He et al.

/ J Zhejiang Univ SCI 2004 5(2):149-156 149

Journal of Zhejiang University SCIENCE

ISSN 1009-3095
[Link]
E-mail: jzus@[Link]

Improved elastase production by Bacillus sp. EL31410

further optimization and kinetics studies of culture
medium for batch fermentation*

HE Guo-qing (何国庆)†, CHEN Qi-he (陈启和), JU Xiao-jie (琚晓捷), SHI Nai-dong (石乃冬)
(Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China)
†
E-mail: gqhe@[Link]
Received May 29, 2002; revision accepted Jan. 24, 2003

Abstract: An efficient culture medium producing a bacterial elastase with high yields was developed further following
preliminary studies by means of response surface method. Central composite design (CCD) and response surface method-
ology were applied to optimize the medium constituents. A central composite design was used to explain the combined effect
of three medium constituents, viz, glucose, K2HPO4, MgSO4x7H2O. The strain produced more elastase in the completely
optimized medium, as compared with the partially optimized medium. The fitted model of the second model, as per RSM,
showed that glucose was 7.4 g/100 ml, casein 1.13 g/100 ml, corn steep flour 0.616 g/100 ml, K2HPO4 0.206 g/100 ml and
MgSO4x7H2O 0.034 g/100 ml. The fermentation kinetics of these two culture media in the flask experiments were analyzed. It
was found that the highest elastase productivity occurred at 54 hours. Higher glucose concentration had inhibitory effect on
elastase production. At the same time, we observed that the glucose consumption rate was slow in the completely optimized
medium, which can explain the lag period of the highest elastase production. Some metal ions and surfactant additives also
affected elastase production and cell growth.

Key words: Elastase, Bacillus sp. EL31410, Culture medium optimization, Central composite design, Response surface
methodology, Batch fermentation, Fermentation kinetics studies
Document code: A CLC number: Q939

INTRODUCTION Shiio, 1975). Reported studies on the reaction mode

between elastin and elastase were rare (Hall and
Elastase is an enzyme that attacks and solubi- Czerkawaki, 1961). The reaction was found to be a
lizes elastin. As elastase can degrade elastin (Mori- complex process of at least three steps (Heather et
hara, 1967) that other proteases cannot; it has broad al., 1999). The mechanism of the reaction is still not
applications in medical therapy, food processing clear (Robert et al., 1997).
and daily use chemicals industry. Considerable eff- Among the microbial elastases, the elastase
orts were made to screen the elastase-producing produced by [Link] had been thoroughly
strains, to study its pathogen effect and its char- studied. [Link] elastase shows considerable
acterizations (Tsuzuki and Oka, 1965; Tsai et al., amino acid sequence homology with other microbial
1988; Sharon et al., 1997; Ozaki and Shiio, 1975). neutral metalloproteinases, especially thermolysin.
Its three-dimensional structure was determined by
*
Project (No. 300024) supported by the Natural Science Founda- Thayer (Thayer, 1991; Joelle et al., 1989) after cry-
tion of Zhejiang Province, China stallization.
150 He et al. / J Zhejiang Univ SCI 2004 5(2):149-156

