Advances in Biomedical Engineering

257 Pages·1979·3.241 MB·English

by  H. Michael Koplove, Charles L. Cooney (auth.)

ADVANCES IN
BIOCHEMICAL
ENGINEERING
Volume 21
Editors: .T K. Ghose, A. Fiechter,
N. Blakebrough
Managing Editor: .A Fiechter
With 99 Figures
Springer-Verlag
Berlin Heidelberg New York 1979
ISBN 3-540-09262-5 Springer-Verlag Berlin Heidelberg New York
ISBN 0-387-09262-5 Springer-Verlag New York Heidelberg Berlin
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Editors
Prof. Dr. T. K. Ghose
Head, Biochemical Engineering Research Centre, Indian Institute of Technology
Hauz Khas, New Delhi 110029/India
Prof. Dr. A.Fiechter
Eidgen. Techn. Hochschule, Mikrobiologisches Institut, WeinbergstraBe 38, CH-8092 Ziirich
Prof. Dr. N. Blakebrough
The University of Reading, National College of Food Technology
Weybridge Surrey KT13 0DE/England
Managing Editor
Professor Dr. A.Fiechter
Eidgen. Techn. Hochschule, Mikrobiologisches Institut, Weinbergstrage 38, CH-8092 Ziirich
Editorial Board
Prof. Dr. .S Aiha Prof. Dr. R.M.Lafferty
Biochemical Engineering Laboratory, Institute of Applied Techn. Hochschule Graz, Institut riK Biochem. Technol.,
Microbiology, The University of Tokyo, Bunkyo-Ku, SchlSgelgasse ,9 A-8010 Graz
Tokyo, Japan
Prof. Dr. M.Moo-Young
Prof. Dr. .B Atkinson University of Waterloo, Faculty of Engineering, Dept. Chem.
University of Manchester, Dept. Chemical Engineering, Eng., Waterloo, Ontario N21 3 GL/Canada
Manchester/England
Dr. .I Nfiesch
Dr. J. BSing Ciba-Geigy, K 4211 B 125, CH-4000 Basel
RShm GmbH, Chem. Fabrik, Postf. ,6614 D-6100 Darmstadt
Prof. Dr. .L K. Nyiri
Prof. Dr. J. R. Bourne Dept. of Chem. Engineering, Lehigh University, Whitaker
Eidgen. Techn. Hochschule, Techn. Chem. Lab., Lab., Bethlehem, PA 18015/USA
Universit~itsstraBe ,6 CH-8092 Ztirich
Prof. Dr. H.J.Rehm
Dr. .E Bylinkina Westf. Wilhelms Universit~it, Institut f'fir Mikrobiologie,
Head of Technology Dept., National Institute of Antibiotika, TibusstraBe 7-15, D-4400 Mtinster
3a Nagatinska Str., Moscow M-105/USSR
Prof. Dr. P. L. Rogers
Prof. Dr. H.Dellweg School of Biological Technology,. The University of New
Techn. Universit~it Berlin, Lehrstuhl fi2r Biotechnologie, South Wales, PO Box ,1 Kensington, New South Wales,
SeestraBe ,31 D-1000 Berlin 56 Australia 2033
Dr. A.L. Demain Prof. Dr. W. Schmidt-Lorenz
Massachusetts Institute of Technology, Dept. of Nutrition Eidgen. Techn. Hochschule, Institut riR Lebensmittelwissen-
& Food Sc., Room 56-125, Cambridge, Mass. 02139/USA schaft, Tannenstrage ,1 CH-8092 Ziirich
Prof. Dr. R.Finn Prof. Dr. H.Suomalainen
School of Chemical Engineering, Director, The Finnish State Alcohol Monopoly, Alko,
Olin Hall, Ithaca, NY 14853/USA P.O.B. 350, 00101 Helsinki 10/Finland
Dr. K. Kieslich Prof. Dr. F.Wagner
Schering AG, Werk Charlottenburg, Max-Dohrn-StraBe, Ges. .f Molekularbiolog. Forschung, Mascheroder Weg ,1
D-1000 Berlin 01 D-3301 StSckheim
Contents
Enzyme Production During Transient Growth
H. M. Koplove, South Charleston, West Virginia (USA)
C. L. Cooney, Cambridge, Massachusetts (USA)
Stabilized Soluble Enzymes 14
R. D. Schmid, Diisseldorf (Germany)
The Use of Coenzymes in Biochemical Reactors 911
.S .S Wang, C.-K. King, Piscataway, New Jersey (USA)
Process Development and Economic Aspects 741
in Enzyme Engineering. Acylase L-methionine System
C. Wandrey, Jfilich (Germany)
E. Flaschel, Lausanne (Switzerland)
