# The Cyclodextrin Glycosyltransferase ofPaenibacillus pabuli US132 Strain: Molecular Characterization and Overproduction of the Recombinant Enzyme

Authors: Sonia Jemli; Ezzedine Ben Messaoud; Sameh Ben Mabrouk; Samir Bejar
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

The gene encoding the cyclodextrin glycosyltransferase (CGTase) ofPaenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the β-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37∘C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19∘C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed Vmax⁡ and Km values of 253±36 μmol of β-cyclodextrin/mg/min and 0.36±0.18 g/L, respectively.


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## Body

## 1. Introduction

Cyclodextrin glycosyltransferases (CGTases; E.C.2.4.1.19) are starch degrading enzymes which
generate cyclic oligosaccharides termed cyclodextrins (CDs). These
oligosaccharides exist as three major types commonly known asα-CD, β-CD, and γ-CD having 6, 7, and 8 glucose residues, respectively. They have
torus-shaped structures able to encapsulate a wide range of molecules, thereby
modifying their physical and chemical properties. Consequently, CDs are
extensively used in pharmaceutical, chemical, agricultural, cosmetic, and food
industries [1, 2].CGTases
belong to the glycoside hydrolase family 13 (α-amylase family) [3]. Members of this family contain a catalytic (β/α)8 barrel and use an α-retaining mechanism but they display a variety of reaction
specificities [4]. Whereas α-amylases typically catalyze starch hydrolysis reaction, CGTases
catalyze mainly three transglycosylation reactions (cyclization, coupling, and
disproportionation) besides a weak hydrolytic activity [5]. Both α-amylases and CGTases share three domains (A, B, and C) while CGTases
possess two additional domains (D and E). Domains A and B form the catalytic
core, domains C and E are involved in substrate binding whereas domain D has
unknown precise function [6, 7].Understanding the enzymatic features has been
explored by cloning, sequencing, and comparing variouscgtase genes
[8–11]. The sequence data analysis has revealed the key amino acids residues
that determine the reaction mechanism and product specificity, thereby
enhancing genetic engineering techniques to provide modified CGTases [7, 12, 13].E. coli is frequently adopted as host strain for
heterologous protein expression since it is well characterised with various
expression systems. However, the formation of inclusion bodies remains a
significant barrier for expression of heterologous proteins in E. coli.
Refolding of inclusion bodies into soluble and active form needs high cost and
tedious jobs. Hence, maximizing the yield of soluble and active recombinant
proteins in vivo by altering the culture conditions is an attractive
alternative [14]. However, no universal approach has been established to
minimize the formation of inclusion bodies and some empirical conditions must
be screened on an individual basis. To date, there are only a few reports that
succeeded the overproduction of CGTase in E. coli by varying
fermentation conditions [15–17].In a
previous study, the CGTase ofPaenibacillus pabuli US132 was reported as
good candidate for cyclodextrins production and efficient antistaling agent
[18]. In this work, we described the molecular cloning of the US132 cgtase gene as well as the amino acid sequence inspection and the comparison with
other related CGTases. We also reported the production enhancement of the recombinant
active enzyme in E. coli by varying
mainly the cultivation conditions.


## 2. Materials and Methods

### 2.1. Bacterial Strains, Plasmids, and Culture Media

Paenibacillus pabuli US132 strain, previously isolated [18], was used as source of chromosomal DNA. E. coli DH5α (F−supE44 ϕ80⁢δlacZΔ M15 Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (rk−,mk+) deoR thi-1 λ−  gyrA96 relA1)
was used for all plasmid constructions. E. coli ER2566 (F−⁢λ−fhu A2
[lon] ompT lacZ:T7 gene1
gal sulA11
Δ(mcrC-mrr)114:IS10 R(mcr-73:miniTn10-TetS)2 R(zgb-210:Tn10) (TetS) endA1 [dcm]) and E.
coli JM109 (F′traD36 proA+B+ lacIqz ΔM15/Δ(lac-proAB e14− (McrA−) recA1 endA1 gyrA96
thi-1 hsdR17   (rk−mk+)
supE44 relA1) were used for protein expression. The
plasmids pSJ8 and pSJ9 (this work) carrying the cgtase US132 gene, derived
from pCR2.1 (Invitrogen) and pTrc99a vector [19], respectively. E. coli recombinant strains were grown on LB, M9, 2TY [20], or M9ZB medium [16] containing
100μg/mL ampicillin. Paenibacillus pabuli US132 strain was grown as
previously described [18].


### 2.2. Cloning of the US132 Cgtase Gene  and Sequence Analysis

To amplify an internalcgtase gene fragment, we used the highly
conserved aa regions previously reported for CGTase enzymes as members of the α-amylase family [4, 21]. Furthermore, we
aligned several cgtases DNA sequences and we located the corresponding
CGTases conserved regions. Hence, a degenerate pair of primers, S209 (5′-GAYTTTGCRCCCAAYCAT-3′)
and S211 (5′-ATCATGATTGTCRATRAA-3′), corresponding
respectively to the conserved regions I (DFAPNH) and IV (FIDNHD), were
designed. The chromosomal DNA isolated from Paenibacillus pabuli US132
strain was used as template for gene amplification by Pfu DNA polymerase
(Fermentas). The PCR
parameters were 94°C for 120 seconds followed by 35 cycles of 94°C for 30 seconds,
50°C for 45 seconds, and 72°C for 120 seconds. The PCR product was purified
using Wizard SV Gel and PCR Clean-Up System (Promega). To amplify the entire
US132 cgtase gene, we have conceived two primers, S260 (5′-TCCAATATTTCTTACGAT-3′)
and S262 (5′-GTTACGTTACTCCGGG-3′), flanking the closely
matching cgtase gene (accession no. L25256). The resulting PCR fragment was
cloned into the pCR2.1 vector to give pSJ8 plasmid harboring the US132 cgtase gene under the control of the IPTG inducible T7 promoter. The pSJ8 was digested with HindIII and EcoRV
and the resultant cgtase gene was subcloned in the pTrc99a, under the control
of the Trc promoter (IPTG inducible), to give pSJ9 plasmid. Nucleotide
sequences of the US132 cgtase gene carried by three independent pSJ8
plasmids obtained from different PCR reactions were determined. DNA sequencing
was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit and the
automated ABI Prism3100-Avant Genetic Analyser (Applied Biosystems). Sequence
analysis and comparison were performed with BioEdit and CLUSTAL W programs. Homology
search was performed using BLAST search algorithm. For the promoter
determination, the prokaryotic promoter prediction program NNPP2.2A
(http://www.fruitfly.org/cgi-bin/seqtools/promoter.pl)
was used. The US132 cgtasesequence was submitted to the EMBL data bank
under the accession no. AM748796.


