereas

t h e

c e l l u l a r

DNA

h a s a

m o l e c u l a r w e i g h t

o f 2 .5 1 0 ( 1 2 ) . T h e

m o l e c u l a r w e i g h t

d i s t r i b u t i o n

o f RNA; 18 i s

25,000,

2 7 i s

500,000,

a n d 5 5 i s

1,000,000 i s s u c h t h a t

most

o f t h e RNA i s a l s o

s i g n i f i c a n t l y

l a r g e r

t h a n

p r o t e i n s ( 1 2 ) .

T h e s e

d i f f e r e n c e s

s u g g e s t

s e v e r a l a d v a n t a g e s o f t h e

c h e m i c a l

p e r m e a b i l i z a t i o n method.

F i r s t

a v o i d i n g c e l l b r e a k a g e s h o u l d

s i m p l i f y t h e

c e l l r e m o v a l

s t e p . S e c o n d , r e t e n t i o n o f t he n u c l e i c

a c i d s i n s i d e t h e

c e l l

s h o u l d e l i m i n a t e t h e

n e e d

f o r a n u c l e i c a c i d

p r e c i p i t a t i o n s t e p . A n o t h e r a d v a n t a g e i s t h a t t he p e r m e a b i l i z a t i o n

p r o c e s s a l s o

k i l l s

t h e c e l l s t h e r e b y e l i m i n a t i n g th e n e e d f o r t h e

f e d e r a l l y mandated c e l l k i l l i n g

s t e p .

F i g u r e 2 showed t h a t 3 5 o f t h e t o t a l c e l l u l a r p r o t e i n i s

r e l e a s e d

upon

t r e a t i n g th e c e l l s w i t h 2M

g u a n i d i n e

a nd 2 T r i t o n . A

more

c o m p l e t e

d e s c r i p t i o n o f ;the e f f e c t o f v a r y i n g t h e

g u a n i d i n e

and

T r i t o n

c o n c e n t r a t i o n s o n t h e

f i n a l amount

o f p r o t e i n r e l e a s e d i s

shown i n F i g u r e 3. Two s e t s o f e x t r a c t i o n s were c o n d u c t e d : one

c o n s i s t e d o f u s i n g 2 T r i t o n

w i t h

a r a n g e o f

g u a n i d i n e

c o n c e n t r a t i o n s , t h e o t h e r c o n s i s t e d o f u s i n g 2M

g u a n i d i n e w i t h

a

r a n g e

o f T r i t o n c o n c e n t r a t i o n s . T h e s e r e s u l t s i n d i c a t e t h a t t h e

g u a n i d i n e - H C l

c o n c e n t r a t i o n i s t he more s e n s i t i v e p a r a m e t e r .

M a n i p u l a t i o n o f t h e

g u a n i d i n e

c o n c e n t r a t i o n i n t h e p r e s e n c e o f 2

T r i t o n

l e a d t o r e l e a s e y i e l d s t h a t

r a n g e d f r o m

6 t o 6 0

whereas

v a r y i n g t he T r i t o n c o n c e n t r a t i o n

f r o m

0 t o 8 i n t h e

p r e s e n c e

of 2M

g u a n i d i n e

o n l y c h a n g e d t h e y i e l d f r o m 2 5 t o 4 0 .

T h e t i m e p r o f i l e s o f t h e 2M/2 , 2M, and 2 t r e a t m e n t s , shown i n

F i g u r e 4, i n d i c a t e a s y n e r g i s t i c e f f e c t

between g u a n i d i n e

and T r i t o n .

Th e p r o t e i n r e l e a s e

p r o f i l e

o f the 2M/2

t r e a t m e n t

i s n o t s i m p l y t h e


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H E T T W E R A N D

W A N G

Protein Release from hemically Permeabilized E . coli

EXTENTOF RELEASE OF CELL COMPONENTS (* ),

CONCENTRATON OF UNDSRUPTED CELLS (

DNA

RNA

IJ

1 2 3 4 5 6 7 8 9 10 11 12

DSRUPTONTME (min.)

F i g u r e 1.

E x t e n t

of

c e l l b r e a k a g e

and

r e l e a s e

of c e l l u l a r

p r o t e i n , DNA, and RNA d u r i n g m e c h a n i c a l d i s r u p t i o n w i t h .1 mm

g l a s s b e a d s .

EXTENTOF RELEASE OF CELL COMPONENTS (* ),

CONCENTRATON OF UNOSRUPTFn fTLLS (*)

TME (hours)

F i g u r e 2. R e l e a s e of

c e l l u l a r

p r o t e i n , DNA, and RNA d u r i n g

t r e a t m e n t w i t h

2M

g u a n i d i n e

HC1and2 T r i t o nX100.


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S E PA R A T I O N R E C O V E R Y A N D P U R IF IC A T I O N IN B I O T E C H N O L O G Y

FINAL PROTEN

RELEASE

(*)

( a l l 2 Tri tonX100)

^^^^Xall 2M GuanidineHC1)

1 2 3 4 5 6 7 8 9 10

TRITON X100 (* v/ v), GUANIDINE HC1 (M)

F i g u r e3. E f f e c tofT r i t o n X 1 0 0andg u a n i d i n eHC1ont h e p r o t e i n

r e l e a s e

y i e l d .

PROTEN

RELEASE (*)

0

2 / 2

-

-

2t1

GUANIDINEHC1

_

2* TRITON X100

1

Q

1 1 1 1

1 2 3 4 5 6

TME hours)

F i g u r e

4

S y n e r g i s t i c e f f e c t

on the


p r o f i l e

b e t w e e n

g u a n i d i n e

HC1an d T r i t o n X 1 0 0 .


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1. H E T T W E R

A N D

W A N G

Protein Release from hemically Permeabilized E

coli 7

a d d i t i o nof thep r o f i l e s o b t a i n e d when2M

g u a n i d i n e

and 2 T r i t o nare

u s e d i n d i v i d u a l l y . Thea c c e l e r a t i o nof ther a t e of p r o t e i n r e l e a s e

by T r i t o nmay be r e l a t e dto thea b i l i t yofT r i t o ntos o l u b i l i ze

l i p i d

membranes.

Onew o u l d

a n t i c i p a t e t h a t

thec o m b i n a t i o nof 2M g u a n i d i n e

an d

2

T r i t o n a l t e r s

the E.

c o l i

i n n e rmembrane

and

o u t e r w a l l

to a

g r e a t e r e x t e n t

t h a n

e i t h e r i n d i v i d u a l

t r e a t m e n t , t h e r e b y pr o d u c i n ga

more p e r m e a b le

c e l l .

T he e f f e c t of v a r y i n g the

c e l l

c o n c e n t r a t i o n on the p r o t e i n

r e l e a s e

p r o f i l e

of 2M/2

t r e a t m e n t s

isshowninF i g u r e 5. The

c e l l

c o n c e n t r a t i o n s are

e x p r e s s e d

int e r m sof thep r o t e i n c o n c e n t r a t i onof

t h e e x t r a c t i o n s o l u t i o n . A l t h o u g hno s i g n i f i c a n t e f f e c twas

o b s e r v e d

on

the

r e l e a s e p r o f i l e

the

r e l e a s e y i e l d

d e c r e a s e d by a

f a c t o r

of

two

upon

i n c r e a s i n g

thec e l l

c o n c e n t r a t i o n f r o m

3.6 g/1 to 43.3 g/1.

