Electronic Journal of Biotechnology 17 (2014) 311316

Contents lists available at ScienceDirect

Electronic Journal of Biotechnology

High-yield production of the human lysozyme by Pichia pastoris SMD1168

using response surface methodology and high-cell-density fermentation
Ying Yu a,1, Xiaoyu Zhou a,1, Sheng Wu b, Tiantian Wei a, Long Yu a,
a
b

State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433, China
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

a r t i c l e

i n f o

Article history:
Received 28 February 2014
Accepted 27 September 2014
Available online 13 October 2014
Keywords:
Expression optimization
LYZ
PlackettBurman design
RSM

a b s t r a c t
Background: Lysozyme plays a crucial role in innate immunity with its well-recognized bacteriolytic activity. In
this study, the inuence of expression parameters (inoculation volume, culture volume, growth time,
induction temperature and time, initial pH and methanol concentration) on human lysozyme (HLZ)
production in recombinant P. pastoris SMD1168 was investigated through PlackettBurman (PB) design and
response surface methodology (RSM).
Results: It was revealed that induction temperature, induction time and culture volume had signicant inuence
(P b 0.01) on HLZ expression level, which were elected for further optimization with three-dimensional
response surface designs for enhanced HLZ production. The highest lysozyme activity reached 3301 U/mL
under optimized conditions (at 23.5C for 90 h with culture volume of 48 mL) in shake ask, which increased
2.2 fold compared with that achieved with the standard protocol (Invitrogen). When high-cell-density
fermentation of the recombinant Pichia pastoris was performed in a 15 L fermenter under optimized
conditions, the extracellular lysozyme activity reached 47,680 U/mL. SDS-PAGE analysis of the product
demonstrated that HLZ was produced as a single major protein with a molecular weight of approximately
14.7 kDa, consistent with its expected size.
Conclusions: The results indicated that the optimized culture conditions using PB design and RSM signicantly
enhanced the expression level of HLZ, and the Pichia expression system for HLZ production was successful and
industrially promising.
2014 Ponticia Universidad Catlica de Valparaso. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction
Traditional antibiotics are facing escalating challenges as the
emergence of multidrug-resistant bacteria. Lysozyme has gained
much attention as an antimicrobial agent due to its ability to cleave the
-(1,4)-glycosidic bond between N-acetylmuramic acid and
N-acetylglucosamine in peptidoglycan of bacterial cell wall, thus
damaging cell wall and causing bacterial cell lysis [1]. Lysozyme plays
an important role in nonspecic immunity and has a direct effect on
gram-positive bacteria [2]. The human lysozyme (HLZ) gene is located
on chromosome 12 and is widely expressed in a variety of tissues and
body uids [3,4]. In addition to its well-characterized antimicrobial
Corresponding author.
E-mail address: longyu@fudan.edu.cn (L. Yu).
Peer review under responsibility of Ponticia Universidad Catlica de Valparaso.

Contributed equally.

activity, HLZ has exhibited other biological functions, including

anti-inammatory [5], anti-HIV [6], anti-tumor [7], anti-proliferative [8]
and immunoregulation [9] activities. As a human-derived protein, HLZ is
considered safer than bactericides or antibiotics from other species in
clinical applications.
Pichia pastoris has emerged as one of the most common and effective
recombinant protein production systems in molecular biology for
producing both secreted and intracellular proteins [10,11]. Up to now,
more than 500 heterologous proteins have been expressed in P.
pastoris [12]. With its powerful inducible alcohol oxidase 1 (AOX1)
promoter, P. pastoris can use methanol as the sole carbon source and
inducer for recombinant proteins in high cell-density fermentation
[13]. As a eukaryotic single-cell-factory, P. pastoris is capable of
post-translational modications such as proteolytic processing,
folding, disulde bridge formation, and glycosylation. Thus, many
proteins expressed in prokaryotic system as inactive forms can be
produced with biological activity in P. pastoris. Other advantages of P.
pastoris include fast growth in a wide temperature ranging from 15C
to 30C, and a relatively broad pH tolerance from 3.0 to 7.0 [12,14].
In our previous studies, a synthetic codon-optimized HLZ cDNA
was inserted into expression plasmid pPIC9K in frame with the

http://dx.doi.org/10.1016/j.ejbt.2014.09.006
0717-3458/ 2014 Ponticia Universidad Catlica de Valparaso. Production and hosting by Elsevier B.V. All rights reserved.

