A cell population structuring model to estimate recombinant strain growth in a closed system

for subsequent search of the mode to increase protein accumulation during protealysin
producer cultivation
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IOP PUBLISHING BIOFABRICATION
Biofabrication 4 (2012) 019601 (1pp) doi:10.1088/1758-5082/4/1/019601
Erratum: A cell population structuring
model to estimate recombinant strain
growth in a closed system for subsequent
search of the mode to increase protein
accumulation during protealysin
producer cultivation
S P Klykov et al 2011 Biofabrication 3 045006
S P Klykov
1,3
, V V Kurakov
1
, V B Vilkov
1
, I V Demidyuk
2
,
T Yu Gromova
2
and D A Skladnev
1
1
PHARM-REGION, Ltd, c.1, b.10, Baryshikha street, Moscow, 125222, Russia
2
Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq. 2, Moscow 123182,
Russia
E-mail: smlk03@mail.ru, pharm-region@mail.ru, duk@img.ras.ru, microb52@mail.ru and
skladda@gmail.com
Received 25 November 2011
Published 23 February 2012
Online at stacks.iop.org/BF/4/019601
There was an error in the published version of gure 4. The
correct gure is shown below.
3
Author to whom any correspondence should be addressed.
Figure 4. Dependence of enzymeactivity on growth time.
Growth time, hours, is the obscissa. Activity P, Units ml
1
, is
the ordinate axis. Experimental activity in Control 2005.
Model calculation of the activity in Control 2005.
Experimental activity in Control 2010. Model calculation
of the activity in Control 2010. Predicted activity in
Experiment 1 according to Control 2010 results. It is assumed
that specic rate of product destruction is equal to the specic
destruction rate in Control 2010. Experimental activity in
Experiment 1. Predicted activity in Experiment 1 according
to Control 2010 results. It is assumed that product destruction
does not occur.
1758-5082/12/019601+01$33.00 1 2012 IOP Publishing Ltd Printed in the UK & the USA
IOP PUBLISHING BIOFABRICATION
Biofabrication 3 (2011) 045006 (12pp) doi:10.1088/1758-5082/3/4/045006
A cell population structuring model to
estimate recombinant strain growth in a
closed system for subsequent search of the
mode to increase protein accumulation
during protealysin producer cultivation
S P Klykov
1,3
, V V Kurakov
1
, V B Vilkov
1
, I V Demidyuk
2
,
T Yu Gromova
2
and D A Skladnev
1
1
PHARM-REGION, Ltd, c.1, b.10, Baryshikha street, Moscow, 125222, Russia
2
Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq. 2, Moscow 123182,
Russia
E-mail: smlk03@mail.ru, pharm-region@mail.ru, duk@img.ras.ru, microb52@mail.ru and
skladda@gmail.com
Received 14 December 2010
Accepted for publication 12 September 2011
Published 25 October 2011
Online at stacks.iop.org/BF/3/045006
Abstract
In this paper we have proposed a new structured population growth model, further developing
a model previously proposed by the authors. Based on this model, optimal growth
characteristics of the recombinant strain Escherichia coli BL-21 (DE3) [pProPlnHis
6
] were
determined, which allowed us to increase the output of metalloproteinase by 300%. We have
experimentally demonstrated the applicability of the new model to cell cultures with implanted
plasmids and the potential practical use for an output increase of a wide variety of biosynthesis
processes.
(Some gures in this article are in colour only in the electronic version)
Notation
LGP logarithmic growth phase;
GIP growth inhibition phase;
S substrate concentration, g l
1
;
X biomass concentration, OD units or
g l
1
;
OD unit of an optical density;
time, hour;
P products-metalloproteinase, units
P ml
1
;
dP/d absolute rate of product synthesis, units
of P(ml h)
1
;
Q = dS/d absolute rate of substrate consumption;
Q
O
2
oxygen mass exchange rate, mmole
O
2
/(volume units per time units);
3
Author to whom any correspondence should be addressed.
J stochiometric factor of energy substrate
(S) oxidation, Joule of S/mmoleO
2
;
specic growth rate of biomass X,
(hour)
1
;
q = Q/X or
q = (1/X)

dP/d specic rate of substrate utilization
or product synthesis, units of S(units
of Xh)
1
or units of P(ml units of
Xh)
1
;
a trophic coefcient, amount of energy
substrate consumed for the synthesis of
a biomass unit, Joule of S/Joule of X or
units of S/units of X;
f amount of energy substrate accumulated
in biomass X during cultivation on a
synthetic medium, Joule of S/Joule of
X or units of S/units of X;
1758-5082/11/045006+12$33.00 1 2011 IOP Publishing Ltd Printed in the UK
Biofabrication 3 (2011) 045006 S P Klykov et al
m energy maintenance coefcient, the
rate of substrate consumption for
maintaining viability of one biomass
unit per a unit of time, Joule of S(Joule
of Xh)
1
or units of S(units of Xh)
1
;
A = m/a (1) Parameter describes a delay of the
biomass growth rate; (2) specic rate of
accumulation of stable cells, h
1
;
X
p
maximum biomass concentration, when
all the energy generated during
cultivation is consumed for cell viability
maintenance;
X
Lim
biomass concentration in the end of
exponential growth phase and beginning
of growth inhibition phase;
X
st
concentration of the biomass of zero age
cells (stable), the content of resting cells,
OD units or g l
1
;
X
div
= X X
st
concentration of proliferation biomass,
OD units or g l
1
;

