Process Biochemistry 35 (2000) 639 – 647

www.elsevier.com/locate/procbio

Statistical optimization of medium for the production of

recombinant hirudin from Saccharomyces cere6isiae using response
surface methodology
K. Jagannadha Rao, Chul-Ho Kim, Sang-Ki Rhee *
Microbial Metabolic Engineering Research Unit, Korea Research Institute of Bioscience and Biotechnology, P.O.Box 115, Yusong,
Taejon 305 -600, South Korea

Received 26 April 1999; received in revised form 27 July 1999; accepted 27 July 1999

Abstract

An efficient fermentation medium producing a colourless product with high yields has been developed for the production of
recombinant hirudin from Saccharomyces cere6isiae. Response surface methodology was applied to optimize the medium
constituents. A 25-1 fractional factorial central composite design has been chosen to explain the combined effect of the six medium
constituents, viz. yeast extract, peptone, casamino acids, ammonium sulphate, potassium phosphate and galactose, and to design
a minimum number of experiments. The optimized medium produced 65.3 mg/l of r-hirudin in shake flask culture, which is 35%
higher than the unoptimized medium. The colour of the purified product was influenced by the colour intensity of culture
supernatant, which in turn depended upon the concentration of complex substrates used in the medium. The concentration of
yeast extract has been reduced to 16 g/l in the optimized medium than other reported media containing 40 g/l yeast extract. The
colour intensity of culture supernatant obtained by the optimized medium was reduced by 60% from the reported media
containing 40 g/l yeast extract. © 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Medium optimization; Central composite design; r-Hirudin; Saccharomyces cere6isiae

1. Introduction have led to the development of microbial recombinant

Hirudin, a 65-amino acid polypeptide derived from
leeches, is a natural potent thrombin-specific inhibitor
and blocks the thrombin-mediated conversion of
fibrinogen into fibrin in blood clot formation [1]. In
animal studies, hirudin, purified from leeches, has
demonstrated efficacy in preventing venous thrombosis,
vascular shunt occlusion and thrombin-induced dissem-
inated intravascular coagulation [2]. Because of these
properties, hirudin might be useful as a therapeutic
agent for cardiovascular diseases, and therefore an
abundant source of highly purified and active hirudin
would be helpful for clinical and model validation
studies [3]. Limited availability and high cost of extrac-
tion and purification of natural hirudin from leeches
* Corresponding author. Tel.: +82-42-8604450; fax: + 82-42-
8604592.
E-mail address: rheesk@kribb4680.kribb.re.kr (S.-K. Rhee)
DNA methods for its large-scale production [4].
To achieve high product yields, it is a prerequisite to
design a proper production medium in an efficient
fermentation process. A medium containing 3–4%
yeast extract along with the other medium components
has been employed for the production of recombinant
proteins from Saccharomyces cere6isiae [5–8]. When a
production medium is designed on a laboratory scale
using high concentrations of complex substrates, little
attention is paid on the limitations associated with the
purification process. One such important limitation is
the colour of the purified end product. Usually a white
or colourless product is recommended, especially when
a protein/peptide is intended for medicinal use. The
colour of the culture supernatant is influenced by the
concentration of the complex substrates used in the
production medium. The use of a high concentration of
yeast extract (4% or more) results in a dark culture
supernatant, which results in a coloured end product