The Rational Design of Affinity Chromatography 912
Separation Processes
D. J. Graves, Philadelphia, Pennsylvania (USA)
Y.-T. Wu, Bound Brook, New Jersey (USA)
Enzyme Production During Transient Growth
Reorganization The of Protein Synthesis
H. Michael Koplove a and Charles L. Cooney b
Department of Nutrition and Food Science
Massachusetts Institute of Technology c
Cambridge, MA 02139, USA
1 Preface ............................................... 2
2 Introduction ............................................ 3
3 Balanced Growth .......................................... 4
3.1 Batch Culture ........................................ 4
3.2 Continuous Culture ..................................... 6
3.3 Enzyme Production during Balanced Growth ...................... 9
3.4 Synchronous Culture .................................... 12
4 Unbalanced Growth ........................................ 13
4.1 Cell Response to Unbalanced Growth .......................... 14
4.2 Maaloe's Model ....................................... 16
4.3 Ribosomal RNA and Ribosomal Proteins ........................ 18
4.4 RibosomaIEfficiency and "Extra" rRNA ........................ 19
4.5 Transfer RNA ........................................ 22
4.6 RNA Chain Elongation Rate and RNA Polymerase ................... 23
4.7 Guanosine Tetraphosphate ................................. 26
4.8 Chromosome Replication ................................. 27
4.9 Enzyme Synthesis ...................................... 28
4.10 Maaloe's Model Revisited .................................. 35
5 Conclusions ............................................. 37
6 References ............................................. 38
The problems of enzyme production are examined in the light of molecular biological events.
An understanding of the dynamics of protein synthesis that occur during transient growth are
reviewed to provide insight into the potentials and the limitationosf differential enzyme syn-
thesis.
a Present address: Union Carbide Corp., South Charleston, West Virginia.
b
Author to whom correspondence should be addressed.
c Publication Number 3359 from the Department of Nutrition and Food Science, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139.
2 .H .M evolpoK dna .C .L Cooney
i Preface
The well-controlled environmental conditions of a laboratory fermentor are, more
often than not, very different from the ecological niche from which the microor-
ganisms in the reactor have evolved. The "natural" environment of many microor-
ganisms is, in fact, one in which the nutrient supply may rapidly change. Therefore,
a discussion of the adaptive response of a microorganism to its environment must in-
clude not only its response to a wide variety of constant environmental factors, such
sa medium formulation, pH, and temperature, but also its manner of reorganizing
macro-molecular synthesis to accommodate environmental fluctuations. Koch )1 has
called this form of ecology a "feast or famine existence" and, in discussing enteric
microorganisms, writes:
The [enteric] microorganisms have not only been selected for ability to grow un-
der chronic starvation, but also for ability to respond quickly to unannounced and
irregular windfalls f o food. Selection is still directed toward growth. However, atone
time, the emphasis is on ability to accelerate growth and, at a later time, the empha-
sis is on coping with a deceleration of growth.
