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Characterization of a yam class IV chitinase produced


by recombinant Pichia pastoris X-33
ab

ce

Muhammad Ali Akond , Yusuke Matsuda , Takayuki Ishimaru , Ken Iwai , Akira Saito ,
c

ad

Akio Kato , Shuhei Tanaka , Jun Kobayashi

ad

ace

& Daizo Koga

The United Graduate School of Agricultural Sciences, Tottori University, Tottori, Japan

Department of Botany, Jahangirnagar University, Savar, Bangladesh

Faculty of Agriculture, Department of Biological Chemistry, Yamaguchi University,


Yamaguchi, Japan
d

Faculty of Agriculture, Department of Biological and Environmental Sciences,


Yamaguchi University, Yamaguchi, Japan
e

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

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Bioscience, Biotechnology, and Biochemistry, 2014

Characterization of a yam class IV chitinase produced by recombinant Pichia


pastoris X-33
Muhammad Ali Akond1,2, Yusuke Matsuda3, Takayuki Ishimaru3, Ken Iwai3,5, Akira Saito3,
Akio Kato3, Shuhei Tanaka1,4, Jun Kobayashi1,4 and Daizo Koga1,3,5,*
1

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

Received September 6, 2013; accepted November 8, 2013

Downloaded by [Muhammad AKOND] at 06:16 28 April 2014

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:

yam (Dioscorea oppsita Thunb); class


IV chitinase; bio-control agent; recombinant Pichia pastoris; lytic activiy

Chitinases [EC 3.2.1.14] are diversied in biological


functions depending on the source. Chitinases, which
hydrolyze chitin, occur in a wide range of organisms,
including viruses, bacteria, fungi, insects, higher plants,
and animals.1) Bacterial chitinases play roles in nutrition and parasitism, while fungal chitinases have autolytic, nutritional, and morphogenetic roles. Viral
chitinases are involved in pathogenesis.2) Invertebrate
chitinases play roles in digestion. In insects and crustaceans, chitinases are associated with ecdysis through
degradation of old cuticle.3) Some plant chitinases are
involved in plant defense mechanisms against fungal
and bacterial pathogens through lytic action against chitin and peptidoglycan (lysozymic action), respectively,4,5) and show in vitro antifungal properties

against several phytopathogenic fungi, inhibiting both


spore germination and hyphal growth either alone or
synergistically with other pathogenesis-related proteins;68) while other chitinases and chitinase isoforms
play important roles in the growth and development of
plants, as in embryonic development, pollination, and
sexual reproduction.911) Some plant chitinases are
reported to play roles in the protection of plants against
environmental stress.11)
Chitinases have immense potential for biotechnological applications, as in preparing pharmaceutically
important chitooligosaccharides and N-acetyl D-glucosamine, which are promising antibacterial agents, lysozyme-inducing elicitors, and immunoenhancers; the
production of single-cell proteins; protoplast isolation
from fungi and yeasts; the development of bio-control
agents for pests and pathogens, and disease resistant
transgenic plants; the control of mosquitoes to interfere
with or block the transmission of diseases such as yellow fever, dengue, and malaria; and chitinous waste
management.1214)
An isozyme of plant chitinase, chitinase E, puried
from yam tuber, was found to have strong lytic activity
against fungal pathogens, indicating its possible application as a bio-control agent.15,16) Hence the genomic
DNA of yam class IV chitinase was cloned along with
its tentative vacuolar targeting signal (VTS) at the Cterminal on the basis of the partial amino acid
sequences of yam chitinase E.17) In the present study,
we made an attempt to produce this yam chitinase in
high yield by means of the recombinant Pichia carrying
its gene. A major advantage of Pichia pastoris over a
bacterial expression system is that the yeast has the
potential to perform many of the post-translational
modications typically associated with higher eukaryotes, such as the processing of signal sequences,
folding, disulde bridge formation, certain types of

*Corresponding author. Email: d.koga@hagi.ac.jp


Abbreviations: Endo H, Endoglycosidase H; PR protein, pathogenesis-related protein; P. pastoris, Pichia pastoris; VTS, vacuolar targeting signal;
PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

M.A. Akond et al.

lipid addition, and O- and N-linked glycosylation.18)


Hence, we investigated the possibility of using this
yam chitinase as a bio-control agent as well as yam
chitinase E.