0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 1 2 9 - 6
640 K.J. Rao et al. / Process Biochemistry 35 (2000) 639–647
upon purification. This problem intensifies further in
scale-up studies, when the industrial grade chemicals
are used for economic reasons. To circumvent this
problem, it is essential to focus on this aspect from the
laboratory scale onwards by designing a suitable
medium that reduces the colour intensity of the culture
supernatant as well as producing better yields. This will
help in adopting simple and economical techniques to
purify the final product with better quality.
The application of statistical experimental design
techniques in fermentation process development can
result in improved product yields, reduced process vari-
ability, closer confirmation of the output response
(product yield or productivity) to nominal and target
requirements and reduced development time and over-
all costs. Conventional practice of single factor opti-
mization by maintaining other factors involved at an
unspecified constant level does not depict the combined
effect of all the factors involved. This method is also a
time consuming process and requires a number of
experiments to determine optimum levels, which are
unreliable. These limitations of a single factor optimiza-
tion process can be eliminated by optimizing all the
affecting parameters collectively by statistical experi-
mental design using response surface methodology
(RSM) [9,10]. RSM can be used to evaluate the relative
significance of several affecting factors even in the
presence of complex interactions [11].
This communication reports an attempt to formulate
a suitable production medium that can substantially
reduce the colour intensity of the culture supernatant
and to optimize the same by statistical experimental
design using RSM to improve the yield of r-hirudin
from S. cere6isiae.
2. Materials and methods
2.1. Strains and plasmids
Haploid S. cere6isiae KCTC 2805 (Mata pep4::HIS3
prb 1 can 1 his3 ura3 – 52) was used as the host for gene
expression and secretion of hirudin. Escherichia coli
HB101 [F-hsdS20(r- m-) recA13 proA2 galK2] or
DH5a [F-lacZdeltaM15 hsdR17(r- m-) gyrA36] was
used for cloning of genes and propagation of plasmids.
Plasmid YEG-HIR525a, in which the synthetic hirudin
gene was placed under the control of the GAL10 pro-
moter and mating factor (MFa1) pre-proleader se-
quence, was used for the further manipulation of
hirudin expression vector [12].
2.2. Media and culti6ation
Minimal YNBCAD medium containing 6.7 g/l yeast
nitrogen base without amino acids, 5 g/l casamino acids
and 20 g/l dextrose was used for the selection of
transformed cells and for the development of seed
culture. A single yeast transformant on the YNBCAD
agar plate was inoculated into 10 ml YNBCAD
medium and incubated overnight at 30°C. For inocu-
lum development, the seed culture at 10% (v/v) level
was transferred into an Erlenmeyer flask containing the
same medium and incubated for 24 h at 30°C. This
culture was used as an inoculum at 10% (v/v) level for
all medium optimization studies.
2.3. Production medium
The unoptimized production medium employed con-
sists of yeast extract 10 g/l; peptone 10 g/l; casamino
acids 5 g/l; (NH4)2SO4 10 g/l; K2HPO4 10 g/l and
galactose 30 g/l. All substrates used in this study were
of laboratory grade. Fermentation was carried out in
the 500-ml shake-flask containing 100 ml of production
medium incubated at 30°C with an agitation speed of
200 rev/min.
2.4. Analytical methods
The hirudin activity in the culture supernatant was
determined using thrombin and a chromogenic sub-
strate, chromozyme TH, as described by Sohn et al [12].
The colour intensity of the culture supernatant was
determined by measuring the absorbance at 580 nm
using UV-Visible spectrophotometer (Pharmacia,
USA).
2.5. Experimental design and optimization
RSM is an empirical statistical modelling technique
employed for multiple regression analysis using quanti-
tative data obtained from properly designed experi-
ments to solve multivariable equations simultaneously.
RSM is used to determine the optimum response of
cells, for the synthesis of recombinant hirudin under a
wide range of nutrient conditions. A full factorial cen-
tral composite experimental design [9] for six indepen-
dent variables was used to obtain the combination of
values that optimizes the response within the region of
three dimensional observation space, which allows one
to design a minimal number of experiments. The exper-
iments were designed using the software, Design Expert
(State Ease, Minneapolis, MN). The significant inde-
pendent variables of the medium components are yeast
extract, peptone, casamino acids, ammonium sulphate,
potassium phosphate, and galactose. Regression analy-
sis was performed on the data obtained from the design
experiments.
Coding of the variables was done according to the
following equation
 K.J. Rao et al. / Process Biochemistry 35 (2000) 639–647
xi = (Xi −Xcp)/DXi, i= 1, 2, 3, . . . , k
where: xi, dimensionless value of an independent vari-
able; Xi, real value of an independent variable; Xcp, real
value of an independent variable at the center point;
and DXi, step change of real value of the variable i