So far, many authors have focused their efforts this approach, concentrations of medium compo-
on isolating and screening of microorganisms for nents are the variables; each variable is referred to
elastase production with higher activity and on pu- some base value and varied in a certain pattern.
rifying and characterizing newly found enzymes Our preliminary studies showed that the most
(Janda and Sharon, 1999; Tsuzuki and Oka, 1965; important factors in the culture medium are the
Ozaki and Shiio, 1975; Shibata et al., 1993; Tsai et casein and corn steep flour. And these two factors
al., 1988). To achieve high product yields, it is a were also optimized with response surface meth-
prerequisite to design a proper production medium odology (Chen and He, 2002). This paper further
in an efficient fermentation process. Little infor- reports an attempt to formulate a suitable production
mation, however, is available in the scientific lit- medium that can substantially reduce the medium
erature on complete optimization of culture media cost and increase the elastase production from Ba-
for elastase production. It is well known that me- cillus sp. EL31410 by using statistical experimental
dium optimization is approached either empirically design such as FFD (fractional factorial design) and
or by statistical methods. But either the classical or RSM (response surface methodology), and also
empirical method has several problems for complete discusses the fermentation kinetics characteristics
optimization. The traditional one-factor at a time of completely optimized culture medium.
approach to optimization is time-consuming and
incapable of reaching the true optimum due espe-
cially to interaction among factors. Moreover, it MATERIALS AND METHODS
assumes that the various fermentation parameters do
not interact and that the process response is a direct Microbial strain
function of the single varied parameter. In contrast, Bacillus sp. EL31410 was isolated from the soil
the observed behavior of fermentation results from at a meat-processing factory in Hangzhou, China.
the interactive influences of the various variables. The modified method for screening microorganism
Unlike conventional optimization, statistical opti- (Shiio et al., 1974) was used. A strain of Bacillus sp.
mization methods can take into account the inter- that produced elastase was screened and identified
action of variables in generating the process re- as EL31410. Several ways such as violet and chemi-
sponse (Haaland, 1989). Factorial design of opti- cal agent were used to mutate the strain and obtain a
mization experiments is especially suitable for positive mutant strain with high elastase activity. It
taking into account the interactions and it is efficient. was maintained on solid LB (g/L) slants (peptone 6,
A combination of factors that generate a certain yeast extract 2, beef extract 4, NaCl 5, agar 2). The
optimum response can be identified through facto- liquid seed medium contained the above compo-
rial design and the use of response surface meth- nents in addition to agar.
odology (Khuri and Cornell, 1987). Response sur-
face methodology, described first by Box and Wil- Shake-flask cultivations
son (1951), is a collection of mathematical and sta- All optimization experiments were carried out
tistical techniques useful for analyzing the effects of in 250-ml Erlenmeyer flasks containing 25 ml of
several independent variables (Box and Hunter, medium with various concentrations of glucose,
1978). This method had been successfully applied in K2HPO4, and MgSO4x7H2O according to the ex-
the optimization of medium compositions (Souza perimental design. Each medium contained a fixed
and Roberto, 1999; Sarra et al., 1993; Roseiro, amount of casein (11.3 g/L) and corn steep flour
1992), conditions of enzymatic hydrolysis (Ma and (6.16 g/L), which were optimized in the previous
Ooraikul, 1986), parameters of food preservation study (Chen and He, 2002). Each flask (250 ml)
(King, 1993), and fermentation processes (Rosi et containing 20 ml of fermentation medium was in-
al., 1987; Ramírez and Anne, 2001). A central oculated with 5% (v/v) seed culture, then cultivated
composite factorial experimental design was also for 24 hours at 37 ºC on the rotary shaker (200
used for the medium optimization reported here. In rev/min). All media were sterilized at 121 ºC for 20
 He et al. / J Zhejiang Univ SCI 2004 5(2):149-156 151