T h e e x a c t

n a t u r e of ther e a s o n

f o r

thed e c r e a s e d

y i e l d

at

h i g h

c e l l

c o n c e n t r a t i o n s

is notknown. However,

d e p l e t i o n

of the g u a n i d i n e

a n d / o r T r i t o n d ur i n g

thep r oc e s s is not

o c c u r r i n g ,

as e v i de n c e dby

t h e

f a c t

t h at t r e a t i n g c e l l s

a s e c o n d t i m e w i t h

f r e s h

g u a n i d i n eand

T r i t o n does

not i n d u c e

a d d i t i o n a l r e l e a s e ( d at a

notshown). If

d e p l e t i o n

of theg u a n i d i n e

a n d / o r T r i t o n

c a u s e d the


t o

c e a s e , one w o u l d e x p e c t

t h a t

a s e c o n d t r e a t me n t w o u l d c a u s e

f u r t h e r

r e l e a s e

of

p r o t e i n

f r o m the

p a r t i a l l y a f f e c t e d

or as yet

u n a f f e c t e d

c e l l s .

hole Broth

r o t i n

Cone

PROTEIN

RELEASE * ) 3.6 g/ 1

12 5 g/ 1

27.4 g/ 1

43.3 g/ 1

TME hours)

F i g u r e

5.

E f f e c t

of c e l l

c o n c e n t r a t i o n

on the


p r o f i l e .


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8

SEPARATION

R EC O V ER Y

A N D PURIFICATION IN

B I O T E C H N O L O G Y

C o n c l u s i o n s

E x p o s u r e of E. c o l i to

g u a n i d i n e - H Cl

and

T r i t o n - X l O O i n d u c e s

the

r e l e a s e of c e l l u l a r p r o t e i n s . The r e l e a s e r a t eand y i e l d were f o u n d

t o be d e p e n d e n t on the g u a n i d i n e , T r i t o n , and c e l l c o n c e n t r a t i o n s .

H i g h e r c o n c e n t r a t i o n s of g u a n i d i n e and T r i t o n and l o w e r c e l l

c o n c e n t r a t i o n s gave g r e a t e r r e l e a s e r a t e sandy i e l d s . G u a n i d i n e a l o n e

i s c a p a b l e of r e l e a s i n g a s i g n i f i c a n t

amount

of p r o t e i n . T r i t o n

r e l e a s e s a v e r y low

l e v e l

ofp r o t e i nbut s u b s t a n t i a l l y i n c r e a s e sthe

r a t e ofr e l e a s ewhen u s e d i nc o n j u n c t i o n w i t h g u a n i d i n e .

T h e

mechanism

of the

r e l e a s e ,

a

p e r m e a b i l i z a t i o n

of the c e l l i s

f u n d a m e n t a l l y

d i f f e r e n t f r o m m e c h a n i c a l

d i s r u p t i o n

w h i c h

i n v o l v e s

e x t e n s i v e f r a g m e n t a t i o n of the c e l l s . The a v o i d a n c e of e x t e n s i v e

c e l l b r e a k a g e s h o u l d s i m p l i f y the c e l l

r e m o v a l

s t e p and r e t e n t i o nof

t he n u c l e i c a c i d s i n s i d e the c e l l s h o u l d e l i m i n a t e the n e e d fo r a

n u c l e i c a c i d

p r e c i p i t a t i o n

s t e p . F u r t h e r m o r e , s i n c e the t r e a t m e n t

k i l l s the c e l l s a s e p a r a t e c e l l k i l l i n g s t e pmay be u n n e c e s s a r y .

Acknowledgment

We

w o u l d

l i k e to

acknowledge p a r t i a l

s u p p o r t

f r o m

the N a t i o n a l

S c i e n c e F o u n d a t i o n .

Literature Cited

1. Edebo, L. In Fermentation Advances ; Perlman, D., Ed.;

AcademicPress: NewYork, 1969; p. 249.

2.

Schutte,

H; Kroner, K. H.; Hustedt, H.; Kula, M. R. Enzyme

Microb. Technol. 1983, 5, 143.

3. Bucke, C. In Principles of Biotechnology ; Wiseman, ., Ed.;

Surrey University

Press:

New

York,

1983; p. 151.

4. Higgins, J. J.; Lewis, D. J.; Daly, W H.; Mosqueira, F. G.;

Dunnill, P.;

Lilly,

M. D. Biotech. Bioeng. 1978, 20, 159.

5. Wang, D. I. C.; Cooney, C. L.; Demain, .; Dunnill, P.;

Humphrey,

.;

Lilly,

M. In Fermentation and Enzyme

Technology ;

JohnWiley

Sons:

New

York,

1979; Chap. 12.

6. Moldow, C. J. MembraneBiol. 1972, 10, 137.

7. Hatefi, Y.; Hanstein, W In Methods in Enzymology ; Fleischer,

S.; Packer, L.; Eds.; Academic

Press:

New

York,

1974; p. 770.

8. Schnaitman, C. J.

Bact.

1971,

108(1),

545.

9. Bradford, M.

Anal.

Bioohem. 1976, 72, 248.

10. Burton, K. Biochem. J. 1956, 62, 315.

11. Herbert, D.; Phipps, P. J.; Strange, R. E. In Methods in

Microbiology ;

Norris, J. R.; Ribbons, D. W.; Eds.; Academic

Press: London, 1971; p. 210.

12. Brock, T. In Biology of Microorganisms ; Prentice-Hall:

Englewood Clifts, NewJersey, 1979; p. 131.

RECEIVEDMarch 26

1986


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2

Structured

and SimpleModelsofEnzymatic Lysis and

DisruptionofYeast

Cells

J .

B.Hunterand J. A.

Asenjo

Biochemical

EngineeringLaboratory,Department

ofChemical

Engineering

andApplied

Chemistry,Columbia

University,NewYork, NY

10027

Microbial

cell-wall-lytic enzymes

are

widely

used

in the

laboratory

for

cell breakage, proto

-plasting

of

yeasts and bacteria, and

forstudies

of

the

structure and composition

of

microbial

cell

walls

1).

Recentlylytic

systems

have come

underconsideration

as a

specific and chemically

mildway

to

rupture microbial cells on an in

dustrial scale (2,3). There appear

to be

attrac

tive commercial applications

of

lytic

systems

for

the

recovery

of

enzymes, antigens and

other

recombinant products accumulatedwithin

cells,

for

upgrading

of

microbial biomass

for

food

and feed

uses 4,5)

and

for

the manufacture

of

functional

biopolymers from

cell

wall carbo

hydrates

6).

This

paper

presents

two models

of

enzymatic

lysis

of

yeast

cells;

a

simplified two-step model,

accountingfor

protein

releaseat

cell lysis

followed

by proteolysis, and

a

more complex mecha

nisticmodelwhichdescribes

the

removal

of the

two layers

of

theyeast

wall

and

the

extrusion

and rupture

of

the protoplast and organelles.

The

use of

these

models

in

predicting

the

release

and breakdown

of

microbial proteins, and

the ap-

plication

of the

structured model

to

enzyme

re-

coverywill also

be

discussed.