corresponding to a variation of a unit for the dimen-
sionless value of the variable i.
The average of the maximum hirudin of the duplicate
values obtained was taken as dependent variable or
response Yi (U). Duplicates are necessary to estimate
the variability of experimental measurements, i.e. the
repeatability of the phenomenon. Replicates at the cen-
tre of the domain in three blocks permit the checking of
the absence of bias between several sets of experiments.
The relationship of the independent variables and the
response was calculated by the second order polyno-
mial equation:
6 6 6 6
Yi = b0 +% bi Xi +%i %j bij Xi Xj +% bii Xi 2i , i "j
1 1 1 1
In this equation, the b values are estimates of polyno-
mial coefficients and Xi values represent coded
variables.
The second order polynomial coefficients (Xi ) were
calculated using the software package Design Expert to
estimate the responses of the dependent variable. Isore-
sponse contour plots were also obtained using Design
Expert. The computation was carried out by multiple
regression analysis making use of the least squares
method and MATLAB sub-routine software (version
4.0, The Math Works, Natick, MA).
3. Results and discussion
Initial fermentation studies were performed with a
medium containing 40 g/l yeast extract apart from
other medium components as reported for yeast fer-
mentation [7,8]. However, this medium produced a
dark culture supernatant, which consequently resulted
in a coloured end product upon purification. The
colour of the culture supernatant was mainly attributed
to the high concentration of yeast extract used in the
medium. Appropriate de-colouration methods were
used to produce colourless final products [13,14]. How-
ever, these efforts resulted in a considerable amount of
product loss (data not shown). The product loss was
much higher especially when industrial grade chemicals
are used as they enhanced further the colour of culture
supernatants than that obtained with the laboratory
grade chemicals. Industrial grade chemicals are gener-
ally used when the process is being scaled-up, from the
economic point of view. When considering the product
cost and medicinal importance of r-hirudin, the product
loss is highly alarming and should be reduced to the
641
(1) maximum possible extent by adopting suitable process
developments. Consequently, studies were conducted to
formulate and screen suitable alternate media on a
laboratory scale. The colour intensity of the culture
supernatant using the medium used in this study has
been reduced by 68% to that of the reported media
containing 40 g/l yeast extract. This could be due to the
reduction of yeast extract (10 g/l), which is a major
contributor to the colour of the culture supernatant.
The yield of r-hirudin was 48.4 mg/l, which is about
92% compared to the medium containing 4% yeast
extract, which yielded about 52.3 mg of r-hirudin/l. The
medium containing 10 g/l yeast extract has been statisti-
cally optimized to improve the yield of r-hirudin.
3.1. Media optimization and empirical modelling
To observe the combined effect of six different
medium components (independent variables), a central
(2) composite design made of fractional factorial matrice of
25-1 plus 10 centre points and 12 star points leading to
a total of 54 experiments were performed. Table 1
presents the levels (design matrix) of the independent
variables in uncoded (natural) units. The mathematical
model relating the production of r-hirudin with the
independent process variables, Xi is given in Eq. (2) and
the second order polynomial coefficient for each term
of the equation determined through multiple regression
analysis using the Design Expert. The experimental and
predicted values of yields of r-hirudin are also given in
Table 1. The coded values of independent variables are
given in Table 2.
The results were analyzed using the analysis of vari-
ance (ANOVA) as appropriate to the experimental
design used (Table 3). The ANOVA of the quadratic
regression model demonstrates that the model is highly
significant, as is evident from the Fisher, F-test (Fmodel,
mean square regression/mean square residual=7.272)
and a very low probability value (Pmodel \ F=0.0001).
The computed value of F exceeding the tabulated value
of F at a probability level of 0.0001 so that the null
hypothesis (H0) is rejected at the 0.0001 level of signifi-
cance. Having rejected H0, it can be inferred that at
least one of the parameters (other than B0) in the fitted
model (Eq. (2)), is not zero. This indicates the com-
bined effects of all the independent variables signifi-
cantly contributed to the enhancement of r-hirudin
production. The goodness of the model was checked by
coefficient of determination, R 2 implies that the sample
variation of 90.1 for r-hirudin production is attributed
to the medium components. The R 2 value also indicates
that only 9.9% of the total variation is not explained by
the model. The value of R, multiple correlation coeffi-
cient, for the production of r-hirudin is 0.949. This
indicates a good agreement between the experimental
and predicted values of r-hirudin production. The
642 K.J. Rao et al. / Process Biochemistry 35 (2000) 639–647

