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Muhammad Ali Akond , Yusuke Matsuda , Takayuki Ishimaru , Ken Iwai , Akira Saito ,
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The United Graduate School of Agricultural Sciences, Tottori University, Tottori, Japan
Faculty of Life Design, Yamaguchi University of Human Welfare and Culture, Hagi,
Japan
Published online: 17 Apr 2014.
To cite this article: Muhammad Ali Akond, Yusuke Matsuda, Takayuki Ishimaru, Ken Iwai, Akira Saito, Akio Kato, Shuhei
Tanaka, Jun Kobayashi & Daizo Koga (2014): Characterization of a yam class IV chitinase produced by recombinant Pichia
pastoris X-33, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.885825
To link to this article: http://dx.doi.org/10.1080/09168451.2014.885825
The United Graduate School of Agricultural Sciences, Tottori University, Tottori, Japan; 2Department of Botany,
Jahangirnagar University, Savar, Bangladesh; 3Faculty of Agriculture, Department of Biological Chemistry,
Yamaguchi University, Yamaguchi, Japan; 4Faculty of Agriculture, Department of Biological and Environmental
Sciences, Yamaguchi University, Yamaguchi, Japan; 5Faculty of Life Design, Yamaguchi University of Human
Welfare and Culture, Hagi, Japan
http://dx.doi.org/10.1080/09168451.2014.885825
A yam (Dioscorea opposita Thunb) class IV chitinase, whose genomic DNA was cloned by Mitsunaga
et al. (2004), was produced by the recombinant Pichia pastoris X-33 in high yields such as 66 mg/L of
culture medium. The chitinase was puried by column chromatography after Endoglycosidase H
treatment and then characterized. It showed properties similar to the original chitinase E puried from
the yam tuber reported by Arakane et al. (2000).
This Pichia-produced chitinase also showed strong
lytic activity against Fusarium oxysporum and Phytophthora nicotianae, wide pH and thermal stability,
optimum activity at higher temperature such as
70 C, and high substrate afnity, indicating that
one can use this Pichia-produced yam chitinase as a
bio-control agent.
Key words:
Table 1.
Primer
For intron removal
Forward primer 1
Forward primer 2
Reverse primer 1
Reverse primer 2
Insert preparation for expression vector
Forward primer 3
Reverse primer 3
Sequence
5-CTCATCAATTTCCAGCCACTC-3
5-GTCACCCATGAAACTGGACATTTATGTTACATTGAAGAAA
GAGATGGACA-3
5-CCATCTCTTTCTTCAATGTAACATAAATGTCCAGTTTCATG
GGTGACATG-3
5-TAGTCGAATTTAAGCCAAGTTC-3
5-GACCTCGAGAAAAGACAAAACTGCCAGTGCGACACC-3
5-GACGCGGCCGCCTAACAAGTGAGATCATTGCCAGG-3
Fig. 1.
Scheme for removing an intron from the genomic yam chitinase sequence.
Fig. 2.
Scheme for removing the C-terminal VTS and replacing the N-terminal signal sequence with the -factor in the cDNA of yam chitinase.
Enzyme assay.
Chitinase activity was measured
with glycolchitin as substrate. For purication, 520 L
of chitinase solution was added to 0.5 mL of 0.05%
(w/v) glycolchitin dissolved in 50 mM sodium phosphate buffer, pH 8.0, and this was incubated at 32 C
for 30 min. The reducing end group produced was measured colorimetrically at 420 nm with a ferri-ferrocyanide reagent by the method of Imoto and Yagishita.21)
Britton-Robinson buffer (pH 2.012.0)22) was also used
to determine the pH optimum and stability. The enzymatic reaction proceeded linearly with time to 20%
completion of the reaction with glycolchitin.
For kinetic analysis, the puried chitinase (nal concentration, 50 nM) was incubated with 0.0250.4 mg/
mL of glycolchitin in 50 mM sodium phosphate buffer,
pH 8.0, at 25 C. The initial velocity was calculated
from the difference in the absorbance at 420 nm
between sample and control experiments assuming a
relationship of 1 A420 = 0.22 mM N-acetylchitooligosaccharides. The kinetic parameters such as Km and
Fig. 3.
Results
Sequence analysis of the yam class IV chitinase gene
introduced into recombinant P. pastoris X-33
The yam chitinase gene carried by recombinant P.
pastoris X-33 was sequenced and compared with the
complete nucleotide sequence of the class IV yam chitinase by homology alignment. The results indicated precise deletion of both intron and VTS and in-frame
fusion of the -factor instead of the signal sequence in
recombinant Pichia carrying yam chitinase gene.