On e p r o b l e m i n p r o d u c t i o nof r e c o mb i n a n t p r o t e i n s i s r e c o v e r yof

t h e f i n i s h e d p r o d u c t f r o m t h e c e l l s w h i c h a c c u mu l a t e i t . T h i s

p r o b l e m i s

p a r t i c u l a r l y

a c u t e i n t h e c a s e

of

y e a s t s

andf u n g i ,

w h i c h

h a v e t o u g h , t h i c k

c e l l

w a l l s w h i c h a r e d i f f i c u l t t or u p t u r e me c h a n i

c a l l y

or by

s o n i c a t i o n . P r o d u c t s e c r e t i o n i s n o t a l w a y s f e a s i b l e ,

e v e n f o r l o w - mo l e c u l a r - w e i g h t p r o d u c t s , a l t h o u g ha n e wl y d e v e l o p e d

s e c r e t i o n p r o c e s s f o r y e a s t (7) a p p e a r s p r o mi s i n g .

0097-6156/

86/

0314-0009 06.50/

0

1986

American Chemical

Society


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10

SEPARATION, R E C O V E R Y , A N D

PURIFICATION

IN

B I O T E C H N O L O G Y

T h e r e a r e n u me r o u s

e x a mp l e s

o f o v e r p r o d u c e d r e c o mb i n a n t p r o t e i n s

w h i c h

p r e c i p i t a t e

i n t r a c e l l u l a r l y

i n E_. c o l i , f o r mi n g d e n s e i n c l u s i o n

b o d i e s ( 8 ) ; t h e s e p r o d u c t s i n c l u d e

i n s u l i n

a n d s o ma t o s t a t i n , b o t h

v e r y s ma l l p r o t e i n s . I n y e a s t ,

r e c o mb i n a n t

v i r a l s u r f a c e a n t i g e n

p r o t e i n s a r e n o t s e c r e t e d , b u t

a s s e mb l e

i n t o p a r t i c l e s ( 9 ) .

S u b

c e l l u l a r

s t r u c t u r e s s u c h a s mi t o c h o n d r i a , l y s o s o me s o r t h e v a c u o l e

mu s t

a l s o b e r e c o v e r e d b y c e l l

b r e a k a g e ,

f o r u s e e i t h e r a s b i o -

c a t a l y s t s ( 1 0 ) o r a s a n i n i t i a l s t e p i n t h e p u r i f i c a t i o n o f e n z y me s

a s s o c i a t e d w i t h s u c h s t r u c t u r e s .

U n t i l

n o w, t h e s e p r o d u c t s h a v e

g e n

e r a l l y b e e n h a r v e s t e d b y me c h a n i c a l l y r u p t u r i n g t h e c e l l s i n a

h o mo g e n i z e r ,

b e a d

m i l l

o r F r e n c h p r e s s . T h e h i g h s h e a r

f i e l d s ,

e l e

v a t e d t e mp e r a t u r e s a nd g a s - l i q u i d

i n t e r f a c e s

g e n e r a t e d i n t h e s e

d e v i c e s c a n d e n a t u r e p r o t e i n s , e s p e c i a l l y mu l t i - e n z y me c o mp l e x e s

a n d me mb r a n e - l i n k e d p r o t e i n s ( 1 1 ) . Mo r e o v e r , t h e s e p a r a t i o n

o f c e l l d e b r i s

f r o m

t h e p r o d u c t s i s e s p e c i a l l y c o mp l i c a t e d i f t h e

p r o d u c t i s p a r t i c u l a t e ,

f r a g i l e

o r me mb r a n e - a s s o c i a t e d .

L y t i c e n z y me s y s t e ms p r o v i d e a c h e mi c a l l y m i l d , l o w- s h e a r

a n d c a t a l y t i c a l l y s p e c i f i c a l t e r n a t i v e t o me c h a n i c a l c e l l d i s

r u p t i o n . De p e n d i n g

o n t h e p a r t i c u l a r

l y t i c s y s t e m e mp l o y e d

a n d

i t s

p u r i t y , t h e

e n z y me s

ma y b e e n g i n e e r e d t o a t t a c k c e l l

w a l l

c o m

p o n e n t s a l o n e , w i t h o u t p r o d u c t d a ma g e . T h e e n z y me l y s o z y me ,

a c t i v e

a g a i n s t

some b a c t e r i a l c e l l w a l l s , h a s b e e n u s e d t o h a r v e s t b o v i n e

g r o w t h h o r mo n e g r a n u l e s f r o m _ E.

c o l i

( 8 ) , a n d a me mb r a n e - a s s o c i a t e d

h y d r o x y l a s e c o mp l e x

f r o m

P . p u t i d a ( 1 1 ) ; u s e o f o t h e r b a c t e r i o

l y t i c e n z y me s

f r o m

a v a r i e t y o f mi c r o b i a l s o u r c e s h a v e a l s o b e e n

r e p o r t e d

( 3 ) .

I n v e s t i g a t i o n s i n t o

t h e s u b c e l l u l a r l o c a t i o n o f

e n z y me a c t i v i

t i e s

i n mi c r o b i a l

c e l l s

s u g g e s t t h a t o n e o r mo r e e n z y me p r o d u c t s

c o u l d

b e

s p e c i f i c a l l y f r a c t i o n a t e d f r o m

a s i n g l e b a t c h o f c e l l s

b y p r o p e r l y c o n t r o l l i n g c e l l d i s r u p t i o n . I n v e r t a s e i n y e a s t i s

p o s s i b l y

t h e b e s t e x a mp l e o f t h i s p r i n c i p l e . T h e s t u d i e s l e a d i n g

t o d i s c o v e r y o f i t s l o c a t i o n ( i n t h e p e r i p l a s mi c

s p a c e ) h a v e

b e e n s u mma r i z e d b y P h a f f ( 1 2 ; p . 1 7 1 - 1 7 3 ) , a n d a s a mp l e p r o c e s s f o r

i t s r e c o v e r y h a s b e e n

p r o p o s e d

( 4 ) . T h e r e c o v e r y o f s e v e r a l

d i f

f e r e n t e n z y me s i n h i g h y i e l d a n d h i g h r e l a t i v e p u r i t y s h o u l d b e

p o s s i b l e

u s i n g a c o mb i n a t i o n o f

l y t i c

e n z y me s , s u r f a c t a n t s a n d o s

mo t i c s u p p o r t b u f f e r s t o

s e l e c t i v e l y

a n d s e q u e n t i a l l y r e l e a s e p r o

t e i n s

f r o m p a r t i c u l a r

s t r u c t u r e s .

C e l l

f r a c t i o n a t i o n

b y me c h a n i c a l r u p t u r e h a s a l r e a d y

come

u n d e r

i n v e s t i g a t i o n . Two s e p a r a t e s t u d i e s o f me c h a n i c a l r u p t u r e o f y e a s t

s h o w e d

d i f f e r e n t r a t e s o f r e l e a s e f o r

e n z y me s

i n d i f f e r e n t c e l l

l o c a t i o n s ( 1 3 , 1 4 ) . W a l l - l i n k e d a n d p e r i p l a s mi c e n z y me s

w e r e

r e

l e a s e d r e l a t i v e l y

f a s t e r t h a n t o t a l p r o t e i n , s o l u b l e c y t o p l a s mi c

e n z y me s a t a b o u t t h e s a me r a t e , a n d t h e mi t o c h o n d r i a l e n z y me f u ma r a s e

l a t e r

t h a n t o t a l p r o t e i n ( 1 3 ) . P r o t e o l y s i s b y t h e y e a s t

1

s own

e n z y me s wa s n o t f o u n d t o b e a p r o b l e m.