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Y. Yu et al. / Electronic Journal of Biotechnology 17 (2014) 311316

Saccharomyces cerevisiae pre-pro -mating factor secretion signal

sequence. The HLZ sequence was under the control of AOX1 promoter,
and the expression of bioactive HLZ in recombinant P. pastoris SMD1168
was successful. In this article, the inuences of environmental
parameters on HLZ production were evaluated employing PB design
and RSM. When high cell-density fed-batch fermentation was
performed in a 15 L fermenter under optimized conditions, the
production of HLZ was improved to 47,680 U/mL, which was much
higher than that achieved under unoptimized conditions.

2.5. Analytical methods

Lysozyme activity was determined by measuring the decrease of
absorbance at 450 nm described by Olmo et al. [15]. 50 L sample
solution was added into 2.95 mL of Micrococcus lysodeikticus cell
suspension (OD 450 approximately 0.7) in 0.05 M potassium
phosphate buffer (pH = 7.0). One unit of lysozyme activity
corresponds to an absorbance decrease of 0.001 at 450 nm/min. SDS
polyacrylamide gel electrophoresis (SDS-PAGE) was performed using
15% gel according to Laemmli [16].

2. Materials and methods

2.6. Experimental design and statistical analysis

2.1. Strain and plasmid

2.6.1. PlackettBurman (PB) design

The experiments were designed by Design-Expert.8.05b software
(Stat Ease Inc., Minneapolis, Minn., USA). The effects of seven factors
(inoculation volume, culture volume, growth time, induction
temperature, induction time, initial pH and methanol concentration)
on HLZ production were investigated using PB design [17]. PB is a
kind of two-level experimental design method, applicable for quickly
and efciently screening for the signicant factors from many using
the least experiments [18]. The high and low levels of the investigated
variables are given in Table 1.

P. pastoris SMD1168 (MutShis4-) was purchased from Invitrogen

(Carlsbad, CA, United States). Recombinant plasmid (pPIC9K-pt-LYC6)
for secreted expression of HLZ in P. pastoris was constructed by our
laboratory previously. HLZ cDNA sequence was optimized according to
the codon preference of P. pastoris to enhance the expression level,
and the recombinant plasmid was transformed into SMD1168 and
integrated into the genome.

2.2. Culture media

Preserved recombinant P. pastoris strain was recovered and cultured
in YPD (10.0 g/L yeast extract, 20.0 g/L peptone and 20.0 g/L glucose. For
plate, 20.0 g/L agar was added to the medium). The BMGY (10.0 g/L
yeast extract, 20.0 g/L peptone, 0.1 M potassium phosphate, pH 6.0,
13.4 g/L YNB, 4 10-4 g/L biotin, 1% glycerol) and BMMY (BMMY
medium with 0.5% methanol instead of glycerol) media were used
for cultivation and expression of HLZ, respectively, in shake ask. The
basal salt medium (0.93 g/L CaSO4, 18.2 g/L K2SO4, 14.9 g/L MgSO4 7H2O,
4.13 g/L KOH, 26.7 mL/L H3PO4, 40.0 g/L glycerol, and 4.0 mL/L PTM1
(6 g/L CuSO4 5H2O, 0.08 g/L NaI, 2 g/L MnSO4 H2O, 20.2 g/L
Na2MoO4, 0.2 g/L H3BO3, 20 g/L CoCl2, 65 g/L FeSO4 7H2O, 0.2 g/L
biotin, and 0.5 g/L H2SO4)) was used for fermentation.
2.3. Shake ask culture
Preserved recombinant strains stored at -80C were streaked onto
YPD plates for recovery and isolation of single colonies. A single
colony was inoculated in 5 mL YPD and incubated overnight at 30C,
220 rpm. The seed culture was transferred to 50 mL BMGY and was
grown for 24 h under the same condition as above. The expression of
HLZ was induced by resuspending the cells in the same volume of
BMMY media after centrifugation, and 100% methanol was added to a
nal concentration of 1% (v/v) every 24 h to maintain induction.
2.4. High cell-density fed-batch fermentation
The fermentation was carried out in a 15 L bioreactor (NC-Bio,
Shanghai, China) under the following conditions: medium volume,
10 L; inoculation volume, 10% (v/v); aeration rate, 1.03.0 vvm;
agitation speed, 200600 rpm; dissolved oxygen (DO), 20%. Growth
and induction temperature were maintained at 30C and 23.5C,
respectively. Growth pH was controlled at 5.5, and induction pH
values were set at 4.5, 5.0, and 5.5 for three parallel experiments to
determine the optimal induction pH. Samples were taken periodically
at 8 h intervals, and the induction period lasted for 96 h. The
fermenter was equipped with oxygen and pH electrodes to monitor
and control DO and pH by adjusting aeration/agitation and addition of
NH4OH (25%, w/v), respectively. Residual ethanol concentration was
determined by gas chromatography.