Table 1
Design matrix of independent variables and their corresponding experimental and predicted yields of r-hirudin

Dsn. id. c Yeast extract Peptone Casamino- (NH4)2SO4 K2HPO4 Galactose Hirudin yield (mg/l)
(g/l) (g/l) acids (g/l) (g/l) (g/l) (g/l)
Experimental Predicted

1 7.5 7.5 4.0 7.5 7.5 25.0 19.1 23.43

2 12.5 7.5 4.0 7.5 7.5 35.0 30.8 28.24
3 7.5 12.5 4.0 7.5 7.5 35.0 20.6 21.48
4 12.5 12.5 4.0 7.5 7.5 25.0 20.6 30.02
5 7.5 7.5 6.0 7.5 12.5 25.0 19.0 16.84
6 12.5 7.5 6.0 7.5 12.5 35.0 31.6 32.95
7 7.5 12.5 6.0 7.5 12.5 35.0 29.0 33.69
8 12.5 12.5 6.0 7.5 12.5 25.0 48.3 44.83
9 7.5 7.5 4.0 12.5 12.5 25.0 18.4 21.40
10 12.5 7.5 4.0 12.5 12.5 35.0 23.9 24.36
11 7.5 12.5 4.0 12.5 12.5 35.0 26.2 30.40
12 12.5 12.5 4.0 12.5 12.5 25.0 33.1 31.69
13 7.5 7.5 6.0 12.5 7.5 25.0 22.6 21.87
14 12.5 7.5 6.0 12.5 7.5 35.0 37.8 35.18
15 7.5 12.5 6.0 12.5 7.5 35.0 37.8 27.42
16 12.5 12.5 6.0 12.5 7.5 35.0 25.4 31.76
17 10.0 10.0 5.0 10.0 10.0 30.0 47.4 45.67
18 10.0 10.0 5.0 10.0 10.0 30.0 47.4 45.67
19 10.0 10.0 5.0 10.0 10.0 30.0 48.4 45.67
20 10.0 10.0 5.0 10.0 10.0 30.0 48.0 45.67
21 7.5 7.5 4.0 7.5 12.5 35.0 23.0 23.14
22 12.5 7.5 4.0 7.5 12.5 25.0 24.9 23.57
23 7.5 12.5 4.0 7.5 12.5 25.0 28.9 32.22
24 12.5 12.5 4.0 7.5 12.5 35.0 39.8 41.23
25 7.5 7.5 6.0 7.5 7.5 35.0 22.8 24.91
26 12.5 7.5 6.0 7.5 7.5 25.0 31.9 28.40
27 7.5 12.5 6.0 7.5 7.5 25.0 23.0 23.24
28 12.5 12.5 6.0 7.5 7.5 35.0 44.9 42.60
29 7.5 7.5 4.0 12.5 7.5 35.0 28.3 32.47
30 12.5 7.5 4.0 12.5 7.5 25.0 30.1 26.11
31 7.5 12.5 4.0 12.5 7.5 25.0 26.9 26.25
32 12.5 12.5 4.0 12.5 7.5 35.0 29.6 32.46
33 7.5 7.5 6.0 12.5 12.5 35.0 24.1 23.58
34 12.5 7.5 6.0 12.5 12.5 25.0 20.0 19.81
35 7.5 12.5 6.0 12.5 12.5 25.0 24.2 27.46
36 12.5 12.5 6.0 12.5 12.5 35.0 48.6 44.97
37 10.0 10.0 5.0 10.0 10.0 30.0 46.4 46.72
38 10.0 10.0 5.0 10.0 10.0 30.0 47.8 46.72
39 10.0 10.0 5.0 10.0 10.0 30.0 48.4 46.72
40 10.0 10.0 5.0 10.0 10.0 30.0 46.9 46.72
41 4.055 10.0 5.0 10.0 10.0 30.0 48.9 38.36
42 15.945 10.0 5.0 10.0 10.0 30.0 45.9 54.47
43 10.0 4.055 5.0 10.0 10.0 30.0 17.0 19.23
44 10.0 15.945 5.0 10.0 10.0 30.0 40.6 36.40
45 10.0 10.0 2.622 10.0 10.0 30.0 47.8 42.41
46 10.0 10.0 7.378 10.0 10.0 30.0 43.6 47.02
47 10.0 10.0 5.0 4.055 10.0 30.0 38.3 37.82
48 10.0 10.0 5.0 15.945 10.0 30.0 37.3 35.80
49 10.0 10.0 5.0 10.0 4.055 30.0 35.6 36.10
50 10.0 10.0 5.0 10.0 15.945 30.0 41.0 38.52
51 10.0 10.0 5.0 10.0 10.0 18.11 23.6 25.60
52 10.0 10.0 5.0 10.0 10.0 41.89 40.0 36.02
53 10.0 10.0 5.0 10.0 10.0 30.0 48.6 53.52
54 10.0 10.0 5.0 10.0 10.0 30.0 48.6 53.52
 K.J. Rao et al. / Process Biochemistry 35 (2000) 639–647 643