A c t i v i t i e s

o f t h e r e l e a s e d

e n z y me s d e c l i n e d s l o w l y o r n o t a t a l l w h e n d i s r u p t i o n wa s c o n

t i n u e d a f t e r t h e e n d o f p r o t e i n r e l e a s e , a n d t h e e f f e c t o f s h e a r wa s

n o t s e p a r a t e d f r o m t h e

e f f e c t

o f p r o t e o l y s i s . S h e t t y a nd K i n s e l l a

( 1 5 ) a l s o f o u n d a l o w r a t e o f

p r o t e o l y s i s a f t e r

me c h a n i c a l

d i s r u p t i o n ,

t h o u g h

t h i o l

r e a g e n t s a d d e d t o we a k e n t h e c e l l w a l l s b e f o r e

d i s r u

p t i o n

c a u s e d

a n i mp o r t a n t i n c r e a s e i n t h e e x t e n t o f p r o t e i n b r e a k

d o w n .


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2. H U N T E R A N D ASENJO Enzymatic Lysis and Disruption of east ells

11

M o d e l B a c k g r o u n d

Y e a s t

c e l l

s t r u c t u r e . The e x t e n s i v e b o d y of l i t e r a t u r e on

c e l l

w a l l c o mp o s i t i o n

and

s t r u c t u r e h a s r e c e n t l y b e e n r e v i e we d

by

B a l l o u

( 1 6 ) and

e a r l i e r

by P h a f f ( 1 2 ) .

A s an e n g i n e e r i n g a p p r o x i ma t i o n , t h e

c e l l

w a l l of y e a s t may be

c o n s i d e r e d

as a t w o - l a y e r s t r u c t u r e . ( F i g u r e 1) The i n n e r w a l l i s

c o mp o s e d of a

mi x t u r e

of

b r a n c h e d 3 ( 1 - 3 )

and

3 ( 1 - 6 ) l i n k e d g l u c a n s ,

g l u c o s e p o l y me r s s i m i l a r t oc e l l u l o s e ( 1 2 ) . Theo u t s i d e o f t he

g l u c a n l a y e r i s c o v e r e d w i t h a ma n n a n - p r o t e i n

c o mp l e x

c o n s i s t i n g

o f

a

c r o s s - l i n k e d

n e t w o r k of

p r o t e i n

mo l e c u l e s , t ow h i c h a r e a t

t a c h e d

t wo

t y p e s

ofma n n a n : ana c i d i c

o l i g o s a c c h a r i d e ,

and a

h i g h e r

mo l e c u l a r we i g h t p h o s p h o ma n n a n h a v i n g a d . p . ofa b o u t 100 ( 1 7 ) .

F r o m

t h e p e r s p e c t i v e of

c e l l l y s i s ,

t h i s ma n n o p r o t e i n l a y e r s e r v e s

t o

p r o t e c t t h e g l u c a n s f r o m h y d r o l y t i c e n z y me s ( 1 8 , 1 9 , 2 0 ) . W i t h i n

t h e t wow a l l

l a y e r s

i s t h e p r o t o p l a s t , c o mp r i s e d of a p l a s ma mem

b r a n e e n c l o s i n g t h e c y t o s o l

and

t h e s u b c e l l u l a r s t r u c t u r e s .

E n z y me s of t h e

l y t i c

s y s t e m. M i c r o b i a l y e a s t - l y t i c

e n z y me

s y s t e ms a r e w i d e l y d i s t r i b u t e d i n n a t u r e ,

and

h a v e b e e n

i s o l a t e d

f r o m

R h i z o c t o n i a s p . , ( 4 ) , B a c i l u s c i r c u l a n s ( 2 1 ) , C o p r i n u s ma c r o r h i z u s

( 2 2 ) ,

andC y t o p h a g a s p . ( 2 3 ) , a m o n g o t h e r s o u r c e s .

C r u d e

y e a s t

l y t i c

e n z y me s y s t e ms c o mp r i s e s e v e r a l h y d r o l y t i c

a c t i v i t i e s , o f t e n i n c l u d i n g c h i t i n a s e , ma n n a n a s e , and av a r i e t yof

p r o t e a s e s andg l u c a n a s e s ( 1 ) . O n l y t wo of t h e s e a c t i v i t i e s , a

l y t i c

p r o t e a s e

and a

l y t i c g l u c a n a s e , a r e e s s e n t i a l f o r

l y s i s

( 1 9 , 2 4 , 2 0 ) .

L y t i c g l u c a n a s e s

u s u a l l y

b i n d p r e f e r e n t i a l l y t o l o n g c h a i n s of 3 ( 1 - 3 )

g l y c o s i d i c

l i n k a g e s , s u c h

as

t h o s e f o u n d i n

m i c r o f i b r i l l a r

y e a s t

w a l l

g l u c a n .

I ng e n e r a l , t h e

l y t i c

g l u c a n a s e s h a v e an e n d o -

a c t i o n

p a t t e r n b u t some a t t a c k e x o - w i s e ,

r e l e a s i n g

o l i g o s a c c h a r i d e s

of

5 g l u c o s e

u n i t s

f r o m t h e s t r u c t u r a l y e a s t g l u c a n . O t h e r g l u c a n a s e s ,

w i t h

d i f f e r e n t

s u b s t r a t e s p e c i f i c i t y and

a c t i o n

p a t t e r n s , a r e

u s u a l l y

p r e s e n t i n t h e

l y t i c

s y s t e m a n d a c t s y n e r g i s t i c a l l y t o d e g r a d e

i n s o l u b l e y e a s t g l u c a n t og l u c o s e andd i s a c c h a r i d e s ( 2 5 ) . L y t i c

p r o t e a s e s h a v e

ac h a r a c t e r i s t i c

h i g h

a f f i n i t y

f o r t h e y e a s t w a l l s u r

f a c e , ando f t e n h a v e a n o ma l o u s l y l o w a c t i v i t i e s a g a i n s t c o n v e n t i o n a l

p r o t e i n

s u b s t r a t e s . T h e i r

r o l e

i n l y s i s of v i a b l e y e a s t c e l l s

c a n n o t be

s u b s t i t u t e d

by o r d i n a r y p r o t e a s e s . ( 2 0 , 2 6 ) .

W

u s e d

a

l y t i c

s y s t e m f r o m O e r s k o v i a

x a n t h i n e o l y t i c a

L L - G1 0 9

f r o m t h e c o l l e c t i o n of M L e c h e v a l i e r ,atR u t g e r s U n i v e r s i t y .