min, and cooled to room temperature prior to use. conditions. In the previous studies (Chen and He,
2002), the optimized culture medium composition
Assays (g/L) was as follows: glucose 40, casein 11.3, corn
Elastolytic activity was assayed by the col- steep flour 6.16, K2HPO4 1, MgSO4x7H2O 0.1, pH
orimetric method of Sachar (1955). Fermentation 7.5 with FFD and RSM experiment. In order to
broth was recovered after batch fermentation and further increase the elastase production and de-
centrifuged at 1 500 g for 15 minutes. Then the crease the medium cost, the following optimization
supernatant was suitably diluted. Enzyme prepara- was carried out.
tion was incubated with 20 mg of Congo-red elastin
in 2 ml of 0.2 mol/L boric acid buffer (pH 7.4) with Further factorial optimization and experimental
shaking for 20 minutes at 37 ºC. The reaction was results
stopped by adding 2 ml of 0.7 mol/L sodium pho- In order to reduce the cost of medium sources,
sphate buffer (pH 6.0), and immediately filtered. we attempted to improve the compositions of the
Absorbency of the filtrate was read at 495 nm medium by comparing different levels of several
against a control (no enzyme). One unit of elastase factors that were found to have lesser influence on
activity was defined as the amount of enzyme re- the production of elastase by Bacillus sp. EL31410
quired to solubilize 20 mg elastin-congo red under in factorial design. The factors included different
the tested conditions. The standard curve was made concentrations of the medium component glucose,
by the method of Sachar (1955). K2HPO4 and MgSO4x7H2O. We used the central
Biomass content was evaluated by dry cell composition design to find the optimal concentra-
weight (g/L). The culture samples (10 ml) were tions of the three factors. The experimental design
centrifuged (1 500 g for 10 min), and the cell pellet and results of the central composite design are given
was washed thoroughly with distilled water, dried to in Tables 1 and 2 (see the next page). The results of
a constant weight at 80 ºC overnight and then cooled the experiment with CCD were fitted with a sec-
and weighed. Reducing sugar was measured with ond-order polynomial function. Six repeats were
the DNS method (Miller, 1959); pH was measured included at the center of the design, and the total
with pH meter. number of this design was 20. The results of the
regression analysis are shown in Table 3.
Experimental designs The statistical significance of the second-order
The experimental designs used were shown in model equation was checked by an F-test. It was
the reference (Chen and He, 2002). found that it adequately approximated to the ex-
perimental data at the 5% probability level. The
coefficient of determination R2, was calculated to be
RESULTS AND DISCUSSIONS 0.9564, which showed that the regression model was
very suitable for describing elastase production. The
The factorial design for medium development results demonstrated that the response surface had a
relies on three stages of experimentation: screening, maximum point at the coded level –1.212894(x1),
optimization and verification. Screening experime- 0.035835(x2) and 0.823588(x3). The optimal medi-
nts include many variables, but provide little in- um composition was calculated to be 7.4% glucose,
formation per variable. Screening aims at reducing a 0.206% K2HPO4 and 0.034% MgSO4x7H2O. As can
determination as to which few process variables be seen from the response surface plot (Figs.1a, 1b
have the greatest impact on performance. Optimi- and 1c), the elastase production can be increased
zation experiments are designed to provide in-depth when the concentration of glucose decreased and
information about a few variables identified during both the magnesium sulfate and K2HPO4 decreased.
screening as having the greatest impact on per- This model was tested for adequacy by the analysis
formance. Finally, verification experiment was used of variance (Table 4). The regression model for elas-
to validate the results under specific experimental tase production was highly significant (P<0.001).
152 He et al. / J Zhejiang Univ SCI 2004 5(2):149-156

Table 1 Definition and levels of independent variables in central composite design

Coded levels
Independent Symbol
variables
−1.682 −1 0 1 1.682
Glucose X1 8.2955 10 12.5 15 16.70450
K2HPO4 X2 0.03182 0.10 0.20 0.30 0.368180
MgSO4x7H2O X3 0.003182 0.01 0.02 0.03 0.036818

Table 2 Design and experimental results of the central composite design

Y1 Y2
Run X1 X2 X3
(EA* U/ml) (Biomass* g/L)
1 −1 −1 1 365 11.6
2 0 0 −1 262 7.7
3 1 −1 1 200 7.2
4 −1 1 1 365 7.9
5 1 1 1 211 8.8
6 0 1.682 0 313 8.2
7 1 1 −1 238 7.2
8 −1 −1 −1 357 7.7
9 0 0 1 297 8.6
10 1 −1 −1 258 6.5
11 0 −1.682 0 312 6.6
12 −1.682 0 0 337 7.9
13 1.682 0 0 193 10.0
14 −1 1 −1 314 6.1
15 0 0 0 329 6.9
16 0 0 0 313 6.9
17 0 0 0 332 6.8
18 0 0 0 320 6.7
19 0 0 0 325 6.8
20 0 0 0 318 6.8
*
EA: elastase activity; Biomass: dry cell weight

