Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 803-811
doi:10.1111/j.1745-7270.2006.00231.x
Design and Expression of a
Synthetic phyC Gene Encoding the Neutral Phytase in Pichia pastoris
Li-Kou ZOU1,3,
Hong-Ning WANG2,1*,
Xin PAN4,
Tao XIE1,
Qi WU5,
Zi-Wen XIE1,
and Wan-Rong ZHOU1
1 Laboratory
of Veterinary and Biotechnology, Sichuan Agricultural University, Ya’an 625014,
China;
2 Sichuan
University Bioengineering Research Center for Animal Disease Prevention and
Control, Chengdu 610065, China;
3 Faculty
of Environment and Resource of Dujiangyan Campus, Sichuan Agricultural
University, Dujiangyan 611830, China;
4 College
of Earth Science, Chengdu University of Technology, Chengdu 610059, China;
5
College of Life Science, Sichuan Agricultural University, Ya’an 625014, China
Received: July 4,
2006
Accepted: September
10, 2006
This work was supported
by the grants from the National Key Technologies R&D Program of China
during the 10th Five-Year Plan Period (No. 2002BA514A-12) and Innovative Fund
for Distinguished Young Scholars of Sichuan Agricultural University
*Corresponding
author: Tel/Fax, 86-28-85471599, 86-835-2886083; E-mail, [email protected]
Abstract The 1074-bp phyCs gene (optimized phyC gene)
encoding neutral phytase was designed and synthesized according to the
methylotrophic yeast Pichia pastoris codon usage bias without altering
the protein sequence. The expression vector, pP9K-phyCs, was linearized
and transformed in P. pastoris. The yield of total extracellular phytase
activity was 17.6 U/ml induced in Buffered Methanol-complex Medium (BMMY) and
18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale,
respectively, improving over 90 folds compared with the wild-type isolate.
Purified enzyme showed temperature optimum of 70 ?C and pH optimum of 7.5. The
enzyme activity retained 97% of the relative activity after incubation at 80 ?C
for 5 min. Because of the heavy glycosylation the expressed phytase had a
molecular size of approximately 51 kDa. After deglycosylation by
endoglycosylase H (EndoHf), the enzyme had an apparent molecular
size of 42 kDa. Its property and thermostability was affected by the
glycosylation.
Key words design; expression; synthetic phyC gene; neutral
phytase; Pichia pastoris
Cereals, legumes, and oilseed crops are grown in over 90% of the
world? harvested
area. These crops serve as a major source of nutrients for human and animals.
An important constituent in these crops is phytic acid (myo-inositol
hexaphosphate). The salt form, phytate, is the major storage form of phosphorus
and accounts for more than 80% of the total phosphorus in cereals and legumes
[1,2]. Simple-stomached animals such as swine and poultry, are incapable of
using phytate phosphorus due to little phytase (myo-inositol
hexaphosphate phosphohydrolase, EC 3.1.3.8 and 3.1.3.26) activity in their
gastrointestinal tracts [3,4], necessitating supplementation of the feed with
inorganic phosphorus. Phytic acid also acts as an antinutritional agent in
simple-stomached animals by chelating various metal ions needed by the animal,
such as calcium, copper, and zinc [5,6].Phytases catalyze the hydrolysis of phytate, thereby releasing
inorganic phosphate [7,8]. Supplemental microbial phytase in corn-soybean meal
diets for swine and poultry effectively improves phytate phosphorus use by
these animals, decreases the addition of phosphorus of feedstuffs and reduces
their fecal phosphorus excretion pollution by up to 50% [9–11].Many phytases, including fungal phytase from Aspergillus spp.
(phyA and phyB) and bacterial phytase from Escherichia coli (appA),
have been cloned, characterized and expressed in the host of Pichia pastoris and Saccharomyces cerevisiae
[12–17]. These phytases are histidine acid phosphatases, which exhibit
an optimum pH from 2.5 to 5.5, and have a pH activity profile ideally suited
for maximal activity in the digestive tract of either pigs or poultry. The
phytases from Bacillus spp. (phyC) are beta-propeller phytases
which exhibit an optimum pH from 6.0 to 9.0, suitable for neutral animal tracts
such as trout and cyprinids, and is more thermostable [18–20]. The phytase
has been expressed in E. coli and B. subtilis [19,20], but the
intracellular expression and endotoxin in E. coli, the instability of
plasmid and the protease from B. subtilis restrict its production.We have cloned the phyC gene from B. subtilis and
expressed it in E. coli and P. pastoris [21,22]. Based on this,
we synthesized the neutral phytase gene without altering the protein sequence
according to the P. pastoris codon usage bias. The recombinant phytase
has been characterized and compared with phytase expressed in E. coli and
P. pastoris. Our aim was to study the property of the enzyme and to
develop high production of phytase.