F i l t e r e d c u l t u r e b r o t h

was

u s e d

as

t h e e n z y me s o u r c e . D e t a i l s

of

t h e e n z y me p r o d u c t i o n a r e g i v e n e l s e wh e r e ( 2 7 , 2 8 ) . The l y t i c a c

t i v i t y

of t h e O e r s k o v i a s y s t e m i s due t o a

l y t i c

p r o t e a s e and an

e n d o 3 ( 1 , 3 ) g l u c a n a s e ( 2 0 ) ,

p o s s i b l y

s u p p l e me n t e d w i t h an e x o 3 ( 1 - 3 )

g l u c a n a s e

r e mo v i n g

a 5 - s u g a r u n i t f r o m t h e c h a i n ( 2 9 ) .

S e q u e n c e of c e l l l y s i s . E n z y ma t i c c e l l

l y s i s

b e g i n s w i t h b i n d

i n g

of t h e

l y t i c

p r o t e a s e t o t h e o u t e r ma n n o p r o t e i n l a y e r of t he

w a l l . Thep r o t e a s e o p e n s up t h e

p r o t e i n

s t r u c t u r e , r e l e a s i n g w a l l

p r o t e i n s

a n d

ma n n a n s , and

e x p o s i n g t h e g l u c a n s u r f a c e

b e l o w

( F i g u r e

2 ) . N e x t , t h e g l u c a n a s e a t t a c k s t h e i n n e r w a l l ands o l u b i l i z e s t h e

g l u c a n ( 1 9 ) . Whent h e

c o mb i n e d a c t i o n

ofp r o t e a s e and g l u c a n a s e

h a s o p e n e d as u f f i c i e n t l y l a r g e h o l e i n t h e

c e l l

w a l l , t h e p l a s ma

me mb r a n e

andi t s c o n t e n t s a r e e x t r u d e d as a p r o t o p l a s t ( 1 ) . I n

o s m o t i c a l l y s u p p o r t e d b u f f e r s c o n t a i n i n g

. 55 t o 1. 2M

s u c r o s e

or


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12

S E PA R A T I O N , R E C O V ER Y , A N D P UR I FI C A T I O N IN B I O T E C H N O L O G Y

Mannoprotein Units

ell

Membrane Structural Glucan Units

F i g u r e 1 D o u b l e - l a y e r e d

s t r u c t u r e

of

t h e

y e a s t w a l l , e n c l o s i n g

t h e c e l l me mb r a n e

F i g u r e 2 S c h e ma t i c

of l y s i n g y e a s t c e l l


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2.

H U N T E R N D ASENJO Enzymatic Lysis and Disruption of east ells

13

m a n n i t o l , t h e p r o t o p l a s t r e ma i n s i n t a c t b u t i n d i l u t e b u f f e r s i t l y -

s e s

i mme d i a t e l y ,

r e l e a s i n g c y t o p l a s m i c p r o t e i n s

and

t h e o r g a n e l l e s

w h i c h may t h e ms e l v e s

l y s e .

Me a n wh i l e ,

p r o t e i n s r e l e a s e d

f r o m

t h e w a l l

and

t h e

c y t o p l a s m

a r e

s u b j e c t

t o

a t t a c k

by

p r o d u c t - d e g r a d i n g p r o t e a s e

c o n t a mi n a n t s

i n t h e

l y t i c s y s t e m

( 2 8 , 3 0 ) .

Mo d e l s

Ma t h e ma t i c a l mo d e l s w i t h d i f f e r e n t l e v e l s

of

s t r u c t u r e a r e

u s e

f u l

f o r t h e

d e s i g n of

r e a c t o r s ,

t o

c a r r y o u t s i m u l a t i o n s t u d i e s , f o r

p r o c e s s

o p t i m i z a t i o n

and

f o r i n c r e a s i n g o u r

u n d e r s t a n d i n g o f t he

m e c h a n i s t i c , b i o l o g i c a l

b e h a v i o r of

b i o c h e m i c a l

s y s t e ms .

H i s t o r i c a l l y

t h e r e h a s b e e n

l i t t l e

p u b l i s h e d

w o r k on

mo d e l s

of

m i c r o b i a l c e l l l y s i s . Themo d e l s

p r o p o s e d

f o r o v e r a l l c e l l

l y s i s

h a v e b e e n e l e me n t a r y and

t h e i r a p p l i c a t i o n h a s

b e e n

l i m i t e d .

F i r s t -

o r d e r

a n d

Mi c h a e l i s - M e n t e n mo d e l s h a v e b e e n

u s e d

t oe s t i ma t e

t h e

p e r f o r ma n c e

of a

s a mp l e

l y s i s p r o c e s s

( 2

S

3 ) , L y s i s

of

f r e e z e - d r i e d

Mi c r o c o c c u s

l y s o d e i k t i c u s c e l l s

by

l y s o z y me

was

mo d e l e d w i t h

a

s e c o n d - o r d e r

r a t e e x p r e s s i o n ( 3 1 ) .

At

t h e

o t h e r e nd of

t h e

s p e c t r u m

o f

ma t h e ma t i c a l c o mp l e x i t y

i s

a mo d e l of l y s o z y me - c a t a l y z e d d e g r a d a

t i o n of

s o l u b l e b a c t e r i a l

c e l l - w a l l

o l i g o s a c c h a r i d e s , f o c u s i n g

on

t h e

d e g r e e

of

p o l y m e r i z a t i o n

of

t h e s u b s t r a t e

and

t h e b i n d i n g m o d e s

of

e n z y me t os u b s t r a t e s ( 3 2 ) .

A c c o u n t i n g

f o r onee n z y me and c a r b o h y

d r a t e

o l i g o me r s up t o

d . p .

9,

i t h a s

n i n e d i f f e r e n t i a l e q u a t i o n sand

t e n

p a r a me t e r s ,

a n d

was

t e s t e d

on

p u r i f i e d r a d i o l a b e l e d o l i g o s a c c h a

r i d e s .

A l t h o u g h

u s e f u l f o r e l u c i d a t i n g e n z y me a c t i o n p a t t e r n s ,

s u c h

mo d e l s

a r e t o o d e t a i l e d

t o be

r e a d i l y a p p l i e d

t o a

mu l t i - e n z y me ,

m u l t i - s u b s t r a t e

s y s t e m.

T h e

t womo d e l s ofy e a s t

l y s i s

p r e s e n t e d h e r e h a v e b e e n

d e v e l o p

e d

t os e r v e t wo

d i f f e r e n t

p u r p o s e s . The s i mp l e mo d e l

i s

a l u mp e d ,

t w o - s t e p mo d e l w h i c h

f o l l o w s t h e

ma j o r

f e a t u r e s

of

t h e

d a t a

a n d

may

p r o v e

u s e f u l f o r d e s i g n

of l y s i s

r e a c t o r s .

The

s t r u c t u r e d mo d e l ,

w h i c h

c a n a c c o u n t f o r t h e

s o u r c e

of p r o t e i n w i t h i n t h e c e l l , was

d e v e l o p e d t o

g a i n

a me c h a n i s t i c

b a s i s f o r p r e d i c t i n g t h e e f f e c t s

of

u n t e s t e d p r o c e s s

c o n d i t i o n s ,

and t o

a i d i n s i g h t i n t o t h e p h y s i c a l

p r o c e s s e s a two r k

d u r i n g

l y s i s .

S i mp l e

mo d e l .