Lim
time of exponential growth phase
termination, hour;
X
Lim
st
concentration of stable cells at the end
of exponential growth phase, OD units
or g l
1
;
R ratio of X
st
to biomass X, relative
content of stable cells in the biomass,
synchronization degree, part of 1;
X
l
initial biomass concentration in LGP
corresponding the beginning of popu-
lation structuring, OD units or g l
1
;
X
nal
nal biomass concentration, at which
R = 1 (when energy consumption is
limited);
k
r
div
, k
r
st
, k
s
div
, k
s
st
constants of metabolite and substrate
biochemical reaction rates, [units of P
(or S)/(ml

units of biomass per one

hour)];
P
Lim
metabolite concentration or ac-
tivity at the end of LGP and
beginning of GIP, units of
P ml
1
;
P
0
metabolite concentration or activity in
LGP, when biomass structuring occurs
at X = X
l
, units of P ml
1
.
1. Introduction
Studies on microorganism growth and biosynthesis of
metabolites have given rise to a great number of
mathematical models describing the dynamics of biomass
growth and nutrient substrate consumption. Many models of
microorganism growth are based on J Monods growth model
or its variants [1, 2]. The basic idea of these models consists in
the restriction or limitation of the specic growth rate of a cell
population either by concentrations of a limiting substrate or
of a biosynthetic end-product. However, limited application
areas and numerous exceptions are the usual drawbacks of
such models that necessitate the construction of a new model.
The basic disadvantage of the J Monod model described in [2]
is an ambiguity about the physical sense of its parameters (or
even absence of this sense).
The works cited above also describe the so-called logistic
curves characterizing all phases of microorganism growth.
However, the model data have no wide practical application in
designing biotechnological production technologies yet.
The Volterra model considers a variant of the logistic curve
describing the phase when all nutrients are completely utilized
and the cell population growth reaches the stationary phase
and/or the phase of cell destruction [2].
The fact that the models mentioned do not consider the
factors inuencing cell growth, for instance oxygen mass-
exchange, is a common disadvantage [2].
A number of mathematical models are also available to
describe product biosynthesis. Among these, the Leudeking
Piret model is one of the most well known [3].
One of the characteristic features of the LeudekingPiret
model is that factors either related or not related to cell
growth contribute to the kinetics of metabolite production.
For the cases when the studied substance is a nal product of
metabolismrelated to energy consumption, the rst member of
the LeudekingPiret equation describes biomass cell growth,
while the second one characterizes the amount of energy
consumed for cell viability maintenance. However, if the
target products are not related to biomass energy metabolism,
the LeudekingPiret equation is difcult to interpret.
Time dependence of metabolite concentration in the
batch process may be rather complicated. In the growth
medium several substances, which then are exposed to further
transformations, can be accumulated. In some cases, a
complicated kinetics of metabolite production may indicate
modications in the mechanism of cell metabolism under the
inuence of growth condition changes. The kinetics of acetate,
butyrate, acetone and butanol production by Clostridium
acetobutilicum[2] serves as a good example of these processes.
The production of metabolites can also be accompanied
by their chemical transformations in the growth medium. Such
complicated processes, thus, should require the inclusion of
these reactions, if they are well studied, into a mathematical
model. Undoubtedly, this would make the mathematical
description more sophisticated.
Currently, the models of biomass growth, substrate
consumption and metabolite synthesis are, as a rule,
subdivided into two groups: structured and unstructured
models [2].
The structured model implies the availability of more than
one component to describe the structure of a cell population
and its viability. The unstructured model suggests that the
cell population is homogeneous and only one component, for
example, biomass X, is used for its characterization.
Since unstructured models are rather simple, they are often
used for research.
In all of the above-mentioned models of cell growth,
energy consumption and metabolite synthesis are calculated
by different equations describing the amounts of consumed
substrates and synthesized products. From our point of view,
this approach is not correct, since both substrates and products
2
Biofabrication 3 (2011) 045006 S P Klykov et al
are substances, which undergo various transformations during
fermentation, and thus should be described by one and the
same equation.
Free access of oxygen to cells during cultivation plays
the key role in aerobic processes. Since oxygen deciency
slows cell growth down resulting in the reduction of aerobic
microorganism biomass output and synthesis of the majority
of metabolites, it is important to provide oxygen inow so
that the concentration of the dissolved oxygen in the substrate
would not be lower than 10% of a complete oxygen saturation
level.
In [4], a biotechnological method providing optimum
conditions for cell growth has been described. The method
suggests that during cultivation microorganism cultures
receive additional feeding from a balanced nutrient medium
concentrate. The inow rate and the amount of feeding
concentrate exclude both the lack of nutritious substrates, and
growth inhibition owing to high concentration of nutrient com-
ponents. In this case, oxygen mass exchange rate in the culture
remains constant and oxygen concentration tends to zero.
The PirtMarr equation,
Q = dS/d = adX/d + mX, (1)
proposed for the calculation of the energy substrate
consumption rate, has been modied for the analysis of energy
consumed by cells for growth and viability maintenance. This
was done by expressing Q through the oxygen mass exchange
rate as follows:
Q = JQ
O
2
. (2)
This yields the initial equation (3) for the unstructured
mathematical model of cell growth limited by oxygen
consumption. All the equation constituents are expressed in
energy units:
JQ
O
2
= adX/dt + mX. (3)
The solution of the basic equation (3) allows for the
formulation of fundamental laws of culture growth under
conditions of a limited oxygen supply rate. Unlike earlier
existing conceptions, it is shown that a linear decrease in
absolute growth rate of the biomass and hyperbolic reduction in
the specic growth rate is a function of biomass concentration,
and that the energy substrate consumption rate specied by
oxygen mass exchange rate is constant. Methods for dening
the parameters of the unstructured model proposed included
growth efciency and energy substrate consumption (m, a and
A=m/a), which were not previously used in any practical way
to estimate periodic culture growth. Parameter A describes a
delay of the total biomass growth rate.
Studies on the effect of Salmonella culture growth rate
on cell survival under adverse external inuences [5] showed
that during GIP, if there is a lack of dissolved oxygen, the
accumulation of stable cells occurs at a constant specic
rate equal to that of the growth delay (A = m/a). The
share of stable cells within the population is obviously equal
to that of nonproliferating cells, which consume energy
only for viability maintenance. Methods for the denition
of parameters of the structured model describing substrate
consumption and metabolite biosynthesis on the basis of
preliminary calculated parameters of the unstructured model
were designed.
Thus, the proposed structured model assumes that within
a growing population there are two groups of cells essentially
differing in their physiology. Group I represents newly
generated (young) cells and group II contains cells being
in a state of active proliferation. Although the cells of
group I are often called resting cells [1], in our opinion these
are the cells of zero age [2], i.e. the cells being in phase G
1
or in phase V as designated for eukaryotes and prokaryotes,
respectively. Group I cells exhibit minimal physiological
functions, and for each cell these functions are constant. A
characteristic feature of the cells is that they consume energy
substrates only for their viability maintenance. In [5] these
cells are called stable.
All these discussions give good theoretical basis for
the dynamics of the structure formation of microbial
populations limited by the lack of oxygen in terms of energy
consumption and cell viability maintenance. An analysis
of the experimental and literature data made it possible to
propose a new structured model of cell population growth
[68]. On the basis of the model, consumption of substrates
used for cell construction and synthesis of metabolites in the
cultures consisting of two groups of cells differing in energy
consumption was described.
The performed experiments and analysis of the literature
data have proved that the proposed model is adequate for
the description of a wide range of microorganisms (from
obligate aerobes up to obligate anaerobes), synthesis of various
products (antibiotics, organic acids, alcohol, poly--butyric
acid, polysaccharides, etc) and consumption of substrates
(glucose, nitrogen and phosphorus) [6, 7]. The present paper
shows the feasibility of the structured and unstructured models
for estimation of the growth of recombinant microorganism
strains and expression of foreign proteins in them.
An analytical solution of constitutive equations (1)(3)
for the unstructured model of biomass growth is shown in
[4]. Equations (1)(6) presented in table 1 describe changes
in the consumption of energy substrate S, growth of biomass
X, absolute growth rate of a total biomass dX/d and specic
growth rate .
In [8], it was shown that quantity of nonproliferating zero
age cells consuming energy only for viability maintenance
changes in direct proportion to the change of energy
consumption for viability maintenance, i.e. adX
st
= dS
st
(3a).
On the other hand, apparently dS
st
/d = mX
st
(3b) since
cells of zero age (stable cells) consume an energy substrate
only for viability maintenance. Superposition of the last two
equations gives equation (28), table 1.
It is also obvious that if m 0, then, according to
equation (1), the rate of energy substrate consumption
Q is proportional to the absolute biomass growth rate
dX/d, and exponential cell population growth is observed.
For GIP, m = 0.
Successful research in the eld of recombinant products
performed during the last 30 years has led to an increase of
their manufacturing. Taking into account all of the above-
mentioned, we have found it interesting to use the proposed
3
Biofabrication 3 (2011) 045006 S P Klykov et al
Table 1. Basic equations for GIP.
Function Equation ##
1 2 3
Q =
dS
d
= f (X) Q = a
dX
d
+ mX (1)
Q = f (Q
O
2
) Q = JQ
O
2
(2)
Q = f (X) JQ
O
2
= adX/d+mX = mX
p
(3)
dS
st
= f (dX
st
) dS
st
= adX
st
(3a)
dS
st
/d = f (X
st
) dS
st
/d = mX
st
(3b)
dX
d
= f (X)
dX
d
= A(X
p
X) (4)
=
dX
d