### 2.3. Heterologous Expression and Recombinant  US132 CGTase Purification

The heterologous
expression of the US132 CGTase was investigated using different recombinant
strains (DH5α/pSJ8, ER2566/pSJ8, DH5α/pSJ9, ER2566/pSJ9 or JM109/pSJ9), various temperature cultivation (37°C
or 19°C during all operating time and 37°C followed by a shift to 19°C after
IPTG induction) and different medium composition (LB, M9, 2TY or M9ZB). An
overnight culture of the studied strain, cultivated at 37°C, was used to
inoculate 50 mL of basal medium (in 250 mL shake flask) with an initial OD600 nm  of 0.1. Protein expression was induced by addition of different IPTG
concentration (16–240μg/mL) at OD600 nm between 0.8 and 1. Effects of the postinduction time on the US132
CGTase production were also investigated. All experiments were performed at
least twice.Batch fermentation was
carried out in a 7 L fermentor INFORS AG CH-4103 (Bottmingen, Switzerland)
containing 4.5 L of 2TY medium. The fermentor was inoculated, with an initial
OD600 nm of 0.1, by an overnight culture of the ER2566/pSJ8 strain
grown at 37°C in 2TY broth. The pH, aeration and agitation were maintained
constant at 7.4, 1.5 vvm and 500 rpm, respectively, during
all cultivation. The temperature was shifted from 37 to 19°C after induction
with 16 mg/L of IPTG when OD600 nm reached 0.8–1.The CGTase extract was
prepared from the periplasm by the modified osmotic-shock procedure of Ausubel et
al. [22]. Cells were harvested by centrifugation at 8000  ×  g for 10 minutes and the pellets were suspended in 30 mM Tris-HCl buffer (pH 8)
containing 20% sucrose and 1 mM EDTA. After agitation for 10 minutes at 25°C,
cells removed by centrifugation were rapidly suspended in ice-cold water,
incubated for 10 minutes at 0°C and clarified by centrifugation at 12000  ×  g
for 30 minutes. The supernatant was recovered as periplasmic fraction and used
for CGTase assay. Residual
cells were sonicated at 4°C for 6 minutes (pulsations 3 s, amplify 90) using a Vibra-Cell 72405
Sonicator and insoluble fraction (cells debris) was recuperated by
centrifugation at 12000  ×  g for 30 minutes.For the purification of
the recombinant US132 CGTase, the ER2566/pSJ8 periplasmic fraction was heat-treated
at 60°C for 15 minutes in presence of 10 mM calcium followed by centrifugation
at 16000  ×  g for 30 minutes at 4°C. The supernatant was then purified using
hydrophobic interaction chromatography and starch adsorption as previously
described for the native enzyme [18].


### 2.4. CGTase Assay and Kinetic  Parameters Determination

The CGTase activity, determined as
dextrinisation activity, was monitored
at 60°C for 10 min as
described in a previous work [18].The kinetic parameters (Km  and
Vmax) were determined by incubating the purified CGTase at 60°C in 50 mM sodium
acetate buffer (pH 6.5) and using various concentrations of soluble starch
ranging from 0.4 to 1.6 g/L. Samples were taken at regular time intervals and the reaction was stopped by boiling for 5
minutes. The amount of generated β-CDs was detected according to a phenolphthalein
method [23]. The initial speed was calculated for
each substrate concentration and then represented according to the Lineweaver-Burk
method [24]. The Km and Vmax were determined graphically using Hyper32 program
(http://homepage.ntlworld.com/john.easterby/hyper32.html).


## 2.1. Bacterial Strains, Plasmids, and Culture Media

Paenibacillus pabuli US132 strain, previously isolated [18], was used as source of chromosomal DNA. E. coli DH5α (F−supE44 ϕ80⁢δlacZΔ M15 Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (rk−,mk+) deoR thi-1 λ−  gyrA96 relA1)
was used for all plasmid constructions. E. coli ER2566 (F−⁢λ−fhu A2
[lon] ompT lacZ:T7 gene1
gal sulA11
Δ(mcrC-mrr)114:IS10 R(mcr-73:miniTn10-TetS)2 R(zgb-210:Tn10) (TetS) endA1 [dcm]) and E.
coli JM109 (F′traD36 proA+B+ lacIqz ΔM15/Δ(lac-proAB e14− (McrA−) recA1 endA1 gyrA96
thi-1 hsdR17   (rk−mk+)
supE44 relA1) were used for protein expression. The
plasmids pSJ8 and pSJ9 (this work) carrying the cgtase US132 gene, derived
from pCR2.1 (Invitrogen) and pTrc99a vector [19], respectively. E. coli recombinant strains were grown on LB, M9, 2TY [20], or M9ZB medium [16] containing
100μg/mL ampicillin. Paenibacillus pabuli US132 strain was grown as
previously described [18].


## 2.2. Cloning of the US132 Cgtase Gene  and Sequence Analysis

To amplify an internalcgtase gene fragment, we used the highly
conserved aa regions previously reported for CGTase enzymes as members of the α-amylase family [4, 21]. Furthermore, we
aligned several cgtases DNA sequences and we located the corresponding
CGTases conserved regions. Hence, a degenerate pair of primers, S209 (5′-GAYTTTGCRCCCAAYCAT-3′)
and S211 (5′-ATCATGATTGTCRATRAA-3′), corresponding
respectively to the conserved regions I (DFAPNH) and IV (FIDNHD), were
designed. The chromosomal DNA isolated from Paenibacillus pabuli US132
strain was used as template for gene amplification by Pfu DNA polymerase
(Fermentas). The PCR
parameters were 94°C for 120 seconds followed by 35 cycles of 94°C for 30 seconds,
50°C for 45 seconds, and 72°C for 120 seconds. The PCR product was purified
using Wizard SV Gel and PCR Clean-Up System (Promega). To amplify the entire
US132 cgtase gene, we have conceived two primers, S260 (5′-TCCAATATTTCTTACGAT-3′)
and S262 (5′-GTTACGTTACTCCGGG-3′), flanking the closely
matching cgtase gene (accession no. L25256). The resulting PCR fragment was
cloned into the pCR2.1 vector to give pSJ8 plasmid harboring the US132 cgtase gene under the control of the IPTG inducible T7 promoter. The pSJ8 was digested with HindIII and EcoRV
and the resultant cgtase gene was subcloned in the pTrc99a, under the control
of the Trc promoter (IPTG inducible), to give pSJ9 plasmid. Nucleotide
sequences of the US132 cgtase gene carried by three independent pSJ8
plasmids obtained from different PCR reactions were determined. DNA sequencing
was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit and the
automated ABI Prism3100-Avant Genetic Analyser (Applied Biosystems). Sequence
analysis and comparison were performed with BioEdit and CLUSTAL W programs. Homology
search was performed using BLAST search algorithm. For the promoter
determination, the prokaryotic promoter prediction program NNPP2.2A
(http://www.fruitfly.org/cgi-bin/seqtools/promoter.pl)
was used. The US132 cgtasesequence was submitted to the EMBL data bank
under the accession no. AM748796.