The

s i mp l e

mo d e l

was

b u i l t

f o r

c o mp a c t

d e s c r i p t

i o n

of

t h e

d a t a

i n

a

p r e - d e t e r mi n e d r a n g e

ofy e a s t and

e n z y me c o n c e n

t r a t i o n s .

I t t r e a t s c e l l

l y s i s and

p r o t e o l y s i s

as

s i n g l e - s t e p

r e a c t i o n s i n s e q u e n c e . B o t h r e a c t i o n s a r e mo d e l e d w i t h M i c h a e l i s -

Me n t e n

k i n e t i c s , e v e n

t h o u g h

y e a s t , t h e s u b s t r a t e

of

t h e

f i r s t

r e a c t i o n ,

i s p a r t i c u l a t e

and

t h e p r o t e i n s a r e s o l u b l e .

The

d i f f e r e n t

e n z y me s

of

t h e l y t i c s y s t e m a r e g r o u p e d t o g e t h e r i n t o

ana l l - i n c l u

s i v e s i n g l e e n z y me ,

E,

b e a r i n g b o t h t h e p r o t e o l y t i c a n d y e a s t - l y t i c

a c t i v i t i e s .

A l l

of

t h e

c e l l

s t r u c t u r e s a r e a l s o c o n s i d e r e d

t o g e t h e r

a s

a

u n i f i e d

y e a s t c e l l ma s s , Y.

When ac e l l

i s a t t a c k e d

by e n z y me s

i t i s

p r e s u me d t o

d i s s o l v e

i n s t a n t a n e o u s l y , r e l e a s i n g i t s e n t i r e

ma s s

as

s o l u b l e p r o t e i n s ,

p e p

t i d e s

andc a r b o h y d r a t e s . Thea s s u mp t i o n of i n s t a n t a n e o u s

s o l u t i o n

o f t h e c e l l ma s s c o n s t r a i n s t h e mo d e l f o r u s e

w h e r e

t h e

l y s i s

me d i u m

i s h y p o - o s mo t i c and

p r o t o p l a s t s c a n n o t s u r v i v e i n t a c t .


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,1986|d

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.1021/bk-1

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14

SEPARATI ON, RECOVERY, A ND P UR I F I C AT I ON I N

BI OTECHNOLOGY

On l y

t wo

i n d e p e n d e n t

v a r i a b l e s a r e u s e d : y e a s t ( Y ) a n d e n z y me

E ) ;

t h e me a s u r e d v a r i a b l e s a r e y e a s t , T C A - i n s o l u b l e p r o t e i n ( ) ,

T C A - s o l u b l e p r o t e i n ( p e p t i d e s , S ) ,

a n d

c a r b o h y d r a t e s

( C ) ;

a l l

a r e

e x p r e s s e d

a s g / 1 d r y

b a s i s . Enz yme c o n c e n t r a t i o n

wa s

e x p r e s s e d

a s

t h e

v o l u me p e r

c e n t

o f c r u d e l y t i c

e n z y me

p r e p a r a t i o n

a d d e d t o t h e

r e a c t i o n mi x t u r e .

P r o t e o l y t i c

a n d o t h e r c a u s e s f o r l y t i c e n z y me

d e a c t i v a t i o n

( e . g . ,

t h e r ma l ) h a v e b e e n a s s u me d t o b e

n e g l i g i b l e

( 2 8 ) ,

d Y

d t

=

- k

a

( Y - Y

d P _ d Y

>

d t

f

p y

d t ,

d S _

- f

d Y /

d t

s y

d t ,

d C _

d t

~ f c y

d Y '

. d t ,

k

r

E

( Y

Y c o )

( Y -

Y c o )

+

k E - P

+ s +

m

P 1+

k

_ J 2

Y *

+ S + K

mp

1 +

-

1 )

( 2 )

3 )

4 )

V a r i a b l e names a n d p a r a me t e r v a l u e s a r e g i v e n i n T a b l e I .

On t h e

r i g h t - h a n d s i d e

o f

e q u a t i o n

1 , t h e

i n i t i a l

t e r m

r e p r e

s e n t s a u t o l y s i s

a n d t h e

s e c o n d

t e r m, e n z y ma t i c l y s i s .

E q u a t i o n

2

d e s c r i b e s p r o t e i n b r e a k d o wn b y p r o d u c t - d e g r a d i n g p r o t e a s e s . T h e

f i r s t

t e r m

o n t h e

r i g h t

s i d e s t a n d s

f o r t h e

p r o t e i n r e l e a s e d

f r o m

l y s i n g c e l l s , a n d t h e s e c o n d t e r m, b r e a k d o wn o f t h e

p r o t e i n a l r e a d y

i n s o l u t i o n . E q u a t i o n

3 s h o ws

t h a t p e p t i d e s

a r e

r e l e a s e d

f r o m

l y s i n g

y e a s t ,

b u t

a l s o a r i s e

f r o m b r e a k d o wn o f

l o n g e r p r o t e i n s ,

P .

S i n c e t h e p r o t e a s e a c t i v i t y a g a i n s t s o l u b l e p r o t e i n s i s

c o n

s i d e r e d

n o n - s p e c i f i c , b o t h l o n g -

a n d

s h o r t - c h a i n p r o t e i n s

w i l l b e

a t t a c k e d

b y t h e e n z y me

w i t h

e s s e n t i a l l y t h e

same

a f f i n i t y p e r g r a m

o f s u b s t r a t e .

He n c e , S w i l l a c t a s a

c o mp e t i t i v e i n h i b i t o r

o f t h e

e n z y me a c t i v i t y a g a i n s t P , wh e r e t h e i n h i b i t i o n c o n s t a n t i s e q u a l

t o

t h e M i c h a e l i s c o n s t a n t K ^ . C a r b o h y d r a t e r e l e a s e i s s h o wn i n

e q u a t i o n

4 .

P a r a me t e r s

f o r

t h e

s i mp l e

mo d e l we r e

d e t e r mi n e d

g r a p h i c a l l y

b y

E a d i e - H o f s t e e p l o t t i n g o f

i n i t i a l

r e a c t i o n r a t e s a n d s u b s t r a t e

c o n

c e n t r a t i o n s .

D e t a i l s

a r e

g i v e n

e l s e w h e r e 3 0 ) . A s h a s b e e n o b

s e r v e d

i n

h y d r o l y s i s

o f

o t h e r s o l i d s u b s t r a t e s ,

a

r e s i d u e

o f n o n -

l y s e d

s u b s t r a t e

wa s f o u n d a t e x t e n d e d

r e a c t i o n t i me s , when

d Y / d t

t e n d e d t o wa r d

z e r o . T h e e x t e n t o f r e a c t i o n wa s s t r o n g l y d e p e n d e n t

o n

i n i t i a l

s u b s t r a t e a n d e n z y me c o n c e n t r a t i o n s 33 , 34 ). A n

e m p i r i c a l

f u n c i t o n

f o r Y ^ wa s f i t t e d t o t h e

u l t i ma t e t u r b i d i t y d a t a

f o r

l y s i s r u n s a t a

v a r i e t y

o f

i n i t i a l y e a s t

a n d e n z y me

c o n c e n

t r a t i o n s u s i n g

a

l e a s t

s q u a r e s me t h o d . T h e

c a l c u l a t e d v a l u e s

f o r

Yoo we r e u s e d i n t h e s i mu l a t i o n s 3 0 ) . F i g u r e 3 s h o ws

r e s u l t s

o f

t h e s i mp l e

mo d e l .

