A two-stage temperature control strategy enhances extracellular secretion of recombinant α-cyclodextrin glucosyltransferase in Escherichia coli
Li et al. AMB Expr
A two-stage temperature control strategy enhances extracellular secretion of recombinant α-cyclodextrin glucosyltransferase in Escherichia coli
Yang Li 0 1
Jia Liu 0 1
Yinglan Wang 0 1
Bingjie Liu 0 1
Xiaofang Xie 0 1
Rui Jia 0 1
Caiming Li 0 1
Zhaofeng Li 0 1 2
0 School of Food Science and Technology, Jiangnan University , 1800 Lihu Avenue, Wuxi 214122, Jiangsu , People's Republic of China
1 School of Food Science and Technology, Jiangnan University , 1800 Lihu Avenue, Wuxi 214122, Jiangsu , People's Republic of China
2 State Key Laboratory of Food Science and Technology, Jiangnan University , Wuxi 214122 , People's Republic of China
The effects of temperature on extracellular secretion of the α-cyclodextrin glucosyltransferase (α-CGTase) from Paenibacillus macerans JFB05-01 by Escherichia coli were investigated. When protein expression was induced at constant temperature, the greatest amount of extracellular recombinant α-CGTase was produced at 25 °C. Higher or lower induction temperatures were not conducive to extracellular secretion of recombinant α-CGTase. To enhance extracellular secretion of α-CGTase by E. coli, a two-stage temperature control strategy was adopted. When expression was induced at 25 °C for 32 h, and then the temperature was shifted to 30 °C, the extracellular α-CGTase activity at 90 h was 45% higher than that observed when induction was performed at a constant temperature of 25 °C. Further experiments suggested that raising the induction temperature can benefit the transport of recombinant enzyme and compensate for the decreased rate of recombinant enzyme synthesis during the later stage of expression. This report provides a new method of optimizing the secretory expression of recombinant enzymes by E. coli.
CGTase; Temperature control; Extracellular secretion; Recombinant enzymes; E; coli
Introduction
The cyclic oligosaccharides α-, β-, and γ-cyclodextrin
consist of 6, 7, and 8 glucose units, respectively, linked
by α-1, 4-glycosidic bonds. Cyclodextrins form inclusion
complexes with many different small, hydrophobic guest
molecules, improving their solubility and stability in
aqueous environments. This property makes it have many
applications in scientific, medical and industrial fields
(Roy et al. 2017)
. The industrial use of α-cyclodextrin is
in its infancy, yet is still expanding because of its small
internal cavity, high water solubility, and resistance to
enzymatic hydrolysis. Previous reports have shown that
α-cyclodextrin can be used as a carrier of active
ingredients, a solubilizer of lipids, a stabilizer of oils, a modifier
of flavors or aromas, and a natural soluble dietary fiber
(Aytac and Uyar 2016; Li et al. 2010b, 2014a)
.
With the expanding use of cyclodextrins on an
industrial scale, the cyclodextrin glucosyltransferases
(CGTases, EC 2.4.1.19), which catalyze the formation of
cyclodextrins, have received increased scientific interest.
Although CGTases can be obtained from a wide range of
bacteria, the characteristics of the CGTases from Bacillus
strains are among the closest to industrial requirements
(Tonkova 1998)
. Early work focused on CGTase
production in Bacillus strains
(Gawande et al. 1998; Rosso et al.
2002)
, and efforts were made to improve CGTase yield by
manipulating environmental factors
(Arce-Vazquez et al.
2016; Es et al. 2016)
. Unfortunately, the strict regulatory
mechanisms present in wild-type strains have limited
productivity enhancements, resulting in high costs and
low yields.
A substantial improvement in CGTase expression
was observed when the overexpression was performed
in recombinant Escherichia coli
(Mana et al. 2015;
Sonnendecker et al. 2017)
. Unfortunately, previous
reports have demonstrated that the CGTases expressed
in E. coli usually accumulated in the cytosol as
biologically inactive inclusion bodies
(Makrides 1996; Choi and
Lee 2004)
, and the refolding processes have been proved
to be inconvenient
(Li et al. 2005)
. Although secretion
into the periplasm is helpful for the rapid isolation of
recombinant proteins, current methods for the selective
release of periplasmic proteins are not suitable for
largescale production
(Yang et al. 1998; Jeang et al. 2005)
.
Therefore, the limitations of cytosolic and periplasmic
expression of CGTase make the extracellular secretion of
CGTases highly needed.
In our previous study, the α-CGTase gene from
Paenibacillus macerans JFB05-01 was cloned into the plasmid
vector pET-20b(+). This plasmid was then inserted into
E. coli BL21(DE3) to form a strain used for the
extracellular expression of α-CGTase by E. coli
(Li et al. 2010a,
b)
. The greatest amount of extracellular recombinant
α-CGTase was produced when expression was induced
at a constant temperature of 25 °C
(Li et al. 2010a, b)
.
Extracellular α-CGTase secretion was inhibited when
expression was induced at temperatures >30 °C, and
very little recombinant enzyme was obtained at 37 (...truncated)