全 文 ：Research Paper 研究报告
微生物学报 Acta Microbiologica Sinica
51(9) :1212 － 1221;4 September 2011
ISSN 0001 － 6209;CN 11 － 1995 /Q
http:/ / journals． im． ac． cn / actamicrocn
Supported by the National High Technology Research and Development Program of China (2005AA213010)and by the National Natural Science
Foundation of China (30270043)
* Corresponding author． Tel / Fax: + 86-20-85228422;E-mail:tdsyao@ jnu． edu． cn
Received:3 March 2011 /Revised:30 April 2011
Cloning，expression，purification and characterization of
an aflatoxin-converting enzyme from Armillaria tabescens
Sixia Wen1，Min Guan1，Tao Zhou1，Hong Cao1，Chunfang Xie1，2，Daling Liu1，2，
Dongsheng Yao1，3*
1 Institute of Microbial Biotechnology，
2 Guangdong Provincial Key Laboratory of Bioengineering Medicine，Jinan University，
3 National Engineering Research Center of Genetic Medicine，Guangzhou 510632，China
Abstract:［Objective］Aflatoxin B1(AFB1)is extremely mutagenic，toxic and a potent carcinogen both to humans and
livestock． Aflatoxin-oxidase (AFO) was an aflatoxin-converting enzyme previously purified by us from Armillaria
tabescens． In order to know better about the molecular characterization of this distinct enzyme，we expressed，purified and
characterized the His6 tag fused aflatoxin-oxidase． ［Methods］ Based on sequences of peptides fragments of AFO
previously obtained by Electrophoresis-Electrospray Ionization tandem mass spectrometry (ESI-MS /MS) ，we cloned the
cDNA of AFO using Switching Mechanism At 5 end of the RNA Transcript (SMART)Rapid Amplification of cDNA Ends
(RACE)technology and expressed this gene as a fusion protein in Pichia pastoris by using pPIC9-afo as vector． We
purified the fusion enzyme using nickel affinity chromatography． We identified the recombinant aflatoxin-oxidase (rAFO)
by both western blot and peptide mass fingerprinting (PMF)． Moreover，we characterized several enzymatic properties of
the rAFO using AFB1 as the substrate including Km value，optimum temperature，optimum pH，thermal stability and pH
stability． ［Results］ The AFO gene is 2321 bp long with a coding region of 2088 bp encoding 695 amino acids． Peptide
mass fingerprinting (PMF)identification showed a 63. 2% coverage of the molecule compared to the theoretical tryptic
cleavage of the rAFO． The recombinant aflatoxin oxidase was purified 5. 99-folds using nickel affinity chromatography． It
has a specific activity of 234 U /mg． Kinetics studies showed that the rAFO converted AFB1 with the Km value of 3. 93 ±
0. 20 × 10 － 6 mol / L under its optimal conditions of pH6. 0 and 30℃ ． Thermostability investigation revealed that the rAFO
had a half-life of 90 min at 30℃，and pH stability results suggested that the rAFO was relatively stable when pH ranged
from 5. 5 to 7. 5． ［Conclusion］ It appears to be the first successful production of the recombinant aflatoxin oxidase
(rAFO)with AFB1 -converting ability from Armillaria tabescens． The purified rAFO with preferably AFB1 -converting
activity confirms that this recombinant aflatoxin oxidase is now ready for further studying．
Keywords:Armillaria tabescens，Aflatoxin-oxidase (AFO) ，Rapid amplification of cDNA ends(RACE) ，Pichia pastoris
CLC number:Q814 Document code:A Article ID:0001-6209 (2011)09-1212-10
温思霞等:假蜜环菌黄曲霉毒素氧化酶的基因克隆、表达、纯化及酶学……(英文)． /微生物学报(2011)51(9)
Aflatoxins are highly toxic secondary metabolites
predominantly produced by Aspergillus flavus and
Aspergillus parasiticus［1 － 4］． The occurrence of
aflatoxins contamination is global with severe problems
especially prevalent in developing countries． Among
them，aflatoxin B1(AFB1) is one of the most potent
naturally occurring mutagens and carcinogens known．
Food contaminated with AFB1 poses a serious health
threat when consumed by human and animals， and
when present at unacceptable levels，the contamination
may also result in severe economic problems
worldwide［5 － 6］．