1
X
= f (X) = A
_
Xp
X
1
_
(5)
X = f () X = X
p
(X
p
X
Lim
) exp[A(
Lim
)] (6)
dX
st
d

1
X
st
dX
st
d

1
X
st
=
m
a
= A (7)
X
st
= f () X
st
= X
st
Lim
exp[A(
Lim
)] (8)
R = f () R =
K
A
2
X
2
p

A
+
A

+ 2
_
(9)
R = f (X) R =
K
A
2
(XpXX
2
)
(10)
K K =
dX
d

dX
st
d
= A
2
X
st
Lim
(X
p
X
Lim
) (11)

nal

nal
=
1
A
ln
XpX
Lim
XpXnal
+
Lim
(12)

nal

nal
=
1
A
ln
Xnal
X
st
Lim
+
Lim
(13)
X
nal
X
nal
=
Xp
2
+
1
2
_
X
2
p
4X
st
Lim
(X
p
X
Lim
) (14)
X
nal
X
nal
=
Xp+
1
A

A
2
X
2
p
4K
2
(15)

nal

nal
= A
_
Xp
Xnal
1
_
(16)
dS
d
= f (X
st
,X
div
) construction substrate consumption rate
dS
d
= k
st
s
X
st
+ k
div
s
X
div
(17)
dP
d
= f (X
st
, X
div
)product synthesis rate
dP
d
= k
st
p
X
st
+ k
div
p
X
div
(18)
P = f (X)
product concentration P = P
Lim
+
_
k
div
p
A
__
X
p
ln
XpX
Lim
XpX
(X X
Lim
)
_
1 +
X
st
Lim
XpX
__
(19)
P = f (X)
product concentration
P = P
Lim
+
_
k
div
p
A
__
X
p
ln
XpX
Lim
XpX
(X X
Lim
)
_
+
+
1
A
(k
st
k
div
)X
st
Lim
XX
Lim
XpX
(20)
S = f (X)construction substrate concentration
S = S
Lim