S t r u c t u r e d mo d e l .

T h i s

mo d e l

c o n s i d e r s

l y s i s o f t h e c e l l f r o m

t h e v i e wp o i n t

o f

p r o g r e s s i v e

b r e a k d o wn o f t h e c e l l

s t r u c t u r e s ,

s t a r t i n g

f r o m

t h e o u t e r w a l l l a y e r a n d p r o g r e s s i n g t o t h e s u b c e l l u l a r

s t r u c t u r e s i n s i d e t h e p r o t o p l a s t 3 5 ) . H e r e t h e c e l l i s d i v i d e d

i n t o


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2. H U N T E R A N D ASENJO Enzymatic Lysis and Disruption of

Yeast

Cells

15

T a b l e

L.

L u mp e d mo d e l

v a r i a b l e s

a nd p a r a me t e r s

V a r i a b l e s -

S i mp l e Mo d e l

Y

Y e a s t , mg/ 1

Y

0

O r i g i n a l

y e a s t c o n c e n t r a t i o n

Yoo

U l t i ma t e y e a s t c o n c e n t r a t i o n ,

mg/ 1 ;

p r o p o r t i o n a l

t o r e s i d u a l t u r b i d i t y .

^= a 4 bE +c Y

0

+I

P r o t e i n

( T C A - i n s o l u b l e ) ,

mg/ 1

S

P e p t i d e s ( T C A - s o l u b l e ) ,

mg/ 1

C Ca r b o h y d r a t e s , mg/ 1

E n z y me , %( v / v ) o f r e a c t i o n

mi x t u r e

P a r a me t e r s - S i mp l e Mo d e l

Yoo c o n s t a n t s : a : 3 . 6 34 2 10

1

b :

- 2 . 6 5 8 4

10 ~

c :

6 . 0 44 2

6

d :

- 9 . 9 6 0 3

10

1

k

a

R a t e

c o n s t a n t

f o r a u t o l y s i s 3 . 9 8 7 1 0 - ^mi n

1

k

r

R a t e

c o n s t a n t

f o r l y s i s

s i mp l e

mo d e l 1 5 . 5 1 mg / L - m n - %e z

K

m

M i c h a e l i s c o n s t a n t

f o r l y s i s 1 9 0 2mg/ L

k p

R a t e

c o n s t a n t f o r

p r o t e o l y s i s ,

4 . 4 41 mg / L - m n - %e z

M i c h a e l i s c o n s t a n t ,

p r o t e o l y s i s , 4 5 9 8mg/ L

I n h i b i t i o n

c o n s t a n t ,

p r o t e o l y s i s , 2 6 0 7 7 mg

y e a s t / L

f

F r a c t i o n of

p r o t e i n

i n y e a s t 0 . 4 0 48

f

g

y

F r a c t i o n

of

p e p t i d e s

i n

y e a s t

0 . 0 7 77

f F r a c t i o n of

c a r b o h y d r a t e s

i n

y e a s t

0 . 3 6 87


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6

SEPARATION, R E C O V E R Y , A N D PURIFICATION IN B I O T E C H N O L O G Y

f o u r r e g i o n s ; t h e o u t e r w a l l o r w a l l p r o t e i n ( WP ) ; i n n e r w a l l o r w a l l

g l u c a n ( WG ) ; t h e c y t o s o l ( C S ) a n d t h e o r g a n e l l e s ,

h e r e g r o u p e d

t o g e t h e r

a s

mi t o c h o n d r i a

( M I ) . T h e

l y t i c

s y s t e m i s a p p r o x i ma t e d a s t h r e e

e n z y me s , a

l y t i c

g l u c a n a s e , E g , wh i c h h y d r o l y z e s t h e i n n e r

c e l l

w a l l

g l u c a n , a

l y t i c

p r o t e a s e ,

E p ,

wh i c h

a t t a c k s o n l y t h e

o u t e r

w a l l l a y e r

a n d a d e s t r u c t i v e p r o t e a s e , E ^ , a c t i v e a g a i n s t s o l u b l e p r o t e i n s .

P r o d u c t i n h i b i t i o n i s i n c l u d e d i n a l l e n z y me r e a c t i o n s . A d s o r p t i o n

a n d d e s o r p t i o n o f t h e

e n z y me s

t o t h e y e a s t w a l l i s n e g l e c t e d , s i n c e

a d s o r p t i o n k i n e t i c s a p p e a r e d

i n s t a n t a n e o u s

o n t h e

t i me

s c a l e o f o u r

me a s u r e me n t s ( 3 5 ) . A s c h e ma t i c o f t h e r e a c t i o n

p a t h wa y s

i s s h o wn i n

F i g u r e 4 .

S p e c i a l v a r i a b l e s .

E G A = ( WG - r - WP )

T h e g l u c a n h y d r o l y s i s r a t e i s n o t r e l a t e d

d i r e c t l y

t o

t o t a l

g l u c a n

c o n c e n t r a t i o n WG, b u t r a t h e r t o t h e a mo u n t o f

g l u c a n

made

a c c e s s i b l e t o a t t a c k t h r o u g h r e mo v a l o f w a l l p r o t e i n f r o m t h e o u t s i d e

o f t h e

c e l l .

E G A ,

e x p o s e d

g l u c a n , a c c e s s i b l e r e p r e s e n t s t h e a mo u n t

o f g l u c a n

u n c o v e r e d

b y

r e mo v a l

o f t h e o u t e r w a l l . T h e p r o p o r t i o n a l i t y

c o n s t a n t r i s t h e

we i g h t r a t i o

o f w a l l g l u c a n t o w a l l p r o t e i n .

T h e o v e r a l l r a t e o f s o l u b l e p r o t e i n h y d r o l y s i s , P B R , p r o t e i n

b r e a k d o wn r a t e ,

a c c o u n t s

f o r d e s t r u c t i o n o f s o l u b l e p r o t e i n b y t h e

d e s t r u c t i v e p r o t e a s e .

T h e r e l e a s e o f c y t o s o l i n t o t h e

me d i u m d e p e n d s

o n t h e o s mo t i c

b r e a k a g e o f t h e p r o t o p l a s t s , wh i c h o c c u r s a t a r a t e a p p r o x i ma t e l y

p r o p o r t i o n a l t o t h e o s mo t i c g r a d i e n t a c r o s s t h e p l a s ma

me mb r a n e

( 3 6 ) .