Elimination and reduction of aflatoxin in foods is a
persistent problem of growing concern to the
agricultural and food industry． But effective
decontamination of foods and feeds via the traditionally
used physical and chemical methods is not
economically feasible． Thus biodegradation of
aflatoxins，using microorganisms or enzymes，can be a
highly promising choice，since it is efficient，specific，
and environmentally friendly．
Some microbial isolates have been reported with
different levels of degradation abilities， and
experimental data had revealed that some of these
active ingredients might be enzymes［7 － 16］． However，
only a few researches reported on purification and
further characterization of these active enzymes［11］，
and there was no report on gene cloning and
recombinant expression of these enzymes．
In our previous study，intracellular extracts from
the edible fungus Armillaria tabescens was comfirmed
with AFB1-converting ability
［10］，moreover，an AFB1-
converting enzyme named aflatoxin-oxidase (AFO)by
the author was purified from the extracts， and
characterization of this AFO showed that it could
effectively concert AFB1 within 30min implying its
promising application prospect［17］．
In present study，in order to obtain large amounts
of this promising enzyme rapidly for its further
researches and potential applications，the gene of AFO
was cloned into Pichia pastoris with a His6-tag coding
sequences at the C termini． The engineered Pichia
pastoris with AFO gene was induced with methanol and
expression of recombinant AFO was confirmed by
western blot． Purification of the His6-tagged AFO was
done by nickel affinity chromatography． Afterwards，
purified enzyme was identified by peptide mass
fingerprinting ( PMF )． Finally， biochemical
characterization of the rAFO was performed including
determination of pH and temperature optima，substrate
kinetics and pH and temperature stability．
1 Materials and methods
1. 1 Materials
Armillaria tabescens (5. 0092) was purchased
from China General Microbiological Culture Collection
Center (CGMCC)． The reagents of RACE and PCR
were using Clontech and Qiagen protocols． Restriction
endonucleases and T4 DNA ligase were from NEB．
pMD18-T vector was bought from TaKaRa． Trizol and
Pichia expression kit were obtained from Invitrogen．
Proteins standard used as sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE )
markers were from Bio-Rad． For the enzyme assay，
AFB1 standard was purchased from Sigma． The
chemicals for HPLC were of HPLC grade and other
chemicals used were all of analytical grade．
1. 2 Total RNA extraction and full-length cDNA
cloning of aflatoxin-oxidase
Armillaria tabescens was cultured as below． The
culture medium used contained 0. 5% (w / v) malt
extract，1% (w / v) yeast extract，0. 1% K2HPO4，
0. 05% MgSO4 and 0. 01% vitamin B1，0. 01%(w / v)
CaCl2 ． And maltose(5%，w / v) ，glucose(5%，w /
v) ，fructose (2%，w / v) ，lactose (2%，w / v) or
sucrose(2%，w / v)was used as the carbon source．
The medium was brought to pH6. 0 with 0. 1N KOH
prior to sterilization． A spore suspension of the fungus
was prepared by adding sterile water (10 mL)to stock
cultures on potato dextrose agar (PDA )． The
suspension was then added to 100 mL medium in a
baffled flask． The latter was shaken at 180 r /min in an
orbital incubator at 28℃ for 7 days． The biomass
concentration was estimated at 18. 37 g /L by a dry
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weight measurement． Then the culture was prepared by
adding about 25 g wet mycelia (obtained by sterile
operation filtrated) to 250 mL medium in a baffled
flask． The flask was shaken at 200 r /min for 7 days at
27℃ ． Then the mycelia were harvested and frozen
under － 80℃ ． The frozen tissue was then ground to a
powder and the total RNA extracted using Trizol．
Since ten peptide fragments of AFO were identified
by Electrophoresis-Electrospray Ionization tandem mass