## 2.3. Heterologous Expression and Recombinant  US132 CGTase Purification

The heterologous
expression of the US132 CGTase was investigated using different recombinant
strains (DH5α/pSJ8, ER2566/pSJ8, DH5α/pSJ9, ER2566/pSJ9 or JM109/pSJ9), various temperature cultivation (37°C
or 19°C during all operating time and 37°C followed by a shift to 19°C after
IPTG induction) and different medium composition (LB, M9, 2TY or M9ZB). An
overnight culture of the studied strain, cultivated at 37°C, was used to
inoculate 50 mL of basal medium (in 250 mL shake flask) with an initial OD600 nm  of 0.1. Protein expression was induced by addition of different IPTG
concentration (16–240μg/mL) at OD600 nm between 0.8 and 1. Effects of the postinduction time on the US132
CGTase production were also investigated. All experiments were performed at
least twice.Batch fermentation was
carried out in a 7 L fermentor INFORS AG CH-4103 (Bottmingen, Switzerland)
containing 4.5 L of 2TY medium. The fermentor was inoculated, with an initial
OD600 nm of 0.1, by an overnight culture of the ER2566/pSJ8 strain
grown at 37°C in 2TY broth. The pH, aeration and agitation were maintained
constant at 7.4, 1.5 vvm and 500 rpm, respectively, during
all cultivation. The temperature was shifted from 37 to 19°C after induction
with 16 mg/L of IPTG when OD600 nm reached 0.8–1.The CGTase extract was
prepared from the periplasm by the modified osmotic-shock procedure of Ausubel et
al. [22]. Cells were harvested by centrifugation at 8000  ×  g for 10 minutes and the pellets were suspended in 30 mM Tris-HCl buffer (pH 8)
containing 20% sucrose and 1 mM EDTA. After agitation for 10 minutes at 25°C,
cells removed by centrifugation were rapidly suspended in ice-cold water,
incubated for 10 minutes at 0°C and clarified by centrifugation at 12000  ×  g
for 30 minutes. The supernatant was recovered as periplasmic fraction and used
for CGTase assay. Residual
cells were sonicated at 4°C for 6 minutes (pulsations 3 s, amplify 90) using a Vibra-Cell 72405
Sonicator and insoluble fraction (cells debris) was recuperated by
centrifugation at 12000  ×  g for 30 minutes.For the purification of
the recombinant US132 CGTase, the ER2566/pSJ8 periplasmic fraction was heat-treated
at 60°C for 15 minutes in presence of 10 mM calcium followed by centrifugation
at 16000  ×  g for 30 minutes at 4°C. The supernatant was then purified using
hydrophobic interaction chromatography and starch adsorption as previously
described for the native enzyme [18].


## 2.4. CGTase Assay and Kinetic  Parameters Determination

The CGTase activity, determined as
dextrinisation activity, was monitored
at 60°C for 10 min as
described in a previous work [18].The kinetic parameters (Km  and
Vmax) were determined by incubating the purified CGTase at 60°C in 50 mM sodium
acetate buffer (pH 6.5) and using various concentrations of soluble starch
ranging from 0.4 to 1.6 g/L. Samples were taken at regular time intervals and the reaction was stopped by boiling for 5
minutes. The amount of generated β-CDs was detected according to a phenolphthalein
method [23]. The initial speed was calculated for
each substrate concentration and then represented according to the Lineweaver-Burk
method [24]. The Km and Vmax were determined graphically using Hyper32 program
(http://homepage.ntlworld.com/john.easterby/hyper32.html).


## 3. Results and Discussion

### 3.1. Identification of the P. Pabuli US132 Cgtase Gene

To clone the
US132cgtase gene, an internal PCR fragment of
about 600 bp was firstly amplified using US132 genomic DNA and two primers
corresponding to the conserved regions I and IV in CGTases as described in
Materials and Methods section. The nucleotide sequence of this internal
fragment (582 bp) showed the highest homology (95% identity) with homologous
region of the putative Bacillus Q cgtase gene (accession no.
L25256). This result incited us to conceive two
primers from the 5′ and
3′ flanking regions of the Bacillus Q cgtase gene in order to amplify the entire US132 cgtase gene using the chromosomal DNA of P. pabuli US132 as template. The PCR amplification, using Pfu DNA polymerase, gave a nucleotide fragment of
approximately 2400 bp. The
cloning of this fragment in pCR2.1 vector, under the control of the T7
promoter, provided pSJ8 plasmid and conferred starch degrading activity for E.
coli host strains.The nucleotide sequence analysis of the
fragment containing the US132cgtase gene revealed the presence of a
single open reading frame (ORF) with two potential initiation ATG codons (Figure 1). A putative Shine-Dalgarno site (5′-AGAAGGGTGG-3′),
exhibiting a good rationally complementarity with the 3′ end of the US132 16S
rRNA gene [18], was 7 bases upstream the first ATG codon. Consequently, this
latter is most likely the true initiator codon. Thus, the US132 cgtase ORF, consisted of 2157 bp, encoded a protein having 718 amino acids. By using
the promoter prediction program, a potential −10 region (5′-TCTGCA-3′) and −35 region (5′-TTGGCG-3′) were found at 101 and 124 bp upstream
the initiator codon, respectively. The first 34 amino acids are considered as signal peptide according to the SignalP server
(http:/www.cbs.dtu.dk/services/signalP). Therefore, the mature enzyme was
consisted of 684 amino acids with an estimated molecular mass of about 74.3 kDa.Figure 1
Nucleotide and deduced amino acid sequences of
the CGTase ofPaenibacillus pabuli US132. The putative ribosome-binding site
(RBS) is double underlined. The putative promoter regions (−10 and −35) are
shaded. The two possible initiator codons are boxed. The underlined amino acid
sequence was the predicted signal peptide and the vertical arrow indicated the
possible signal peptide cleavage site. Full nucleotide and amino acid sequences
data reported in this paper were submitted to the GenBank under Accession number AM748796.The alignment of the mature US132 CGTase sequence
to the data bank showed 93, 92, 74, and 73% identity with the CGTases ofBacillus sp. (accession no. CAA46901), Bacillus licheniformis (accession no. CAA33763), Bacillus circulans strain 251 (accession no. CAA55023), and Bacillus sp.1011 (accession no. 1I75A), respectively. The highest homology (97% similarity
and 94% identity) was found with the CGTase of Bacillus circulans strain
no. 8 (accession no. 1CGT). The alignment showed a difference of 39/684 amino
acids which does not
radically affect the product specificity. Indeed, both enzymes are specific for
the β-CD production and are classified as β-CGTases since they generated a
mixture of CDs composed of  α-, β-, and γ-CD 20 : 58 : 13 and
10 : 64 : 20 ratios for the CGTase
of strain US132 and B. circulans strain no. 8 [25], respectively. However, as far as we know, no data was available
concerning biochemical properties of the CGTase of B. circulans strain
no. 8 (pH, thermoactivity, thermostability) allowing the comparison of these
two enzymes.From the multiple sequence alignment (Figure2), the five structural domains (A, B, C, D, and E) [11, 26] could be identified
in the US132 CGTase. Furthermore, four highly conserved regions labelled I–IV
and located in the catalytic domain were found. These conserved regions would
constitute the active center of the enzyme since it contained the three
catalytic residues Asp229, Glu257, and Asp328 referring to the CGTase of B.
circulans strain no. 8 numbering. The conserved amino acids in the acceptor
binding site, namely Lys47, Tyr89, Asn94, Phe183, Asn193, Leu194, Tyr195, Asp196,
Phe259, Phe283, and Asp371, reported as the main determinants for cyclization
reaction [12, 27], were also identified.Figure 2
Comparison of the deduced amino acid sequence of
US132 CGTase with those of related CGTases. The five domains found in CGTase
proteins are indicated by letters A to E under the sequences. The four highly
conserved regions are boxed and the catalytic residues are shaded. Arrows
indicate the main amino acid residues of cyclization activity Lys47, Tyr89,
Asn94, Phe183, Asn193, Leu194, Tyr195, Asp196, Phe259, Phe283, and Asp371. The
numbering starts after the respective signal sequence, with identity (*),
strongly similar (:), and weakly similar (⋅). PP132: CGTase of Paenibacillus
pabuli US132 strain; BC8: CGTase of Bacillus circulans strain no. 8;
Bli: CGTase of Bacillus licheniformis; BC251: CGTase of Bacillus
circulans strain 251; Bsp1011: CGTase of Bacillus sp.1011.