Materials and Methods
Strains, plasmids and medium
E. coli JM109 was used as a host for
sub-cloning. P. pastoris GS115 was used as a host for expression, and
GS115 albumin and b-gal as controls. Plasmid pMD18-T was used as a vector for cloning
and sequencing, and plasmid pPIC9K was used for expression. E. coli was
cultured in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, 0.5%
NaCl) or on LB agar plate. When needed, ampicillin/kanamycin was added at a
concentration of 100/80 mg/ml. The expression strains were screened in minimal dextrose (MD)
medium [1.34% yeast nitrogen base (YNB), 0.00004% biotin, 2% dextrose] and
minimal methanol (MM) medium (1.34% YNB, 0.00004% biotin, 0.5% methanol). Yeast
was cultured in yeast extract peptone dextrose (YPD) medium (1% yeast extract,
2% peptone, 2% dextrose, 2% agar), buffered glycerol-complex (BMGY) medium (1%
yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB,
0.00004% biotin, 1% glycerol) and induced in buffered methanol-complex (BMMY)
medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34%
YNB, 0.00004% biotin, 1% methanol).Wheat Bran extract grown (WBEG) and wheat Bran extract induction
(WBEI) media were our substitute media for growing and induction. WBEG medium was
prepared by dissolving 100 g of malt powder and 15 g of wheat bran in 600 ml of
distilled water, incubated at 65 ?C with constant stirring every 15 min until
the starch hydrolyzed completely. The extract was filtered through a piece of
cheesecloth, and the volume was adjusted to 600 ml with distilled water and was
followed by autoclaving at 121 ?C for 20 min.WBEI medium was prepared by dissolving 100 g of malt powder and 15 g
of wheat bran in 600 ml of distilled water, boiled for about 5 min, incubated
at 90 ?C with constant stirring every 15 min for about 3 h. The extract was
filtered through a piece of cheesecloth, and the volume was adjusted to 600 ml
with distilled water and was followed by autoclaving at 121 ?C for 20 min.
After cooling to room temperature, methanol was added to a final concentration
of 1.5% (V/V).
Design and synthesis of the phyCs
gene
Design and synthesis of the phyCs
gene
The nucleic acid sequence of the synthetic gene was designed from
the amino acid sequence of neutral phytase based on the P. pastoris
preferred codons [23,24]. The DNAStar program was used to analyze the GC
content, AT-rich stretches and the restriction enzyme sites of the resulting
DNA sequence. In addition, the RNAstructure 4.2 program, Rensselaer
bioinformatics system (http://www.bioinfo.rpi.edu), Apache (http://rna.tbi.univie.ac.at),
GeneBee service (http://www.genebee.mus.su) and CodonW (http://bioweb.pasteur.fr/seqanal/interfaces/codonw.html)
were used to analyze the mRNA structure, mRNA 5‘– and 3‘-untranslated
region (UTR) and DG of the folding mRNA. The synthetic gene was designed with SnaBI
and NotI restriction enzyme sites at the 5‘ and 3‘
terminal, respectively. The resulting sequence with the full length of 1074 bp
(Fig. 1) was successfully synthesized by Shanghai Sangon Bioengineering
Company (Shanghai, China) and cloned into pMD18-T named pMD-phyCs. The
final synthetic gene, phyCs, was sequenced.
Construction of expression
vector pP9K-phyCs
The DNA manipulations were carried out by using standard procedures.
