Download Identification and expression of GH

RESEARCH LETTER
Identi¢cation and expression of GH-8 family chitosanases from
several Bacillus thuringiensis subspecies
Han-Seung Lee1,2, Jun Sung Jang1, Soo-Keun Choi1,3, Dong-Woo Lee4, Eui-Joong Kim1,
Heung-Chae Jung1,3 & Jae-Gu Pan1,3
1
National Research Laboratory of Microbial Display, GenoFocus Inc., Daejeon, Korea; 2Department of Bio-Food Materials, Silla University, Busan, Korea;
Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea; and 4Department of Biology,
University of Pennsylvania, Philadelphia, PA, USA
3
Correspondence: Jae-Gu Pan, National
Research Laboratory of Microbial Display,
GenoFocus Inc., Daejeon 305-811, Korea.
Tel.: 182 42 862 4483; fax: 182 42 862
4484; e-mail: [email protected]
Received 9 June 2007; accepted
24 August 2007.
First published online November 2007.
DOI:10.1111/j.1574-6968.2007.00944.x
Editor: Andre Klier
Keywords
chitosanase; Bacillus thuringiensis ; highthroughput screening; chitosanoligosaccharides; GH-8.
Abstract
The Gram-positive spore-forming bacterium, Bacillus thuringiensis, a member of the
Bacillus cereus group, produces chitosanases that catalyze the hydrolysis of chitosan
to chitosan-oligosaccharides (COS). Although fungal and bacterial chitosanases
belonging to other glycoside hydrolase (GH) families have been characterized in a
variety of microorganisms, knowledge on the genetics and phylogeny of the GH-8
chitosanases remains limited. Nine genes encoding chitosanases were cloned from
29 different serovar strains of B. thuringiensis and they were expressed in Escherichia
coli. The ORFs of the chitosanases contained 1359 nucleotides and the protein
products had high levels of sequence identity (496%) to other Bacillus species GH-8
chitosanases. Thin-layer chromatography and HPLC analyses demonstrated that
these enzymes hydrolyzed chitosan to a chitosan-trimer and a chitosan-tetramer as
major products, and this could be useful in the production of COS. In addition, a
simple plate assay was developed, involving a soluble chitosan, for high-throughput
screening of chitosanases. This system allowed screening for mutant enzymes with
higher enzyme activity generated by error-prone PCR, indicating that it can be used
for directed chitosanase evolution.
Introduction
Chitosanases (EC 3.2.1.132) are glycosyl hydrolases that
catalyze the hydrolysis of the b-1,4-glycosidic linkage of
chitosan (a linear biopolymer of b-1,4-linked glucosamine,
GlcN) to yield chitosan-oligosaccharides (COS). The oligomers produced by the enzymatic hydrolysis of chitosan are
very attractive for use in food, and in agricultural and
pharmaceutical applications, because of their various biological activities, including antitumor (Harish Prashanth &
Tharanathan, 2005) and antibacterial effects (Tsai et al.,
2004; Moon et al., 2007). Although acid-based chemical
hydrolysis of chitosan is possible, it produces unfavorable
byproducts and short-chain oligosaccharides (Rupley, 1964;
Su et al., 2006). Hence, the production of COS using
glycolytic enzymes is more efficient and desirable. Accordingly, chitosanases have been isolated from bacteria (Choi
et al., 2004; Cruz Camarillo et al., 2004; Yun et al., 2005;
Su et al., 2006), fungi (Nogawa et al., 1998; Cheng & Li, 2000;
FEMS Microbiol Lett 277 (2007) 133–141
Zhang et al., 2000) and plants (Osswald et al., 1994), with a
view to using them in the production of COS.