### 3.2. Production Enhancement of the
Active Recombinant US132 CGTase

The purification of the US132 CGTase
from the native strain (Paenibacillus pabuli) was extremely hampered by
the viscosity of the crude supernatant, probably due to the presence of
polysaccharides of unknown nature (data not shown). In the aim to overcome this
problem and also to overproduce the US132 CGTase, we have tested the enzyme
production from the initial recombinant E. coli strain (DH5α/pSJ8). This study showed that this latter strain, cultivated in LB medium at 37°C and induced by 16μg/mL IPTG for 18 hours,
exhibited a very low activity of about only 0.3 U/mL. For this reason, we have
studied the effect of several parameters on the production enhancement of the
active recombinant CGTase.


#### 3.2.1. Effect of the Association
Promoter/Host Strain

Using the same culture conditions (LB medium, 37°C and induction by 16μg/mL IPTG
for 18 hours), the association E. coli ER2566/pSJ8 increased the production of the
active recombinant enzyme by 3-fold (Table 1) since the ER2566 strain contains
a chromosomal IPTG inducible copy of the T7 RNA polymerase gene. Despite this
improvement, the production of the active US132 CGTase remained negligible (0.92 U/mL) compared to that obtained by the native strain (18 U/mL) [18]. To further
enhance this production, the gene was placed downstream the Trc promoter in
pSJ9 and the enzyme production was investigated in various strains (DH5α, ER2566 and JM109). As shown in Table 1, the association E. coli ER2566/pSJ9 gave the highest activity of only about 1 U/mL which is nearly the
same with that obtained by ER2566/pSJ8 strain.Table 1
Effect of the
association promoter/host strain on the production of the active recombinant
US132 CGTase.
Plasmids (promoters)pSJ8 (T7)pSJ9 (Trc)E. coli strainsDH5αER2566DH5αER2566JM109Enzyme production (U/mL)0.32±0.080.92±0.080.35±0.051.07±0.030.85±0.05This study showed that the use of different
promoters andE. coli host strains did not significantly enhance the production
of the active recombinant
CGTase, which
is probably due to the formation of inclusion bodies often reported for E.
coli heterologous expression [14, 28].


#### 3.2.2. Effect of the Operating Temperature

A well-known technique
to prevent aggregation of recombinant proteins in vivo consists to reduce the
operating temperature [15, 16, 29]. Therefore, in order to improve the recombinant
US132 CGTase production, ER2566/pSJ9 and ER2566/pSJ8 strains were cultivated at
different temperatures and were induced by 16μg/mL of IPTG for 18 hours: (a)
37°C during all operating time, (b) 37°C followed by a temperature shift to
19°C after IPTG induction, and (c) 19°C during all operating time. This study
showed that the conditions (b) and (c) allowed the enhancement of the active enzyme
production in the periplasm for the two constructions pSJ8 and pSJ9 (Table 2). The
highest production (15 U/mL) was reached by E. coli ER2566/pSJ8 when the
culture was performed at 37°C followed by temperature shift to 19°C after IPTG induction.
SDS-PAGE analysis of periplasmic and insoluble fractions, obtained from E.
coli ER2566/pSJ8, showed that the cultivation of the strain at 37°C
expressed most the US132 CGTase as insoluble inclusion bodies with a small amount
of soluble protein (Figure 3) explaining the low activity detected. However,
the decrease of the operating temperature (37–19°C or 19°C)
strongly increased the expression of the US132 CGTase as periplasmic soluble
form (Figure 3). The enhancement of the production of soluble and active enzyme
at low cultivation temperature could be explained by: (i) the reduction of the
high rate of protein synthesis, which prevents misfolding [14], (ii) the enhancement of
chaperones expression, which allows
correct protein folding [30].Table 2
Effect of the operating temperature on the
production of the active US132 CGTase byE. coli ER2566/pSJ8 and
ER2566/pSJ9.
StrainsOperating temperature37°C37–19°C19°CER2566/pSJ80.93 U/mL15 U/mL13 U/mLER2566/pSJ91 U/mL4 U/mL3.6 U/mLFigure 3
Influence of the operating temperature on the production
of soluble US132 CGTase byE. coli ER2566/pSJ8 strain. Protein samples
loaded onto the 10% SDS-polyacrylamide gel were stained with Coomassie
Brilliant Blue. Lanes 1, 2, and 3: periplasmic fractions obtained from cells
cultivated at 37, 19, and 37–19°C, respectively. Lane M: low molecular weight
marker. Lanes 4, 5 and 6: insoluble fractions obtained when cells are
cultivated at 37–19, 19, and 37°C, respectively.Based on all obtained
data, we retained for further investigation the strainE. coli ER2566/pSJ8
and a culture temperature at 37°C followed by a shift to 19°C afterinduction.