_
k
div
s
A
__
X
p
ln
XpX
Lim
XpX
(X X
Lim
)
_

_
1
A
_
(k
st
s
k
div
s
)X
st
Lim
XX
Lim
XpX
(21)
q = f(R) q = k
div
+
_
k
st
k
div
_
R (22)
X
st
Lim
X
st
Lim
= 2
_
X
2
Lim
_
X
2
p
_
(X
p
X
Lim
) (23)
X
st
Lim
X
st
Lim
= (X
p
X
nal
)X
nal
/(X
p
X
Lim
) (24)
models for the description and intensication of the growth of
genetically modied strains and biosynthesis of recombinant
metabolites.
For testing the proposed model, E. coli strain BL-21
(DE3) [pProPlnHis
6
] [9], a producer of protealysin
(metalloproteinase belonging to the family of thermolysins)
[1012] was used. Enzymes of this group can serve as potential
drugs; in addition, they have a considerable innovative
potential for biotechnological application [13]. Currently,
recombinant producers [1419] are used for thermolysin-like
proteases. In the proposed paper, an attempt to increase
the efciency of metalloproteinase expression by recombinant
E. coli strain BL-21 (DE3) [pProPlnHis
6
] owing to the increase
of oxygen mass exchange rate in a reactor followed by a
consequent correction of nutrient medium composition has
been made. The correction of mediumcomposition is required
due to a signicant increase of the rates of all metabolic
processes in the reactor. In this case, additional nutrients are
required. This is also stipulated by the increase of energy used
for maintaining the producer viability during biosynthesis of
substances genetically foreign for a cell host.
Thus, the objective of the proposed paper is to demonstrate
novel structured and unstructured models of recombinant
strain growth and metabolite biosynthesis with the purpose
to intensify cultivation processes and to increase the target
product output.
2. Materials and methods
2.1. Escherichia coli strain BL-21 (DE3) [pProPlnHis
6
] a
producer of protealysin
This was previously constructed on the base of E. coli strain
BL-21 (DE3) widely used for recombinant protein production
[9]. This strain exhibits lysogenic properties with respect to
4
Biofabrication 3 (2011) 045006 S P Klykov et al
prophage DE3 bearing prophage T7 RNA pol gene under
the control of promotor lacUV5, IPTG, galactose and glucose
being the inducers and repressors, respectively.
2.1.1. Total proteolytic activity. TPA was determined from
the ability of the strain to hydrolyse of azocasein [10]. For this
purpose 50 l of the enzyme solution were added to 100 l
of 1% azocasein solution in 20 mM Tris-HCl (pH 8,0). The
mixture was incubated at 37

S for 15 min. The reaction was
ceased by adding 200 l of 10% trichloracetic acid solution.
The sediment was separated by centrifuging at 9500 g for
5 min. 50 l of 5 M NaOH were added to 250 l of the
supernatant. Light absorption was measured at 450 nm on a
plate photometer.
The amount of the enzyme increasing the light absorption
by 1 ODunits per 1 min was considered as 1 unit of the activity.
Enzyme activity was determined after the destruction of the
biomass and metalloprotease formation. For this purpose,
the cells were centrifuged and then resuspended in 20 mM
Tris-HCl, pH 8,0. The cells suspension at a concentration
of approximately 30 g l
1
was exposed to ultrasonication for
1.52 min 57 times and incubated at 4

S for 2448 h.
2.2. Cultivation of Escherichia coli BL-21 (DE3)
[pProPlnHis
6
]
A standard scheme of culture preparation was used: producer-
collection culture on Petri dishes-inoculation asks-reactor.
The cultivation was performed in a reactor with a working
volume of 55.5 l, in a liquid medium, containing (g l
1
):
tripton15; yeast extract7.5; sodium chloride1.0;
potassium di-phosphate5.0; potassium monophosphate
2.5; glucose-3.0; ampicillin0.1; antifoam1.0 ml, at
pH7,0 0,2. At this stage 2 control cultivations (Control 2005
and Control 2010) were performed. Experimental cultivation
(experiment 1) was conducted at high oxygen inow rate
under stirring and broken glucose feeding according to pH
and pO
2
detector signals. Flasks were inoculated with a small
amount of inoculate taken with a microbiological loop from
the surface of the bacterium layer in Petri dishes. Each of
the ve asks containing 0.2 l of the inoculation material was
used to inoculate the reactor containing 44.5 l of the nutrient
medium with all the necessary additives.
Stirrer rotations and the amount of oxygen consumed were
adjusted automatically according to the signal of pO
2
detector.
Prior to inoculation, a small amount (2 ml l
1
) of 50%
glucose solution was poured into the reactor. Before the initial
amount of glucose had been consumed, pH was maintained
at 7,0 with 15% NaOH solution according to the call of the
pH detector. After the consumption of the initial amount of
glucose, it was fed in low doses through a peristaltic pump
according to the pH detector signal. The rate of pumping was
adjusted to provide glucose feeding (dry weight) within the
range from 1 to 3 g (l h)
1
. Alkaline feeding was stopped to
avoid glucose overdose. IPTG (0.01 g l
1
) was introduced
into Control 2005 and Control 2010 reactors after microbial
suspension had reached optical density equal to 2.5 and
2 OD units, respectively. The inductor was placed into
experiment 1 reactor at OD units equal to 7 OD units.
2.3. Description of a model for the estimation of Escherichia
coli strain BL-21 (DE3) [pProPlnHis
6
] growth and
metalloproteinase biosynthesis
The basic equations of the proposed model are presented in
table 1.
2.3.1. Unstructured model equations. Equations (1)(6),
except for (3a) and (3b), deduced in [5] represent basic
equations of the unstructured model of microorganismgrowth.
The presented equations are used for
analysis of oxygen mass exchange in the cultural medium,
k
d
;
analysis of physiological producer constants, m, a, A,

max
,
Lim
, X
Lim
, X
p
.
Oxygen mass exchange can be measured by other well-
known methods and selected for cultivation. If this parameter
is unknown, it can be calculated using equations (2) and (3) of
the unstructured model.
Parameters of the unstructured model
Lim
, X
Lim
, X
p
depend on the selected oxygen mass exchange. All the
parameters depend on nutrient medium composition and
cultivation conditions.
Then all the parameters of the unstructured model are
used for calculating the parameters of the structured model
and biosynthesis constants.
Experimental biomass concentration parameters are used
for the estimation of biomass growth according to the
unstructured model. Sampling is performed at equal time
intervals =
i +1

i
= const, at which biomass
concentration changes, X

= X
+
X

.
According to equation (3) for GIP the following is
deduced:
X
+
= X

+ X (25)
X

+ X = X
p
(X
p
X

exp(A

), (26)
or
X
+
= X
p
(X
p
X

exp(A

) (27)
X = X
p
X

(X
p
X

exp(A

) (28)
X = (X
p
X

)(1 exp(A

)) (29)
at =const (30) equation (29) represents a linear regression
function of X

X = (1 exp(A

))X
p
(1 exp(A

))X

.
(31)
In the point of intersection with the ordinate axis, at X = 0,
equation (31) is converted as follows:
X = (1 exp(A

))X
p
=
0
X, (32)
where
0
X is the point where the regression line crosses the
ordinate axis (31).
From this it follows that
1 X/X
p
= exp(A

