Biotechnol Lett (2015) 37:899906

DOI 10.1007/s10529-014-1755-3

ORIGINAL RESEARCH PAPER

Enhanced extracellular production of a-amylase

in Bacillus subtilis by optimization of regulatory elements
and over-expression of PrsA lipoprotein
Jingqi Chen Yuanming Gai Gang Fu
Wenjuan Zhou Dawei Zhang Jianping Wen

Received: 8 November 2014 / Accepted: 10 December 2014 / Published online: 17 December 2014
Springer Science+Business Media Dordrecht 2014

Abstract a-Amylase was used as a heterologous

model protein to investigate the effects of promoters,
signal peptides and over-expression of an extracytoplasmic molecular chaperone, PrsA lipoprotein,
on enhancing the secretion of a-amylase in Bacillus
subtilis. Four promoters and six signal peptides were
compared, successively, and the highest yield of
a-amylase was achieved under the promotion mediated by PAprE, a strong constitutive promoter, and
secretion by SPnprE, a signal peptide from B. subtilis.
Moreover, under conditions of overexpressed PrsA
Electronic supplementary material The online version of
this article (doi:10.1007/s10529-014-1755-3) contains supplementary material, which is available to authorized users.
J. Chen  J. Wen
Department of Biological Engineering, School of
Chemical Engineering and Technology, Tianjin
University, Tianjin 300072, Peoples Republic of China
J. Chen  Y. Gai  G. Fu  W. Zhou  D. Zhang (&)
Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, Peoples Republic
of China
e-mail: zhang_dw@tib.cas.cn
J. Chen  Y. Gai  G. Fu  W. Zhou  D. Zhang
Key Laboratory of Systems Microbial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, Peoples
Republic of China
G. Fu  D. Zhang
National Engineering Laboratory for Industrial Enzymes,
Tianjin 300308, Peoples Republic of China

lipoprotein, the secretion production and activity of aamylase increased to 2.5-fold. The performance of the
recombinant B. subtilis 1A751PL31 was evaluated
with a fed-batch fermentation in a 7.5 l fermentor.
Optimization of regulatory elements and over-expression of PrsA lipoprotein had a significant effect on
enhancing the production of a-amylase in B. subtilis.
Keywords a-Amylase  Bacillus subtilis  Fed-batch
fermentation  Promoter  PrsA  Regulatory elements 
Signal peptide

Introduction
Production of heterologous proteins of interest at high
levels is crucial for both basic research and practical
applications. Bacillus subtilis is a widely used host for
the expression of heterologous proteins. Compared to
Escherichia coli, B. subtilis is considered as a
generally recognized as safe (GRAS) organism (Westers et al. 2004). It has a naturally high secretory
capacity and exports proteins directly into the extracellular medium (Simonen and Palva 1993). However,
there are several bottlenecks in the B. subtilis expression system (i.e., transcription, protein folding, translocation across the membrane, signal peptide
processing and proteolysis) that limit its application
potential (Li et al. 2004).
To obtain large amounts of secreted protein, the use
of strong promoters and optimal signal peptides are

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necessary. The promoters PHpaII (Westers et al. 2006),