Chitosanases have been classified into five glycoside
hydrolase (GH) families i.e., GH-5, GH-8, GH-46, GH-75,
and GH-80, based on the amino acid sequence similarity of
their catalytic domains. The GH-75 chitosanases are mainly
fungal enzymes from Aspergillus fumigatus (Cheng & Li,
2000) and Aspergillus oryzae (Zhang et al., 2000), with a few
from Streptomyces (GenBank accession numbers
AM238663, AP005026, AP005028, and AL391039); the
chitosanases from bacteria such as Streptomyces sp. strain
N174 (Masson et al., 1994) and Bacillus circulans MH-K1
(Saito et al., 1999) mainly belong to the GH-46 family while
those from Bacillus sp. strain K17 (Yatsunami et al., 2002),
Bacillus sp. strain KCTC 0377BP (Choi et al., 2004), and
Bacillus sp. strain S65 (Su et al., 2006) are GH-8 enzymes. A
few bacterial chitosanases from Mitsuaria chitosanitabida
(Park et al., 1999; Yun et al., 2005) and Sphingobacterium
multivorum (Matsuda et al., 2001) have also been classified
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134
H.-S. Lee et al.
as GH-80 enzymes. A variety of glycoside hydrolases including chitosanases, cellulases, xylanases, and licheninases are
classified as GH-5 and GH-8 enzymes. On the other hand,
the GH-46, GH-75, and GH-80 glycosyl enzymes are exclusively chitosanases. To date, the three dimensional (3D)
structures of GH-46 chitosanases from Streptomyces sp.
strain N174 (Marcotte et al., 1996), B. circulans MH-K1
(Saito et al., 1999), and GH-8 from Bacillus sp. K17 (Adachi
et al., 2004) have been determined. The catalytic residues of
these chitosanases are Glu and Asp. However, GH-5 and
GH-75 chitosanases from Streptomyces griseus HUT6037
(Tanabe et al., 2003) and M. chitosanitabida (Yun et al.,
2005) are involved in a retaining mechanism in which one of
the two essential residues functions as a nucleophile and the
other as a general acid/base. Knowledge of the 3D structures
and amino acid sequences of chitosanases has led to the idea
that most microbial chitosanases are GH-46 enzymes whose
active sites are closely related to those of GH-80 enzymes.
However, the different substrate specificities and relatively
low levels of sequence similarity between GH-5 and GH-8
chitosanases indicate that these microbial enzymes are
highly diverse. Although a few bacterial GH-8 chitosanases
have been purified and characterized (Adachi et al., 2004;
Choi et al., 2004; Su et al., 2006), in general they remain
poorly characterized.
For these and related reasons, a variety of chitosanase
genes have been isolated from different Bacillus thuringiensis
strains (Shin et al., 1995), and their amino acid sequences
have been compared with other known microbial enzymes.
A very efficient and simple-to-use plate assay with a specific
soluble chitosan has also been developed to assess chitosanase activities linked to the growth rate of cells. The activity
of enzymes mutated by error-prone PCR has also been
evaluated using the plate assay to test whether this method
could enable one to direct the evolution of chitosanase.
Materials and methods
Bacterial strains and culture conditions
The 29 different serovar types of B. thuringiensis strains used
in this study are listed in Table 1 (Shin et al., 1995). To test
whether they exhibit chitosanase activity, they were grown
Table 1. Relevant properties of Bacillus thuringiensis strains
Chitosanase activity
Strain
Abbreviation
LB
GYS
PCR products
Bacillus thuringiensis var. aizawai
Bacillus thuringiensis var. alesti
Bacillus thuringiensis var. berliner
Bacillus thuringiensis var. canadensis