T h e i n t e r n a l o s m o l a l i t y o f t h e c e l l s w a s e s t i ma t e d t o b e 0 . 6 1 7 O s / L

( 3 5 ) , wh e r e 1 O s / L i s e q u i v a l e n t t o 1

Mo l / L

o f a n i d e a l s o l u t e . T h e

e x t e r n a l o s m o l a l i t y i s t h e s u m o f t h e c o n t r i b u t i o n f r o m t h e b u f f e r

s y s t e m i n t h e me d i u m ( a b o u t 0. 02M i n o u r e x p e r i me n t s ) a n d t h e

s u b s t a n c e s

r e l e a s e d b y l y s i n g p r o t o p l a s t s . T h e

s t a b i l i z a t i o n

o f t h e

r e ma i n i n g

c e l l s

b y t h e s e s u b s t a n c e s i s f a r s t r o n g e r t h a n c o u l d b e

e x p e c t e d s o l e l y o n t h e b a s i s o f o s mo t i c e f f e c t s , a n d c o u l d r e s u l t

f r o m t h e r e l e a s e o f c a t i o n s wh i c h i n t e r a c t w i t h s p e c i f i c r e c e p t o r s

o n t h e p l a s ma

me mb r a n e

( 3 7 ) . T h e r e l e a s e o f s o l u b l e p r o d u c t s o f

g l u c a n a n d p r o t e i n h y d r o l y s i s a r e a l s o e x p e c t e d t o a d d t o t h e

s t a b i l i

z i n g e f f e c t o f t h e l y s a t e .

T h e e f f e c t i v e o s m o l a l i t y o f

c e l l

l y s a t e w a s f i t t o a L a n g mu i r

e x p r e s s i o n , wh e r e

SM

L

i s t h e maxi mum

s t a b i l i z i n g

e f f e c t a n d i s

t h e e q u i l i b r i u m c o n s t a n t f o r i n t e r a c t i o n o f t h e

s t a b i l i z e r s

w i t n t h e

p r o t o p l a s t s . T h e r e s u l t i n g e q u a t i o n ,

e x p r e s s e s

t o t a l

e f f e c t i v e o s m o l a l i t y i n t h e

l y s i s

me d i u m.

B

Q

i s t h e

o r i g i n a l

o s m o l a l i t y o f t h e

l y s i s

b u f f e r a n d C S * i s t h e s u m o f p r o t e i n ,

p e p t i d e s a n d c a r b o h y d r a t e s p r e s e n t a t t h e s t a r t o f r e a c t i o n .

P B R

SM

x

= B

0

+

Q S

V

K

o s J

C S

* +

C S

o ~

C S

>

+ ( C S * + C S - C S )

o s m


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H U N T E R

AND A S EN JO Enzymatic LysisandDisruptionof Yeast Cells

TME

MINUTES

F i g u r e 3 S i mp l e mo d e l s i m u l a t i o n of y e a s t l y s i s

Y e a s t

c e l l

ma s s , mg 1

mg/ 1

P e p t i d e s ,

mg 1

h y d r a t e s , mg 1

0. 78 g/ 1

y e a s t c o n c e n t r a t i o n ;

10%en z y me

e n

s

5

P r o t e i n ,

-

C a r b o -

Oligopeptides

Amino

Acids

Wall

Protein

Wall

Enzymes

WallMannan

Soluble Proteins

Cytoplasmic Enzymes

1Proteaseattack

2. Glucanase attack

3 Release

of

cell

contents

4. Lysis of

organelles

5.Glucanhydrolysis

6

7. >Productproteolysis

;

/ - \

Proteins

O r g a n e l l e s - < > -

Organellar Enzymes

Carbohydrates

fl l-3)

Oligosaccharides

Glucose

F i g u r e 4 R e a c t i o n p a t h wa y s f o r s t r u c t u r e d mo d e l


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18

S E P A R A T I O N , R E C O V E R Y , A N D P U R I F IC A T I O N IN B I O T E C H N O L O G Y

B a s e d o n t h e p r o d u c t OS M [ / K

c

t h e s t a b i l i z i n g e f f e c t o f c e l l l y

s a t e a t l o w c o n c e n t r a t i o n s i s e q u i v a l e n t t o 4. 4 1 0

H

Os/ mg c y t o s o l

r e l e a s e d ( 3 5 ) .

W a l l h y d r o l y s i s e q u a t i o n s .

d ( WP )

d t

wp ( Km

WP

w j y _

( 1 )

1 +

WP

Km

wp

WP - WP

K i

wp

d( WG)

d t

k

wg g ( Km

EGA

wg

( 2 )

l + # ^ +

Km

WG - WG

wg

Km

s g

R e l e a s e

o f c y t o s o l a n d m i t o c h o n d r i a . T h e

o s mo t i c

g r a d i e n t b e

t we e n p r o t o p l a s t s a n d b u f f e r o r m i t o c h o n d r i a a n d b u f f e r d r i v e s t h e

r e l e a s e o f p r o t e i n i n t o t h e me d i u m. I f t h e o s m o l a l i t y o f t h e e x t e r n a l

me d i u m

e x c e e d s t h e

i n t e r n a l

o s m o l a l i t y o f t h e p r o t o p l a s t o r o r g a n e l l e ,

n o r u p t u r e o c c u r s . T h e o s m o l a l i t y

d e c r e a s e s

i n t e r n a l l y , a n d i n c r e a s e s

e x t e r n a l l y , a s m a t e r i a l i s r e l e a s e d f r o m t h e p r o t o p l a s t . I n a d d i t i o n ,

t h e r e l e a s e

o f

c y t o s o l

i s

p r o p o r t i o n a l

t o t h e

s i z e

o f t h e

o p e n i n g

i n

t h e w a l l g l u c a n ,

u p t o a ma x i mu m

h o l e s i z e

o f 1/ 3 o f t h e

c e l l ^ s u r

f a c e a r e a .

d ( C S )

d t

-

( C S ) . k

f l

( C S ) k ^ ma x ^ O S M ^

CS

CS

- 0 S M

x

) ]

ma x ( . 3 3 ,

1

WG

WG

) ( 3 )

d ( M I )

d t C S ,

d ( C S )

' d t

k

r m

[ ma x ( 0 ,

0 . 3 - OS K . )

]

( 4 )

S o l u b l e p r o d u c t s .

V a l u e s

f o r T C A - i n s o l u b l e p r o t e i n , p e p t i d e s a n d

c a r b o h y d r a t e s r e l e a s e d we r e e s t i m a t e d b y s u mmi n g t h e c o n t r i b u t i o n t o

e a c h p o o l

f r o m

t h e b r e a k d o wn o f e a c h c e l l u l a r s t r u c t u r e .

^ - - f

d t

d ( WP )

p wp [ d t

- f

' d ( C S ) '

p e s

I

d t

f

k

[ ma x ( 0 ,

0 . 3 - OS M

) ]

- P B R

pm r m

( 5 )


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2. HUNTER

A N D ASENJO

Enzymatic

ysis

and Disruption ofYeast Cells

19

- - f -

d t s wp

' d ( WP ) '

- f

[ d t s e s

d C S l

d t

f - k [ ma x ( 0 , 0. 3 - OSM) ] - M I + PBR ( 6)

s m

r m

d

m

_ d ( WG) _ f - d ( C S )

( 7 )

d t

d t ' d t

T o t a l

y e a s t c e l l

mas s

was e s t i ma t e as t h e s um o f WG WP, CS,a n d MI

( s t r u c t u r e s r e ma i n i n g w i t h t h e c e l l ) , w i t h an a d d e d f a c t o r a c c o u n t i n g

f o r n o n - p r o t e i n , n o n - c a r b o h y d r a t e s u b s t a n c e s i n t h e c e l l . T h e s e sums

g e n e r a t e v a l u e s f o r y e a s t ,

p r o t e i n ,

p e p t i d e s

Separation Recovery and Purification in Biotechnology 1986 Recent Advances and Mathematical Modeling

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