Table 2
Design matrix of independent variables in coded form

Dsn. Id. c Yeast extract (g/l) Peptone (g/l) Casamino-acids (g/l) H(NH4)2SO4 (g/l) K2HPO4 (g/l) Galactose (g/l)

1 −1 −1 −1 −1 −1 −1
2 +1 −1 −1 −1 −1 0
3 −1 +1 −1 −1 −1 0
4 +1 +1 −1 −1 −1 −1
5 −1 −1 +1 −1 +1 −1
6 +1 −1 +1 −1 +1 0
7 −1 +1 +1 −1 +1 0
8 +1 +1 +1 −1 +1 −1
9 −1 −1 −1 +1 +1 −1
10 +1 −1 −1 +1 +1 0
11 −1 +1 −1 +1 +1 0
12 +1 +1 −1 +1 +1 −1
13 −1 −1 +1 +1 −1 −1
14 +1 −1 +1 +1 −1 0
15 −1 +1 +1 +1 −1 0
16 +1 +1 +1 +1 −1 0
17 0 0 0 0 0 0
18 0 0 0 0 0 0
19 0 0 0 0 0 0
20 0 0 0 0 0 0
21 −1 −1 −1 −1 +1 0
22 +1 −1 −1 −1 +1 −1
23 −1 +1 −1 −1 +1 −1
24 +1 +1 −1 −1 +1 0
25 −1 −1 +1 −1 −1 0
26 +1 −1 +1 −1 −1 −1
27 −1 +1 +1 −1 −1 −1
28 +1 +1 +1 −1 −1 0
29 −1 −1 −1 +1 −1 0
30 +1 −1 −1 +1 −1 −1
31 −1 +1 −1 +1 −1 −1
32 +1 +1 −1 +1 −1 0
33 −1 −1 +1 +1 +1 0
34 +1 −1 +1 +1 +1 −1
35 −1 +1 +1 +1 +1 −1
36 +1 +1 +1 +1 +1 0
37 0 0 0 0 0 0
38 0 0 0 0 0 0
39 0 0 0 0 0 0
40 0 0 0 0 0 0
41 −a 0 0 0 0 0
42 +a 0 0 0 0 0
43 0 −a 0 0 0 0
44 0 +a 0 0 0 0
45 0 0 −a 0 0 0
46 0 0 +a 0 0 0
47 0 0 0 a 0 0
48 0 0 0 +a 0 0
49 0 0 0 0 −a 0
50 0 0 0 0 +a 0
51 0 0 0 0 0 −a
52 0 0 0 0 0 +a
53 0 0 0 0 0 0
54 0 0 0 0 0 0

confidence level of \ 99% validates the model used in
the present study.
Isoresponse contour plots (Figs. 1 – 6) were obtained
using the Design Expert. The response surfaces are
inverted paraboloids and they can be used to predict
the hirudin yields for different values of the test vari-
ables and to identify the major interactions between the
test variables from the circular or elliptical nature of
contour [10]. The coordinates of the central point
within the highest contour level in each of these figures
644 K.J. Rao et al. / Process Biochemistry 35 (2000) 639–647