All endonucleases were from TaKaRa (Dalian, China) unless stated otherwise.The 1074-bp synthetic gene sequence fragment, phyCs, was
prepared by digestion of pMD-phyCs with restriction endonucleases SnaBI
and NotI, followed by agarose gel electrophoresis resolution and
purification using the TaKaRa agarose gel DNA purification kit version 2.0. The
expression vector was prepared by analogous procedures. The purified phyCs
fragment was ligated to the purified SnaBI-NotI double-digested
secretory expression vector pPIC9K with the correct orientation using TaKaRa
DNA ligation kit version 2.1. E. coli strain JM109 was transformed with
the ligation products. Bacterial transformants were selected for their ability
to grow in LB medium in the presence of both 100 mg/ml ampicillin and 80 mg/ml kanamycin.
The bacterial transformants were screened by colony-PCR using the primers phyCsA
(5‘–TACGTAAAGCACAAGTTGTCTGACC-3‘)
and phyCsB (5‘-GCTTACTTACCAGATCTGTCAGTCAAC-3‘). The recombinant expression vector,
pP9K-phyCs, was prepared using a plasmid miniprep kit (W), identified by
SnaBI-NotI double-digested restriction analysis and sequenced by
TaKaRa.
Transformation and selection
of P. pastoris expression strains
pP9K-phyCs used for transformation, was linearized by SacI.
P. pastoris GS115 strains were made competent and transformed with SacI-linearized
pP9K-phyCs by electroporation following the manufacturer’s
recommendations (Manual of multi-copy Pichia expression kit, Invitrogen,
Carlsbad, USA). About 10 mg of linearized DNA pP9K-phyCs and 80 ml of competent
GS115 cells were used for each transformation. Transformants were plated onto
MD plate and incubated at 28 ?C for 3–4 days. All plates were checked daily. Using a
sterile toothpick, a single colony was selected and the His+ transformant was patched in a regular pattern on both MM plates and
MD plates, ensuring to patch the MM plate first. For testing the effectiveness
of expression conditions, GS115 albumin and GS115 b-gal were grown as a
control.
Expression of recombinant
neutral phytase
Single colonies and recombinant P. pastoris expression
strains were grown at 28 ?C in 10 ml BMGY/WBEG medium in 100-ml flasks for 16–20 h with
vigorous shaking. Next, 3% (V/V) cells were inoculated into 100
ml BMGY/WBEG medium. Cells were grown at 28 ?C for 17–20 h and shaken at 225–250 rpm until an
A600 value of approximately 20 had been reached, then harvested by
centrifugation at 3000 g and 4 ?C for 5 min. Supernatant was decanted
and the cell pellet was washed with potassium phosphate buffer (100 mM, pH
6.0). The pellet was resuspended in 25 ml induction medium BMMY/WBEI in
separate 250 ml flasks. Methanol (1.5%, V/V) was added to the
flask every 12 h in order to induce phytase production during the induction
period. Supernatants were taken after induction for 24 h, 48 h, 72 h, and 96 h
for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
analysis.
Purification of recombinant
neutral phytase
All purification steps were carried out at 4 ?C unless otherwise
stated.Recombinant P. pastoris strains induced in WBEI were
collected by centrifugation at 5000 g for 15 min. CaCl2 was added to a final concentration of 2 mM in the collected
supernatant. Initial purification was achieved by mixing the culture
supernatant with three volumes of cold (–20 ?C) enthanol. Constant
stirring was carried out for 30 min, and the precipitation was continued
overnight at –20 ?C. The precipitate was collected by centrifugation at 1800 g
for 20 min. The collected precipitate was washed once with cold ethanol and
once with cold acetone. The drying was completed at room temperature. Dried
precipitate was dissolved in 50 mM Tris-HCl (2 mM CaCl2, 10%
glycerol, pH 7.5). Then ammonium sulfate was added slowly with constant
stirring to give 45% saturation. The solution was incubated overnight and
centrifuged at 10,000 g for 15 min, and the supernatant was collected.
The supernatant was dialyzed against 25 mM Tris-HCl (pH 8.1, 2 mM CaCl2) overnight and concentrated by PEG20000. Aliquots of enzyme
preparation were stored at –20 ?C. Final purification of the enzyme preparation was performed by
Chromatography Systems BioLogic LP and BioFracTM Fraction
Collector (Bio-Rad, California, USA). Three milliliters of the thawed sample
was passed through an Econo-PacR High Q Cartridge column
equilibrated with buffer A (25 mM Tris-HCl, 2 mM CaCl2, pH 8.1).