P43 (Zhang et al. 2014) and PAprE (Jan et al. 2001)
have been claimed to bring about high levels of protein
secretion. The optimization of the targeted signal
peptide was also carried out by non-rational
approaches, such as single specific verification,
random mutagenesis or high-throughput screening
(Brockmeier et al. 2006; Caspers et al. 2010; Degering
et al. 2010; Harwood and Cranenburgh 2008). To date,
the adaptation mechanism between signal peptide and
the target protein is still unknown though it might
involve many aspects, such as molecular mass, folding
rate and charge of the target protein (Zhang et al.
2013). At the cytoplasmic membrane-cell wall interface of B. subtilis, the PrsA lipoprotein, which belongs
to the family of parvulin-type PPIases (Rahfeld et al.
1994), acts as a folding factor for exoproteins. When
an increased amount of PrsA lipoprotein is introduced
by over-expression, several model proteins are
secreted at increased levels (Kakeshita et al. 2011;
Kontinen and Sarvas 1993; Vitikainen et al. 2001).
This suggests that the PrsA lipoprotein is the ratelimiting component of the secretion machinery in the
cases above.
In this study, the regulatory elements of a-amylase
were investigated and optimized. Four constitutively
active promoters were tested, and the strong promoter
PAprE was eventually selected. We screened and
analyzed six signal peptides from 15 candidates with
a semi-rational approach, compared to the native
signal peptide SPAmyL. With the over-expression of
PrsA lipoprotein, the secretion of a-amylase was
further enhanced substantially. Furthermore, the production of a-amylase by B. subtilis was conducted in a
7.5 l fermentor. The results obtained here may be
useful for the achievement of industrial a-amylase
production.

Materials and methods

Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in this study are
described in Supplementary Table 1. Escherichia coli
DH5a served as a host for cloning and plasmid preparation, and was grown in LB medium (1 % tryptone,
0.5 % yeast extract, and 0.5 % NaCl) at 37 C. Bacillus
subtilis 1A751 was used as the expression host for the

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Biotechnol Lett (2015) 37:899906

plasmid-encoded amyl (a-amylase gene from Bacillus

licheniformis), and was cultivated at 37 C in SR medium
(1.5 % tryptone, 2.5 % yeast extract, and 0.3 %
K2HPO4). Antibiotics were: ampicillin, 100 lg/ml;
kanamycin, 50 lg/ml; and chloramphenicol, 12.5 lg/
ml. The E. coli/B. subtilis shuttle plasmid pMA5 was
kindly provided by Bacillus Genetic Stock Center
(BGSC). All vectors for a-amylase expression in B.
subtilis are pMA5 derivatives. The plasmid pDL is an
integration vector which has a promoterless bgaB flanked
by both the front and back parts of amyE.
Construction of expression vectors containing
different promoters and signal peptides
All vectors were constructed by a sequence-independent
simple cloning method without the need for restriction and ligation enzymes (You et al. 2012). All primers
used in this study are listed in Supplementary Table 2.
The assembly of pMAL1 is used as an example below to
illustrate the procedure for the construction of the
vectors. The complete amyl gene with its own signal
peptide SPAmyL was amplified from purified B. licheniformis CICC 10181 genome (Liu et al. 2008), using
primers PAmyL-F and PAmyL-R. The pMAL1 vector
backbone was amplified from pMA5, with primers
PMA5-F1 and PMA5-R1. The two linear DNA fragments both contained 30 - and 50 - 2550 bp overlapping
termini. The DNA multimer is generated based on these
DNA templates by prolonged overlap extension PCR
(POE-PCR). Eventually, the POE-PCR products (DNA
multimer) are transformed into competent E. coli DH5a
directly, yielding the desired plasmid pMAL1. Two
promoters (P43 and PAprE) were cloned from the
chromosome of B. subtilis 168 using primer pairs
PP43-F/R and PPAprE-F/R, respectively. The other one
promoter PAmyL, the native promoter of amyl gene, was
amplified from purified genomic B. licheniformis CICC
10181 DNA with primer pairs PPAmyL-F/R. Then
pMAL2 (P43), pMAL3 (PAprE) and pMAL4 (PAmyL)
were constructed on the basis of pMAL1 (PHpaII), as
described previously.
As predicted by the SignalP 3.0 Server [http://www.
cbs.dtu.dk/services/SignalP/], a 87 bp signal peptide
sequence was found in the amyl open reading frame.
To investigate the influence of signal peptides on the
expression of a-amylase in B. subtilis, the native signal peptide of a-amylase was replaced by six different
signal peptides (SPnprE, SPAprE, SPwapA, SPyncM,