(A)
(B)
centrifugation, showed ve main active bands by activity staining after SDS-PAGE with molecular masses of
about 58, 48, 38, 32, and 28 kDa (Fig. 4(A)). After
deglycosylation by treatment with Endo H, the pattern
of the chitinase active band shifted to smaller molecular
masses, at 32 and 28 kDa. In order to obtain 32 kDachitinase, which is similar in molecular size to yam
chitinase E (33.5 kDa), indicating its possible application as a bio-control agent,15,16) the following course of
purication was applied. The deglycosylated crude
enzyme after dialysis against 10 mM sodium phosphate
buffer, pH 8.0, was applied sequentially to DEAEToyopearl 650(M), Fractogel EMD DEAE 650(M),
DEAE-Toyopearl 650(M), Sephacryl S-100, and
DEAE-Toyopearl 650(M) resins. The active peaks of
the rst chromatographic separation on DEAE-Toyopearl 650(M) were obtained at sodium chloride conductivities between 7.5 and 18 mS (Fig. 5(A)). The active
fractions, indicated by the horizontal bar, were combined and applied to Fractogel EMD DEAE 650(M),
and the active fractions were obtained between 15 and
27 mS of sodium chloride conductivity. These were
combined and applied again to DEAE-Toyopearl 650
(M), followed by Sephacryl S-100 (Fig. 5(B)). The
active fractions of small molecular sizes of about 32
kDa were pooled by gel ltration chromatography and
eluted again by DEAE-Toyopearl 650(M) with a gradient of 00.3 M sodium chloride (Fig. 5(C)). Purity analysis by both SDS-PAGE and native PAGE showed a
single homogeneous band with a molecular mass of 32
kDa (Fig. 6). The quantitative results for purication
steps are presented in Table 2. A 3% yield with 10-fold
overall purication was achieved at the nal step with
a specic activity of 374 A420/h/mg.
N-Terminal amino acid sequencing
Automated Edman degradation of the chitinase produced by recombinant P. pastoris was unsuccessful.
When digested with pyroglutamate amino peptidase,
the N-terminals were read to be SYD, the same as the
amino acid sequence of the introduced yam chitinase
from the 21st. This result was obtained repeatedly, suggesting that appearance of the SYD sequence from the
21st amino acid is due to the action of peptidase contaminating in this pyroglutamate amino peptidase.
(A)
(B)
Step
Crude extract
Ammonium sulfate precipitation
DEAE-Toyopearl 650(M) (1)
Fractogel EMD DEAE 650(M)
DEAE-Toyopearl 650(M) (2)
Sephacryl S-100
DEAE-Toyopearl 650(M) (3)
Total activity
(A420/h)
Protein (mg)
Specic activity
(A420/h/mg)
Overall yield
(%)
Overall
purication
34,800
34,200
12,300
9070
6610
5680
1010
915
760
89.0
40.3
25.4
19.3
2.71
38.0
45.0
138
225
260
294
374
100
98.4
35.3
26.1
19.0
16.3
2.92
1.00
1.18
3.63
5.92
6.84
7.74
9.84
(A)
Discussion
As shown in Fig. 3, a family 19 Class IV acidic yam
chitinase without VTS was produced increasingly with
time by recombinant P. pastoris X-33. The results of
SDS-PAGE for Pichia-produced chitinase in 5-d culture
medium, however, showed several chitinase active
bands of various molecular sizes from 28 to 58 kDa
(Fig. 4). On the other hand, the molecular weight of
(B)
Fig. 8. Temperature optimum and thermal stability of the yam chitinase puried from recombinant P. pastoris X-33.
Notes: Panels: (A) The optimum temperature of the puried chitinase was measured by incubation of 50 nM chitinase with 0.05% glycolchitin in Britton-Robinson buffer, pH 8.0, for 560 min at
temperatures of 1080 C; (B) the thermal stability of the puried chitinase was determined by measuring the remaining activity after incubation of 50 nM chitinase in Britton-Robinson buffer, pH 8.0, for 15
min at temperature range of 1080 C.
Enzyme used
Pichia-produced yam chitinase
Yam chitinase E15)
Table 4.
kcat (1/s)
Km (mg/mL)
kcat/Km (mL/mg/s)
50
50
0.0250.400
0.0250.400
0.200
0.645
0.233
0.518
0.858
1.25
Parameters
Molecular weight
Optimum pH toward glycol chitin
Optimum temperature toward glycol chitin
pH stability
Thermal stability
Drying stability
Kinetics:
For glycol chitin
Km (mg/mL)
kcat (1/s)
kcat/Km (mL/mg/s)
Lytic activity
Fusarium oxysporum
Phytophthora nicotianae
Action mechanism against powdery mildew
Glycosyl hydrolase family
Yam chitinase E
32.0 kDa
5, 8
70 C
312
70 C
68.4%
33.5 kDa
4.0, 7.5
70 C
511
70 C
ND
0.233
0.200
0.858
167 105
229 105
ND
19
0.518
0.645
1.25
104 105
ND
Break-down
19
Supplemental material
The supplemental material for this paper is available
at http://dx.doi.org/10.1080/09168451.2014.885825.
Acknowledgment
We are thankful to Mr Minoru Iwase for his generous help in constructing the cDNA of chitinase by
deletion of the intron.
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