The bound protein was eluted with buffer B (25 mM Tris-HCl, 2 mM CaCl2, 0.5 M NaCl, pH 8.1) using the following gradient: 0–10 min, 100% A;
10–22
min linearly increased from 0% to 50% B; 22–27 min, 50%–100% B; and all
steps were at a flow rate of 1.5 ml/min. The collected protein fraction was
stored at –20 ?C for the next step analysis.
Recombinant phytase activity
assay
The crude enzyme preparations and culture supernatant after
induction were assayed for phytase activity. All chemicals were analytical
reagents and purchased from Shanghai Sangon Bioengineering Company or TaKaRa.
Phytase activity was assayed by measuring the rate of increase in inorganic
orthophosphate (Pi) using the method as described by Mikio Shimizu [25], Kim et
al. [26] and Bae et al. [27]. One unit of enzyme activity was
defined as the amount of enzyme hydrolyzing 1 mmol of Pi per minute under
assay conditions. The crude enzyme was diluted in suitable volume of 50 mM
Tris-HCl buffer (2 mM CaCl2, 10% (v/v)
glycerol, pH 7.5). All enzyme assays were run in duplicate. A reaction
mixture containing 100 ml enzyme preparation and 400 ml 2 mM sodium phytate in
100 mM Tris-HCl buffer (2 mM CaCl2, pH 7.0) was incubated at 37
?C for 30 min. The reaction was stopped by adding 500 ml of 15% (W/V)
trichloroacetic acid (TCA). The released inorganic phosphate was measured by
adding 1 ml of a coloring reagent (freshly prepared by mixing four volumes of
1.5% (W/V) ammonium molybdate in a 5.5% (V/V)
sulfuric-acid solution and one volume of a 2.7% (W/V) ferrous
sulfate solution). The control was incubated with TCA at 37 ?C for 30 min. And
the solution’s absorbance at 700 nm was measured.
Property of recombinant
neutral phytase
Enzyme activity assays were performed in defined buffers for various
pH and temperature tests. The optimal pH of the expressed phytase was
determined (37 ?C) using buffers of 0.1 M glycin-HCl (pH 2.0 and 2.5), 0.1 M
sodium citrate (pH 3.5, 4.0, 5.0 and 5.5), 0.1 M Tris-maleate (pH 6.0 and
6.5), 0.1 M Tris-HCl (pH 7.0, 7.5, 8.0 and 9.0), and 0.1 M glycin-NaOH (pH
10.0, 11.0, 12.0 and 13.0). The purified enzyme was diluted in assay buffers
with 2 mM CaCl2. The optimal temperature (pH 7.0) was
determined at 16, 26, 37, 45, 50, 55, 60, 65, 70, 75, 80, 90 and 100 ?C. For
enzyme thermostability tests, the enzyme was incubated at seven different
temperatures (37, 75, 80, 85, 90, 95 and 100 ?C) in buffer of 50 mM Tris-HCl [2
mM CaCl2, 10% (V/V) glycerol, pH 7.5] for 5 min and 10 min and
then cooled to room temperature before the enzyme assay.
Deglycosylation and SDS-PAGE
analysis
Endoglycosylase H (EndoHf, New England Biolabs,
Beijing, China) was used to deglycosylate the phytase. The reaction was carried
out by incubating purified phytase with 100 U EndoHf in 0.5 M
sodium citrate (pH 5.5) for 5 h at 37 ?C and stopped by chilling at 4 ?C. After
the reaction, the mixture was subjected to SDS-PAGE. The molecular weight was
determined by using 4.4%–12.5% gradient SDS-PAGE. The properties of the enzyme before and
after deglycosylation were compared.
Results
Design and synthesis of phyCs
gene
The synthetic gene and expression vector inserted the synthetic gene
encoding phytase are shown in Fig. 2. The synthesized 1074-bp phyCs
gene showed 73.5% homology with the wild type phyC gene (Fig. 1).
All codons in phyCs were designed to be preferential for methylotrophic
yeast P. pastoris. The GC content was improved from 46.2% to 49.1%
compared with the original phyC gene. AT-rich stretches were eliminated
to avoid premauture termination. The mRNA secondary structure around the AUG start codon was
adjusted, so that AUG is relatively free of the secondary structure to ensure
efficient translation of mRNA as predicated by the RNA fold software analysis (Fig.