Bacillus thuringiensis var. colmeri
Bacillus thuringiensis var. dakota
Bacillus thuringiensis var. darmstadiensis
Bacillus thuringiensis var. entomocidus
Bacillus thuringiensis var. entomocidus/subtoxicus
Bacillus thuringiensis var. finitimus
Bacillus thuringiensis var. galleriae
Bacillus thuringiensis var. indiana
Bacillus thuringiensis var. israelensis
Bacillus thuringiensis var. kenyae
Bacillus thuringiensis var. kumamotoensis
Bacillus thuringiensis var. kurstaki
Bacillus thuringiensis var. kyushuensis
Bacillus thuringiensis var. morrisoni
Bacillus thuringiensis var. pakistani
Bacillus thuringiensis var. ostriniae
Bacillus thuringiensis var. san diego
Bacillus thuringiensis var. sotto
Bacillus thuringiensis var. sotto/dendrolimus
Bacillus thuringiensis var. thompsoni
Bacillus thuringiensis var. thuringiensis
Bacillus thuringiensis var. tochigiensis
Bacillus thuringiensis var. tohokuensis
Bacillus thuringiensis var. tolworthi
Bacillus thuringiensis var. toumanoffi
BTAI
BTAL
BTBE
BTCA
BTCO
BTDA
BTDM
BTEN
BTES
BTFI
BTGA
BTIN
BTIS
BTKE
BTKU
BTKS
BTKY
BTMO
BTPA
BTOS
BTSA
BTSO
BTSD
BTTH
BTTR
BTTO
BTTH
BTTW
BTTM
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
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FEMS Microbiol Lett 277 (2007) 133–141
135
Bacillus thuringensis chitosanase
on solid Luria–Bertani (LB) and glucose–yeast–salts (GYS)
[0.1% glucose, 0.2% yeast extract, 0.05% K2HPO4, 0.2%
(NH4)2SO4, 0.002% MgSO4, 0.005% MnSO4, 0.008%
CaCl2] media supplemented with 0.2–0.5% (w/v) soluble
chitosan (EZ Life Science Co. Ltd, Seoul, Korea) and 1.5%
(w/v) agar (Junsei chemicals, Tokyo, Japan).
Cloning of chitosanase genes
Based on the sequences of five GH-8 chitosanase genes from
Bacillus sp. (GenBank accession numbers AF334682,
AB051575, AB006819, M68872 and X52880), two degenerate primers, forward primer NEST-F (5 0 -GCNACA
GATGGRGATNTNGAYATTGC-3 0 ) and reverse primer
NEST-R (5 0 -GCAATRTCNANATCYCCATCNGTNGC-3 0 ),
were designed for nested PCR (Fig. 1). First, the genomic
DNA of B. thuringiensis serovar morrisoni (BTMO), which
showed the highest chitosanase activity on LB medium, was
extracted with a Wizards Genomic DNA purification kit
(Promega, WI). It was partially digested with Sau3AI to
yield DNA fragments of 1–10 kb that were ligated into the
BamHI site of pUC19 vector treated with calf intestinal
alkaline phosphatase (Takara, Japan); the resulting genomic
library was introduced into Escherichia coli JM109 [F 0 traD36
proA1B1 lacIq D(lacZ)M15/ D(lacproAB) glnV44 e14
gyrA96 recA1 relA1 endA1 thi hsdR17].
Next, nested PCR was performed using pairs of either
NEST-F and M13 reverse primers or NEST-R and M13
forward primers to amplify DNA fragments containing
partial chitosanase genes. For the PCR reactions, a Bioneer
AccuPower PCR Premix kit was used (Bioneer, Inc.,
Daejeon, Korea). The thermal cycler parameters were as
follows: 35 cycles of 30 s at 94 1C, 30 s at 45 1C, and 2 min at
72 1C. The PCR products were sequenced, and based on
these sequences, two additional primers, BTMO-F (5 0 -GA
ACTGCAGATGAATGGAAAAAGAAAT-3 0 ) and BTMO-R
(5 0 -CCCGGTACCTTAATTATCGTATCCTTCATAAAT-3 0 ),
were synthesized to amplify DNA fragments corresponding
to the complete chitosanase-coding sequences. Each reaction
mixture (50 mL) contained 1.5 mM MgCl2, 0.25 mM of
each dNTP, and 2.5 U of Bioneer AccuPower Taq DNA polymerase. PCR was carried out for 30 cycles consisting of 30 s
of denaturation at 94 1C, 45 s of annealing at 55 1C, and 90 s
of extension at 72 1C. The PCR products were sequenced.