Biotechnol Lett (2015) 37:899906

901

SPamyE, and SPsacB), respectively. The six signal

peptides were amplified using B. subtilis 168 genome
as the template (Primers are listed in Supplementary
Table 1). The vector backbone was amplified from
pMAL3 with primers PMA5-F3 and PMA5-R3. The
expression vectors with different signal peptides were
then constructed.
Construction of the over-expression PrsA strain
To construct recombinant strains 1A751P and 1A751PL31,
an additional expressing PrsA with chromosomally
encoding PrsA, two DNA fragments containing promoter Pgrac with ribosome bind site (RBS) sequence or
PrsA coding region were amplified using plasmid
pHT43 or B. subtilis 168 genome as the template, and
primer pairs Pgrac-F/R or PprsA-F/R, respectively.
A mixture of the two fragments was used as a template
and amplified by POE-PCR with primers PgracF/PprsA-R to generate the fusion fragment PgracprsA. The pDLP vector backbone was cloned from
pDL using primers PpDL-F/R. As stated before, the
pDLP was constructed, and then transformed into
B. subtilis 1A751 and 1A751L31 by competence
transformation after digestion by PstI.
Fed-batch fermentation
The recombinant B. subtilis 1A751PL31 containing an
additional expressing PrsA and harboring the plasmid
pMAL31 was used to scale up fermentation in 7.5 l
fermentor (BioFlo 310; New Brunswick Scientific Co
Inc., USA), with a fed-batch strategy. The air-flow rate
was 6 l/min, and dissolved O2 tension was maintained
between 20 and 40 % air saturation by automatic
adjustment of speed of the stirrer. The temperature
was kept at 37 C and the pH was controlled at pH 7.0.
Foam was controlled by the addition of a siliconebased anti-foaming agent. The fermentation medium
(1.5 % tryptone, 2.5 % yeast extract, and 0.3 %
K2HPO4) was supplemented with 50 lg kanamycin/
ml and 100 lg chloramphenicol/ml. The fermentation
was performed with an initial working volume of 3.5 l.
When the cell growth rate was constant, the substrate
fed-batch mode was started by adding 5 % yeast
extract at a constant flow rate of 20 ml/h from 24 to
72 h. Cell growth was monitored by measuring dry
cell weight. The activity of a-amylase was determined
by measuring the supernatant of fermentation broth.

Fig. 1 Comparison of a-amylase production in recombinant

strains with different promoters. a Analysis of a-amylase
activity. The samples were collected at incubation of 48 h in a
250 ml shake-flask and centrifuged at 10,0009g and 4 C for
10 min. After extraction, the supernatant was used for analysis
of a-amylase activity. Data represent the mean of three parallel
experiments, and error bars represent standard error. b SDSPAGE analysis of a-amylase distribution in culture supernatants. 8 ll culture supernatant of the recombinants were
analyzed. Lane M molecular weight marker. Lane C starting
strain 1A751, served as negative control. Specifically, samples
14 in a and b were in correspondence which represented
1A751L3 (PAprE), 1A751L1 (PHpaII), 1A751L2 (P43), and
1A751L4 (PAmyL), respectively

SDS-PAGE analysis
Culture samples (1 ml) were harvested and the
supernatant was separated from the culture medium
by centrifugation (12,0009g, 10 min, 4 C). After
adding 59 SDS-PAGE sample buffer, the supernatants were boiled for 10 min and proteins were
separated in SDS-PAGE using the NuPAGE 12 %
BisTris Gel (Novex by Life Technologies, USA) in
combination with MOPS SDS Running Buffer (Invitrogen Life Technologies, USA). PageRuler Prestained
Protein Ladder (Invitrogen Life Technologies, USA)
was used to determine the apparent molecular weight
of separated proteins. Proteins were visualized with
Coomassie Brilliant Blue.