Table 3 Regression coefficient, standard error, and student’s t-test results for the composite design
(CCD-U) with six center points
Parameter
Parameter DF Standard error P>T
estimates
Intercept 1 0.8792430 0.015869 0
x1 1 −0.1376440 0.010528 0
x2 1 −0.0097610 0.010528 0.3757
x3 1 0.0061780 0.010528 0.5703
x1 x1 1 −0.0490640 0.010248 0.0007
x2 x1 1 0.0108750 0.013756 0.4476
x2 x2 1 −0.0064720 0.010248 0.5418
x3 x1 1 −0.0456250 0.013756 0.0078
x3 x2 1 0.0236250 0.013756 0.1167
x3 x3 1 −0.0363400 0.010248 0.0053
 He et al. / J Zhejiang Univ SCI 2004 5(2):149-156 153

Table 4 Analysis of variance (ANOVA) for regression model of elastase production obtained from the re-
sponse surface experiments
Regression DF Sum of square R2 F-ratio P
Linear 3 0.260590 0.7511 57.377 0.000
Quadratic 3 0.049142 0.1416 10.820 0.0018
Crossproduct 3 0.022064 0.0636 4.858 0.0245
Total regression 9 0.331796 0.9564 24.352 0.0263

(a) (b) (c)

Fig.1 Response surface for the effects of (a) glucose (x1) and K2HPO4 (x2); (b) K2HPO4 (x2) and magnesium sulfate (x3);
and (c) glucose (x1) and magnesium sulfate (x3) on elastase production (y1)

Table 4 also suggests that elastase production was partially optimized medium.
primarily determined by the linear terms and quad-
ratic terms of the three factors. There existed in- Effects of various inorganic salts on enzyme
teractions between the three factors. The results also production and cell growth
showed that the response surface had a maximum The effects of various inorganic salts are listed
point based on the response surface plot and the in Table 5. It was reported that phosphates are usu-
response model. The effects of the three variables on ally used as buffering reagents in order to keep the
biomass were also seen according to the dry cell pH constant in culture medium, Ca2+, Zn2+ and Fe2+
weight (data not shown). The effect was the same as could increase the elastase production in the culture
that for elastase production. It was considered as the
substrate inhibition during the elastase fermentation
Table 5 Effect of various inorganic salts on elastase
when the medium contained higher glucose. The production and cell growth
K2HPO4 and MgSO4x7H2O all had positive effects Concentr Relative
Metal EA Biomass
on enzyme production, which may be due to the ions
ation
(U/ml)
activity
(g/L)
(g/100ml) (%)
constant pH and the enzyme structure. It had been 2+
Ca 0.01 295 112 7.35
reported in other elastase-producing strains. The
0.05 129 52 7.40
maximum response predicted by the model was 369
0.10 46 22 7.50
U/ml. The response equations obtained are as follows, 2+
Fe 0.01 294 111 7.05
and represent a suitable model for describing the
Zn2+ 0.01 264 101 7.35
effects of these three factors on elastase production.
0.05 180 70 8.90
To validate these predictions, flask cultivation
using the completely optimized medium composi- 0.10 153 60 7.60
2+
tion was done. The measured elastase activity (mean Cu 0.01 272 104 7.25
of three replicates) was 42% higher than that of the Control 277 100 7.40
154 He et al. / J Zhejiang Univ SCI 2004 5(2):149-156