Table 3
Analysis of variance (ANOVA) and regression analysis for the production of r-hirudin: optimization of medium constituentsa

Source Sum of squares Degree of freedom Mean square F-value Prob\F

Blocks 547.398 2
Model 4804.137 27 177.93098 7.272 0.0001
Error 587.195 24 24.46648
Corrected total 5938.730 53

a
R 2, coefficient of determination =0.9011; R, multiple correlation coefficient =0.949. Root mean square error, RMSE = 4.94636 and corrected
value, CV =14.42%.

will correspond to the optimum concentrations of the The colour intensity of the culture supernatant was
respective components. substantially reduced by 60% using the optimized
The Student t distribution and the corresponding P medium than the medium containing 40 g/l yeast ex-
values, along with the parameter estimate, are given in tract. But this reduction in colour intensity was about
Table 4. The P values were used as a tool to check the
significance of each of the coefficients, which, in turn,
are necessary to understand the pattern of the mutual
interactions between the test variables. The smaller the
magnitude of the P, the more significant is the corre-
sponding coefficient [9]. The parameter estimate and
the corresponding P values (Table 4) suggests that,
among the test variables, yeast-extract and peptone
(PB 0.0001) produce largest effect on r-hirudin produc-
tion, followed by the galactose (PB 0.0075). Both lin-
ear and quadratic relationships between yeast-extract
and r-hirudin yield and that between peptone and
r-hirudin yield are highly significant (P B 1 × 10 − 4 –
9× 10 − 4). (NH4)2SO4, K2HPO4 and galactose also infl-
uences the production of r-hirudin significantly, their
quadratic effect (P B 0.0001) is more pronounced than
the linear effect. Also, the effect of casamino acids is
considerably important, its quadratic effect (PB
0.0221) is more pronounced than the linear effect (PB Fig. 1. Isoresponse contour plot of r-hirudin yield (mg/l): effect of
yeast extract and peptone on r-hirudin production. Other variables
0.2091). These observations can be interpreted as a
are held at zero level.
consequence of a proportional relationship between the
different medium constituents and r-hirudin produc-
tion. The mutual interaction between yeast-extract and
peptone (PB0.1103), yeast-extract and casamino acids
(PB 0.0628), and that between peptone and potassium
phosphate (PB 0.0051) are also important. Other inter-
actions are insignificant.
Maximization of the regression equation (Eq. (2))
was carried out using an iterative procedure [15] to
obtain optimum concentrations of independent vari-
ables (Table 5). By substituting the corresponding
coded concentration levels of the factors into the re-
gression equation, the maximum predictable response
for r-hirudin production was calculated. It was experi-
mentally verified as shown in Table 3. The maximum
production of hirudin obtained using the optimized
medium was 65.3 mg/l, it was in correlation with the
predicted value. After optimization, the production of
r-hirudin was enhanced by 35% (Table 5). These find- Fig. 2. Isoresponse contour plot of r-hirudin yield (mg/l): effect of
ings strongly support the use of response surface model yeast extract and galactose on r-hirudin production. Other variables
for the medium optimization. are held at zero level.
 K.J. Rao et al. / Process Biochemistry 35 (2000) 639–647 645

efficient fermentation medium was developed for the

synthesis of r-hirudin.

4. Conclusion

An efficient fermentation medium producing a

colourless end product, r-hirudin, with high yields has
been developed. Response surface methodology was
used to optimize the medium constituents. The opti-
mized medium produced 35% higher yields of r-hirudin
than the unoptimized medium.

Fig. 3. Isoresponse contour plot of r-hirudin yield (mg/l): effect of

peptone and galactose on r-hirudin production. Other variables are
held at zero level.

Fig. 5. Isoresponse contour plot of r-hirudin yield (mg/l): effect of

galactose and ammonium sulphate on r-hirudin production. Other
variables are held at zero level.

Fig. 4. Isoresponse contour plot of r-hirudin yield (mg/l): effect of

galactose and potassium phosphate on r-hirudin production. Other
variables are held at zero level.