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Biotechnol Lett (2015) 37:899906

Table 1 Comparison of different signal peptide sequences used for a-amylase production in B. subtilis
Protein

Signal peptide

Charge
N-regiona

Hydrophobic
amino acidb

yncM

MAKPLSKGGILVKKVLIAGAVGTAVLFGTLSSGIPGLPAADAQVAKA

35

YbdN

MVKKWLIQFAVMLSVLSTFTYSASA

15

YrvJ

MNKKYFVLIVCIIFTSALFPTFSSVTA

16

wapA

MKKRKRRNFKRFIAAFLVLALMISLVPA

18

nprE

MGLGKKLSVAVAASFMSLSISLPGVQA

19

NprB

MRNLTKTSLLLAGLCTAAQMVFVTHASA

16

amyE

MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA

26

SleB

MKSKGSIMACLILFSFTITTFINTETISAFS

16

SacB

MNIKKFAKQATVLTFTTALLAGGATQAFA

19

YqzG

MMIKQCVICLSLLVFGTTAAHA

14

YjcM

MKKELLASLVLCLSLSPLVSTNEVFA

15

aprE

MRSKKLWISLLFALTLIFTMAFSNMSVQA

18

LytE

MKKQIITATTAVVLGALFA

13

YobV

MKLERLLAMVVLLISKKQVQA

14

TyrA

MNQMKDTILLAGLGLIGGSIALA

17

The signal peptide highlighted in bold were chosen for further studies to improve a-amylase production. The cleavage sites of signal
peptides were underlined
a

The netto charge of the N-region was calculated with amino acids aspartate and glutamate defined as -1; arginine and lysine
defined as ?1 and any other amino acid defined as 0

The hydrophobic amino acids of the each signal sequence was calculated with amino acids G, A, V, L, I, M, F, W and P defined as
hydrophobic, any other amino acid being characterized as hydrophilic

a-Amylase activity assay

The definition of a-amylase is described below. One
unit of enzyme is the amount of amylase needed to
complete the hydrolysis of starch into dextrin per min
at 70 C and pH 6.0. The measurement was done
according to Chinese Industrial Standard (GB
8275-2009). The calculation of the enzyme activity
was based on the formula below. X = c 9 n 9 16.67,
where X is the enzyme activity of the sample (U/ml),
c is the concentration of the control enzyme (U/ml)
corresponded with the absorbance and n is the dilution
fold.

Results and discussion

Effect of different promoters on a-amylase
expression
The activity of the promoter directly influences the
expression efficiency of heterologous protein, and
strong promoters are usually used to improve gene

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expression. Four widely used promoters (PHpaII, PP43,

PAprE and PAmyL) were employed to construct the
expression vectors (pMA5L1, pMA5L2, pMA5L3,
and pMA5L4), and then the four recombinant plasmids were transformed into B. subtilis 1A751. PHpaII is
a widely used promoter from Staphylococcus aureus.
The promoters P43 and PAprE are both strong and
constitutively expressed promoters in B. subtilis.
PAmyL is the native promoter of a-amylase from B.
licheniformis.
To verify the transcriptional efficiency of the
selected four promoters PHpaII, PP43, PAprE, and
PAmyL, extracellular a-amylase activity assay of the
recombination strains were conducted after 48 h
incubation in SR medium. As shown in Fig. 1a, the
activity of a-amylase expressed by the recombinant
strain 1A751L3 was the highest (150 U/ml), which
was approx. 100 % higher than that of the other
recombinants. To provide further evidence supporting
the result described above, the SDS-PAGE analysis
was performed to compare the production of aamylase. As shown in Fig. 1b, distinct bands with a
molecular mass of about 60 kDa were observed which

Biotechnol Lett (2015) 37:899906

903

is in good agreement with the deduced value. The

a-amylase transcribed by promoter PAprE had a
stronger band than that of promoters PHpaII, PP43,
and PAmyL. As a result, it could be concluded that
PAprE is the most effective promoter among these four
promoters.