Materials and methods

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Plasmids, vectors, and host strains.


pBluescript
SK+ (Stratagene Inc.) and pDrive (Qiagen K.K.) were
used as cloning vectors, and were transformed into the
Escherichia coli JM 109 as host. Vector pPICZA
(Invitrogen Corp.) was used as expression vector for the
chitinase gene. The methylotrophic yeast P. pastoris X33 (Invitrogen Corp.) was used as expression host for
the production of recombinant chitinase.
Cloning of the yam class IV chitinase gene without
intron and VTS.
Molecular cloning of a genomic
DNA fragment containing the full-length open reading
frame of the family 19 yam class IV chitinase gene
(accession no. AB102714), which included one intron
124 bp long, was done previously.17) First, the intron
was removed by three consecutive rounds of polymerase chain reaction (PCR) with the following primer
pairs: forward primer 1 and reverse primer 1, forward
primer 2 and reverse primer 2, and forward primer 1
and reverse primer 2 (Table 1), respectively, as summarized in Fig. 1. Then removal of the C-terminal VTS of
24 bp and replacement of the N-terminal signal
sequence of yam chitinase with -factor of the expression vector pPICZA were done as shown in Fig. 2 by
PCR with forward primer 3 and reverse primer 3
(Table 1). These primers contained the site for XhoI
and a partial sequence for -factor, respectively, and a
NotI digestion site along with stop codon. PCR amplication of the gene was done with Elongase Enzyme
Mix (Life Technologies Corp.). Thus, we obtained the
yam class IV chitinase gene without intron and VTS.
Construction of expression vector and recombinant
P. pastoris X-33.
Construction of a recombinant
expression vector (pPICZA) containing the functional
yam chitinase gene and electroporation of the expression cassette into competent P. pastoris X-33 were
done by the method of Saito et al.19)

Table 1.

Large-scale expression of recombinant Pichia


strain. A 3 mL preculture of recombinant P. pastoris
was developed in YPDM + ZeocineTM medium (1%
yeast extract, 2% peptone, 2% dextrose, 0.5% methanol, and 100 g/mL of ZeocinTM) in a 50 mL test tube
at 30 C for 48 h at 140 strokes/min in a shaker water
bath. For the production of chitinase, the preculture
was transferred to 200 mL of SM medium in a 500-mL
ask and incubated for 8 d at 30 C at 180 rpm in a
rotary shaker incubator (Bio-shaker BR300LF; Taitec
Corp.). The SM medium contained 0.2% (NH4)2SO4,
1% KH2PO4, 0.01% CaCl2, 0.2% MgSO4/7H2O, 0.2%
KCl, 0.01% NaCl, 0.01% ZnSO4/7H2O, 0.0005%
CuSO4/5H2O, 0.01% FeCl3/6H2O, 106% biotin, 5
105% thiamine hydrochloride, 5 105% pyridoxine
hydrochloride, 5 105% sodium pantothenate, 2
103% inositol, and 100 mM sodium phosphate buffer,
pH 7.5. For chitinase induction, methanol was added to
the culture medium every 24 h as carbon source to
maintain a nal concentration of 0.5%. Chitinase was
harvested from culture supernatant by centrifugation at
8000 g for 15 min at 4 C. The harvested chitinase in
culture supernatant was desalted by dialysis against 10
mM sodium phosphate buffer, pH 8.0, in a cellulose
tube with a cut-out molecular mass of 10,000 Da.
Activity assay and activity staining were performed
before and after dialysis.

Purication of P. pastoris-produced yam chitinase.


The crude enzyme harvested in the supernatant of the
culture medium was deglycosylated by treatment with
Endoglycosidase H (Endo H) (New England Biolabs
Inc.) at a rate of 4 L of Endo H (500 unit/L) for 400
mL of culture supernatant with incubation at 27 C for
24 h. The occulated carbohydrate moieties released
from the Pichia-produced chitinase by the action of
Endo H were removed by centrifugation at 8000 g over
20 min. The treated enzyme was applied to an anionexchange resin, DEAE-Toyopearl 650(M) (Tosoh
Corp.), which was packed in a glass column (2 50
cm) and pre-equilibrated with 10 mM sodium phosphate
buffer, pH 8.0. The column was washed with the same
buffer to elute the unbound proteins, and then the bound
chitinase was eluted with a linear gradient of NaCl from
0 to 0.5 M in the same buffer. The selected active fractions were applied to Fractogel EMD DEAE 650(M)
(Merck KGaA) in a glass column (1.2 20 cm), and

Oligonucleotide primer sequences used in PCR.