2.6.2. BoxBehnken design (BBD) and response surface methodology

Signicant factors were chosen for further optimization by RSM
based on the results of PB design. BBD and RSM are the combination
of mathematical and statistical methods exploring the relationships
between several explanatory variables and one or more response
variables, normally using a second-degree polynomial model to do
this. The ultimate goal is to optimize the response values through
modeling and analysis of multiple independent variables affecting the
response [19]. The mathematical model is as follows [Equation 1]:

Y 0 i Xi i j Xi X j ii Xi :

Equation 1

Y is the predicted response value, 0 is the constant term, i is the

factor inuence coefcient, ij is the interactive response coefcient
between factors, and ii is the factors of the second order inuence
coefcient. Xi and Xj are the levels of the independent variables.
3. Results
3.1. Screening the major factors for HLZ expression with PB design
A 7-factor and a 2-level method were used in PB design, and the high
levels of the variables were 25% higher than the low levels. Table 2
showed the experimental design using Design Expert software and
the results. All the experiments were carried out in 500 mL shake
asks, triplicates from three independent experiments were measured
and means were calculated. The signicant factors were determined
and the analysis of variance was performed (Table 3).
Table 1
Factors and levels of PB design.
Coded

Factors

X1
X2
X3
X4
X5
X6
X7

Inoculation volume (%)

Culture volume (mL)
Growth time (h)
Induction temperature (C)
Induction time (h)
Initial pH
Methanol concentration (%)

Levels
-1

+1

5
40
24
24
72
5
1

6.25
50
30
30
90
6.25
1.25

Y. Yu et al. / Electronic Journal of Biotechnology 17 (2014) 311316

Table 2
Experimental design and results of PB.
Run

Table 4
BBD and RSM model predicted values of lysozyme activity.

Factors

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

X1

X2

X3

X4

X5

X6

X7

-1
1
-1
1
-1
1
1
1
-1
-1
-1
1

1
1
-1
1
-1
-1
-1
-1
1
-1
1
1

1
-1
-1
1
-1
-1
1
1
-1
1
1
-1

-1
1
-1
-1
1
-1
1
1
1
-1
1
-1

1
1
-1
-1
-1
1
1
-1
1
1
-1
-1

-1
1
-1
1
1
-1
-1
1
1
1
-1
-1

1
-1
-1
1
1
1
-1
1
1
-1
-1
-1

Lysozyme activity
Y (U/mL)
2631
2039
3066
2095
1808
2979
2279
1519
2172
3229
1630
2431