to screen a matched signal peptide for the a-amylase,

we used a semi-rational approach (Zhang et al. 2013).
Fifteen candidate signal peptides (Table 1) of B.
subtilis were compared based on its compositions,
especially on the positively charged N-domain, the
hydrophobic H-domain and the C-domain. Many
studies have proposed that signal peptides which
containing more positively charged N-domain, more
hydrophobic H-domain and more conserved C-domain
would be crucial for efficient secretion. As a result, six
different signal peptides, nprE, aprE, wapA, yncM,
amyE and SacB, were chosen for further studies to
improve the secretion of a-amylase.
On the basis of the effect of four promoters above, we
used PAprE to drive expression of a-amylase fused to
different signal peptides in B. subtilis. The plasmids
pMA5L31 (SPnprE), pMA5L32 (SPaprE), pMA5L33
(SPwapA), pMA5L34 (SPyncM), pMA5L35 (SPamyE),
pMA5L36 (SPSacB) were transformed into B. subtilis
1A751, resulting recombinants 1A751L31, 1A751L32,
1A751L33, 1A751L34, 1A751L35, 1A751L36, respectively. As shown in Fig. 2a, the extracellular a-amylase
activity of seven recombinant strains, 1A751L31,
1A751L32, 1A751L3, 1A751L33, 1A751L34, 1A75
1L35, and 1A751L36 were accumulated to 260, 180,
145,140, 132, 107 and 77 U/ml, respectively, after 48 h
incubation. Obviously, the recombinant strain
1A751L31 produced a highest extracellular a-amylase
titer. In order to further verify the above results, the
SDS-PAGE analysis was carried out to compare the
production of a-amylase. As shown in Fig. 2b, obviously, the recombinant strain 1A751L31 showed a
thicker band than the others, which was consistent with
the results of a-amylase activity. Therefore, the results
indicated that the signal peptide SPnprE was an excellent
candidate for a-amylase production.

Optimization of the signal peptide for enhancing aamylase production

Over-expression of PrsA lipoprotein enhanced

the secretion of a-amylase

Application of an appropriate signal peptide was a

critical point for achieving highly efficient extracellular expression of the target protein (Brockmeier et al.
2006). Although numerous attempts have been made
to study the features of signal peptides that are
required for efficient protein secretion (Leloup et al.
1999; Nakamura et al. 1989; Zanen et al. 2005), there
has been no specific approach to identifying the
optimal signal peptide to a specific partner. Therefore,

PrsA lipoprotein is an extracellular folding chaperone

that influences protein folding in the periplasmic
space. Several model proteins are secreted at increased
level when PrsA lipoprotein is overexpressed. To
overexpress PrsA lipoprotein in B. subtilis, we
constructed the integration plasmid pDLP (Fig. 3a).
After a double crossing-over event, the gene prsA
under the control of the strong expressed B. subtilis
promoter Pgrac was inserted into the chromosome of

Fig. 2 Comparison of a-amylase production in recombinant

strains with different signal peptides. a Analysis of a-amylase
activity. Data represent the mean of three parallel experiments,
and error bars represent standard error. b SDS-PAGE analysis
of a-amylase distribution in culture supernatants. Lane M
molecular weight marker. Lane C starting strain 1A751, served
as negative control. Specifically, samples 17 in a and b were in
correspondence which represented 1A751L31 (SPnprE),
1A751L32 (SPaprE), 1A751L3 (SPAmyL), 1A751L33 (SPwapA),
1A751L34 (SPyncM), 1A751L35 (SPamyE), and 1A751L36
(SPSacB), respectively

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Fig. 3 Comparison of a-amylase secretion in recombinant

1A751L31 and 1A751PL31 which is PrsA lipoprotein overexpressing strain. a Schematic representation of the integration
plasmid pDLP containing prsA expression cassette. Pgrac and
prsA represent the B. subtilis groESL promoter and B. subtilis
prsA, respectively; RBS, ribosome binding site; cat, chloramphenicol-resistant gene. The gene prsA inserted into the
plasmid pDL under control of the Pgrac promoter and integrated