#### 3.2.3. Effect of Medium Composition  and Postinduction Time

The composition of growth medium could have
significant metabolic effects on both cells growth and protein production [31].
To investigate the effect of this parameter, the strain E. coli ER2566/pSJ8
was cultivated in 2TY, M9ZB, and M9 besides LB medium used in all previous
experiences. The US132 CGTase production monitored after 18 hours of induction
showed that the use of M9ZB and 2TY medium increased the production by about
1.1-fold (16.5 U/mL) and 1.3-fold (20 U/mL), respectively, in comparison to that
obtained by LB broth. However, the use of M9 medium decreased the production to
attain only 8 U/mL. The enhancement of the US132 CGTase production was likely
correlated with cell density since the OD600 nm reached about 3.7,
4.8, 6, and 2 for the LB, M9ZB, 2TY, and M9 medium, respectively (Figure 4). Moreover, this difference in the production level of
the recombinant active enzyme, using different medium, could be also influenced by the growth phase at which induction
took place on.Figure 4
Influence of the growth medium on the active
US132 CGTase production and the cell density ofE. coli ER2566/pSJ8
strain. The temperature was shifted from 37 to 19°C after induction with 16 mg/L of IPTG when OD600 nm reached 0.8–1.Otherwise, extensive attempts to improve the
enzyme production using different IPTG concentrations and stressing additives
(sorbitol, mannitol, and ethanol) did not increase the US132 CGTase production (data
not shown) as reported for other
recombinant proteins [16, 28, 32].The effect of postinduction
time on the US132 CGTase production was examined by analysis of samples taken,
from ER2566/pSJ8 culture performed in 2TY medium, at different times after IPTG
addition. Following the IPTG induction, the culture temperature is shifted from
37 to 19°C. The evolution of both cell growth and active enzyme production (Figure 5) showed that the CGTase production reached its maximum (22 U/mL) after 22 hours
of induction when cells are at full stationary phase. When culture attained the
lysis phase the production of the active enzyme decreased by about 27% probably
due to the proteases release.Figure 5
Evolution of bacterial cell growth
and the active US132 CGTase production generated byE. coli ER2566/pSJ8 cultivated in 2TY medium. Induction was performed by 16 mg/L IPTG when
culture reached OD600 nm
between 0.8 and 1. Following the
IPTG induction, the culture temperature is shifted from 37 to 19°C.


#### 3.2.4. Batch Fermentation Scale

The fermentation, using the ER2566/pSJ8, was
scaled up in 7 L fermentor strain under the conditions previously optimised: 2TY
medium and temperature shift from 37 to 19°C after IPTG induction. Interestingly,
the production of the US132 CGTase in fermentor reached a maximum (35 U/mL) after
only 18 hours of induction while this maximum (22 U/mL) was obtained after 22 hours
in Erlenmeyer. It should be mentioned that the cell density obtained in the two
cases was almost the same (about OD600 nm  =  6). This finding
suggested that the improvement of the active enzyme production was not only
related to the cell growth but it could also depend on the stability of
fermentation parameters (pH and pO2), which probably affect
translation and correct folding of recombinant proteins as well as proteolysis
according to Makrides [28].


### 3.3. Purification and Kinetics Parameters Determination of the Recombinant US132 CGTase

For the purification of the
recombinant US132 CGTase, we have used the periplasmic fraction of the
ER2566/pSJ8 strain cultivated in the fermentor in the cultivation conditions
described above. This periplasmic fraction was firstly heat-treated at 60°C in
order to remove E. coli thermolabile proteins. The extract was then
purified using hydrophobic interaction chromatography (HIC) and starch
adsorption as described for the native enzyme [18]. The recombinant enzyme was
purified to homogeneity 25-fold with a yield of 20% (Table 3, Figure 6). This
recombinant US132 CGTase retained the same biochemical properties (data not
shown) as the native enzyme (thermoactivity, thermostability, pH stability, CDs
production) [18].Table 3
Purification steps of the recombinant US132
CGTase.
Purification stepTotal activity (U)Total protein (mg)Specific activity (U/mg)Yield (%)Purification (fold)Crude extract700035200100—Heat treatment665014.8450952.2HIC*43401.5528006214Starch adsorption14000.2850002025*HIC: Hydrophobic Interaction Chromatography.Figure 6
SDS-PAGE analysis of the purified
US132 CGTase from the recombinantE. coli ER2566/pSJ8 strain. Lane 1:
purified US132 CGTase, Lane 2: molecular weight marker.The kinetic parameters were
determined by incubating the purified recombinant US132 CGTase in presence of
various soluble starch concentrations and using the Lineweaver-Burk method. TheVmax and Km values were evaluated to be 253 ± 36μmol of β-cyclodextrin/mg/min and 0.36±0.18 g/L, respectively. Km
values
determined, using also soluble starch as substrate, for CGTase from Bacillus
agaradhaerens [33], Bacillus circulans E192 [34], Bacillus firmus [35], and Bacillus sp. TS1-1 [11] were 21.2, 5.7, 1.21, and
0.52 g/L,
respectively. Since a small value of Km  exhibited a high affinity for the substrate, the values shown above suggested that US132 CGTase was more specific towards starch than the other reported CGTases.