The values of Prob N F (P-value) indicated the signicant levels of

the model and the factors. The inuences of seven investigated factors
on HLZ production were ranked as: X4 N X5 N X2 N X7 N X1 N X3 N X6.
X2, X4 and X5 (induction temperature, induction time and culture
volume) had signicant effects (P b 0.01) on HLZ production in tested
range and were chosen for further optimization.
3.2. Optimization of the signicant factors using RSM
As revealed by the PB experiments, induction temperature, induction
time and culture volume, which were encoded as A, B, and C, had
signicant inuence on the response values. Subsequently, BBD was
used to investigate the interactions and the optimal levels of the three
factors. The mathematical equation was shown below in [Equation 2]:
Y 3194 228:56A 158:56 B 175:75C

Equation 2

368:13AB309:25 AC
2

313

420:25BC677:19A 555:19B 216:06C :

Y was the predicted lysozyme activity, A, B and C were coded

values for induction temperature, induction time and culture volume,
respectively. Each factor had three coded levels (1, 0, -1, see Table 4)
and a total of 17 runs were performed.
Analysis of variance (ANOVA) was carried out to verify the signicant
level of the parameters and the regression model, and the results were
shown in Table 5. The Model F-value of 80.58 implied that the model
was signicant. There was only a 0.01% chance that a Model F-value
this large could occur due to noise. The value of Prob N F less than
0.05 indicates that the model term is signicant. In this case, A, B, C, AB,
AC, BC, A2, B2, and C2 were signicant terms. Simulation loss curve (lack
of Fit) indicates the probability that model prediction does not t the
actual value. The lack of Fit F-value of 0.51 implied that the lack of Fit
was not signicant compared to the pure error. There was a 69.90%
chance that a lack of Fit F-value this large could occur due to noise.
Non-signicant lack of t is good. Coefcient of variation (CV) reects
the condence of the model, and the lower the CV value is, the higher
the condence of the model will be [20]. The CV value of 3.69%
indicated that the model equation could reect the real experimental
Table 3
Analysis of the results of PB design.
Factor

F value

P value (Prob N F)

Signicant

X1
X2
X3
X4
X5
X6
X7

9.93
24.66
8.61
151.26
53.81
6.37E-05
13.14

0.0345
0.0077
0.0426
0.0003
0.0018
0.9940
0.0222

5
3
6
1
2
7
4

Run

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

Factors

Lysozyme activity
Y (U/mL)

A: Induction
temperature (C)

B: Induction
time (h)

C: Culture
volume (mL)

Actual
value

Predicted
value

23 (0)
26 (1)
23 (0)
23 (0)
26 (1)
26 (1)
20 (-1)
23 (0)
23 (0)
20 (-1)
23 (0)
23 (0)
26 (1)
23 (0)
20 (-1)
20 (-1)
23 (0)

84 (0)
72 (-1)
84 (0)
84 (0)
96 (1)
84 (0)
72 (-1)
84 (0)
72 (-1)
84 (0)
72 (-1)
84 (0)
84 (0)
96 (1)
96 (1)
84 (0)
96 (1)

40 (0)
40 (0)
40 (0)
40 (0)
40 (0)
30 (-1)
40 (0)
40 (0)
50 (1)
50 (1)
30 (-1)
40 (0)
50 (1)
30 (-1)
40 (0)
30 (-1)
50 (1)

3021
1665
3194
3227
2716
2708
1944
2540
3302
2512
2462
3227
2350
1941
1522
1633
3224

3194
1664
3194
3194
2716
2663
1943
2457
3194
2557
2509
3194
2396
1985
1524
1587
3177