1A751 and 1A751L31 at the amyE locus, generating

the strain 1A751P and 1A751PL31.
To investigate the effect of over-expression of PrsA
lipoprotein on enhancing the secretion of a-amylase,
the activity of strains 1A751L31 and 1A751PL31 after
48 h of incubation was analyzed. As shown in Fig. 3b,
the activity of secreted a-amylase in 1A751PL31
(640 U/ml) was about 1.5-fold higher than that of
1A751L31 (252 U/ml). That is, under the conditions
of overexpressed PrsA lipoprotein, the activity of
secreted a-amylase was increased compared to that
under the normal expression level of PrsA. As shown
in Fig. 3c, the a-amylase secreted by 1A751LP31 had
a stronger band than that of 1A751L31, which was
consistent with the results of a-amylase activity. These
results indicated that over-expression of PrsA lipoprotein could significantly enhance the secreted
a-amylase in B. subtilis.
Production of a-amylase with fed-batch strategy
The expression efficiency of the recombinant strain
1A751PL31 was further explored in a 7.5 l fermentor.
The fermentor was inoculated with 5 % (v/v) freshly

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into the bacterial chromosome via a double crossing-over event

at the amyE locus. b Analysis of a-amylase activity. Data
represent the mean of three experiments, and error bars
represent standard error. c SDS-PAGE analysis of a-amylase
distribution in culture supernatants. Lane M molecular weight
marker. Lane C1 starting strain 1A751. Lane C2 1A751P.
Specifically, samples 12 in b and c were in correspondence
which represented 1A751L31 and 1A751PL31, respectively

Fig. 4 Production of a-amylase in the recombinant strain

1A751PL31 by fed-batch fermentation in a 7.5 l fermentor.
White square cell growth; black square extracellular a-amylase
activity; solid line DO concentration

cultured 1A751PL31 grown in SR medium at 37 C

for 18 h. To maintain cell growth and a-amylase
production, we choose a fed-batch strategy. When the
cell growth rate was constant, 5 % (w/v) yeast extract
was added at a constant flow rate of 20 ml/h from 24 to
72 h. As shown in Fig. 4, during the growth phase, the
maximum dry cell weight in the fermentor reached
6.8 g/l at 24 h. The expression of a-amylase was

Biotechnol Lett (2015) 37:899906

continuously increased and reached the maximum

yield of 1,089 U/ml at 66 h with a high productivity of
16.5 U/ml h. The high activity of a-amylase indicated
that B. subtilis was a suitable host for the industrial
production of a-amylase.

Conclusion
The gene of a-amylase (amyl) from B. licheniformis
was cloned and expressed in B. subtilis 1A751. PAprE
were chosen to improve the transcriptional level of
amyl from four strong promoters (PHpaII, PP43, PAprE,
and PAmyL). In order to optimize and strengthen the
expression level, six signal peptides (SPnprE, SPaprE,
SPwapA, SPyncM, SPamyE, and SPSacB) were screened
and explored, the results of which indicated that SPnprE
was the most suitable for the production of a-amylase
in B. subtilis. In addition, over-expression of PrsA
lipoprotein further significantly enhanced the secretion of a-amylase. The secretory capacity of the
recombinant 1A751PL31 was evaluated by fed-batch
fermentation in a 7.5 l fermentor and a high activity of
a-amylase (1,089 U/ml) with a productivity of
16.5 U/ml h was achieved. The work presented herein
illustrates a useful method to enhance the extracellular
production of a heterologous protein in B. subtilis.
Acknowledgments This research was supported by grants
from National Nature Science Foundation of China (31200036,
31370089), the State Key Development Program for Basic
Research of China (973 Program, 2013CB733600), and the Key
Projects in the Tianjin Science & Technology Pillar Program
(14ZCZDSY00065).
Supporting information Supplementary Table 1Strains
and plasmids used in this study.
Supplementary Table 2Primers used in this study.

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