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

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Characterization of Pichia-produced yam class IV chitinase

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.

elution was done with an NaCl linear gradient from 0 to


0.5 M in the same buffer. The active fractions were again
subjected to DEAE-Toyopearl 650(M) chromatography.
The active fractions from the second DEAE-Toyopearl
650(M) were dialyzed against 50 mM sodium phosphate
buffer, pH 8.0, containing 0.2 M sodium chloride and put
on a Sephacryl S-100 (GE Healthcare Japan Corp.) column (2 95 cm) equilibrated with the same buffer. The
active fractions were pooled, dialyzed against 10 mM
sodium phosphate buffer, pH 8.0, and chromatographed
on a DEAE-Toyopearl 650(M) column (1 30 cm)
with a sodium chloride linear gradient from 0 to 0.3 M in
the same buffer.

Protein measurement. To monitor proteins during


chromatographic separation, absorbance was detected at
280 nm. The protein concentration was measured by
the method of Lowry et al.20) with bovine serum albumin as standard.

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

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Fig. 3.

M.A. Akond et al.

Time course of chitinase production by recombinant P. pastoris X-33 in culture medium.

Vmax (= kcat E0) were obtained by double reciprocal


plots by the method of Lineweaver and Burk.23)
To determine stability to dryness, 100 L of the puried chitinase (8 g) was kept in an open Eppendorf
tube in an incubator at 25 C for 7 d. The control sample was kept closed at 4 C. After 7 d, the residual dry
mass at the bottom of the tube was recovered by solubilization with 100 L of distilled water. Then the
remaining activity of the dried chitinase was measured
and compared with that of the control.
To determine lytic activity, the puried chitinase was
incubated with mycelia of fungal pathogen in 25 mM
sodium phosphate buffer, pH 8.0, containing 3% Zymolyase 20T (Seikagaku Corp.), whose main component is 1,3-glucanase, and 0.4% magnesium sulfate, which was
used to stabilize the protoplasts, at 30 C for 8 h. The protoplasts released were counted under a light microscope.
Polyacrylamide gel electrophoresis (PAGE) and
activity staining.
SDS-PAGE was performed with a
10% polyacrylamide slab gel containing 0.1% sodium
dodecyl sulfate (SDS) by the method of Laemmli.24)
Molecular weight was measured by SDS-PAGE (10%
gel) with standard marker proteins (Takara Bio Inc.)
including rabbit muscle phosphorylase b (97.2 kDa),
bovine serum albumin (66.4 kDa), hen egg white ovalbumin (44.3 kDa), bovine carbonic anhydrase (29 kDa),
soybean trypsin inhibitor (20.1 kDa), and hen egg white
lysozyme (14.3 kDa). Native PAGE was done with a
7.5% polyacrylamide gel pH 9.5 by the method of
Davis.25) Chitinase activity staining was done with
0.02% uorescent brightener 28 (Sigma) by the method
of Koga et al.26) After electrophoresis, the proteins in
the gel (85 45 1 mm) were transblotted electrophoretically on to a similar polyacrylamide gel containing
0.01% glycolchitin as substrate with a semi-dry type
blotting apparatus (AE-6670P/N; Atto Corp.) at 90 mA
for 30 min after SDS-PAGE or for 10 min after native
PAGE. The chitinase active band was observed as a
black band under UV irradiation.
N-Terminal sequence analysis. The puried chitinase was checked as to its N-terminal sequence by the
automated Edman degradation method. For this

purpose, the chitinase was treated with pyroglutamate


amino peptidase and then separated by SDS-PAGE followed by transfer to a PVDF membrane (Immobilon P;
Millipore Corp.) by electroblotting and stained with
Coomassie Brilliant Blue R-250. Then the excised protein bands were subjected to N-terminal sequence analysis with PPSQ-21A (Shimadzu Corp.).

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)

Fig. 4. SDS-PAGE of culture medium of recombinant P. pastoris


X-33 before and after treatment with Endoglycosidase H (Endo H).
Notes: Panels: (A) Chitinase activity staining; (B) protein staining.
Lanes: 1, before treatment with Endo H; 2, after treatment with Endo
H; M, molecular markers.

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Characterization of Pichia-produced yam class IV chitinase

Fig. 5. Purication of yam chitinase from recombinant P. pastoris X-33.


Notes: Panels: (A) The rst DEAE-Toyopearl 650(M) chromatography of Endo H-treated crude chitinase; (B) Sephacryl S-100 gel ltration of
the selected active fraction obtained by the second round of DEAE-Toyopearl 650(M) chromatography following Fractogel EMD DEAE 650(M);
(C) the third round of DEAE-Toyopearl 650(M) chromatography of the selected active fraction obtained by Sephacryl S-100 gel ltration. Active
fractions indicated by the horizontal bar were used in the following experiment.

Purication of the chitinase from recombinant P.


pastoris culture
Chitinase secretion in the culture medium almost
reached a plateau after 5 d of incubation (Fig. 3). The
crude enzyme, extracted from culture medium by

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

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M.A. Akond et al.

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.

Fig. 6. PAGE of the yam chitinase puried from recombinant P.


pastoris X-33.
Notes: Lanes: A, B, and M, SDS-PAGE; C and D, native PAGE; A
and D, chitinase activity staining; B, M, and C, protein staining; A,
B, C, and D, puried chitinase; M, molecular markers.

(A)

(B)

Fig. 7. pH optimum and pH stability of yam chitinase puried from


recombinant P. pastoris X-33.
Notes: Panels: (A) The optimum pH of the puried chitinase was
determined by incubation of 50 nM chitinase with 0.05% glycolchitin
in Britton-Robinson buffer, pH 2.012.0, at 32 C for 560 min; (B)
the pH stability of the puried chitinase was determined by measuring the remaining activity after incubation of 50 nM chitinase in Britton-Robinson buffer, pH 2.012.0, at 4 C for 120 h.

pH optimum and pH stability of yam chitinase


puried from recombinant P. pastoris X-33
To determine the optimum pH, the activity of the
puried chitinase was measured with glycolchitin in
Britton-Robinson buffer, pH 212, at 32 C. The result
is shown in Fig. 7(A). Pichia-produced yam chitinase
showed highest activity at pH 5 and a second peak at
pH 8.0 with comparatively low activity. To determine
pH stability, the puried chitinase was treated with
Britton-Robinson buffer, pH 212, for 12 h at 4 C.
The result is shown in Fig. 7(B). The Pichia-produced
yam chitinase was stable over a wide range of pH
from 3 to 12. Even at pH 2, it retained activity at
38%.
Temperature optimum and thermal stability of yam
chitinase puried from recombinant P. pastoris X-33
To determine the optimum temperature, the activity
of the puried chitinase was measured with glycolchitin
at 1080 C in Britton-Robinson buffer, pH 8.0. The
result is shown in Fig. 8(A). Pichia-produced yam chitinase showed highest activity at 70 C. To determine
thermal stability, the puried chitinase was treated at
1080 C for 15 min in Britton-Robinson buffer, pH
8.0. The result is shown in Fig. 8(B). The Pichia-produced yam chitinase was stable at up to 70 C with
remaining activity of more than 60%. The stability with
above 90% was observed up to 60 C, but at 75 C it
retained activity at only 10%.

Characterization of Pichia-produced yam class IV chitinase


Table 2.

Purication of P. pastoris-produced yam chitinase.

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)

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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

Stability of the yam chitinase puried from


recombinant P. pastoris X-33 to dryness
To determine stability to dryness, the puried chitinase was exposed to air at 25 C for 7 d. Remaining
activity was measured with glycolchitin and was compared with the control kept closed at 4 C. The stability
of the Pichia-produced yam chitinase was up to 68.4%
of the remaining activity.
Kinetic analysis of the yam chitinase puried from
recombinant P. pastoris X-33
To investigate the enzymatic action, kinetic analysis
was done with glycolchitin as substrate. The Km, kcat,
and kcat/Km values were obtained by double reciprocal
plot.23) They are shown in Table 3.

(A)

Lytic activity of the yam chitinase puried from


recombinant P. pastoris X-33
The lytic activity of the puried chitinase against
pathogens was investigated by counting the protoplasts
released from the pathogen under conditions stabilizing
the released protoplasts. As pathogens, Phytophthora
nicotianae and Fusarium oxysporum were used. The
results are shown in Fig. 9. In both cases, the numbers
of protoplasts released increased depending on the concentration of puried chitinase, indicating that protoplast release was due to the chitinase action. The lytic
activity of the Pichia-produced yam chitinase was
higher against Phytophthora than against Fusarium.

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.

Fig. 9. Lytic activity of yam chitinase puried from recombinant P.


pastoris X-33 against fungal pathogens.
Notes: The lytic activity of the puried chitinase was measured
under a light microscope by counting the protoplasts released from
mycelia of fungal pathogens such as F. oxysporum (solid circle) and
P. nicotianae (solid square) after incubation of 39 M chitinase with
2 mg pathogen mycelium in 0.2 mL of 25 mM sodium phosphate buffer, pH 8.0, containing 3% Zymolyase and 0.4% magnesium sulfate
at 30 C for 8 h.

M.A. Akond et al.


Table 3.

Kinetic parameters of P. pastoris-produced yam chitinase and yam chitinase E.

Enzyme used
Pichia-produced yam chitinase
Yam chitinase E15)

Table 4.

Enzyme concentration (nM)

Substrate concentration (mg/mL)

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

Comparative characteristics of P. pastoris-produced yam chitinase and yam chitinase E.

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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

Pichia-produced yam chitinase

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

Note: ND, Not done.

yam chitinase E puried from yam tuber has been


reported to be 33.5 kDa,15) while results for molecular
cloning of it indicated that the molecular mass is 28
kDa.17) Therefore, the extra mass over 28 kDa must be
due to carbohydrate moiety. After treatment of the culture medium with Endo H, chitinase active bands
shifted as expected mainly to 32 kDa, which is similar
to the molecular weight of yam chitinase E (33.5 kDa).
Regarding chitinase activity toward glycolchitin, no
effect of Endo H treatment was observed. After treatment with Endo H, the 32-kDa chitinase was puried
by column chromatography (Table 2). The specic
activity of the puried chitinase was 374A420/h/mg.
By this specic activity, the production of chitinase in
an 8-d culture medium was calculated to be 66 mg/L.
This is considerably high production.
In order to compare the Pichia-produced yam chitinase with yam chitinase E, the puried chitinase was
investigated as to optimum activity and stability. The
results are summarized and compared with those of
yam chitinase E in Table 4. With respect to physicochemical properties, as shown in Fig. 6, as to SDSPAGE and native PAGE, the puried chitinase was an
acidic chitinase with molecular mass of 32 kDa. The Nterminal amino acid was not detected from both Pichia-produced yam chitinase and yam chitinase E by the
Edman degradation method. With respect to activity,
Pichia-produced yam chitinase showed two pH optima,
in acidic and alkaline ranges, which is one of the characteristics of yam chitinase E. Both chitinases showed
an optimum temperature of 70 C. On the other hand,
the stability of chitinase is important for its application
as a bio-control agent. With respect to pH stability, the
Pichia-produced yam chitinase showed a wider range
of pH than yam chitinase E, and they showed the same
thermal stability. Furthermore, the Pichia-produced
yam chitinase showed high stability against dryness.
With respect to kinetic behavior, the Pichia-produced
yam chitinase was found to be better in terms of sub-

strate afnity due to its lower Km value (0.233) toward


glycolchitin than yam chitinase E (Km = 0.518), indicating that the Pichia-produced yam chitinase, with a
smaller Km value, had more substrate afnity than yam
chitinase E, but kcat (0.200) was lower than that of yam
chitinase E (kcat = 0.645). Thus, the overall reaction
(kcat/Km = 0.858) of Pichia-produced yam chitinase
toward glycolchitin was found to be 31.4% less strong
than that of yam chitinase E (1.25). Taking account of
the differences in glycolation degree of glycolchitin
manually prepared for each study, the differences
within single gures in the values of kinetic parameters
such as Km and kcat might be negligible. Accordingly
Pichia-produced yam chitinase proved to be similar to
yam chitinase E as to kinetic behavior.
On the basis of the kinetic data, lytic activity was
also expected to be high, similarly to yam chitinase E.
As expected, the Pichia-produced yam chitinase
showed strong lytic activity against Fusarium and Phytophthora (Fig. 9 and Table 4). The number of protoplasts released from F. oxysporum mycelia by the action
of 6.0 M Pichia-produced yam chitinase at pH 8.0 at
30 C for 8 h of incubation was 167 105, while it was
104 105 in case of yam chitinase E under the same
experimental conditions, as obtained by recalculation of
the data obtained by Arakane et al.15) from 6 to 8 h
(Table 4). Thus, it appears that the Pichia-produced
yam chitinase was better than yam chitinase E in terms
of protoplast formation. Taking into account of the different homogeneities of pathogenic mycelia as substrates for each study, however, such a difference in the
lytic activity might be negligible. In conclusion, the Pichia-produced yam chitinase might be useful as a biocontrol agent, as well as yam chitinase E.
It should be noted for further study that P. pastoris
X-33 might have a capacity to modify through cyclization. The N-terminal of the recombinant protein produced by itself like the original plants such as yam,
because the N-terminus was found to be blocked.

Characterization of Pichia-produced yam class IV chitinase

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.

Downloaded by [Muhammad AKOND] at 06:16 28 April 2014

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