Fig. 1. Alignment of the amino acid sequences of nine Bacillus thuringiensis chitosanases and other microbial chitosanases. Residues of GH-8
chitosanases involved in catalysis and substrate recognition are indicated in boldface type. The amino acid sequence used to prepare degenerate primers
(NEST-F and NEST-R) for nested PCR is boxed. GenBank accession number AE016877, Bacillus cereus ATCC 14579; NC_005945, Bacillus anthracis str.
Sterne; AF334682, Bacillus sp. KCTC 0377BP.
FEMS Microbiol Lett 277 (2007) 133–141
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136
H.-S. Lee et al.
Cloning and expression of chitosanases in E. coli
Assay of enzyme activity
On the basis of the DNA sequences obtained above, primers
BTC-F (5 0 -GGAATTCCATAT-GAATGGAAAAAGAAAT-3 0 )
and BTC-R (50 -AACTGCAGTTAATTATCGTATCCTTCAT-30 ),
incorporating NdeI and PstI sites (underlined), were
used to amplify the complete chitosanase sequences, which
were cloned into the auto-inducible pACE vector (GenoFocus
Inc, Daejeon, Korea) digested with NdeI and PstI to yield
pACE-BTC. These plasmids were transformed into E. coli
JM109 or Top10. For expression, transformants were
grown overnight at 37 1C in LB medium. To check the
expression levels of the chitosanases, a BTC-RH primer
(5 0 -AACTGCAGTTAATGATGATGATGATGATGATTATCGT
ATCCTTCAT-3 0 ) was also designed containing a 6 His tag.
Chitosanase activity was determined by measuring the
amount of reducing sugar product using the dinitrosalicylic
acid method as described previously (Miller, 1959; Lee et al.,
2000). The standard reaction mixture (300 mL) contained
100 mL of 2% (w/v) soluble chitosan, 100 mL of 100 mM
sodium phosphate buffer (pH 6.0), and 100 mL of enzyme
preparation (periplasmic fractions of E. coli expressing
B. thuringiensis chitosanase) at a suitable dilution, and was
incubated at 50 1C for 1 h. After this, 0.9 mL of a dinitrosalicylic acid reagent was added, and the mixture was boiled
for 10 min, chilled on ice, and centrifuged at 10 000 g for
10 min. The absorbance of the resulting supernatant at
540 nm was determined. One unit of chitosanase activity
was defined as the amount of enzyme that produced
1 mmol reducing sugar min1 under the assay conditions.
Selective screening of the engineered
chitosanases in E. coli
Error-prone PCR (error rate = 6 bp kb1) (Cadwell & Joyce,
1992) was performed of the chitosanase gene from
B. thuringiensis serovar israelensis (BTIS), as an example, to
introduce random mutations throughout the gene using a
Diversify PCR Random Mutagenesis Kit (Clontech Laboratories Inc., CA). The error-prone PCR mixture (50 mL)
contained the chitosanase gene as a template (50 ng),
primers (10 pmol each) BTC-F and BTC-R, 0.2 mM of each
dNTP, 0.64 mM MnSO4, and 1 mL of 50 TITANIUMTM
Taq DNA polymerase. PCR was performed at 94 1C for 30 s,
with 25 cycles of 94 1C for 30 s, and 68 1C for 1 min, and a
final extension of 68 1C for 1 min. The randomized PCR
products (1.4 kb) were cloned into pACE after double
digestion of both vector and insert with NdeI and PstI. The
resulting plasmid library was transformed into E. coli JM109
and E. coli Top10-competent cells by electroporation.
The transformants were plated on LB medium containing
0.3% (w/v) soluble chitosan. Each transformant colony was
inoculated into three consecutive wells of a 96-deep-well
plate containing 1 mL of LB medium per well. The wild-type
clones were also inoculated into nine wells each at three
different locations. In addition, E. coli JM109 harboring the
pACE vector was inoculated into three wells as a negative
control. The plates were incubated overnight at 30 1C. Each
culture (200 mL) was then transferred to a new 96-well plate
containing 800 mL of 2% soluble chitosan in 20 mM sodium
phosphate buffer (pH 7.0) and incubated at 50 1C for 3 h.