In balanced growth, microorganisms have supplies of ribosomal RNA, ribosomal
proteins, RNA polymerase and perhaps transfer RNA which increase sa a function of
growth rate. This pattern of response si referred to as "growth-associated". DNA,
protein, and messenger RNA concentrations are growth rate independent. Following
a change in the microbial environment, which allows the microorganism to grow more
rapidly, the cell will preferentially synthesize growth-associated components in a
short period of time. An increase in "general" protein synthesis, however, often lags
the initial burst of ribosome synthesis by 15-45 min and the DNA synthesis rate may
remain at the pre-shift rate for 60 rain or longer.
The purpose of this article si to present to the biochemical engineering audience
selected investigations which relate to the phenomenon of temporal organization.
Studies will be cited from both fundamental molecular biology and from investiga-
tions of a more general scope, with a focus upon enzyme synthesis during unbal-
anced growth. It si hoped that this presentation will help the reader to establish a
background of relevant physiologicala nd molecular biological studies as he reads
this Literature Review and enable him/her to conceptualize the events which occur
during microbial adaptations to rapidly changing environments. eW believe that the
regulatory mechanisms which are involved in temporal organization have been rela-
tively untapped with regards to their process development potential and we hope
that this review will stimulate thinking along these lines. With an improved under-
standing of the molecular biology and the regulatory mechanisms underlying enzyme
synthesis it should be possible to better direct via process control the overproduc-
tion of desired enzymes. In other words, it si time to now capitalize on our understand
ing of the molecular biology of the gene and its expression.
Production Enzyme gniruD tneisnarT htworG
2 Introduction
The terms "balanced growth" and "steady-state" growth are often used interchange-
ably although they really refer to somewhat different phenomena. Steady state, in
the engineering sense, is defined as the condition in which derivatives of all system
parameters with respect to time are now, and in principle, will remain zero. This defi-
nition can apply in a rigorous sense only to continuous cultures. Nagai et .la )2 have
incorporated this concept of steady state into a definition of balanced growth by
stating that, for balanced growth,
d(Ci)f
-0 (1)
dt
in which (Ci)f si the fraction of ,iC the concentration of the i th component of cell
material, and t is time.
The "steady state" discussed by Maaloe and Kjeldgaard )3 and many other investi-
gators who do not utilize chemostats is actually the balanced growth of microorgan-
isms in a batch culture carried out in a particular medium at a time at which lla nu-
trients are present in excess. During certain periods of time which may last several
cell doubling times, the steady-state criterion of Eq. (1) may be satisfied while the
chemical environment is changing. In the case of bacteria, both the cell mass and
number increase exponentially. In this situation, which si often called "unrestricted
growth", the cells are growing at their maximum specific growth rate permitted by
the physical-chemical environment. In order to change growth rates in a batch cul-
ture, investigators have changed a nutrient or other environmental parameter in the
medium. Typically, investigators of exponential growth have formulated media with
a constant amount of the necessary nitrogen, sulfur, phosphate, magnesium, and
trace salts and have varied the carbon source. For instance, in a culture of Escherichia
coli, a change from alanine to glucose wiclhla nge the growth rate from 0.014 to
1.0 -1 . h In some cases, amino acids are added to the growth medium; this type of
addition si called a nutritional enrichment. Throughout this review, to avoid confu-
sion, a medium which is referred to as "succinate medium" or "glucose medium"
will mean a medium whose sole carbon source si succinate or glucose and the remain-
der of the medium is a mineral salts solution which supplies the necessary quantity
of non-organic nutrients for growth.
A change in the carbon source to alter the specific growth rate probably causes de-
repression or induction of a region of the genome not previously transcribed and
translated. On the other hand, alterations in the specific growth rate in continuous
culture may be achieved by altering the flow rate of medium through the system .)4
This change in growth rate may be mediated through physiologicalr egulation, i.e.
by the control of enzyme activity or concentration, as opposed to molecular biologi-
cal events such sa enzyme synthesis s, )6
Throughout the following discussion, a persistant question may be raised by the
reader: "Are the authors quoted here truly observing what they claim to be key reg-
.H .M and Koplove .C Coone3 L.
ulatory events or are they seeing the end molecular biological result of very complex
physiological interactions?" This is an important and complex query which focuses
on the relationship of the regulation of protein synthesis and the physiological effects
of the regulation.