). (33)
5
Biofabrication 3 (2011) 045006 S P Klykov et al
Expression (33) can be presented in the following form:
A = [ln(1
0
X/X
p
)]/. (34)
Similarly to formula (34) for A of logarithmic growth phase
(LGP), equations for
max
can be deduced:
X
+
= X

+ X = X

exp(
max

), (35)
from which
X = [exp(
max

) 1]

, (36)
and

max
= [Ln(1 + M)]/, (37)
where M = [exp(
max

) 1] is the inclination angle
tangent of the straight line X = f (X) for LGP.
In the work given,
max
was calculated by a standard
technique [8]. In this case, dependence of LGP natural
logarithms, X, on time was built and the tangent of the
inclination angle of the obtained straight line was determined.
These values were compared to the results obtained with
equation (37).
For the analysis of biomass growth, the following
parameters are determined rst: biomass concentration,
X
Lim
, and time,
Lim
, corresponding to LGP termination
and beginning of GIP; hypothetical maximum biomass
concentration, X
p
, when all energy transformed by the system
is consumed for biomass viability maintenance; A = m/a is
the specic growth delay rate of the biomass during GIP.
2.3.2. Equations for the structured model. Equation (7)
was theoretically proved in [68]. This equation indicates
that in any population limited in energy consumption, X
st
,
a specic rate of the accumulation of nonproliferating cells
consumed energy only for viability maintenance is constant
and equal to A. This is due to the fact that the energy consumed
for cell viability maintenance causes a total reduction in the
growth rate of proliferating cells, X
div
, and, as a consequence,
of the population growth rate as a whole. In this case,
nonproliferating cell accumulation is directly proportional to
concentration X
st
at any instant time.
Equation (8) represents an integrated formof equation (7).
This equation shows exponential character of X
st
accumulation
in time.
Equations (9)(16) were deduced in [58].
Equations (9) and (10) represent a share of
nonproliferating cells in the population. In [8], it was shown
that R =X
st
/X also describes the degree of synchronization of
zero age cultures. All physiological functions of zero age cells
are minimal, and cell resistance to adverse external inuences
is maximal owing to temporarily inhibited metabolism. It is
shown [5, 7, 8] that if growth of the biomass is limited only by
energy inow, all proliferating cells are converted into stable
ones after which further proliferation ceases. Thus, the whole
amount of energy is consumed for cell viability maintenance.
It is obvious that equality R = 1 is true for this case.
Physical meaning of parameter Kin equation (11) consists
in the fact that with growth limitation strengthening (increase
the duration of growth phase), the effect fromone and the same
work of biochemical mechanisms underlying cell processes
continuously decreases, i.e. the number of proliferating cells
reduces over time. At the same time, there is an accelerated
increase in the quantity of stable cells consumed energy only
for viability maintenance (see equations (7) and (8)). The
latter statement should probably be understood to reect the
increase of stable cell numbers due not only to the termination
of early cell proliferation cycles, but also to the progressive
failure of stable cells to proliferate; otherwise, the cells could
start proliferation again in the absence of limits.
Equations (12)(16) describe the time of culture growth
termination and specic growth rate at the moment, when the
discussed unstructured model of cell growth and biosynthesis
during GIP, as it is presented in table 1, does not work. In this
case, other equations are required (see below).
Equations (17), (18) and (19)(21) were considered in
[68]. The physiological processes occurring in proliferating
and stable cells differ greatly and are diametrically opposed.
Therefore, subdivision of population cells into proliferating
(X
div
) and stable (X
st
) ones makes it possible to use
equations (17), (18), and table 1 to describe the consumption
rate of substrates utilized for cell construction and metabolite
synthesis:
dP(or S)/d = k
div
P,S
X
div
+ k
st
P,S
X
st

We assume that metabolites are synthesized only by

proliferating cells. Nonproliferating cells, as a rule, destroy
these products. Therefore, signs of the constants for metabolite
synthesis and degradation are opposite. The same should be
stated for substrates utilized for cell construction.
If stable cells do not inuence synthesis of metabolites
(and substrate utilization), i.e. k
st
P,S
= 0, then the synthesis is
carried out by proliferating cells and can be described by the
integrated equation (19). If both proliferating cells and zero
age cells participate in the synthesis, then the accumulation
of metabolites is described by equation (20). Similarly
equation (21) is proposed for the consumption of substrates
used for cell construction. An analysis of equations (17)(21)
shows that metabolite synthesis proceeds with rate constants,
whose signs are opposite to each other if the inuence of the
stable cells on the product output is not equal to 0. This means
that groups of proliferating and stable cells behave differently
during product biosynthesis: the former group synthesizes
metabolites, the latter destroys them.
It was found out that if cell growth process is limited
not only by energy consumption but also by any substrate
participating in cell construction, then the increase of biomass
yield may suddenly stop. Studies on the dynamics of
biomass accumulation limited by substrates utilized for cell
construction [8] showed that this parameter can be presented
as
R
nal
= k
div
/(k
div
k
st
). (38)
The required nal value of R
nal
is expressed by an identical
equation, if it is necessary to terminate the biosynthesis process
in order to avoid the destruction of metabolite products by
active cells when k
st
P
= 0. The situation, when maximum
of biomass accumulation does not coincide with that of
metabolite accumulation, i.e. the latter occurs earlier than the
6
Biofabrication 3 (2011) 045006 S P Klykov et al
former, is actually rather common. For this case, equation (15)
would have the following appearance:
X
nal
=
X
p
+
1
A
_
A
2
X
2
p
(1/R
nal
)