The plates were centrifuged at 6000 g for 15 min, and the
resulting supernatants (50 mL) were transferred to a 96-well
plate, each well of which contained 150 mL of dinitrosalicylic
acid solution. After a 5-min incubation at 95 1C, the samples
were transferred to another 96-well plate and the absorbance
of each sample at 640 nm was determined using a plate
reader. A QUADRA3 liquid handler (Tomtec, CT) was used
for transferring all liquid samples.
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Analysis of chitosan hydrolysates
Thin-layer chromatography (TLC) of chitosan hydrolysates
was performed in n-propanol–30% ammonia (2/1, v/v) by
the ascending technique using 0.2-mm silica gel-coated
aluminum plates (Kiesel gel 60 F254; Merck, NJ). The plates
were sprayed with 10% H2SO4 in ethanol and heated to
visualize the carbohydrate spots.
HPLC was carried out with an Asahipak NH2P-50 4E
column (Shodex, Kawasaki, Japan) equipped with an RI
detector. COS were separated by isocratic elution in 70%
acetonitrile at a flow rate of 1 mL min1. COS (n = 2–6)
standards were purchased from Seikagaku Co. (Tokyo,
Japan).
Nucleotide sequence accession numbers
The GenBank/EMBL accession numbers of the chitosanase
genes from B. thuringiensis strains reported here are as
follows: BTAL, EF143917; BTSA, EF143918; BTTH,
EF143919; BTMO, EF143920; BTDM, EF143921; BTIS,
EF143922; BTSO, EF143923; BTCA; EF143924; and BTTO,
EF143925.
Results
Screening strains of B. thuringiensis for
chitosanase production
As shown in Table 1, 29 B. thuringiensis strains were
collected. To test whether the strains had chitosanase
activity, they were grown on two solid media (LB and GYS)
supplemented with 0.2% (w/v) soluble chitosan. Eight
strains formed clear halos on both media and five more
strains exhibited medium-dependent halo formation (Table 1).
Nevertheless, all 29 strains were screened by PCR because the
possibility could not be excluded that they possessed
FEMS Microbiol Lett 277 (2007) 133–141
137
Bacillus thuringensis chitosanase
chitosanase genes that gave only low levels of expression (or
induction), or harbored different types of iso-enzymes.
Identification of the chitosanase genes in
B. thuringiensis strains
Using degenerate primers (NEST-F and NEST-R) based on
the conserved region of five GH-8 chitosanase genes from
Bacillus species (Fig. 1), nested PCR was performed with a
chromosomal DNA library of strain BTMO, which had the
highest chitosanase activity in the plate assays (see ‘Materials
and methods’). The sequence of the PCR product revealed a
putative ORF sequence (1359 bp) with 95% identity to the
ORFs of Bacillus sp. No. 7-M (Izume et al., 1992) and KCTC
0377BP (Choi et al., 2004), which are classed as GH-8
chitosanases. Based on this result, two further primers
(BTMO-F and BTMO-R) were designed and PCR reactions
were performed with the chromosomal DNAs of all 29
B. thuringiensis strains. As summarized in Table 1, PCR
products were obtained from each of the strains that had
chitosanase activity on either LB or GYS medium, except for
BTIN and BTPA. Interestingly, chitosanase genes were also
amplified from BTDM, BTEN, BTSA, BTSO, and BTTO,
which failed to exhibit chitosanase activity on either LB or
GYS medium. These discrepancies between phenotype and
genotype suggest that the levels of expression of chitosanases
depend on nutritional conditions or that there are different
types of glycosidases.