3). The DG of the folding mRNA was increased to 235.7 kkcal/mol, which is
higher than that of the original mRNA –244.0 kkcal/mol.
Activity of recombinant
phytase produced from P. pastoris strains
From hundreds of transformants of P. pastoris, one colony
that exhibited the highest phytase activity among the colonies examined was
selected for shake-flask expression. As shown in Fig. 4, after 120 h of
methanol induction, the yield of total extracellular phytase activity was 17.6
U/ml induced in BMMY and 18.5 U/ml in WBEI medium at the flask scale,
respectively, increased over 90-fold compared to the wild-type isolate B.
subtilis WHNB02 [28].
Purification and property of
recombinant phytase
The expressed protein was obtained in gradual resolved fractions,
four of these demonstrated high phytase activity (18 to 21; Fig. 5).
Therefore, the four fractions were taken together for enzymatic property
analysis. Before and after deglycosylation, the enzymatic activity of the
expressed phytase both showed temperature optima of 70 ?C as shown in Fig.
6(A). They both showed high relative activity at pH between 6.0 and 9.0 and
optima pH of 7.5, as shown in Fig. 6(B). However, less phytase activity
was detected at pH below 4.0 and above 11.0. The glycosylated and
deglycosylated enzyme activity retains 97% and 77% respectively, of the
relative activity after incubation at 80 ?C for 5 min. At 80 ?C for 10 min the
phytase lost 17% and 31% of its activity, as shown in Fig. 6(C).
SDS-PAGE analysis of culture
supernatant and deglycosylated product
The molecular mass of the mature phyCs phytase was determined
by SDS-PAGE. As shown in Fig. 7(A), phyCs phytase demonstrated an
apparent molecular size of nearly 51 kDa in SDS-PAGE. Three major bands were
found at 47.95 kDa, 50.23 kDa and 51.17 kDa after 24 h induction. The
glycosylation of P. pastoris phytase appears to have increased with
induction time. After deglycosylation by EndoHf, the
enzyme had an apparent molecular size of 42 kDa [Fig. 7(B)]. Thus, the
percentage of glycosylation was approximately 14.17%, 19.6% and 21.83%.
Discussion
Although phytase genes have been cloned from plants, animals, bacteria
and fungi, and expressed in different hosts, many of these are acidic phytases
belonging to the histidine acid phosphatases families and exhibit an optimum pH
of below 7.0, which is only suitable for animal acid digestive tracts such as
swine and poultry. The phytases from Bacillus spp. are beta-propeller
phytases, which exhibit an optimum pH around 6.0 to 9.0 and is suitable for
neutral animal tract such as trout and cyprinids and are more thermostable.
However, low expression levels in wild type isolates restrict its production.The synthesized 1074-bp phyCs gene showed 73.5% homology with
the wild type phyC gene. In synthetic gene, 246 bases were changed
compared with the original phyC gene. Almost 202 amino acid codons were
substituted by the P. pastoris optimal codons including 32 amino acid
codons whose relative synonymous codon usage (RSCU) was 0 in the original phyC
gene [23]. To eliminate the AT-rich stretches, the GC content was improved from
46.2% to 49.1% in comparison to the original phyC gene. In addition to
codon usages, the free energy and secondary structure also affected the
expression level. The more stable the mRNA second structure, the lower the mRNA
free energy. Therefore the free energy of mRNA was increased compared with the
original gene. Secondary structure in the mRNA has a negative effect on
expression of the recombinant protein, so online service was adopted to
analyze the 5‘-UTR of the mRNA for secondary structure formation to
ensure efficient translation of mRNA. An AUG sequence should be avoided in the
loop of the 5‘-UTR to ensure efficient translation of mRNA from the
actual translation initiation site. This was accomplished by redesigning the
initial portion of the coding sequences with yeast preferential codons.Kerovuo and Tynkkyen [29] reported that the phyC gene was
expressed in Lactobacillus plantarum 755 using L. amylovorus a-amylase
secretion signals, but the recombinant phytase was secreted at a lower rate in
comparison to the native proteins of L. plantarum 755. Xiong et al.