4K
2
. (39)
Equations (12)(14) and (16) in this case are transformed
according to (39).
In the studies of growing cultures, especially, of
Pseudomonas and Yersinia pestis, by cytorefractometric
methods [20], we have repeatedly noticed that a certain number
of nonproliferating cells are always present in the exponential
growth phase. As the cell growth reaches GIP, the number of
such cells increases signicantly.
Equations (12)(14) and (16) then can be transformed
according to (39).
In the given work, biosynthesis parameters were
determined for cells in GIP. The calculated values were
used to estimate the conformity of the data obtained for the
cells in GIP. As is well known, biosynthesis of metabolites
often occurs during GIP. In [8], the factors affecting
cell population structuring during LGP were investigated.
Equations describing metabolite accumulation in this growth
phase are as follows:
X
st
= 2X
l
(1 X
l
/X), (40)
R = 2X
l
(1/X X
l
/X
2
) (41)
and
P = P
0
+ (k
div
/
max
)(X X
l
)
+2[(k
st
k
div
)/
max
]X
l
[ln(X/X
l
) + X
l
/X 1] (42)
where
X
l
= X
2
Lim
/X
p
(43)
is the initial biomass concentration in LGP, corresponding to
the beginning of cell population structuring.
2.4. Technique for the estimation of bacterium growth and
metabolite biosynthesis
Parameters X
theor
for LGP were calculated according
to standard exponential equation (1, 2): X
theor
=
X
0

exp[
max

],where
max
and X
0
were determined from
LnX
experiment
= f () calculated from the data presented in
gure 1.
max
was also calculated from equation (37) and
compared with the parameter calculated according to the
above equations. For this purpose, X for the cells in LGP
was estimated according to experimental data X
experiment
from
which corresponding values X were calculated for each of
the specied sampling interval. For each previous value of
the experimental biomass concentration change, X, i.e. X

= X
(+)
X

., dependence X

= f (X

) was then built

according to equation (36) and
max
was determined from
(37) (see gure 2).
Parameters X
theor
for GIP were calculated on the base of
experimental data X
experiment
(gure 1), according to which
values X for sampling within specied time , were
obtained as a difference between previous and subsequent X,
i.e. X

= X
(+)
X

. Then, dependence X

= f (X

)
was drawn, and corresponding constants
0
X, X
p
, A were
Table 2. Estimated model parameters.
Control Control Experiment
Parameter 2005 2010 2010
X
0
,[OD units] 0,20 0,65 1,1

max,
[1,2], [h
1
] 0,740 0,550 0,649

max
,(37),[h
1
] 0,821 0,438 0,622
A,[h
1
] 0,330 0,381 0,395
X
Lim
,[OD units] 0,75 4 7

Lim,
[h] 4,5 4 3
X
p
,[OD units] 4,33 13,33 24,5
,[h] 1 1 1

0
X,[OD units] 1,224 4,210 7,90
X
Lim
St
,[OD units] 0,21 1,68 2,86
K,[(OD units)
2
h
2
] 0,0840 2,2792 7,7942
P
Lim
,[units ml
1
] 0,04 0,7 1,225
k
div
,[units (ml OD units h)
1
] 0,0535 0,200 0,200
k
st
,[units (ml OD units h)
1
] 0 0,164 0,045
X
l
,[OD units] 0,13 1,20 2,0
P
0
,[units ml
1
] 0 0,22 0,20
R
nal
= R
forPmax
(38) 1 0,549 0,826
determined.
0
X is an intersection of the regression line (31)
and ordinate axis; X
p
is an intersection of the regression line
(31) and abscissa axis; A is calculated from formula (34) (see
gure 2). The boundary point of two growth phases (X
Lim
,

Lim
) is determined from the intersection of straight lines (31)
and (36) (see gure 2).
Biosynthesis parameters are calculated from
equations (10) and (22). For this purpose X
st
Lim
is pre-
liminarily estimated by equation (23). Then the values
of the biosynthesis specic rate are calculated. (q
P
)

is
estimated from [P/]

= [P
(i+)
P

]/ and (q
p
)

= (1/X

)[P/]

. On the basis of X
p
, X
Lim
, A,
Lim
previously estimated from equation (8) dependence X
theor
=
f () is deduced (gure 1). X
st
Lim
calculated above makes it
possible to obtain dependence R = f
1
() = f
2
(X). Then the
straight line (q
P
)

= k
div
P
+
_
k
st
P
k
div
P
_
R

(gure 3), at R =
0, cuts off a line segment equal to k
P
div
on the ordinate axis.
Inclination angle tangent of this line is equal to k
st
P
k
div
P
,
from which k
st
P
can be calculated.
The integrated accumulation of metabolite P
theor
is
estimated according to equation (19) or (20): if k
st
P
= 0, then
equation (19) is used, if k
st
P
= 0, equations (20) and (21) are
suitable.
Experimental results on E. coli strain BL-21 (DE3)
[pProPlnHis
6
], X, metalloproteinase P synthesis and the
corresponding calculated parameters X
theor
and P
theor
for
LGP were compared with the model values. The latter
were obtained from the calculated parameters for GIP,
X
p
, X
Lim
, X
st
Lim
, A, k
div
P
, k
st
P
, and from
max
determined
preliminary for LGP. Equation (40) for calculating the
number of stable cells was used. For the description of
metabolite accumulation during LGP, equation (42), where
P
0
is the product concentration at the moment, when biomass
structuring supposed to occur during LGP, is used.
Parameters P
0
and P
Lim
were calculated from the results
of cell cultivation in fermentations Control 2005 and Control
2010 and are presented in table 2.
As was noted above, often there is a situation when
biomass growth stops growing while metabolite biosynthesis
7
Biofabrication 3 (2011) 045006 S P Klykov et al
Figure 1. Control 2005 cultivation. Experimental biomass, X, and biomass calculated according to unstructured model X
theor
, stable
(nonproliferating) cells, X
st
, proliferating cells, X
div
. Growth time, hours, is the abscissa axis. X, OD units, is the ordinate axis.
X, X
theor
, X
st
, X
div
.
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
1,1
1,2
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
Figure 2. Control 2005 cultivation. Determination of unstructured model parameters, A,
max,