As shown in Fig. 1, the chitosanase genes cloned from the
fifteen B. thuringiensis strains consist of 1359 bp and encode
proteins of 453 amino acids. Their putative amino acid
sequences are very similar (495.6% identity), and are also
virtually identical (496% identity) to the GH-8 chitosanases of Bacillus sp. KCTC 0377BP (Choi et al., 2004),
Bacillus cereus (Han et al., 2006), and Bacillus anthracis. In
addition, amino acid sequence alignment showed that their
putative catalytic residues, Glu122 and Glu309 (proton
donor and an activator of water molecule, respectively), are
conserved in GH-8 chitosanases, but not in any other
GH families. To compare the evolutionary relationships
among the glycosyl hydrolases of the various GH families, a
phylogenetic tree was constructed. Figure 2 shows that the
chitosanases of B. thuringiensis as well as those of B. cereus,
B. anthracis, and Bacillus sp. strains KCTC 0377BP and No.
7-M form a large GH-8 group distinct from the chitosanases
of the other GH families. Therefore, it was concluded that all
the B. thuringiensis strain chitosanases in this study were
GH-8 enzymes.
Expression of B. thuringiensis chitosanases in
E. coli
To express the B. thuringiensis chitosanases in E. coli, and to
facilitate subsequent Western blot analysis, the chitosanase
FEMS Microbiol Lett 277 (2007) 133–141
Fig. 2. Phylogenetic tree of Bacillus thuringiensis and other microbial
chitosanases. Bootstrap values are indicated at the branch points. The
bar indicates a branch length equivalent to 0.2 changes per amino acid.
Amino acid sequences were aligned with CLUSTAL X software, version 1.81.
Phylogenetic trees were constructed by the neighbor-joining method
(Saitou & Nei, 1987), using MEGA software, version 3.0 (Kumar et al.,
2004). The p-distance correction substitution model was used in the
tree-building analysis. Bootstrap values were calculated based on 100
replicates of the data (Felsenstein, 1996). All sequences were obtained
from GenBank (see also Fig. 1). GenBank accession number NC_000964,
Bacillus subtilis; L07779, Streptomyces sp. N174; D10624, Bacillus
circulans MH-K1; AY190324, Aspergillus fumigatus; D85388, Fusarium
solani; AB010493, Mitsuaria chitosanitabida.
genes were amplified by PCR from the genomic DNAs of the
B. thuringiensis strains and cloned into the autoinducible
pACE expression vector to yield pACE-BTCs. To determine
whether the B. thuringiensis chitosanases were expressed in
E. coli, Western blotting of the E. coli extracts was performed
with 6 His tag antibody. All the expected recombinant
B. thuringiensis chitosanases that showed halo formation on
LB medium were detected, and their apparent molecular
weights were estimated to be 46 000, consistent with the
molecular weight (47 911) calculated from their presumptive amino acid sequences. It was also confirmed that the
chitosanases had chitosan-degrading activity using the solid
plate assay (Fig. 3). The halo-forming activities of the E. coli
recombinants were consistent with the expression seen by
Western blotting. For example, the BTTO chitosanase that
had no activity in the plate assay was also not detected by
immunoblotting (data not shown). Thus, those chitosanase
genes that were expressed in E. coli were expressed without
induction.
Chitosanases of the GH-8 family can hydrolyze carboxymethylcellulose as well as chitosan (Pedraza-Reyes &
Gutierrez-Corona, 1997; Mitsutomi et al., 1998). Nine of the
B. thuringiensis chitosanases expressed in E. coli were tested
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138
H.-S. Lee et al.
Fig. 3. Assay of Bacillus thuringiensis chitosanase
expressed in Escherichia coli JM109 on solid medium with different concentrations of carboxymethyl cellulose (a) and soluble chitosan (b).
Escherichia coli JM109 harboring the pACE vector
was used as a negative control.
for activity for carboxymethylcellulose as well as chitosan.
As shown in Fig. 3, all the E. coli strains expressing
B. thuringiensis chitosanases, except BTCA, gave clear halos
on both media, indicating that the chitosanases are bifunctional, with activity as chitosanases and cellulases, as expected for the GH-8 family enzyme. On the other hand,
although BTCA had chitosanase activity on LB and GYS
media, E. coli harboring pACE-BTCA had no activity for
chitosan or carboxymethylcellulose. This might be due to
lack of expression in E. coli.