8% less than that obtained with the unoptimized

medium. The concentration of yeast extract (16 g/l) was
increased in the optimized medium compared to the
unoptimized medium (containing only 10 g/l yeast ex-
tract), suggesting that yeast-extract was the major con-
tributor to the intensity of the culture supernatant.
Even though, the colour intensity of the culture super-
natant was a little higher using the optimized medium
compared to the unoptimized medium, the optimized
medium was highly preferable as it enhanced the yield
of r-hirudin by 35% compared to the unoptimized
medium. When this culture supernatant was subjected
to purification using simple methods, a colourless end Fig. 6. Isoresponse contour plot of r-hirudin yield (mg/l): effect of
product, r-hirudin, was obtained without significant potassium phosphate and ammonium sulphate on r-hirudin produc-
amount of product loss (unpublished results). Thus, an tion. Other variables are held at zero level.
646 K.J. Rao et al. / Process Biochemistry 35 (2000) 639–647

Table 4
The least-squares fit and the parameter estimates (significance of regression coefficients)a

Model term Parameter estimate Degree of freedom Computed t (t) P (P\t)

Intercept 48.63487 (1.56696) 1 31.04 –

x1 3.38644 (0.75161) 1 4.506 0.0001
x2 3.60937 (0.75161) 1 4.802 0.0001
x3 0.97004 (0.75161) 1 1.291 0.2091
x4 −0.42434 (0.75161) 1 −0.5646 0.5746
x5 0.50892 (0.75161) 1 0.6771 0.5048
x6 2.19348 (0.75161) 1 2.918 0.0075
x1x1 −1.25648 (0.63606) 1 −1.975 0.0598
x2*x2 −4.54567 (0.63606) 1 −7.417 0.0001
x3*x3 −1.55711 (0.63606) 1 −2.448 0.0221
x4*x4 −2.95413 (0.63606) 1 −4.644 0.0001
x5*x5 −2.86571 (0.63606) 1 −4.505 0.0001
x6*x6 −4.01516 (0.63606) 1 −6.313 0.0001
x1*x2 1.45000 (0.87440) 1 1.658 0.1103
x1*x3 1.70625 (0.87440) 1 1.951 0.0628
x1*x  −1.16875 (0.87440) 1 −1.337 0.1939
Ax1*x5 0.03125 (0.87440) 1 0.0357 0.9718
x1*x6 0.66875 (0.87440) 1 0.7648 0.4518
x2*x3 0.91875 (0.87440) 1 1.051 0.3039
x2*x4 −0.63125 (0.87440) 1 −0.7219 0.4773
x2*x5 2.69375 (0.87440) 1 3.081 0.0051
x2*x6 −0.51875 (0.87440) 1 −0.5933 0.5586
x3*x4 −0.53750 (0.87440) 1 −0.6147 0.5445
x3*x5 0.03750 (0.87440) 1 0.0429 0.9661
x3*x6 1.0000 (0.87440) 1 1.144 0.2641
x4*x5 −1.12500 (0.87440) 1 −1.287 0.2105
x4*x6 0.58750 (0.87440) 1 0.6719 0.5081
x5*x6 0.08750 (0.87440) 1 0.1001 0.9211

a
Standard error is given in parenthesis; standard error of mean =0.67311.

Table 5
Experimental verification of combined effect of optimized medium on the response of r-hirudin

Variables Range studied Levels before Levels after optimization Hirudin yield (mg/l)
(g/l) optimization (g/l)
Coded Uncoded (g/l) Before opti- After optimization
mization
Predicted Experimental

Yeast extract 4.055–15.945 10 2.3780 15.945

(X1)
Peptone (X2) 4.055–15.945 10 1.3378 13.34
Casamino-acids 2.622–7.378 5 2.3962 7.24 48.4 63.42 65.3
(X3)
(NH4)2SO4 (X4) 4.055–15.945 10 −1.0239 7.44
K2HPO4 (X5) 4.055–15.945 10 0.9567 12.39
Galactose (X6) 18.11–41.89 30 0.6194 33.10

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