Lim
, X
Lim
, X
p
,
0
X. Parameter values are
presented in table 2. X, OD units, is the abscissa axis. X, OD units/hour, is the ordinate axis. Experimental data for LGP,
Linearization for GIP, Linearization for LGP, Calculation data for GIP. X
LGP
= 1,273X; X
GIP
= 0,2814X + 1,224.
continues for some time. It was found that the absolute
rate of product synthesis can be described also by equation
(18). However, if no changes of R occur, then X, X
st
and X
div
remain constant. The integrated accumulation of biosynthesis
metabolites in this case can be expressed by the following
equation:
P = P
nalGIP
+ k
div
X
div
nalGIP
(
nalGIP
)
+k
st
X
st
nalGIP
(
nalGIP
), (44)
where P
nalGIP
is the nal concentration of the product, which
is determined fromequation (20), table 1, X
div
nalGIP
and X
st
nalGIP
are the corresponding nal concentrations that can either be
calculated using equations (6), (8), (38) and (39) or determined
experimentally.
3. Results and discussion
The possibility to increase the output of the target product
synthesized by E. coli strain BL-21 (DE3) [pProPlnHis
6
] is
determined by the presence of the following genes:
(1) ompT responsible for the inhibition of protease activity
for product output increase.
(2) lon responsible for the inhibition of intracellular ATP-
dependent protease for product output increase.
(3) lacUV5 being a promoter repressed by glucose.
Gene 1 and 2 determine the selection of nitrogen component
of the nutrient medium, acid casein hydrolysate. Amino acids
8
Biofabrication 3 (2011) 045006 S P Klykov et al
-0,10
-0,05
0,00
0,05
0,10
0,15
0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0
Figure 3. Control 2005 cultivation. Determination of parameters k
div
and k
st
according to the structured model. Parameter values are
presented in table 2. R, portion 1, is the abscissa axis. q, units P/(ml

OD

hour), is the ordinate axis. Experimental data for GIP,

Linearization for LGP. q = 0,0476R + 0.0535.
are the source of nitrogen in the caseine hydrolysate. lacUV5
determines the mode of glucose feeding of the producer with
induced product expression. It is efcient to introduce glucose
into the nutrient medium at small doses (13 g (1 h)
1
) in a
formof 50%solution according to the pHdetector signal. Such
mode of glucose feeding allows for the producer to remain in
a stated of glucose limitation. In this state, glucose does not
induce lac-operon repression.
Experimental results obtained during the cultivation
of producer X, metalloproteinase P biosynthesis and the
corresponding parameters X
theor
and P
theor
are presented
in gures 14. Parameters of producer biomass and
metalloproteinase changes calculated according to the model
are also depicted in the gures.
The parameters of growth and biosynthesis shown in
table 2 were calculated from experimental data X (gure 1)
and P (gure 4), according to the technique described above.
A mode of calculating the parameters presented in table 2
is shown in gures 2 and 3, the Control 2005 experiment being
taken as an example. Parameters of the other processes were
calculated by the similar mode.
The producer growth estimation was performed from a
limited number of points (from 4 to 6). This provides the
limited accuracy of the related analyses possible for these
cases. For LGP, calculation of
max
by two well-known modes
showed practically similar values (table 2). However, even at
the accuracy given, the description of growth and biosynthesis
both during GIP and LGP has appeared comprehensible to
sum up the results allowing for forecasting metalloproteinase
biosynthesis processes. Nutrient medium composition and
cultivation conditions have been respectively corrected.
Enzyme activity in the intermediate samples of all three
experiments is depicted in gure 4. The area of gray color in
the center of gure 4 represents an expectation value of the
enzyme activity for experiment 1 performed at high oxygen
mass exchange rate and increased content of nutrients.
The upper curve limiting this area is built using a product
synthesis constant corresponding to the Control 2010 process
on the assumption that product degradation does not take place.
The rest of the parameters correspond to the characteristics
of the experiment 1 process. The lower curve differs from
the upper one because degradation constant k
st
equal to the
value determined for the Control 2010 process is taken into
consideration.
The enzyme activity values in the control experiments,
Control 2005 and Control 2010, were obtained when
cultivation was performed at a low oxygen exchange rate,
and the decreased content of nutrients is depicted in gure 4.
These values were used as the basis for modeling experiment
1 processes.
As is seen from gure 4, the data for experiment
1 satisfactorily fall into the area marked in gray. This
correspondence has been forecasted. Results of mathematical
description of three points of experiment 1 were obtained
by a trial and error method using the constants of product
synthesis and destruction (see table 2) at the minimized root-
mean-square deviation. Denition by the method described in
section 2.4 is unsuitable for the given case, since quantity of
points for the analysis (three points) is not enough. In order
to receive a comprehensible result by the method proposed, a
minimal quantity of the points should be no less than 4.
For the Control 2005 process, product destruction
constant is equal to 0. For the Control 2010 process, product
destruction constant, k
st
, makes about half of the product
synthesis constant, k
div
. This means that more intensive growth
9
Biofabrication 3 (2011) 045006 S P Klykov et al
Figure 4. Dependence of enzyme activity on the growth time. Growth time, hours, is the abscissa. Activity P, units ml
1
, is the ordinate
axis. Experimental activity in Control 2005. Model calculation of the acvitity in Control 2005. Experimental activity in Control
2010. Model calculation of the activity in Control 2010. Predicted activity in Experiment 1 according to Control 2010 results.
It is assumed that specic rate of product destruction is equal to the specic destruction rate in Control 2010. Experimental activity in
Experiment 1. Predicted activity in Experiment 1 according to Control 2010 results. It is assumed that product destruction does not
occur.
(a) (b) (c)
Figure 5. (a) The change of decimal logarithm of the concentration of mammalian cell HUVEC-population corresponding to the
experimental and model data (according to the proposed model); (b) experimental data on the quantity of cells of different age cycle;
(c) model data (according to the proposed model) on the quantity of cells of different age cycle. Reproduced by permission [25],
coordinated by Dr J Tyson.
of the producer in Control 2010 has not been provided with
the required additional amount of nutrients that has caused
degradation of target protein being a potential source of carbon
and nitrogen for cells.
During experiment 1, a successful attempt to prevent
the undesirable degradation of the protein by increasing the
content of nutrients in the initial medium and duly addition of
glucose as an energy source has been undertaken. Thus, the
product destruction constant, k
st
, was lowered approximately
four-fold.
Knowing the amount of energy inow to the system,
which can be calculated from equations (1)(3), using the
dynamics of glucose solution inow expressed in terms of
heat of combustion and Xp expressed in the units of biomass
heat combustion, it is possible to calculate m(3) and a values
easily.
If the oxygen mass exchange rate has not been
preliminarily determined by any other way, it is possible to
calculate this parameter using equations (2) and (3).
Equations (1)(6) for the estimation of total biomass
are used to be applied within the unstructured model of cell
population growth and substrate consumption. In our paper,
we pioneered the use of equations (7) and (8) for the structured
model with the purpose of obtaining a combined solution of
equations (17), (18) and (22) (also shown for the rst time), in a
form of equations (19)(21). The pioneer solution of equation
(22) had been conrmed with international patent PCT [21].
10
Biofabrication 3 (2011) 045006 S P Klykov et al
One of the reviewers questions is as follows: Why do we
use the biosynthesis of a recombinant strain to demonstrate
our model? The point is that biosynthesis processes, which
are described in the above-stated international Patent and the
Patent of the Russian Federation, are ordinary biosynthetic
processes carried out by usual not recombinant strains of
bacteria, yeasts and fungi. Within the last 30 years, the role
of recombinant strains has sharply increased in science and
industry. Indeed, the recombinant strains are mainly used now.
This circumstance has determined the choice of the research
object.
The experimental and model data were analyzed using
f -tests included in the Excel Program [2224]. For this
purpose, experimental data for Control 2005 and Control
2010 were divided by the corresponding data, calculated
according to the model for Control 2005 and Control 2010. If
the model adequately describes the experiment, the obtained
average value of the ratio of the experimental and model data
(their quotients) will be randomly grouped near 1. Indeed,
according to the f -test for two experiments carried out by
a conventional method (Control 2005, Control 2010), the
ratio of the average value is equal to 0.96 0.42, condence
probability being 95%.
Thus, the Fisher criterions is equal to 1.644, 3.695 and
2.248 for various combinations of the number sequences for
the analysis that is less than the critical criterion equal to 5.05,
the discretion range number being equal to 5. Hence, the
model is adequate for the experimental data.
For the model and experimental data of experiment 1,
a similar analysis was made using two-selective f -tests at
various dispersions as well. For this purpose, experimental
data for experiment 1 were divided by the corresponding data
calculated according to the model for experiment 1 and were
compared with the number sequence of the ratio of the average
values of the experiment/model for Control 2005 and Control
2010. The Fisher criterion is equal to 7.37, that is less than
the critical criterion equal to 19.3, the discretion range number
being equal to 2. The ratio of the average value is equal to
0.99 0.15, the condence probability being 95%.
Thus, there is no reason to consider the observed
difference between the number sequences statistically
signicant.
This provides evidence to the fact that both the values
belong to one and the same general data set and that the model
is adequate.
Experimental conrmation of the conclusions of our
theory was published in February 2011 by the American
researchers Dr John J Tyson et al [25]; it is shown in the last
drawing in this work and gure 5 of this paper. Figure 5 fully
conrms the predictions obtained by our model for changes in
the proportion of non-dividing (resting) cells (see equation for
R). It is clear from the graph, which is designated by letter B
in the gure, with our proposed model describing the authors
data better than the model offered by the authors themselves.
The fundamental equation for the unstructured model is
the MarrPerth equation (1) for the phase of slower growth
in the integral form (6). It can be easily obtained if the
relevant work is done with equations (1) and (4) under our
methodology.
The basic equations for the non-structured model are the
following: (7) integral equation for X, expressed by equation
(6), and equation (17) and/or (18).
All other equations are auxiliary, derived from the basic
equations, and are applied depending on the objectives set by
a researcher or an engineer.
Moreover, from equations (1), (4), (6), (7) a general
equation was obtained:
d
n
X
div
d(X
st
)
n
=
K
A
2