Fig. 4b, HPLC analysis demonstrated that the main products
of chitosan hydrolysis by purified BTSO chitosanase were
chitosan-trimer (37.5%) and chitosan-tetramer (33.2%),
but no monomers were formed. These results suggest that
the B. thuringiensis chitosanase is an endo-b-D-glucosaminidase. It could therefore be more useful for the production
of COS than the GH-46 chitosanase from B. subtilis, which
yields chitosan-dimer and chitosan-trimer as the main end
products (Chianga et al., 2003).
Screening of chitosanase-positive clones
Analysis of the reaction products
To identify the products generated by recombinant
B. thuringiensis chitosanases with chitosan as a substrate, TLC
was used (Fig. 4a). After a 15-min incubation of 2% (w/v)
soluble chitosan with the periplasmic fractions of E. coli
JM109 cells expressing B. thuringiensis chitosanases at 50 1C,
soluble chitosan was hydrolyzed mainly into chitosan-tetramer and chitosan-pentamer. After 1 h of incubation, the
main products were chitosan-timer and chitosan-tetramer.
In fact, the patterns of hydrolysis of chitosan by all the
B. thuringiensis chitosanases were very similar. Therefore the
BTSO chitosanase, which had the highest activity in E. coli
was selected, to purify further using Ni21 affinity chromatography in order to analyze the hydrolysates in detail by
HPLC. The purified recombinant enzyme showed maximal
activity at 60 1C and pH 7.0 (data not shown). As shown in
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To test whether the plate assay using soluble chitosan can be
used for high-throughput screening to perform directed
evolution, error-prone PCR was carried out preliminarily
to obtain variants of the B. thuringiensis chitosanases. It was
first required to optimize the concentration of soluble
chitosan because chitosan has antibacterial activity as well
as low solubility, which could interfere with high-throughput selective screening. In fact, the soluble chitosan that
has been used in this study is more suitable for turbidity
assays on solid plates than the chitosans from other
commercial companies, because of its relatively high solubility in an aqueous solution and suitable molecular mass
of 20–100 kDa. Various concentrations of soluble chitosan
(0.2–0.5%, w/v) were added to LB medium to detect
any inhibition of the growth of E. coli cells expressing
B. thuringiensis chitosanases (Fig. 3). Apart from BTCA,
FEMS Microbiol Lett 277 (2007) 133–141
139
Bacillus thuringensis chitosanase
Discussion
Fig. 4. Analysis of enzyme hydrolysates. COS produced by Bacillus
thuringiensis chitosanase were analyzed by TLC (a) and HPLC (b).
Periplasmic fractions of Escherichia coli cells expressing B. thuringiensis
chitosanases were used for the TLC analysis, and the purified BTSO
chitosanase was used for HPLC analysis. Analyses were performed as
described in the ‘Materials and methods’.
which did not form any halo due to lack of expression, all
the other eight chitosanase-expressing E. coli cells grew on
0.3% soluble chitosan. However, cells expressing BTAL and
BTIS did not grow on 0.5% chitosan–LB medium due to
either low levels of expression or inhibitory effects, implying
that such levels of chitosanase expression are not enough to
sustain cell growth. Based on these experiments with the
wild-type enzymes, 0.3% soluble chitosan was chosen in LB
medium as the basic system for screening chitosanases for
enzyme activity.
A mutant library of BTIS was generated by error-prone
PCR and transformed into two E. coli strains: JM109 and
Top10. Mutants with altered activity on 0.3% chitosan–LB
medium were detected as described above (see ‘Materials
and methods’). As a result, more than 60% of the clones had
higher activity than the wild-type enzyme but a few clones
had lower activity (data not shown). The plate assay with
soluble chitosan is thus very effective for screening chitosanase clones with altered hydrolytic activity.