spectrometry (ESI-MS /MS)［17］，degenerated primers:
sense P1:5’-TGGGARGGNTTYACNGC-3’，antisense
G2:5’-GCNGTRAANCCYTCCCA-3’，sense G1:5’-
CARGAYGCNAAYGGNGA-3’ and antisense P2:5’-
TCNCCRTTNGCRTCYTG-3’ were designed based on
two of them， whose amino acid sequences were
WEGFTA and QDANGE，respectively． Then an internal
fragment of AFO gene termed as E1 was amplified by
reverse transcriptase-polymerase chain reaction (RT-
PCR)with total RNA as a template using the OneStep
RT-PCR kit (Qiagen)according to the manufacturers
instructions． The amplication product E1 extracted from
1% agarose gel was cloned by T /A ligation in the
pMD18-T vector (Takara ) and transformation of
Escherichia coli DH5α for DNA sequence confirmation．
Two specific primers: the primer S1 (5 ’-
TAGGCGAAGTGTCGTCGTCAATGGAA-3 ’) and the
primer S3 (5’-GAAGTTATCGGCTTTCCAGTCAGAGGGT
-3’)were designed based on the sequence of E1 for
amplification of the 5’- and the 3’- ends of AFO gene．
Then the 5’and 3’ends of AFO gene termed as E2 and
E3 respectively were obtained by using a SMARTTM RACE
cDNA Amplification Kit (Clontech，Austria)［18］． Both
5’- and 3’- amplification products were ligated into
pMD18-T vector (Takara ) and transformed into
Escherichia coli DH5α for sequencing． The full-length
cDNA sequence of AFO was obtained by jointing E1，E2
and E3 via the software DNAMAN． The AFO full-length
cDNA was composed of the 5’- noncoding region，the
open reading frame(ORF)and the 3’-noncoding region．
1. 3 Construction of yeast expression vectors
containing the ORF of AFO
The open reading frame (ORF) of AFO fused
with an extra His6-tag at the C-terminus was amplified
by PCR using the forward primer P3 (5 ’-
GTCGAATTCATGGCCACCACAACTGTC-3’， EcoRI
site underlined，and initiation codon in bold)and the
reverse primer P4 (5’-AAGGAAAAAAGCGGCCGCT
CAATGGTGGTGGTGATGGTGCAATCGTCTCTCAATG
AAACTTTC-3’，NotI site underlined，a His6 tag in
italic and termination codon in bold)． The PCR
reactions were carried out using Pfu DNA polymerase
with AFO 5’-ready cDNA as template under the
following conditions: initial denaturation at 94℃ for
5 min followed by 5 cycles at 94℃ for 30 s，72℃ for
4 min and 35 cycles at 94℃ for 30 s，68℃ for 4 min，
with a final extension at 72℃ for 10 min． Then the
PCR product designated as afo was cloned into yeast
vector pPIC9 downstream of α-factor signal peptide
sequence by EcoRⅠ and NotⅠ restriction sites． And
the recombinant vector after sequence confirmation was
designated pPIC9-afo．
1. 4 Recombinant AFO expression and Western
Blotting identification
For expression of recombinant AFO， histidine-
deficient Pichia pastoris strain GS115 was
electrotransformed with pPIC9-afo linearized by SalⅠ
according to the manual of Pichia expression kit
(Invitrogen)． His + -transformed methylotropic (His +，
Mut + ) yeast was selected using histidine-absent
medium containing dextrose (MD)or methanol (MM)
as the only carbon source．
Three randomly chosen transformants as well as an
empty vector control were precultivated in buffered
complex glycerol medium (BMGY) at 30℃ with
shaking at 200 r /min to early logarithmic growth
phase． The cells were harvested by centrifugation at
5000 × g for 15 min at 4℃ ． Then the cell pellets were
resuspended in 10 mL of buffered complex methanol
medium (BMMY)and added to 100 mL with BMMY
in a baffled flask to an initial OD600 of 2 － 6． The
production of recombinant AFO was conducted at 30℃
for 4 days with shaking at 200 r /min． Methanol was
added to the culture medium daily to a final
concentration of 0. 5% ．
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After 4-day methanol induction， the culture
supernatant was obtained by centrifugation at 5000 × g
for 15 min at 4℃ for sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and
western blotting identification． Supernatant samples
were separated on 12% SDS-PAGE according to the
method of Laemmli［19］． The gel was stained with
Coomassie brilliant blue R-250 and a low molecular
marker (Bio-Rad) was used for the estimation of
protein size．
For western-blotting， supernatant samples were
subjected to a SDS-PAGE as described above， then
proteins in the gel without stain were transferred onto
polyvinylidene difluoride (PVDF) membrane and
blocked by 5% dried skim milk． Subsequently，the
membrane was probed with horse-radish peroxidase
(HRP)-conjugated anti-penta-His Antibody (Qiagen)
and following washing steps， protein bands were
revealed by exposure to diaminobenzidine (DAB)
substrate (Qiagen)．
1. 5 Enzyme assay and protein determination
The activity of the rAFO was expressed by the
decrease of AFB1 between the reaction group and the
control group． For the reaction group，AFB1 (final
concentration，100 μg /L)was incubated at 30℃ for
30 min in a 200 μL assay mixture containing
0. 04 mol /L Na2HPO4-0. 02 mol /L citric acid buffer
(pH6. 0)and 10 μl of suitably diluted enzyme． The
reaction was terminated by mixing with 300 μL
methanol． For the control group，equal enzyme was
added after the reaction was pre-terminated by
methanol． For both reaction and control groups，the
residual AFB1 in the terminated mixture were extracted
using 500 μL chloroform for three times，then all the
extract were transformed to a new eppendorf tube and
evaporated under nitrogen gas． The evaporated residue
was dissolved in 200 μL mobile phase for the
quantitative analysis with HPLC． Meanwhile，
0. 125 μg － 2. 5 μg AFB1 standard were also dissolved
in 200 μL mobile phase and analyzed by HPLC for
quantitation of AFB1 ． HPLC analysis was performed on
a Shimadzu HPLC System (LC-20AT Prominence
Liquid Chromatograph，Japan)using a Shim-pack VP-
ODS C18 (150 mm × 4. 6 mm，4. 6 μm) column．
The mobile phase was methanol-acetonitrile-aquafer
(60 /150 /490，V /V /V)at a flow rate of 1ml /min and
the sample temperature was set at 40℃ ． AFB1 was
detected using a RF-10AXL fluorescence detector
(Shimadzu， Japan) with excitation wave length of
365 nm and emission wave length of 425 nm． Raw
data were evaluated by the HPLC LCsolution software
system (Shimauzu，Japan)．
One unit of the rAFO activity was defined as the
amount of enzyme that transforming 1 pmol of AFB1
equivalent per minute． Specific activities are expressed
as units per milligram of protein．
Protein concentrations were measured by the
method of Bradford with bovine serum albumin (BSA)
as the standard．
1. 6 Purification of recombinant AFO
For purification of the recombinant AFO，
transformant whose supernant with higher special
activity was subjected to a high cell density
fermentation using a 5L jar fermenter (BTF-5L，
Biotop，China)． The whole process of fermentation
was consist of three stages; (i)a glycerol batch phase
generating biomass; (ii) a glycerol feed phase for
continued biomass generation and derepression of the
alcohol oxidase promoter AOX1;and (iii)a methanol
feed phase for induction of expression［20］． The
transformant was cultivated at 30℃ in 2 L of basal salt
medium (BSM ) containing 0. 01% antifoam．
Methanol induction fermentation was maintained for
77 h after glycerol batch and fed-batch phases．
Purification procedures were carried out at 4℃
unless otherwise stated． Broth were harvested after
nearly 4 days of fermentation (centrifugation，10000 × g，
4℃， 30 min) ，and the supernatant was filtrated
through membrane filters with pore size of 0. 45 μm
(CFP-4-E-4X2MA，GE USA)． The filtrate with the
volume of 1800 mL was concentrated about 20-fold and
buffer-exchanged into equilibrium buffer (0. 05 mol /L
Tris-Cl pH8. 0)using a Kvick Lab SCU (molecular
weight cutoff of 10 kDa，GE，USA)． The concentrated
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enzyme was applied to AKTA explore100 (GE
Healthcare)equipped with a HisTrap HP 1 mL column
pre-equilibrated with equilibrium buffer (0. 05 mol /L
Tris-Cl pH8. 0)． After sample was loaded on， the
column was washed with equilibrium buffer (10 mL)
followed by the same buffer containing 10 mmol /L
imidazole (10 mL)at a fiow rate of 1 mL /min． Then
His6-tag fused rAFO were eluted with equilibrium
buffer containing 500 mmol /L imidazole． Active
fractions were combined and concentrated using an
Amicon Ultra-15 (molecular weight cutoff of 30 kDa，
Millipore)．
1. 7 Peptide mass fingerprinting (PMF )
identification of purified rAFO
The purified rAFO was subjected to peptide mass
fingerprinting ( PMF ) analysis for further
identification． The electrophoresis strip of the rAFO
was excised from the Coomassie stained gel and the gel
pieces were subjected to DTT reduction and alkylation．
Digestion was performed with trypsin，and aliquots of
the supernatants were then incorporated into a fast
evaporation nitrocellulose matrix with α-cyano-4-
hydroxy cinnamic acid as the matrix substance． The
mass spectrometric measurements were performed on a
Reflex MALDI-TOF mass spectrometer (Bruker，
Germany)equipped with an ion gate and pulsed ion
extraction． The acceleration voltage was set to 20 kV
and the reflector voltage 23 kV． This work was
performed by the National Center of Biomedical
Analysis， Academy of Military Medical Sciences，
China．
1. 8 Biochemical characterization of purified rAFO
Investigation to find out the optimum pH for the
rAFO activity was carried out in Na2HPO4-citric acid
buffers with various pH values (pH4. 0 － 8. 0)． While
the optimum temperature for the rAFO activity was
determined by incubating the enzyme in 0. 04 mol /L
Na2HPO4 － 0. 02 mol /L citric acid buffer (pH 6. 0)at
different temperatures (20℃ － 50℃) . Other
procedures involved in activity determination were all
the same as the standard assay．
Then activities of the rAFO were measured under
the optimal conditions determined above at different
AFB1 concentrations range from 100 μg /L －
300 μg /L． The Vmax and Km value were calculated
using a Lineweaver-Burk plot．
On stability studies，the rAFO was incubated in
Na2HPO4-citric acid buffers with various pH values
(pH4. 0 － 8. 0)at 30℃ for 90 min to determine its pH
stability，and then remaining activities of these treated
enzymes were measured by the standard assay
procedure． For thermal stability determination， the
purified rAFO in 0. 04 mol /L Na2HPO4 － 0. 02 mol /L
citric acid buffer (pH 6. 0)was incubated at different
temperatures(20℃ － 50℃)for 90 min． These treated
enzymes were subject to the standard enzyme assay
after being cooled on ice for 30 min．
2 Results
2. 1 Full-length cDNA and ORF cloning of the AFO
gene
Fig． 1 PCR amplification of the open reading frame (ORF)of
AFO． M:DNA ladder;AFO:product of the open reading frame
(ORF)amplification by PCR．
An 803 bp fragment of the AFO cDNA named E1
was isolated by RT-PCR using the degenerated primers
and total RNA as the template． Based on the sequence
information of E1，specific primers for the enzyme were
designed and used for RACE-PCR to obtain the 5’-
and 3’-cDNA ends． And PCR products of 3’- and 5’
RACE termed as E2 and E3 were 750 bp and 1530 bp
respectively． By joining E1，E2 and E3， the full-
length cDNA of AFO was 2321 bp long with a coding
region of 2088 bp，corresponding to a protein of 695
amino acid residues． This cDNA sequence of AFO was
deposited in GenBank (Accession No． AY941095)．
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ORF cloning of AFO gene fused with an extra His6-tag
at the C-terminus revealed a PCR product of 2105 bp
(Fig． 1)whose sequence was completely in accordance
with that of joined full-length cDNA．
2. 2 Expression，and Western Blotting identification
of the rAFO
Since the recombinant secretion vector pPIC9-afo
was introduced into Picha． pastoris by homologous
recombination at the chromosomal AOX1 promoter
locus． The secretive expression of the rAFO was
performed under the induction of methanol in shaking
flask culture． The culture supernatants of three
randomly selected transformants were sampled for the
western blotting identification． As illustrated in Fig． 2，
a single band was detected in two of the three
transformants，while it was absent in both the empty
vector control and transformant 3． The special activities
of the supernatant of the transformant 1 and 2 were
determined as described in section 2. 5． Quantitation
analysis showed that AFB1 has a good linear relation
with peak area score when its concentration was within
the range of 25 μg /L － 500 μg /L (Y = 0. 0003X －
4. 0801，R2 = 0. 999)． Activity determination revealed
that the specific activities of the supernatant of the
transformant 1 and 2 were 6. 1 U /mg and 4. 3 U /mg
respectively． Thus transformant 1 with higher specific
activity was chosen for further experiments．
Fig． 2 Identification of rAFO by western blotting． Cell-free
supernatant of transformant 1 (lane 1) ;transformant 2(lane 2) ;
transformant 3(lane 3) ;empty vector control(lane 4) ;M:protein
marker． The rAFO-sepecific bands are indicated by an arrow
(approximately 76 kDa)． For each sample 1 μg ． supernatant was
loaded in the tracks of the gel．
2. 3 Purificaion and PMF identification of the rAFO
The transformant 1 was cultivated in a jar
fermenter for the rAFO production． Supernatant of
1800mL was harvested after 77-hour methanol
induction fermentation and then concentrated about 20-
fold for subsequent affinity chromatography
purification． Table 1 summarizes the results of a typical
purification procedure． The rAFO was purified 5. 99-
folds from culture supernatant with a yield of 61. 3% to
a specific activity of 234 U /mg． The apparent
molecular mass of the purified rAFO was estimated to
be 76 kDa by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) (Fig． 3)．
Fig． 3 Purification of rAFO． M:protein marker;Lane 1:cell-free
supernatant induced by methanol for 77 h;Lane 2:Eluant from
HisTrap FF column with equilibrium buffer;Lane 3:Eluant from
HisTrap FF column with 10 mmol / L imidazole;Lane 4:10-fold
concentrated eluant from HisTrap FF column with 500 mmol / L
imidazole． For each sample，1 μg was loaded in the tracks of the gel．
The purified rAFO was analyzed by peptide mass
fingerprinting (PMF) after trypsin digestion． The
peptide mass fingerprint was submitted to protein
database and searching results revealed a 63. 2%
coverage of the molecule when compared to the
theoretical tryptic cleavage of the rAFO，indicating the
correct expression of the rAFO．
Table 1 Summary of the purification of recombinant AFO
Step
Total
activity
(U)
Total
protein
(g)
Specific
activity
(U /mg)
Recovery
(%)
Purification
factor
Crude extract 46566 1. 19 39 100 1
Concentrated
crude extract
38044 0. 87 44 81. 7 1. 13
Histrap FF 28545 0. 12 234 61. 3 5. 99
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2. 4 Biochemical characterization of purified rAFO
Enzyme kinetics of purified rAFO was investigated
since it was correctly expressed with activity． Firstly，
activity of the rAFO was determined under various pH
values (pH4. 0 － 8. 0) and temperatures (20℃ －
50℃)with AFB1 as the substrate． Results suggested
that the optimal pH and temperature of the rAFO were
pH6. 0 (Fig． 4A)and 30℃ (Fig． 4B)respectively．
Then enzymatic kinetics of the rAFO was investigated
under the optimal conditions． Enzyme activity was
measured under different AFB1 concentrations range
from 100 μg /L － 300 μg /L． Vmax was determined as
1. 096 ± 0. 06 pmol /min and the Km value was 3. 93 ±
0. 20 × 10 － 6 mol / L calculated by the Lineweaver and
Burk plotting method．
On stability of the enzyme，the assays of enzyme
resistance to various pH indicated that rAFO could
preserve half or more of its activity in weak acid
solution (pH5. 5 － 6. 5) at 30℃ for 90 min (Fig.
4C)． And the thermostability investigation revealed
that this recombinant enzyme had a half-life of 90 min
at 30℃ but was deactivated when treated at 45℃ for
90min (Fig． 4D)．
Fig． 4 Biochemical characterization of purified rAFO． (A)Optimum pH，10 μL purified enzyme
was applied for activity determination under buffers with different pH value (pH4． 0 － 8. 0)at 30℃;
(B)Optimum temperature，10 μL purified enzyme was applied for activity determination under
different temperatures (20℃ － 50℃) at pH6. 0; the special activity was calculated; (C) pH
stability; (D) thermal stability． For each stability experimental，purified enzyme was was treated
under different conditions for 90 min，then the treated enzyme was subjected to enzyme assay under
standard conditions． Residual activity was expressed as percentage of activity of untreated enzyme． All
experiments were performed in duplicate．
3 Discussion
Recently，interests in biotransformation of aflatoxin
B1(AFB1)have greatly increased． Some active extracts
or enzymes had been reported with AFB1 degradation
function． However， many of them were limited to
practical applications by either cofactor requirement［21］
or long incubation time of 24 h or longer［7 － 8］．
Aflatoxin-oxidase (AFO ) isolated from Armillaria
tabescens was firstly named aflatoxin-detoxifizyme
(ADTZ ) for its AFB1-converting ability by our
group［17］． Unlike other reported AFB1-convergting
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enzymes，AFO could effectively convert AFB1 without
any cofactors within 30min revealing a greatly potential
application of this enzyme． Thus gene cloning and
heterogenous expression of AFO were done for further
research on its structure and function．
After the gene of AFO was cloned，a pairwise
alignment was done between the amino acid sequences
of the ten native AFO peptides and of the recombinant
AFO，results indicated that six of these ten peptides
were well matched． However，activity determination of
the recombinant aflatoxin-oxidase (rAFO)provided the
evidence that the rAFO could convert AFB1，and the
peptide mass fingerprinting (PMF)identification of the
rAFO was in accordance with the pairwise alignment
result． It could be considered that the rAFO had been
successfully cloned and expressed． Since sample of
native AFO for mass spectrometry (MS)identification
was obtained from the native polyacrylamide gel
electrophoresis (PAGE)［17］，this meant that the purity
of the purified native AFO used for MS identification
was approximate 95% ． Therefore，the four peptides of
AFO poorly matched in the pairwise sequence
alignment with the rAFO might be due to the impurity
proteins of the native AFO sample．
On the other hand，the amino acid sequence of
the rAFO was submitted to protein databases of NCBI
for a protein blast． And result showed that the enzyme
was similar to predicted basidiomycete dipeptidyl
peptidases III with identities above 70% ． However，
electrochemical experiments using the rAFO-modified
electrode as detecting element showed that rAFO could
oxidize AFB1 accompanying with hydrogen peroxide
formation［22］． This leds us to believe that this enzyme
was an oxidase，thus it was renamed aflatoxin-oxidase
(AFO)．
Unfortunately， we encountered difficulties in
isolating and identifying the oxidized product of AFB1
by the rAFO． For clearly interpretation of the catalytic
mechanism of the rAFO，attempts are still being made
to obtain and identify this oxidized product
notwithstanding．
In addition，the rAFO could convert AFB1 under
mild conditions within relatively short time，indicating
its promising application． But the pH and temperature
stability of this enzyme was inadequate to meet
practical application conditions． Thus we are also
making efforts on enzymatic feature improvement by
molecular directed evolution．
In conclusion， A gene encoding an enzyme
afiatoxin-oxidase (AFO) with aflatoxin conversion
ability was successfully cloned from Armillaria
tabescens，and expressed in P． pastoris． This has made
it much easily to produce this enzyme in an industrial
scale． And characterization of the rAFO suggested its
potential application． Further researches on elucidating
the enzymatic reaction mechanism and on improving
enzymatic features by directed evolution might be
greatly helpful in its industrial application．
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0221
温思霞等:假蜜环菌黄曲霉毒素氧化酶的基因克隆、表达、纯化及酶学……(英文)． /微生物学报(2011)51(9)
假蜜环菌黄曲霉毒素氧化酶的基因克隆、表达、纯化及酶学性
质分析
温思霞1，管敏1，周涛1，曹红1，谢春芳1，2，刘大玲1，2，姚冬生1，3*
1暨南大学微生物技术研究所，2 广东省生物工程药物重点实验室，3 基因工程药物国家工程研究中心，广州 510632
摘要:【目的】黄曲霉毒素氧化酶(aflatoxin-oxidase，AFO)来源于假蜜环菌(Armillariella tabescens)的细胞内提
取物，具有转化黄曲霉毒素 B1(Aflatoxin B1，AFB1)的特性。为更进一步了解该酶的性质，我们克隆了 AFO
的基因，并进行了重组 AFO 蛋白的表达、纯化和酶学性质分析。【方法】本研究利用基质辅助激光解吸飞行
时间质谱(MALDI-TOF-MS)获得的 AFO 短肽序列设计简并引物进行逆转录，再通过 cDNA 末端快速扩增
(rapid-amplification of cDNA ends，RACE)技术获得了 AFO 基因的全长 cDNA 序列。构建重组表达载体
pPIC9-afo，在毕赤酵母中进行重组 AFO(rAFO)的融合分泌表达，用 Ni 离子螯合层析进行 rAFO 的纯化，获得
有活性的 rAFO 后，对其进行肽质量指纹(peptide mass fingerprinting，PMF)鉴定和酶学性质分析。【结果】黄
曲霉毒素氧化酶(AFO)基因的开放阅读框为2088 bp，编码 695 个氨基酸;肽质量指纹鉴定结果显示重组
AFO 的肽片段序列覆盖率为 63. 2%。活性测定表明纯化后的重组 AFO(rAFO)比活力为234 U /mg;对 rAFO
进行酶学性质分析表明，对于底物黄曲霉毒素 B1，rAFO 的 Km 值为 3. 93 ± 0. 20 × 10
－ 6 mol / L;反应最适温度
为 30℃，最适 pH 为 6. 0;30℃放置90 min后酶活力下降 50%;rAFO 在 pH5. 5 － 7. 0 之间酶活力较稳定，相对
活力维持在 51% － 65%之间。【结论】本文第一次成功克隆并重组表达了一种具有黄曲霉毒素 B1 转化功能
的酶———黄曲霉毒素氧化酶(aflatoxin-oxidase，AFO) ，纯化后的重组 AFO(rAFO)具有较好的黄曲霉毒素 B1
转化活性，为进一步研究和应用奠定了基础。
关键词:假蜜环菌，黄曲霉毒素氧化酶，cDNA 末端快速扩增，毕赤酵母
中图分类号:Q814 文献标识码:A 文章编号:0001-6209 (2011)09-1221-10
(本文责编:王晋芳)
基金项目:国家“863 计划”(2005AA213010) ;国家自然科学基金(30270043)
* 通信作者。Tel / Fax:+ 86-20-85228422;E-mail:tdsyao@ jnu． edu． cn
作者简介:温思霞(1984 －) ，女，广东梅州人，硕士研究生，研究方向为分子微生物学。E-mail:wensixia2008@ 163． com
收稿日期:2011-03-03;修回日期:
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