value well. The correlation coefcient R2 of the model was 98.09%

(R2 N 90%), indicating that the predicted and actual values tted well.
Thus, this model can be applied to predict HLZ production effectively.
In order to gain a better understanding of the effects of the variables
on HLZ production, the 2D contour plots and the 3D response surface
were constructed (Fig. 1, Fig. 2 and Fig. 3). The contour map reects the
cross-interaction between two variables by keeping the other variable
at constant and middle level. An elliptical contour plot (as in Fig. 1a,
Fig. 2a and Fig. 3a) implies a signicant interaction between variables.
All of the three contour plots exhibit similar relationships with respect
to the inuence of each variable. The statistically optimal values of
variables were achieved by moving along the major and minor axes of
the contour. The highest predicted value is obtained in the smallest
ellipse in the contour diagram. The optimal solution was found to be
induction temperature 23.36C, induction time 89.76 h and culture
volume 47.89 mL for a predicted response of 3315 U/mL. Taking into
account the actual situation, induction temperature 23.5C, induction
time 90 h and culture volume 48 mL were employed. Under the
optimized expression parameters, the highest lysozyme activity
production reached 3301 U/mL.
3.3. Cultivation in a 15 L bioreactor under the optimal conditions
To conrm the model feasibility for predicting maximum HLZ
production, fermentation was carried out in a 15 L bioreactor under the
Table 5
ANOVA for response surface quadratic model.
Source

Sum of squares

df

Mean square

F value

P value
(Prob N F)

Model
A
B
C
AB
AC
BC
A2
B2
C3
Residual
Lack of t
Pure error
Cor total

6.25E+06
4.18E+05
2.01E+05
2.47E+05
5.42E+05
3.83E+05
7.06E+05
1.93E+06
1.30E+06
1.97E+05
60,279.56
16,574.06
43,705.5
6.31E+06

9
1
1
1
1
1
1
1
1
1
7
3
4
16

6.94E+05
4.18E+05
2.01E+05
2.47E+05
5.42E+05
3.83E+05
7.06E+05
1.93E+06
1.30E+06
1.97E+05
8611.37
5524.69
10,926.38

80.58
48.53
23.36
28.70
62.95
44.42
82.04
224.22
150.71
22.83
0.51

b0.0001
0.0002
0.0019
0.0011
b0.0001
0.0003
b0.0001
b0.0001
b0.0001
0.0020
0.6990

R = 0.9904, R2 = 0.9809, R2 (adjust) = 0.9567, R2 (predicted) = 0.8970, coefcient of

variation (CV) = 3.69%.

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Y. Yu et al. / Electronic Journal of Biotechnology 17 (2014) 311316

Fig. 1. 2D contour plot and 3D response surface for the effect of cross-interaction between A (induction phase temperature) and B (induction time) on the lysozyme activity.

Fig. 2. 2D contour plot and 3D response surface for the effect of cross-interaction between A (induction phase temperature) and C (culture volume) on the lysozyme activity.

optimal conditions determined by RSM. In addition, three parallel

experiments were carried out to investigate the inuence of pH on HLZ
production, with pH controlled at 4.5, 5.0, and 5.5, respectively. The
time-course proles of HLZ production in fermentation were shown
in Fig. 4, resulting in the maximum HLZ production of 47,680 U/mL at 88
h of induction under pH 4.5. SDS-PAGE analysis for the samples of the
fermentation supernatants revealed the expression of HLZ, shown as a
single predominant protein band of approximately 14.7 kDa (Fig. 5,
lanes 36), consistent with its expected size and the control (Fig. 5, lane 2).
4. Discussion
A notable problem in high-cell-density fermentation is the
degradation of recombinant proteins due to release of proteases from

host cells. P. pastoris SMD1168 is a protease-decient strain, which

could reduce the degree of potential proteolysis [21].
Wei et al. [22] performed a one-factor-at-a-time method study in
which the expressed HLZ activity was up to 533 U/mL by P. pastoris
GS115. However, the one-factor-at-a-time strategy is unable to reveal
the interaction between multiple factors. Ercan et al. [23] used a
three-factor BBD of response surface method in terms of temperature,
pH and aeration levels to nd the optimum combination of growth
parameters of Kluyveromyces lactis K7 for human lysozyme production
in biolm reactor and the highest lysozyme production was 141 U/mL.
By evaluating the inuence of fermentation parameters on HLZ
production using PB design, we found that induction temperature,
induction time and culture volume were highly signicant factors
(P b 0.01). Then a series of experiments and RSM analysis concerning

Fig. 3. 2D contour plot and 3D response surface for the effect of cross-interaction between B (induction time) and C (culture volume) on the lysozyme activity.