FEMS Microbiol Lett 277 (2007) 133–141
In this study, nine genes encoding chitosanase from different
serovar type strains of B. thuringiensis were cloned and they
were expressed in E. coli. In addition, they were characterized with respect to their sequences and reaction products,
and it was shown that they are endo-b-1,4-glycoside hydrolases belonging to the GH-8 family. Until now, only a few
bacterial GH-8 chitosanases have been isolated, mainly from
the genus Bacillus, e.g. Bacillus sp. S65 (Su et al., 2006),
Bacillus sp. KCTC 0377BP (Choi et al., 2004), and Bacillus
sp. K17 (Yatsunami et al., 2002). Compared with the enzyme
of Bacillus sp. K17, whose 3D structure has been solved
(Adachi et al., 2004), all the chitosanases from B. thuringiensis
strains have very similar characteristics with respect to
sequence. The amino acid sequence data revealed that they
are very similar to the GH-8 chitosanase of Bacillus species.
Besides the conserved Glu122 and Glu309 residues thought
to be the catalytic residues, the four acidic residues (Asp179,
Glu309, Asp183, and Glu107) involved in substrate recognition and the hydrophobic residues (Trp235, Trp166, Phe413,
and Tyr318) that bind the hexose ring are highly conserved
(Fig. 1). A phylogenetic tree also clearly showed that the
chitosanases from B. thuringiensis form a distinct cluster
together with those of the B. cereus family as well as the GH8 chitosanases from Bacillus strains reported so far (Fig. 2).
Recently, the genome sequences of three B. anthracis strains,
three B. cereus strains and one B. thuringiensis strain
(B. thuringiensis serovar konkukian str. 97-27) have been
deposited in genome databases. These strains are members
of the B. cereus group and are closely related. However, there
seems to be no chitosanase gene in the genome of
B. thuringiensis serovar konkukian str. 97-27. Nevertheless,
there is a chitosanase gene (RBTH_01383) in the published
partial genome sequence of B. thuringiensis serovar
israelensis, and its deduced amino acid sequence is identical
to that of BTIS. Thirteen chitosanases could be isolated from
B. thuringiensis strains and it is proposed that B. thuringiensis,
as a member of the B. cereus family, be considered a
representative microorganism for GH-8 chitosanase. This is
also supported by analysis of hydrolysates of soluble chitosan as shown in Fig. 4 (Choi et al., 2004; Su et al., 2006).
A number of B. thuringiensis strains have been used as
biopesticides, and their fermentation products, levels of
protein production, and procedures for genetic manipulation are well established for application on an industrial
scale. In addition, the B. thuringiensis chitosanases could be
very useful for the production of COS for industrial
applications, because the B. thuringiensis GH-8 chitosanase
are more suitable for high-level chitosan-oligosaccharide
production than the GH-46 chitosanases from B. circulans
MH-K1 (Fukamizo et al., 2005), B. subtilis (Chianga et al.,
2003), and Streptomyces strains (Fukamizo et al., 1995) that
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140
produce significant amounts of chitosan-dimer as their
main product. Nevertheless, in order to use B. thuringiensis
chitosanases in practical applications, several of their physiological properties (i.e. acidic stability, higher specific
activity, etc.) need to be improved. For this purpose,
directed evolution is one of the best strategies. However,
protein engineering of chitosanases by directed evolution is
not straightforward because of limitations in the use of
soluble chitosan as a substrate. Chitosan does not dissolve at
neutral pHs and can form a precipitate with agar, so that it is
very difficult to make a solid medium of uniform turbidity
for screening purposes. However, the soluble chitosan used
in this study seems to lack these undesirable properties and
enabled preparation of solid media with (40.3%, w/v)
chitosan, when a particular agar was also used. After many
attempts, a practicable chitosan plate assay could be developed that allowed identification of improved chitosanases
from a mutant pool generated by error-prone PCR. This
approach could be used to obtain enzymes with acidophilic
properties, which favor the hydrolysis of chitosan to COS.
The authors are currently characterizing a number of
mutant chitosanases.
Acknowledgements
The authors thank Julian Gross for helpful discussions and
editing the manuscript.
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