[30] synthesized an acidic phytase gene and the expressed phytase activity
increased 14.5 folds compared with the wild type phytase of the P. pastoris
strain. But the recombinant phytase was a histidine acid phosphatase that
had two pH optima (pH 2.5 and pH 5.5) and was less thermostable. In 2004 and
2005 [21,22], Wu et al. reported the expression of B. subtilis WHNB02
phyC gene in E. coli and P. pastoris. The recombinant
phytase expressed in E. coli showed optimum temperature and pH of 50 ?C
and 7.0 and the residual activity with treatment at 80 ?C for 5 min was about
50%. The phytase activity expressed in P. pastoris reached 2.4 U/ml
whose activity was also low and not suitable for production. The purified
enzyme revealed temperature and pH optima of 65 ?C and 7.0 and the residual
activity with treatment at 80 ?C for 5 min was about 95%. In this study, we
successfully expressed the synthetic neutral phytase gene (phyCs) in P.
pastoris, producing phytase at a level of 18.5 U/ml at shake-flask scale,
90-fold over the wild-type isolate B. subtilis WHNB02 [28]. The
recombinant neutral phytase secreted showed maximal activity at 70 ?C and pH
7.5, and the enzyme activity retained 97% of the relative activity after
incubation at 80 ?C for 5 min. Compared with the phyC gene expressed in E.
coli, the optimum temperature of purified phytase (phyCs)
improved from 50 ?C to 70 ?C, and the phytase was more thermostable. Compared
with the phyC gene expressed in P. pastoris, the optimum
temperature of purified phytase (phyCs) improved from 65 ?C to 70 ?C.
Although the enzyme revealed optimum pH of 7.5, it also showed high activity
around pH 6.0 to 9.0, just as phyC gene expressed in E. coli and P.
pastoris. It seemed that the increased production affected the property of
the recombinant phytase. This is the first report of a synthetic neutral
phytase gene in methylotrophic yeast P. pastoris which has been shown to
be suitable for high-level expression of various heterologous proteins either
intracellulary or secretory. Although P. pastoris secretes low levels of
endogenous proteins, it has the capacity to secrete grams per liter of foreign
proteins [31]. In addition, protein expression levels in P. pastoris can
be scaled up by 100-fold when the cells are grown in a fermenter [32,33].
Expression of recombinant proteins in P. pastoris is based on the use of
the alcohol oxidase gene. So there is a huge potential for the fermentation of
synthetic neutral phytase. Compared with the expensive BMMY medium, WEBG is an
excellent medium for the growth of P. pastoris and WEBI is also suitable
for protein expression.The expressed protein was obtained by chromatography systems
without a sharp peak (Fig. 4) due to the glycosylation and the low flow
eluted rate. But the four fractions (18 to 21) all showed high phytase
activity. Therefore, the four were taken together for phytase activity
analysis. Three N-glycosylated potential motifs that occurred within the
Asn-Xaa-Ser/Thr sequon were found by the online analysis software NetNglyC 1.0,
and a prediction score larger than 0.5 was recommended (http://www.cbs.dtu.dk/services/NetNGlyc).
Three major bands were found at 47.95 kDa, 50.23 kDa and 51.17 kDa after 24 h
induction. After deglycosylation by EndoHf, the
enzyme had an apparent molecular weight of 42 kDa, the same as the phytase
from the wild-type isolate [21,28]. The glycosylated and deglycosylated phytase
both revealed a temperature optimum of 70 ?C and a pH optimum of 7.5 [Fig.
5(A,B)]. As shown in Fig. 5(C), the glycosylated phytase resulted in
the enhancement of thermostability. Before deglycosylation, the enzyme activity
retained 97% of the relative activity after incubation at 80 ?C for 5 min;
enough for the pelleting of feedstuff. However, after deglycosylation the
residual activity of phytase was 77%. The purified phytase showed streaking [Fig.
6(B)] in SDS-PAGE, but after deglycosylation the streaking was eliminated.In conclusion, the phyCs gene was highly expressed as an
active, thermostable and extracellular phytase in P. pastoris, and its
enzyme property and thermostability was affected by glycosylation.
Acknowledgements
We are
grateful to Prof. Wen-Jun LIU, Mr. Wei-Jie DENG, Ms. Wen SHAO and Dr. Yan LUO
for critically reading and correcting the manuscript. Many thanks to Chang-Tai
LU, