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Characterization of a yam class IV chitinase producedby
recombinant Pichia pastoris X-33Muhammad Ali Akondab, Yusuke
Matsudac, Takayuki Ishimaruc, Ken Iwaice, Akira Saitoc,Akio Katoc,
Shuhei Tanakaad, Jun Kobayashiad & Daizo Kogaacea The United
Graduate School of Agricultural Sciences, Tottori University,
Tottori, Japanb Department of Botany, Jahangirnagar University,
Savar, Bangladeshc Faculty of Agriculture, Department of Biological
Chemistry, Yamaguchi University,Yamaguchi, Japand Faculty of
Agriculture, Department of Biological and Environmental
Sciences,Yamaguchi University, Yamaguchi, Japane Faculty of Life
Design, Yamaguchi University of Human Welfare and Culture,
Hagi,JapanPublished online: 17 Apr 2014.
To cite this article: Muhammad Ali Akond, Yusuke Matsuda,
Takayuki Ishimaru, Ken Iwai, Akira Saito, Akio Kato, ShuheiTanaka,
Jun Kobayashi & Daizo Koga (2014): Characterization of a yam
class IV chitinase produced by recombinant Pichiapastoris X-33,
Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2014.885825
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http://dx.doi.org/10.1080/09168451.2014.885825
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Characterization of a yam class IV chitinase produced by
recombinant Pichiapastoris 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,*
1The 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
EnvironmentalSciences, Yamaguchi University, Yamaguchi, Japan;
5Faculty of Life Design, Yamaguchi University of HumanWelfare and
Culture, Hagi, Japan
Received September 6, 2013; accepted November 8, 2013
http://dx.doi.org/10.1080/09168451.2014.885825
A yam (Dioscorea opposita Thunb) class IV chiti-nase, whose
genomic DNA was cloned by Mitsunagaet al. (2004), was produced by
the recombinant Pi-chia pastoris X-33 in high yields such as 66
mg/L ofculture medium. The chitinase was purified by col-umn
chromatography after Endoglycosidase Htreatment and then
characterized. It showed proper-ties similar to the original
chitinase E purified fromthe yam tuber reported by Arakane et al.
(2000).This Pichia-produced chitinase also showed stronglytic
activity against Fusarium oxysporum and Phy-tophthora nicotianae,
wide pH and thermal stability,optimum activity at higher
temperature such as70 C, and high substrate affinity, indicating
thatone can use this Pichia-produced yam chitinase as abio-control
agent.
Key words: yam (Dioscorea oppsita Thunb); classIV chitinase;
bio-control agent; recombi-nant Pichia pastoris; lytic activiy
Chitinases [EC 3.2.1.14] are diversified in biologicalfunctions
depending on the source. Chitinases, whichhydrolyze chitin, occur
in a wide range of organisms,including viruses, bacteria, fungi,
insects, higher plants,and animals.1) Bacterial chitinases play
roles in nutri-tion and parasitism, while fungal chitinases have
auto-lytic, nutritional, and morphogenetic roles. Viralchitinases
are involved in pathogenesis.2) Invertebratechitinases play roles
in digestion. In insects and crusta-ceans, chitinases are
associated with ecdysis throughdegradation of old cuticle.3) Some
plant chitinases areinvolved in plant defense mechanisms against
fungaland bacterial pathogens through lytic action against chi-tin
and peptidoglycan (lysozymic action), respec-tively,4,5) and show
in vitro antifungal properties
against several phytopathogenic fungi, inhibiting bothspore
germination and hyphal growth either alone orsynergistically with
other pathogenesis-related pro-teins;68) while other chitinases and
chitinase isoformsplay important roles in the growth and
development ofplants, as in embryonic development, pollination,
andsexual reproduction.911) Some plant chitinases arereported to
play roles in the protection of plants againstenvironmental
stress.11)
Chitinases have immense potential for biotechnologi-cal
applications, as in preparing pharmaceuticallyimportant
chitooligosaccharides and N-acetyl D-glucosa-mine, which are
promising antibacterial agents, lyso-zyme-inducing elicitors, and
immunoenhancers; theproduction of single-cell proteins; protoplast
isolationfrom fungi and yeasts; the development of
bio-controlagents for pests and pathogens, and disease
resistanttransgenic plants; the control of mosquitoes to
interferewith or block the transmission of diseases such as yel-low
fever, dengue, and malaria; and chitinous wastemanagement.1214)
An isozyme of plant chitinase, chitinase E, purifiedfrom yam
tuber, was found to have strong lytic activityagainst fungal
pathogens, indicating its possible appli-cation as a bio-control
agent.15,16) Hence the genomicDNA of yam class IV chitinase was
cloned along withits tentative vacuolar targeting signal (VTS) at
the C-terminal on the basis of the partial amino acidsequences of
yam chitinase E.17) In the present study,we made an attempt to
produce this yam chitinase inhigh yield by means of the recombinant
Pichia carryingits gene. A major advantage of Pichia pastoris over
abacterial expression system is that the yeast has thepotential to
perform many of the post-translationalmodifications typically
associated with higher eukary-otes, such as the processing of
signal sequences,folding, disulfide bridge formation, certain types
of
*Corresponding author. Email: [email protected]:
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.
Bioscience, Biotechnology, and Biochemistry, 2014
2014 Japan Society for Bioscience, Biotechnology, and
Agrochemistry
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lipid addition, and O- and N-linked glycosylation.18)
Hence, we investigated the possibility of using thisyam
chitinase as a bio-control agent as well as yamchitinase E.
Materials and methods
Plasmids, vectors, and host strains. pBluescriptSK+ (Stratagene
Inc.) and pDrive (Qiagen K.K.) wereused as cloning vectors, and
were transformed into theEscherichia coli JM 109 as host. Vector
pPICZA(Invitrogen Corp.) was used as expression vector for
thechitinase gene. The methylotrophic yeast P. pastoris X-33
(Invitrogen Corp.) was used as expression host forthe production of
recombinant chitinase.
Cloning of the yam class IV chitinase gene withoutintron and
VTS. Molecular cloning of a genomicDNA fragment containing the
full-length open readingframe of the family 19 yam class IV
chitinase gene(accession no. AB102714), which included one
intron124 bp long, was done previously.17) First, the intronwas
removed by three consecutive rounds of polymer-ase chain reaction
(PCR) with the following primerpairs: forward primer 1 and reverse
primer 1, forwardprimer 2 and reverse primer 2, and forward primer
1and reverse primer 2 (Table 1), respectively, as summa-rized in
Fig. 1. Then removal of the C-terminal VTS of24 bp and replacement
of the N-terminal signalsequence of yam chitinase with -factor of
the expres-sion vector pPICZA were done as shown in Fig. 2 byPCR
with forward primer 3 and reverse primer 3(Table 1). These primers
contained the site for XhoIand a partial sequence for -factor,
respectively, and aNotI digestion site along with stop codon. PCR
amplifi-cation of the gene was done with Elongase EnzymeMix (Life
Technologies Corp.). Thus, we obtained theyam class IV chitinase
gene without intron and VTS.
Construction of expression vector and recombinantP. pastoris
X-33. Construction of a recombinantexpression vector (pPICZA)
containing the functionalyam chitinase gene and electroporation of
the expres-sion cassette into competent P. pastoris X-33 weredone
by the method of Saito et al.19)
Large-scale expression of recombinant Pichiastrain. A 3 mL
preculture of recombinant P. pastoriswas developed in YPDM +
ZeocineTM medium (1%yeast extract, 2% peptone, 2% dextrose, 0.5%
metha-nol, and 100 g/mL of ZeocinTM) in a 50 mL test tubeat 30 C
for 48 h at 140 strokes/min in a shaker waterbath. For the
production of chitinase, the preculturewas transferred to 200 mL of
SM medium in a 500-mLflask and incubated for 8 d at 30 C at 180 rpm
in arotary shaker incubator (Bio-shaker BR300LF; TaitecCorp.). 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, 10
6% biotin, 5 105% thiamine hydrochloride, 5 105%
pyridoxinehydrochloride, 5 105% sodium pantothenate, 2 103%
inositol, and 100 mM sodium phosphate buffer,pH 7.5. For chitinase
induction, methanol was added tothe culture medium every 24 h as
carbon source tomaintain a final concentration of 0.5%. Chitinase
washarvested from culture supernatant by centrifugation at8000 g
for 15 min at 4 C. The harvested chitinase inculture supernatant
was desalted by dialysis against 10mM sodium phosphate buffer, pH
8.0, in a cellulosetube with a cut-out molecular mass of 10,000
Da.Activity assay and activity staining were performedbefore and
after dialysis.
Purification of P. pastoris-produced yam chitinase.The crude
enzyme harvested in the supernatant of theculture medium was
deglycosylated by treatment withEndoglycosidase H (Endo H) (New
England BiolabsInc.) at a rate of 4 L of Endo H (500 unit/L) for
400mL of culture supernatant with incubation at 27 C for24 h. The
flocculated carbohydrate moieties releasedfrom the Pichia-produced
chitinase by the action ofEndo H were removed by centrifugation at
8000 g over20 min. The treated enzyme was applied to an
anion-exchange resin, DEAE-Toyopearl 650(M) (TosohCorp.), which was
packed in a glass column (2 50cm) and pre-equilibrated with 10 mM
sodium phosphatebuffer, pH 8.0. The column was washed with the
samebuffer to elute the unbound proteins, and then the
boundchitinase was eluted with a linear gradient of NaCl from0 to
0.5 M in the same buffer. The selected active frac-tions were
applied to Fractogel EMD DEAE 650(M)(Merck KGaA) in a glass column
(1.2 20 cm), and
Table 1. Oligonucleotide primer sequences used in PCR.
Primer Sequence
For intron removalForward primer 1
5-CTCATCAATTTCCAGCCACTC-3Forward primer 2
5-GTCACCCATGAAACTGGACATTTATGTTACATTGAAGAAA
GAGATGGACA-3Reverse primer 1
5-CCATCTCTTTCTTCAATGTAACATAAATGTCCAGTTTCATG
GGTGACATG-3Reverse primer 2 5-TAGTCGAATTTAAGCCAAGTTC-3Insert
preparation for expression vectorForward primer 3
5-GACCTCGAGAAAAGACAAAACTGCCAGTGCGACACC-3Reverse primer 3
5-GACGCGGCCGCCTAACAAGTGAGATCATTGCCAGG-3
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elution was done with an NaCl linear gradient from 0 to0.5 M in
the same buffer. The active fractions were againsubjected to
DEAE-Toyopearl 650(M) chromatography.The active fractions from the
second DEAE-Toyopearl650(M) were dialyzed against 50 mM sodium
phosphatebuffer, pH 8.0, containing 0.2 M sodium chloride and puton
a Sephacryl S-100 (GE Healthcare Japan Corp.) col-umn (2 95 cm)
equilibrated with the same buffer. Theactive fractions were pooled,
dialyzed against 10 mMsodium phosphate buffer, pH 8.0, and
chromatographedon a DEAE-Toyopearl 650(M) column (1 30 cm)with a
sodium chloride linear gradient from 0 to 0.3 M inthe same
buffer.
Protein measurement. To monitor proteins duringchromatographic
separation, absorbance was detected at280 nm. The protein
concentration was measured bythe method of Lowry et al.20) with
bovine serum albu-min as standard.
Enzyme assay. Chitinase activity was measuredwith glycolchitin
as substrate. For purification, 520 Lof chitinase solution was
added to 0.5 mL of 0.05%(w/v) glycolchitin dissolved in 50 mM
sodium phos-phate buffer, pH 8.0, and this was incubated at 32 Cfor
30 min. The reducing end group produced was mea-sured
colorimetrically at 420 nm with a ferri-ferrocya-nide reagent by
the method of Imoto and Yagishita.21)
Britton-Robinson buffer (pH 2.012.0)22) was also usedto
determine the pH optimum and stability. The enzy-matic reaction
proceeded linearly with time to 20%completion of the reaction with
glycolchitin.For kinetic analysis, the purified chitinase (final
con-
centration, 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 calculatedfrom the difference in the
absorbance at 420 nmbetween sample and control experiments assuming
arelationship of 1 A420 = 0.22 mMN-acetylchitooligo-saccharides.
The kinetic parameters such as Km and
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.
Characterization of Pichia-produced yam class IV chitinase 3
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Vmax (= kcat E0) were obtained by double reciprocalplots by the
method of Lineweaver and Burk.23)
To determine stability to dryness, 100 L of the puri-fied
chitinase (8 g) was kept in an open Eppendorftube in an incubator
at 25 C for 7 d. The control sam-ple was kept closed at 4 C. After
7 d, the residual drymass at the bottom of the tube was recovered
by solu-bilization with 100 L of distilled water. Then theremaining
activity of the dried chitinase was measuredand compared with that
of the control.
To determine lytic activity, the purified chitinase wasincubated
with mycelia of fungal pathogen in 25mMsodium phosphate buffer, pH
8.0, containing 3% Zymoly-ase 20T (Seikagaku Corp.), whose main
component is -1,3-glucanase, and 0.4% magnesium sulfate, which
wasused to stabilize the protoplasts, at 30 C for 8 h. The
pro-toplasts released were counted under a light microscope.
Polyacrylamide gel electrophoresis (PAGE) andactivity staining.
SDS-PAGE was performed with a10% polyacrylamide slab gel containing
0.1% sodiumdodecyl 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 oval-bumin (44.3 kDa), bovine carbonic anhydrase (29
kDa),soybean trypsin inhibitor (20.1 kDa), and hen egg
whitelysozyme (14.3 kDa). Native PAGE was done with a7.5%
polyacrylamide gel pH 9.5 by the method ofDavis.25) Chitinase
activity staining was done with0.02% fluorescent brightener 28
(Sigma) by the methodof Koga et al.26) After electrophoresis, the
proteins inthe gel (85 45 1 mm) were transblotted
electrophoret-ically on to a similar polyacrylamide gel
containing0.01% glycolchitin as substrate with a semi-dry
typeblotting apparatus (AE-6670P/N; Atto Corp.) at 90 mAfor 30 min
after SDS-PAGE or for 10 min after nativePAGE. The chitinase active
band was observed as ablack band under UV irradiation.
N-Terminal sequence analysis. The purified chiti-nase was
checked as to its N-terminal sequence by theautomated Edman
degradation method. For this
purpose, the chitinase was treated with pyroglutamateamino
peptidase and then separated by SDS-PAGE fol-lowed by transfer to a
PVDF membrane (Immobilon P;Millipore Corp.) by electroblotting and
stained withCoomassie Brilliant Blue R-250. Then the excised
pro-tein bands were subjected to N-terminal sequence anal-ysis with
PPSQ-21A (Shimadzu Corp.).
ResultsSequence analysis of the yam class IV chitinase gene
introduced into recombinant P. pastoris X-33The yam chitinase
gene carried by recombinant P.
pastoris X-33 was sequenced and compared with thecomplete
nucleotide sequence of the class IV yam chiti-nase by homology
alignment. The results indicated pre-cise deletion of both intron
and VTS and in-framefusion of the -factor instead of the signal
sequence inrecombinant Pichia carrying yam chitinase gene.
Fig. 3. Time course of chitinase production by recombinant P.
pastoris X-33 in culture medium.
(A) (B)
Fig. 4. SDS-PAGE of culture medium of recombinant P.
pastorisX-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
EndoH; M, molecular markers.
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Purification of the chitinase from recombinant P.pastoris
culture
Chitinase secretion in the culture medium almostreached a
plateau after 5 d of incubation (Fig. 3). Thecrude enzyme,
extracted from culture medium by
centrifugation, showed five main active bands by activ-ity
staining after SDS-PAGE with molecular masses ofabout 58, 48, 38,
32, and 28 kDa (Fig. 4(A)). Afterdeglycosylation by treatment with
Endo H, the patternof the chitinase active band shifted to smaller
molecular
Fig. 5. Purification of yam chitinase from recombinant P.
pastoris X-33.Notes: Panels: (A) The first DEAE-Toyopearl 650(M)
chromatography of Endo H-treated crude chitinase; (B) Sephacryl
S-100 gel filtration 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
filtration. Activefractions indicated by the horizontal bar were
used in the following experiment.
Characterization of Pichia-produced yam class IV chitinase 5
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masses, at 32 and 28 kDa. In order to obtain 32 kDa-chitinase,
which is similar in molecular size to yamchitinase E (33.5 kDa),
indicating its possible applica-tion as a bio-control agent,15,16)
the following course ofpurification was applied. The deglycosylated
crudeenzyme after dialysis against 10 mM sodium phosphatebuffer, pH
8.0, was applied sequentially to DEAE-Toyopearl 650(M), Fractogel
EMD DEAE 650(M),DEAE-Toyopearl 650(M), Sephacryl S-100,
andDEAE-Toyopearl 650(M) resins. The active peaks ofthe first
chromatographic separation on DEAE-Toyo-pearl 650(M) were obtained
at sodium chloride conduc-tivities between 7.5 and 18 mS (Fig.
5(A)). The activefractions, indicated by the horizontal bar, were
com-bined and applied to Fractogel EMD DEAE 650(M),and the active
fractions were obtained between 15 and27 mS of sodium chloride
conductivity. These werecombined and applied again to
DEAE-Toyopearl 650(M), followed by Sephacryl S-100 (Fig. 5(B)).
Theactive fractions of small molecular sizes of about 32kDa were
pooled by gel filtration chromatography andeluted again by
DEAE-Toyopearl 650(M) with a gradi-ent of 00.3 M sodium chloride
(Fig. 5(C)). Purity anal-ysis by both SDS-PAGE and native PAGE
showed asingle homogeneous band with a molecular mass of 32kDa
(Fig. 6). The quantitative results for purificationsteps are
presented in Table 2. A 3% yield with 10-foldoverall purification
was achieved at the final step witha specific activity of 374
A420/h/mg.
N-Terminal amino acid sequencingAutomated Edman degradation of
the chitinase pro-
duced by recombinant P. pastoris was unsuccessful.When digested
with pyroglutamate amino peptidase,the N-terminals were read to be
SYD, the same as theamino acid sequence of the introduced yam
chitinasefrom the 21st. This result was obtained repeatedly,
sug-gesting that appearance of the SYD sequence from the21st amino
acid is due to the action of peptidase con-taminating in this
pyroglutamate amino peptidase.
pH optimum and pH stability of yam chitinasepurified from
recombinant P. pastoris X-33To determine the optimum pH, the
activity of the
purified chitinase was measured with glycolchitin
inBritton-Robinson buffer, pH 212, at 32 C. The resultis shown in
Fig. 7(A). Pichia-produced yam chitinaseshowed highest activity at
pH 5 and a second peak atpH 8.0 with comparatively low activity. To
determinepH stability, the purified chitinase was treated
withBritton-Robinson buffer, pH 212, for 12 h at 4 C.The result is
shown in Fig. 7(B). The Pichia-producedyam chitinase was stable
over a wide range of pHfrom 3 to 12. Even at pH 2, it retained
activity at38%.
Temperature optimum and thermal stability of yamchitinase
purified from recombinant P. pastoris X-33To determine the optimum
temperature, the activity
of the purified chitinase was measured with glycolchitinat 1080
C in Britton-Robinson buffer, pH 8.0. Theresult is shown in Fig.
8(A). Pichia-produced yam chi-tinase showed highest activity at 70
C. To determinethermal stability, the purified chitinase was
treated at1080 C for 15 min in Britton-Robinson buffer, pH8.0. The
result is shown in Fig. 8(B). The Pichia-pro-duced yam chitinase
was stable at up to 70 C withremaining activity of more than 60%.
The stability withabove 90% was observed up to 60 C, but at 75 C
itretained activity at only 10%.
Fig. 6. PAGE of the yam chitinase purified 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, purified chitinase; M, molecular
markers.
(A)
(B)
Fig. 7. pH optimum and pH stability of yam chitinase purified
fromrecombinant P. pastoris X-33.Notes: Panels: (A) The optimum pH
of the purified chitinase was
determined by incubation of 50 nM chitinase with 0.05%
glycolchitinin Britton-Robinson buffer, pH 2.012.0, at 32 C for 560
min; (B)the pH stability of the purified chitinase was determined
by measur-ing the remaining activity after incubation of 50 nM
chitinase in Brit-ton-Robinson buffer, pH 2.012.0, at 4 C for 120
h.
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Stability of the yam chitinase purified fromrecombinant P.
pastoris X-33 to dryness
To determine stability to dryness, the purified chiti-nase was
exposed to air at 25 C for 7 d. Remainingactivity was measured with
glycolchitin and was com-pared with the control kept closed at 4 C.
The stabilityof the Pichia-produced yam chitinase was up to 68.4%of
the remaining activity.
Kinetic analysis of the yam chitinase purified fromrecombinant
P. pastoris X-33
To investigate the enzymatic action, kinetic analysiswas done
with glycolchitin as substrate. The Km, kcat,and kcat/Km values
were obtained by double reciprocalplot.23) They are shown in Table
3.
Lytic activity of the yam chitinase purified fromrecombinant P.
pastoris X-33The lytic activity of the purified chitinase
against
pathogens was investigated by counting the protoplastsreleased
from the pathogen under conditions stabilizingthe released
protoplasts. As pathogens, Phytophthoranicotianae and Fusarium
oxysporum were used. Theresults are shown in Fig. 9. In both cases,
the numbersof protoplasts released increased depending on the
con-centration of purified chitinase, indicating that proto-plast
release was due to the chitinase action. The lyticactivity of the
Pichia-produced yam chitinase washigher against Phytophthora than
against Fusarium.
Discussion
As shown in Fig. 3, a family 19 Class IV acidic yamchitinase
without VTS was produced increasingly withtime by recombinant P.
pastoris X-33. The results ofSDS-PAGE for Pichia-produced chitinase
in 5-d culturemedium, however, showed several chitinase activebands
of various molecular sizes from 28 to 58 kDa(Fig. 4). On the other
hand, the molecular weight of
Table 2. Purification of P. pastoris-produced yam chitinase.
StepTotal activity(A420/h) Protein (mg)
Specific activity(A420/h/mg)
Overall yield(%)
Overallpurification
Crude extract 34,800 915 38.0 100 1.00Ammonium sulfate
precipitation 34,200 760 45.0 98.4 1.18DEAE-Toyopearl 650(M) (1)
12,300 89.0 138 35.3 3.63Fractogel EMD DEAE 650(M) 9070 40.3 225
26.1 5.92DEAE-Toyopearl 650(M) (2) 6610 25.4 260 19.0 6.84Sephacryl
S-100 5680 19.3 294 16.3 7.74DEAE-Toyopearl 650(M) (3) 1010 2.71
374 2.92 9.84
(A)
(B)
Fig. 8. Temperature optimum and thermal stability of the yam
chiti-nase purified from recombinant P. pastoris X-33.Notes:
Panels: (A) The optimum temperature of the purified chiti-
nase was measured by incubation of 50 nM chitinase with 0.05%
gly-colchitin in Britton-Robinson buffer, pH 8.0, for 560 min
attemperatures of 1080 C; (B) the thermal stability of the purified
chi-tinase was determined by measuring the remaining activity after
incu-bation of 50 nM chitinase in Britton-Robinson buffer, pH 8.0,
for 15min at temperature range of 1080 C.
Fig. 9. Lytic activity of yam chitinase purified from
recombinant P.pastoris X-33 against fungal pathogens.Notes: The
lytic activity of the purified chitinase was measured
under a light microscope by counting the protoplasts released
frommycelia of fungal pathogens such as F. oxysporum (solid circle)
andP. nicotianae (solid square) after incubation of 39 M chitinase
with2 mg pathogen mycelium in 0.2 mL of 25 mM sodium phosphate
buf-fer, pH 8.0, containing 3% Zymolyase and 0.4% magnesium
sulfateat 30 C for 8 h.
Characterization of Pichia-produced yam class IV chitinase 7
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yam chitinase E purified from yam tuber has beenreported to be
33.5 kDa,15) while results for molecularcloning of it indicated
that the molecular mass is 28kDa.17) Therefore, the extra mass over
28 kDa must bedue to carbohydrate moiety. After treatment of the
cul-ture medium with Endo H, chitinase active bandsshifted as
expected mainly to 32 kDa, which is similarto the molecular weight
of yam chitinase E (33.5 kDa).Regarding chitinase activity toward
glycolchitin, noeffect of Endo H treatment was observed. After
treat-ment with Endo H, the 32-kDa chitinase was purifiedby column
chromatography (Table 2). The specificactivity of the purified
chitinase was 374A420/h/mg.By this specific activity, the
production of chitinase inan 8-d culture medium was calculated to
be 66 mg/L.This is considerably high production.
In order to compare the Pichia-produced yam chiti-nase with yam
chitinase E, the purified chitinase wasinvestigated as to optimum
activity and stability. Theresults are summarized and compared with
those ofyam chitinase E in Table 4. With respect to
physico-chemical properties, as shown in Fig. 6, as to SDS-PAGE and
native PAGE, the purified chitinase was anacidic chitinase with
molecular mass of 32 kDa. The N-terminal amino acid was not
detected from both Pichi-a-produced yam chitinase and yam chitinase
E by theEdman degradation method. With respect to
activity,Pichia-produced yam chitinase showed two pH optima,in
acidic and alkaline ranges, which is one of the char-acteristics of
yam chitinase E. Both chitinases showedan optimum temperature of 70
C. On the other hand,the stability of chitinase is important for
its applicationas a bio-control agent. With respect to pH
stability, thePichia-produced yam chitinase showed a wider rangeof
pH than yam chitinase E, and they showed the samethermal stability.
Furthermore, the Pichia-producedyam chitinase showed high stability
against dryness.
With respect to kinetic behavior, the Pichia-producedyam
chitinase was found to be better in terms of sub-
strate affinity due to its lower Km value (0.233)
towardglycolchitin than yam chitinase E (Km = 0.518), indi-cating
that the Pichia-produced yam chitinase, with asmaller Km value, had
more substrate affinity than yamchitinase E, but kcat (0.200) was
lower than that of yamchitinase E (kcat = 0.645). Thus, the overall
reaction(kcat/Km = 0.858) of Pichia-produced yam chitinasetoward
glycolchitin was found to be 31.4% less strongthan that of yam
chitinase E (1.25). Taking account ofthe differences in glycolation
degree of glycolchitinmanually prepared for each study, the
differenceswithin single figures in the values of kinetic
parameterssuch as Km and kcat might be negligible.
AccordinglyPichia-produced yam chitinase proved to be similar toyam
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 chitinaseshowed strong lytic
activity against Fusarium and Phy-tophthora (Fig. 9 and Table 4).
The number of protop-lasts released from F. oxysporum mycelia by
the actionof 6.0 M Pichia-produced yam chitinase at pH 8.0 at30 C
for 8 h of incubation was 167 105, while it was104 105 in case of
yam chitinase E under the sameexperimental conditions, as obtained
by recalculation ofthe data obtained by Arakane et al.15) from 6 to
8 h(Table 4). Thus, it appears that the Pichia-producedyam
chitinase was better than yam chitinase E in termsof protoplast
formation. Taking into account of the dif-ferent homogeneities of
pathogenic mycelia as sub-strates for each study, however, such a
difference in thelytic activity might be negligible. In conclusion,
the Pi-chia-produced yam chitinase might be useful as a bio-control
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 cycliza-tion. The
N-terminal of the recombinant protein pro-duced by itself like the
original plants such as yam,because the N-terminus was found to be
blocked.
Table 3. Kinetic parameters of P. pastoris-produced yam
chitinase and yam chitinase E.
Enzyme used Enzyme concentration (nM) Substrate concentration
(mg/mL) kcat (1/s) Km (mg/mL) kcat/Km (mL/mg/s)
Pichia-produced yam chitinase 50 0.0250.400 0.200 0.233 0.858Yam
chitinase E15) 50 0.0250.400 0.645 0.518 1.25
Table 4. Comparative characteristics of P. pastoris-produced yam
chitinase and yam chitinase E.
Parameters Pichia-produced yam chitinase Yam chitinase E
Molecular weight 32.0 kDa 33.5 kDaOptimum pH toward glycol
chitin 5, 8 4.0, 7.5Optimum temperature toward glycol chitin 70 C
70 CpH stability 312 511Thermal stability 70 C 70 CDrying stability
68.4% NDKinetics:For glycol chitin Km (mg/mL) 0.233 0.518
kcat (1/s) 0.200 0.645kcat/Km (mL/mg/s) 0.858 1.25
Lytic activity Fusarium oxysporum 167 105 104 105
Phytophthora nicotianae 229 105 NDAction mechanism against
powdery mildew ND Break-downGlycosyl hydrolase family 19 19
Note: ND, Not done.
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Supplemental material
The supplemental material for this paper is availableat
http://dx.doi.org/10.1080/09168451.2014.885825.
Acknowledgment
We are thankful to Mr Minoru Iwase for his gener-ous help in
constructing the cDNA of chitinase bydeletion of the intron.
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Characterization of Pichia-produced yam class IV chitinase 9
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http://dx.doi.org/10.1080/09168451.2014.885825
Abstract Materials and methods Plasmids, vectors, and host
strains Cloning of the yam class IV chitinase gene without intron
and VTS Construction of expression vector and recombinant P.
pastoris X-33 Large-scale expression of recombinant Pichia strain
Purification of P. pastoris-produced yam chitinase Protein
measurement Enzyme assay Polyacrylamide gel electrophoresis (PAGE)
and activity staining N-Terminal sequence analysis
Results Sequence analysis of the yam class IV chitinase gene
introduced into recombinant P. pastoris X-33 Purification of the
chitinase from recombinant P. pastoris culture N-Terminal amino
acid sequencing pH optimum and pH stability of yam chitinase
purified from recombinant P. pastoris X-33 Temperature optimum and
thermal stability of yam chitinase purified from recombinant P.
pastoris X-33 Stability of the yam chitinase purified from
recombinant P. pastoris X-33 to dryness Kinetic analysis of the yam
chitinase purified from recombinant P. pastoris X-33 Lytic activity
of the yam chitinase purified from recombinant P. pastoris X-33
Discussion Supplemental materialAcknowledgmentReferences