## 3.1. Identification of the P. Pabuli US132 Cgtase Gene

To clone the
US132cgtase gene, an internal PCR fragment of
about 600 bp was firstly amplified using US132 genomic DNA and two primers
corresponding to the conserved regions I and IV in CGTases as described in
Materials and Methods section. The nucleotide sequence of this internal
fragment (582 bp) showed the highest homology (95% identity) with homologous
region of the putative Bacillus Q cgtase gene (accession no.
L25256). This result incited us to conceive two
primers from the 5′ and
3′ flanking regions of the Bacillus Q cgtase gene in order to amplify the entire US132 cgtase gene using the chromosomal DNA of P. pabuli US132 as template. The PCR amplification, using Pfu DNA polymerase, gave a nucleotide fragment of
approximately 2400 bp. The
cloning of this fragment in pCR2.1 vector, under the control of the T7
promoter, provided pSJ8 plasmid and conferred starch degrading activity for E.
coli host strains.The nucleotide sequence analysis of the
fragment containing the US132cgtase gene revealed the presence of a
single open reading frame (ORF) with two potential initiation ATG codons (Figure 1). A putative Shine-Dalgarno site (5′-AGAAGGGTGG-3′),
exhibiting a good rationally complementarity with the 3′ end of the US132 16S
rRNA gene [18], was 7 bases upstream the first ATG codon. Consequently, this
latter is most likely the true initiator codon. Thus, the US132 cgtase ORF, consisted of 2157 bp, encoded a protein having 718 amino acids. By using
the promoter prediction program, a potential −10 region (5′-TCTGCA-3′) and −35 region (5′-TTGGCG-3′) were found at 101 and 124 bp upstream
the initiator codon, respectively. The first 34 amino acids are considered as signal peptide according to the SignalP server
(http:/www.cbs.dtu.dk/services/signalP). Therefore, the mature enzyme was
consisted of 684 amino acids with an estimated molecular mass of about 74.3 kDa.Figure 1
Nucleotide and deduced amino acid sequences of
the CGTase ofPaenibacillus pabuli US132. The putative ribosome-binding site
(RBS) is double underlined. The putative promoter regions (−10 and −35) are
shaded. The two possible initiator codons are boxed. The underlined amino acid
sequence was the predicted signal peptide and the vertical arrow indicated the
possible signal peptide cleavage site. Full nucleotide and amino acid sequences
data reported in this paper were submitted to the GenBank under Accession number AM748796.The alignment of the mature US132 CGTase sequence
to the data bank showed 93, 92, 74, and 73% identity with the CGTases ofBacillus sp. (accession no. CAA46901), Bacillus licheniformis (accession no. CAA33763), Bacillus circulans strain 251 (accession no. CAA55023), and Bacillus sp.1011 (accession no. 1I75A), respectively. The highest homology (97% similarity
and 94% identity) was found with the CGTase of Bacillus circulans strain
no. 8 (accession no. 1CGT). The alignment showed a difference of 39/684 amino
acids which does not
radically affect the product specificity. Indeed, both enzymes are specific for
the β-CD production and are classified as β-CGTases since they generated a
mixture of CDs composed of  α-, β-, and γ-CD 20 : 58 : 13 and
10 : 64 : 20 ratios for the CGTase
of strain US132 and B. circulans strain no. 8 [25], respectively. However, as far as we know, no data was available
concerning biochemical properties of the CGTase of B. circulans strain
no. 8 (pH, thermoactivity, thermostability) allowing the comparison of these
two enzymes.From the multiple sequence alignment (Figure2), the five structural domains (A, B, C, D, and E) [11, 26] could be identified
in the US132 CGTase. Furthermore, four highly conserved regions labelled I–IV
and located in the catalytic domain were found. These conserved regions would
constitute the active center of the enzyme since it contained the three
catalytic residues Asp229, Glu257, and Asp328 referring to the CGTase of B.
circulans strain no. 8 numbering. The conserved amino acids in the acceptor
binding site, namely Lys47, Tyr89, Asn94, Phe183, Asn193, Leu194, Tyr195, Asp196,
Phe259, Phe283, and Asp371, reported as the main determinants for cyclization
reaction [12, 27], were also identified.Figure 2
Comparison of the deduced amino acid sequence of
US132 CGTase with those of related CGTases. The five domains found in CGTase
proteins are indicated by letters A to E under the sequences. The four highly
conserved regions are boxed and the catalytic residues are shaded. Arrows
indicate the main amino acid residues of cyclization activity Lys47, Tyr89,
Asn94, Phe183, Asn193, Leu194, Tyr195, Asp196, Phe259, Phe283, and Asp371. The
numbering starts after the respective signal sequence, with identity (*),
strongly similar (:), and weakly similar (⋅). PP132: CGTase of Paenibacillus
pabuli US132 strain; BC8: CGTase of Bacillus circulans strain no. 8;
Bli: CGTase of Bacillus licheniformis; BC251: CGTase of Bacillus
circulans strain 251; Bsp1011: CGTase of Bacillus sp.1011.


## 3.2. Production Enhancement of the
Active Recombinant US132 CGTase

The purification of the US132 CGTase
from the native strain (Paenibacillus pabuli) was extremely hampered by
the viscosity of the crude supernatant, probably due to the presence of
polysaccharides of unknown nature (data not shown). In the aim to overcome this
problem and also to overproduce the US132 CGTase, we have tested the enzyme
production from the initial recombinant E. coli strain (DH5α/pSJ8). This study showed that this latter strain, cultivated in LB medium at 37°C and induced by 16μg/mL IPTG for 18 hours,
exhibited a very low activity of about only 0.3 U/mL. For this reason, we have
studied the effect of several parameters on the production enhancement of the
active recombinant CGTase.


### 3.2.1. Effect of the Association
Promoter/Host Strain

Using the same culture conditions (LB medium, 37°C and induction by 16μg/mL IPTG
for 18 hours), the association E. coli ER2566/pSJ8 increased the production of the
active recombinant enzyme by 3-fold (Table 1) since the ER2566 strain contains
a chromosomal IPTG inducible copy of the T7 RNA polymerase gene. Despite this
improvement, the production of the active US132 CGTase remained negligible (0.92 U/mL) compared to that obtained by the native strain (18 U/mL) [18]. To further
enhance this production, the gene was placed downstream the Trc promoter in
pSJ9 and the enzyme production was investigated in various strains (DH5α, ER2566 and JM109). As shown in Table 1, the association E. coli ER2566/pSJ9 gave the highest activity of only about 1 U/mL which is nearly the
same with that obtained by ER2566/pSJ8 strain.Table 1
Effect of the
association promoter/host strain on the production of the active recombinant
US132 CGTase.
Plasmids (promoters)pSJ8 (T7)pSJ9 (Trc)E. coli strainsDH5αER2566DH5αER2566JM109Enzyme production (U/mL)0.32±0.080.92±0.080.35±0.051.07±0.030.85±0.05This study showed that the use of different
promoters andE. coli host strains did not significantly enhance the production
of the active recombinant
CGTase, which
is probably due to the formation of inclusion bodies often reported for E.
coli heterologous expression [14, 28].


### 3.2.2. Effect of the Operating Temperature

A well-known technique
to prevent aggregation of recombinant proteins in vivo consists to reduce the
operating temperature [15, 16, 29]. Therefore, in order to improve the recombinant
US132 CGTase production, ER2566/pSJ9 and ER2566/pSJ8 strains were cultivated at
different temperatures and were induced by 16μg/mL of IPTG for 18 hours: (a)
37°C during all operating time, (b) 37°C followed by a temperature shift to
19°C after IPTG induction, and (c) 19°C during all operating time. This study
showed that the conditions (b) and (c) allowed the enhancement of the active enzyme
production in the periplasm for the two constructions pSJ8 and pSJ9 (Table 2). The
highest production (15 U/mL) was reached by E. coli ER2566/pSJ8 when the
culture was performed at 37°C followed by temperature shift to 19°C after IPTG induction.
SDS-PAGE analysis of periplasmic and insoluble fractions, obtained from E.
coli ER2566/pSJ8, showed that the cultivation of the strain at 37°C
expressed most the US132 CGTase as insoluble inclusion bodies with a small amount
of soluble protein (Figure 3) explaining the low activity detected. However,
the decrease of the operating temperature (37–19°C or 19°C)
strongly increased the expression of the US132 CGTase as periplasmic soluble
form (Figure 3). The enhancement of the production of soluble and active enzyme
at low cultivation temperature could be explained by: (i) the reduction of the
high rate of protein synthesis, which prevents misfolding [14], (ii) the enhancement of
chaperones expression, which allows
correct protein folding [30].Table 2
Effect of the operating temperature on the
production of the active US132 CGTase byE. coli ER2566/pSJ8 and
ER2566/pSJ9.
StrainsOperating temperature37°C37–19°C19°CER2566/pSJ80.93 U/mL15 U/mL13 U/mLER2566/pSJ91 U/mL4 U/mL3.6 U/mLFigure 3
Influence of the operating temperature on the production
of soluble US132 CGTase byE. coli ER2566/pSJ8 strain. Protein samples
loaded onto the 10% SDS-polyacrylamide gel were stained with Coomassie
Brilliant Blue. Lanes 1, 2, and 3: periplasmic fractions obtained from cells
cultivated at 37, 19, and 37–19°C, respectively. Lane M: low molecular weight
marker. Lanes 4, 5 and 6: insoluble fractions obtained when cells are
cultivated at 37–19, 19, and 37°C, respectively.Based on all obtained
data, we retained for further investigation the strainE. coli ER2566/pSJ8
and a culture temperature at 37°C followed by a shift to 19°C afterinduction.