(1)
(n1)
n!
(X
st
)
(n+1)
C, (45)
where n are the whole numbers, order of the derivative of a
function. Moreover, C = 1 if n = 1 and C = 0 if n 2.
The factor C has the following physical sense: in GIP
each stable cell can become dividing again 1 time only,
i.e. C
(n=1)
= 1; each such subsequent transition for each
separate line of cell is impossible, i.e. C
(n=2, ... )
= 0. This
expression is a generalizing equation for any component of
the biomass and for total biomass and, thus, unies structured
and unstructured models. To describe the biomass growth for
any proposed model, the major differential equation is (45). To
describe the cycle of the main substances a major differential
equation is (17) and/or (18).
These equations describe all the known diversity of the
processes with S-like growth curves and changes in the
concentrations of substances in closed systems, which is an
entirely new and previously unknown fact. It is known
that a physical law means a generalization of a numerical
relationship between the objects of the real physical world
that is running under specied conditions for the class of
the objects and does not follow from any of the previously
discovered laws. There is no reason not to admit the two
described equations for the GIP as laws for GIP.
The data obtained were used for the selection of
techniques to increase the protein expression by genetically
modied microorganisms.
4. Conclusions
(1) Growth characteristics of the recombinant strain and
metalloproteinase biosynthesis were calculated on the
basis of structured and unstructured population models.
The results obtained allowed for the selection of
cultivation conditions providing the increase of target
product output in order to perform one more series of
the experiment to design new more intensive cultivation
regimens;
(2) It was shown that the producer cultivated in the
presence of different nitrogen sources and at different
modes of glucose feeding exhibits different growth and
biosynthetic properties. Also, it was demonstrated
that metalloproteinase shows different biosynthetic
properties during cultivation on nutrient media of different
compositions.
(3) Preferable substrates and cultivation regimens were
selected to optimize growth properties of the producer and
to increase the target product (metalloproteinase) output.
11
Biofabrication 3 (2011) 045006 S P Klykov et al
Acknowledgments
This work was supported in part by the Russian Foundation
for Basic Research (project no 09-04-00734). The authors
cordially thank Dr V V Derbyshev for valuable advice given
during the designing of the unstructured model and Dr V A
Samoilenko and Ms T VYakshina for assistance in experiment
performance.
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