medium, especially for gram-negative bacteria, as Table 6 Effect of surfactants and polymeric additives on
the Ca2+ and Zn2+ ions can keep the elastase stable. elastase production and cell growth
Mg2+ ion can increase the permeability of the cell Concentration EA
Relative
Biomass
Surfactants activity
wall, especially for the gram-positive bacteria. To (g/100ml) (U/ml) (g/L)
(%)
find out the effects of Zn2+, Ca2+ and Fe2+ ions on the Tween80 0.02 373 111 8.35
cultivation of Bacillus sp. EL31410, a medium 0.04 351 104 9.65
containing only the basic medium components that 0.2 342 102 9.95
included casein/1.13% and corn steep flour/0.616% SDS 0.1 268 81 10.90
was tested. Results demonstrated that there was no Polymeric
significant difference in enzyme production and cell additives
PEG 0.1 291 87 8.10
growth, but the morphology of bacterial growth
changed greatly (data not shown). The enzyme 0.3 338 101 8.15
production in the tested medium was in some ways Gelatin 0.1 356 106 9.60
increased, especially the effects of 0.01% Ca, Fe, Zn 0.3 252 77 6.75
and Cu ions on the enzyme produced. When the Control 332 100 8.10
concentration of several metal ions was above
0.01%, elastase production all decreased but the cell mer or surfactants could affect the rheological
growth increased in some way. It was confirmed properties of the medium, availability of nutrients
that Ca2+ and Zn2+ are required for catalysis and and gases, and physiological functions of the cell.
stabilization of elastase; reducing the availability of The chemical structure, positioning of groups, de-
these ions should restrict autoprocessing of proe- gree of ionization in the medium, and charge density
lastase (Joan and Dennis, 1992). This had been in the macromolecule would determine the effect of
supported in the [Link] strain. The low con- these changes (Chen and Li., 1996).
centrations of Ca and Zn could induce elastase In order to verify its effect on cell growth and
production and proelastase processing. Other metal enzyme production, verifying experiment was per-
ions increased the elastase activity in some way at formed. From the experimental results (data not
the 0.01% concentration. shown), the elastase activity increased a little after
the addition of these surfactants and polymeric ad-
Effects of surfactants and polymeric additives ditives. Consequently, in later studies, we did not
The effects of various polymer and surfactant add these substances to the culture medium.
additives on elastase production by Bacillus sp.
EL31410 are shown in Table 6. It was found that Batch fermentation
0.02% Tween80 had the maximal effect on elastase Shake-flask fermentation was carried out using
production. Both 0.1% SDS and 0.1% PEG had the completely optimized media and partially op-
negative effect on elastase activity. It is possible that timized medium to compare its cultivation kinetics.
these substances can denature the elastase in the The time course of typical cultivation using the
higher concentration. The cell growth also increased optimized medium is shown in Fig.2.
when the concentration of Tween80 increased. It From Fig.2, it can be concluded that the com-
may be thought that Tween80 can improve the gas pletely optimized medium produced more elastase
availability. However, PEG and Gelatin showed di- than the partially optimized culture medium during
fferent effects on cell growth. It was explained that 72 hours fermentation.
the higher concentration of polymeric additives is Based on the kinetics analysis results (Table 7),
not helpful for the bacteria growth, mainly owing to it was obvious that the 18 hours cultivation was the
the high viscosity of polymeric compounds. The best point for enzyme production with the two me-
function of the surfactants and the polymeric addi- dia. But the qp of the later medium was higher than
tives in the fermentation broth is known to be very that of that the former, which means the completely
complicated. It was reported that addition of a poly- optimized medium could increase the enzyme pro-
 He et al. / J Zhejiang Univ SCI 2004 5(2):149-156 155

demonstrated that the bacterium at 6 hours cultiva-

tion time grew faster than at other fermentation time.
At the same time, it was obvious that the reducing
sugar in the partially optimized medium decreased
more slowly than that in the completely optimized
medium. And the elastase production was also
prolonged in the completely optimized medium.
That showed the glucose could provide the full
carbon source and extend the cell growth and en-
zyme production. In contrast, the partially opti-
mized medium was not really appropriate for en-
zyme production due to its low content of glucose
and other inorganic salts that were verified to be
beneficial for cell growth and elastase production.

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