### 3.2.3. Effect of Medium Composition  and Postinduction Time

The composition of growth medium could have
significant metabolic effects on both cells growth and protein production [31].
To investigate the effect of this parameter, the strain E. coli ER2566/pSJ8
was cultivated in 2TY, M9ZB, and M9 besides LB medium used in all previous
experiences. The US132 CGTase production monitored after 18 hours of induction
showed that the use of M9ZB and 2TY medium increased the production by about
1.1-fold (16.5 U/mL) and 1.3-fold (20 U/mL), respectively, in comparison to that
obtained by LB broth. However, the use of M9 medium decreased the production to
attain only 8 U/mL. The enhancement of the US132 CGTase production was likely
correlated with cell density since the OD600 nm reached about 3.7,
4.8, 6, and 2 for the LB, M9ZB, 2TY, and M9 medium, respectively (Figure 4). Moreover, this difference in the production level of
the recombinant active enzyme, using different medium, could be also influenced by the growth phase at which induction
took place on.Figure 4
Influence of the growth medium on the active
US132 CGTase production and the cell density ofE. coli ER2566/pSJ8
strain. The temperature was shifted from 37 to 19°C after induction with 16 mg/L of IPTG when OD600 nm reached 0.8–1.Otherwise, extensive attempts to improve the
enzyme production using different IPTG concentrations and stressing additives
(sorbitol, mannitol, and ethanol) did not increase the US132 CGTase production (data
not shown) as reported for other
recombinant proteins [16, 28, 32].The effect of postinduction
time on the US132 CGTase production was examined by analysis of samples taken,
from ER2566/pSJ8 culture performed in 2TY medium, at different times after IPTG
addition. Following the IPTG induction, the culture temperature is shifted from
37 to 19°C. The evolution of both cell growth and active enzyme production (Figure 5) showed that the CGTase production reached its maximum (22 U/mL) after 22 hours
of induction when cells are at full stationary phase. When culture attained the
lysis phase the production of the active enzyme decreased by about 27% probably
due to the proteases release.Figure 5
Evolution of bacterial cell growth
and the active US132 CGTase production generated byE. coli ER2566/pSJ8 cultivated in 2TY medium. Induction was performed by 16 mg/L IPTG when
culture reached OD600 nm
between 0.8 and 1. Following the
IPTG induction, the culture temperature is shifted from 37 to 19°C.


### 3.2.4. Batch Fermentation Scale

The fermentation, using the ER2566/pSJ8, was
scaled up in 7 L fermentor strain under the conditions previously optimised: 2TY
medium and temperature shift from 37 to 19°C after IPTG induction. Interestingly,
the production of the US132 CGTase in fermentor reached a maximum (35 U/mL) after
only 18 hours of induction while this maximum (22 U/mL) was obtained after 22 hours
in Erlenmeyer. It should be mentioned that the cell density obtained in the two
cases was almost the same (about OD600 nm  =  6). This finding
suggested that the improvement of the active enzyme production was not only
related to the cell growth but it could also depend on the stability of
fermentation parameters (pH and pO2), which probably affect
translation and correct folding of recombinant proteins as well as proteolysis
according to Makrides [28].


## 3.2.1. Effect of the Association
Promoter/Host Strain

Using the same culture conditions (LB medium, 37°C and induction by 16μg/mL IPTG
for 18 hours), the association E. coli ER2566/pSJ8 increased the production of the
active recombinant enzyme by 3-fold (Table 1) since the ER2566 strain contains
a chromosomal IPTG inducible copy of the T7 RNA polymerase gene. Despite this
improvement, the production of the active US132 CGTase remained negligible (0.92 U/mL) compared to that obtained by the native strain (18 U/mL) [18]. To further
enhance this production, the gene was placed downstream the Trc promoter in
pSJ9 and the enzyme production was investigated in various strains (DH5α, ER2566 and JM109). As shown in Table 1, the association E. coli ER2566/pSJ9 gave the highest activity of only about 1 U/mL which is nearly the
same with that obtained by ER2566/pSJ8 strain.Table 1
Effect of the
association promoter/host strain on the production of the active recombinant
US132 CGTase.
Plasmids (promoters)pSJ8 (T7)pSJ9 (Trc)E. coli strainsDH5αER2566DH5αER2566JM109Enzyme production (U/mL)0.32±0.080.92±0.080.35±0.051.07±0.030.85±0.05This study showed that the use of different
promoters andE. coli host strains did not significantly enhance the production
of the active recombinant
CGTase, which
is probably due to the formation of inclusion bodies often reported for E.
coli heterologous expression [14, 28].


## 3.2.2. Effect of the Operating Temperature

A well-known technique
to prevent aggregation of recombinant proteins in vivo consists to reduce the
operating temperature [15, 16, 29]. Therefore, in order to improve the recombinant
US132 CGTase production, ER2566/pSJ9 and ER2566/pSJ8 strains were cultivated at
different temperatures and were induced by 16μg/mL of IPTG for 18 hours: (a)
37°C during all operating time, (b) 37°C followed by a temperature shift to
19°C after IPTG induction, and (c) 19°C during all operating time. This study
showed that the conditions (b) and (c) allowed the enhancement of the active enzyme
production in the periplasm for the two constructions pSJ8 and pSJ9 (Table 2). The
highest production (15 U/mL) was reached by E. coli ER2566/pSJ8 when the
culture was performed at 37°C followed by temperature shift to 19°C after IPTG induction.
SDS-PAGE analysis of periplasmic and insoluble fractions, obtained from E.
coli ER2566/pSJ8, showed that the cultivation of the strain at 37°C
expressed most the US132 CGTase as insoluble inclusion bodies with a small amount
of soluble protein (Figure 3) explaining the low activity detected. However,
the decrease of the operating temperature (37–19°C or 19°C)
strongly increased the expression of the US132 CGTase as periplasmic soluble
form (Figure 3). The enhancement of the production of soluble and active enzyme
at low cultivation temperature could be explained by: (i) the reduction of the
high rate of protein synthesis, which prevents misfolding [14], (ii) the enhancement of
chaperones expression, which allows
correct protein folding [30].Table 2
Effect of the operating temperature on the
production of the active US132 CGTase byE. coli ER2566/pSJ8 and
ER2566/pSJ9.
StrainsOperating temperature37°C37–19°C19°CER2566/pSJ80.93 U/mL15 U/mL13 U/mLER2566/pSJ91 U/mL4 U/mL3.6 U/mLFigure 3
Influence of the operating temperature on the production
of soluble US132 CGTase byE. coli ER2566/pSJ8 strain. Protein samples
loaded onto the 10% SDS-polyacrylamide gel were stained with Coomassie
Brilliant Blue. Lanes 1, 2, and 3: periplasmic fractions obtained from cells
cultivated at 37, 19, and 37–19°C, respectively. Lane M: low molecular weight
marker. Lanes 4, 5 and 6: insoluble fractions obtained when cells are
cultivated at 37–19, 19, and 37°C, respectively.Based on all obtained
data, we retained for further investigation the strainE. coli ER2566/pSJ8
and a culture temperature at 37°C followed by a shift to 19°C afterinduction.