3 Balanced Growth
3.1 Batch Culture
Maaloe and Kjeldgaard )3 are considered the founding fathers of the branch of mo-
lecular biology which focuses upon the regulation of macromolecular synthesis in
response to growth rate changes, a set of regulatory events which has been also called
temporal organization )7 or metabolic regulation 8). In 1966, Maaloe and Kjeldgaard
compiled the primer of the field, a monograph entitled The Control of Macromolec-
ularSynthesis, in which they summarized and discussed the literature concerned with
balanced and unbalanced growth 3).
During balanced exponential growth of Salmonella typhimurium, Maaloe and
Kjeldgaard )3 reported that the amount of total stable RNA per unit of cell mass was
a linear function of specific growth rate, whereas the DNA content per unit of cell
mass decreased slightly with increasing growth rate. Of the total stable RNA, the ribo-
somal RNA (rRNA) concentration increased linearly with growth rate, while the
transfer RNA (tRNA) concentration was essentially independent of growth rate. To-
tal protein per unit of cell mass decreased slightly with increasing growth rate and
the number of genomes or nuclei per cell was shown to increase linearly at growth
rates exceeding 0.85 h -I , up to a maximum of 4.5 genomes/cell. (This number was
later confirmed by Chai and Lark )9 in an elegant autoradiography experiment.) A sum-
mary of results of this key experiment is shown in Table .1 Maaloe and Kjeldgaard 3),
using a variety of assumptions, proceeded to calculate the peptide chain elongation
rate per ribosome, often termed the ribosomal efficiency, and determined that the
elongation rate was constant at 14 amino acids per ribosome per second at growth
rates below 0.85 h -1 and increased slightly to 16 amino acids per ribosome-second
at a growth rate of 1.6 h -1 .
The data which they presented has been corroborated for the most part by more
recent investigations. For instance, Rosset et al. )°1 separated the RNA constituents
of Escherichia coli, A erobacter aerogenes, and S. typhimurium on methylated serum
albumin columns and found that the tRNA content is indeed constant as a function
of growth rate and comprises a larger fraction of the total RNA at lower growth
rates. On the basis of these results, Rosset et al.io) postulated that tRNA and rRNA
are regulated by different control mechanisms.
Dennis and Bremer I )1 compiled a large number of facts regarding the macromo-
lecular constituents ofE. coli during balanced, exponential growth. Some of their
more interesting observations were that both the RNA/DNA and the RNA/protein
Enzyme Production During Transient Growth
Table 1 (see ref. 3 for further details)
A
u Cells Total a
(h -l) (XIO -12)
per g DNA RNA rRNA tRNA Protein b
dry weight
(mg.g -1) (mg.g -1 ) (mg.g -1 ) (mg.g -1) (mg.g -1)
2.4 1.3 30 310 250 60 670
1.2 3.1 35 220 135 85 740
0.6 4.8 37 180 90 90 780
0.2 6.3 40 120 35 85 830
a Per g dry weight
b To a certain extent, membranes and ceil-wall material are included in this figure. We have not
attempted to estimate these quantities separately in .S typhimurium
zt Genome 70 S ribo- tRNA Amino acids b Amino acids c
(h -l) equiva- somes (per molecules (per genome (per 70 S ri-
lents a genome) (per genome X 10 -8) bosome per
(per cell) X 10 -5) second)
2.4 4.5 15,500 2.4 5.4 16
1.2 2.4 6,800 2.8 4.9 17
0.6 1.7 4,200 2.7 4.7 14
0.2 1.4 1,450 2.3 4.5 14
a Calculated from Eq. (3-4a)
b Based on an average molecular weight of 125 for the amino acids
c Calculated on the basis of the average number of ribosomes present in the cell during the cycle,
i.e., the figures of Column 3 multiplied by 1.5
fractions were linearly growth-associated, but that the protein/DNA ratio, paradoxi-
cally, was also growth-associated although only in certain regions at growth rates less
than 0.7 h- .1 "Growth associated" in this case, and in all future references, will be
used to indicate a positive correlation between growth-rate and the value of the para-
meters. A linearly growth-associated RNA/DNA ratio thus means that the ratio in-
creases linearly with growth rate.