Y. Yu et al. / Electronic Journal of Biotechnology 17 (2014) 311316

50000

lysozyme acvity (U/ml)

45000
40000
35000
30000
25000
20000
15000
10000
5000
0

16

24

32

40

48

56

64

72

80

88

96

Time(h)
pH 4.5

pH 5.0

pH 5.5

Fig. 4. Time-course prole of lysozyme production with different pH during fed batch
fermentation.

these factors were performed. The highest lysozyme activity obtained

under optimized conditions (at 23.5C for 90 h with culture volume of
48 mL in 500 mL shake ask) was 3301 U/mL, which was 2.2 fold
higher than that under standard conditions (1515 U/mL) and was in
good accordance with the predicted value (3315 U/mL) by RSM.
Higher temperature is generally benecial to the growth and
metabolism of Pichia and protein expression. However, over
temperature can lead to premature aging of the host strain and
shorten the fermentation period thus reducing heterologous protein
production. The optimal temperature for Pichia growth is 30C and
employed induction temperature range from 20C to 28C. It has been
reported that lower temperature was benecial to extracellular
expression, as it is advantageous to the correct processing and
secretion of recombinant proteins [24,25]. The induction period also
plays a vital role in the production of recombinant proteins for
expression level increase over time during induction, while the
products may suffer severe degradation under some circumstances if
the induction period is overdue. Therefore, it is necessary to
determine the appropriate temperature and induction time during
expression, and a temperature-changing fermentation strategy was
adopted in this study. Huang et al. [26] used K. lactis K7 as the
host strain to produce recombinant human lysozyme, and it was
demonstrated that 25C with no pH control (initial pH 6.0) resulted in
better production (64.1 U/mL) than with pH controlled near neutrality.
Growth of Pichia and expression of heterologous proteins are
oxygen-consuming processes, so the DO is an important factor during
the production of HLZ. In shake ask, oxygen supply is enhanced by

Fig. 5. SDS-PAGE analysis of the culture supernatants of the recombinant P. pastoris. Lane 1,
protein marker; lane 2, positive control: HLZ standard (Sigma-Aldrich Trading Co., Ltd.,
Shanghai, China); lane 3, HLZ expressed under optimized conditions in ask; lines 46,
HLZ production in 15 L fermenter under induction pH of 5.5, 5.0 and 4.5, respectively.

315

increasing shaking speed or reducing culture volume. However, in

this study, lysozyme activity was not increased as the reduction of
culture volume. It could be that the methanol evaporated quickly as
the culture volume went to low, leaving the culture lack of carbon
source and inducer. In our study, the optimal culture volume was
approximately 10% of the shake ask volume, which is in good
accordance with a previous report [27].
The results of batch-fermentation experiments in a 15 L fermenter
demonstrated that the optimized expression conditions could also
improve HLZ production during high-cell-density fermentation, and
the induction pH value of 4.5 was more preferable for HLZ expression
than a pH of 5.0 or 5.5 (Fig. 4). In addition, the relatively low
temperature (23.5C) and acidic condition (pH 4.5) employed in this
study could help to limit the protease activity, according to previous
studies [28,29].
To the best of our knowledge, this is the rst study concerning the
optimization of process parameters for HLZ production in Pichia
SMD1168 combining PB design and RSM, and the extracellular
lysozyme activity (47,680 U/mL) was the highest by far. The
high-yield production of HLZ using fed-batch fermentation under
optimized conditions was successful and industrially promising, which
could shed some light on the scale-up production of HLZ with higher
activity in a cost-effective manner and provide enough quantity of
protein for future studies and applications.
Financial support
This work was nancially supported by the National Science and
Technology Major Projects for Major New Drugs Innovation and
Development, China. (No. 2011ZX09102-007).
Author contributions
Proposed the theoretical frame: YY, XZ, LY; Conceived and designed
the experiments: YY, XZ; Contributed reagents/materials/analysis tools:
SW; Wrote the paper: YY, XZ; Performed the experiments: YY, XZ, TW;
Analyzed the data: XZ.
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