## 3.2.3. Effect of Medium Composition  and Postinduction Time

The composition of growth medium could have
significant metabolic effects on both cells growth and protein production [31].
To investigate the effect of this parameter, the strain E. coli ER2566/pSJ8
was cultivated in 2TY, M9ZB, and M9 besides LB medium used in all previous
experiences. The US132 CGTase production monitored after 18 hours of induction
showed that the use of M9ZB and 2TY medium increased the production by about
1.1-fold (16.5 U/mL) and 1.3-fold (20 U/mL), respectively, in comparison to that
obtained by LB broth. However, the use of M9 medium decreased the production to
attain only 8 U/mL. The enhancement of the US132 CGTase production was likely
correlated with cell density since the OD600 nm reached about 3.7,
4.8, 6, and 2 for the LB, M9ZB, 2TY, and M9 medium, respectively (Figure 4). Moreover, this difference in the production level of
the recombinant active enzyme, using different medium, could be also influenced by the growth phase at which induction
took place on.Figure 4
Influence of the growth medium on the active
US132 CGTase production and the cell density ofE. coli ER2566/pSJ8
strain. The temperature was shifted from 37 to 19°C after induction with 16 mg/L of IPTG when OD600 nm reached 0.8–1.Otherwise, extensive attempts to improve the
enzyme production using different IPTG concentrations and stressing additives
(sorbitol, mannitol, and ethanol) did not increase the US132 CGTase production (data
not shown) as reported for other
recombinant proteins [16, 28, 32].The effect of postinduction
time on the US132 CGTase production was examined by analysis of samples taken,
from ER2566/pSJ8 culture performed in 2TY medium, at different times after IPTG
addition. Following the IPTG induction, the culture temperature is shifted from
37 to 19°C. The evolution of both cell growth and active enzyme production (Figure 5) showed that the CGTase production reached its maximum (22 U/mL) after 22 hours
of induction when cells are at full stationary phase. When culture attained the
lysis phase the production of the active enzyme decreased by about 27% probably
due to the proteases release.Figure 5
Evolution of bacterial cell growth
and the active US132 CGTase production generated byE. coli ER2566/pSJ8 cultivated in 2TY medium. Induction was performed by 16 mg/L IPTG when
culture reached OD600 nm
between 0.8 and 1. Following the
IPTG induction, the culture temperature is shifted from 37 to 19°C.


## 3.2.4. Batch Fermentation Scale

The fermentation, using the ER2566/pSJ8, was
scaled up in 7 L fermentor strain under the conditions previously optimised: 2TY
medium and temperature shift from 37 to 19°C after IPTG induction. Interestingly,
the production of the US132 CGTase in fermentor reached a maximum (35 U/mL) after
only 18 hours of induction while this maximum (22 U/mL) was obtained after 22 hours
in Erlenmeyer. It should be mentioned that the cell density obtained in the two
cases was almost the same (about OD600 nm  =  6). This finding
suggested that the improvement of the active enzyme production was not only
related to the cell growth but it could also depend on the stability of
fermentation parameters (pH and pO2), which probably affect
translation and correct folding of recombinant proteins as well as proteolysis
according to Makrides [28].


## 3.3. Purification and Kinetics Parameters Determination of the Recombinant US132 CGTase

For the purification of the
recombinant US132 CGTase, we have used the periplasmic fraction of the
ER2566/pSJ8 strain cultivated in the fermentor in the cultivation conditions
described above. This periplasmic fraction was firstly heat-treated at 60°C in
order to remove E. coli thermolabile proteins. The extract was then
purified using hydrophobic interaction chromatography (HIC) and starch
adsorption as described for the native enzyme [18]. The recombinant enzyme was
purified to homogeneity 25-fold with a yield of 20% (Table 3, Figure 6). This
recombinant US132 CGTase retained the same biochemical properties (data not
shown) as the native enzyme (thermoactivity, thermostability, pH stability, CDs
production) [18].Table 3
Purification steps of the recombinant US132
CGTase.
Purification stepTotal activity (U)Total protein (mg)Specific activity (U/mg)Yield (%)Purification (fold)Crude extract700035200100—Heat treatment665014.8450952.2HIC*43401.5528006214Starch adsorption14000.2850002025*HIC: Hydrophobic Interaction Chromatography.Figure 6
SDS-PAGE analysis of the purified
US132 CGTase from the recombinantE. coli ER2566/pSJ8 strain. Lane 1:
purified US132 CGTase, Lane 2: molecular weight marker.The kinetic parameters were
determined by incubating the purified recombinant US132 CGTase in presence of
various soluble starch concentrations and using the Lineweaver-Burk method. TheVmax and Km values were evaluated to be 253 ± 36μmol of β-cyclodextrin/mg/min and 0.36±0.18 g/L, respectively. Km
values
determined, using also soluble starch as substrate, for CGTase from Bacillus
agaradhaerens [33], Bacillus circulans E192 [34], Bacillus firmus [35], and Bacillus sp. TS1-1 [11] were 21.2, 5.7, 1.21, and
0.52 g/L,
respectively. Since a small value of Km  exhibited a high affinity for the substrate, the values shown above suggested that US132 CGTase was more specific towards starch than the other reported CGTases.


## 4. Conclusion

The molecular characterization of
the gene encoding the US132 CGTase showed that the enzyme is a novelβ-CGTase
exhibiting 95% of identity with the β-CGTase from Bacillus circulans strain no. 8.The production of the
active US132 CGTase byE. coli DH5α under usual culture conditions (37°C and LB) was very low (0.3 U/mL)
suggesting the formation of inclusion bodies. The investigation of different
culture parameters showed that mainly the shift of the operating temperature
from 37 to 19°C increased the production of the active CGTase to reach 22 U/mL.
This production was further enhanced to reach 35 U/mL by using batch
fermentation while the native strain produced only 18 U/mL.The recombinant US132
CGTase, purified to homogeneity, shared the same biochemical properties with
the native enzyme. The determination of the kinetic parameters (Vmax and Km) of this recombinant enzyme showed that the
US132 CGTase had the highest affinity towards soluble starch in comparison to
other reported CGTases.



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*Source: 692573-2008-08-10.xml*