Dennis and Bremer )11 also observed that the rRNA/total RNA fraction was con-
stant at 0.85; this observation implies that tRNA must also be a function of growth
rate, a result which appears to contradict Maaloe and Kjeldgaard .)3 Thus, there ap-
pears to be a discrepancy in the literature regarding the correlation between tRNA
and growth rate. More discussion will be presented regarding tRNA and this discrep-
ancy in Sect. 4.5 dealing with the response of tRNA to unbalanced growth.
.H .M evolpoK dna .C .L ~enooC
Based upon their data, Dennis and Bremer calculated a constant ribosome effi-
ciency in the growth rate range of 0.5-0.85 -1 h of 13.5 amino acids per ribosome-
second. At lower growth rates, they claimed that the ribosome efficiency, which si
called also the peptide chain elongation rate, decreased. Perhaps the most interesting
statement they make, which si a concept overlooked or at least rarely addressed by
many other molecular biologists, is that the difference in observations between high
and low growth rates may reflect a change in the nature of the metabolic limitation:
at low growth rates (e.g. <0.7 h -1 ), the limitation may result from an enzyme defi-
ciency (a physiological deficiency), and at higher growth rates, the limitation may
result from ribosomal efficiency (a molecular biological limitation).
Waldron and LaCroute )~l extended balanced growth observations to eucaryotes
by studying Saccharomyces cerevisiae. The RNA/DNA fraction of the cell was found
to increase linearly from 0.02 to 0.12 in the growth rate range of 0.14 to 0.5 h -1.
At 0.5 h -l, rRNA comprised 85% of the total stable RNA. Waldron and LaCroute
determined that the ribosomal efficiency varied linearly from 0.5 to 6.0 amino acids
per ribosome-second in the growth rate range of 0.035-0.42 h -1. They concluded
from this result that in eucaryotes, unlike bacteria, ribosomal efficiency si a function
of growth rate.
3.2 Continuous Culture
Most of the research concerned with the dependence of macromolecular composition
and regulatory mechanisms on growth rates has been done with balanced growth in
batch cultures. Several studies in chemostats, however, have explored the same phe-
nomena and are highlighted in this section. The primary objective of this section and
the previous one si to present a picture of macromolecular synthesis as a function
of growth rate during balanced growth. In later sections, the data presented here will
be used to develop a conceptual model of unbalanced growth.
Herbert )31 studied the effect of dilution rate on the cellular RNA, DNA and mean
mass cell ofA. aerogenes and Bacillus megaterium. His results, shown in Fig. ,1 were
qualitatively similar to those reported by Maaloe and Kjeldgaard3): the RNA fraction
per dry cell weight and the mean cell mass increased with increasing growth rate (or
dilution rate) while both the protein and DNA weight fraction decreased slightly sa
the growth rate increased. Similar results were obtained with A. aerogenes with both
glycerol and ammonia limitations from which evidence Herbert concluded that
growth rate, rather than the type of growth limitation, was the prime controlling
factor of cellular composition.
Dean andR ogers )41 studied sciezlel and macromolecular composition ofA. aero-
genes under a variety of growth limiting conditions in chemostats and found that the
RNA/DNA fraction increased linearly with growth rate, the protein/RNA decreased
with growth rate, and the DNA/cell mass was independent of growth rate for glucose,
nitrogen, and phosphate limitations. The data obtained from sulfate limitation varied
significantly from the above results and the authors claimed that anomalous RNA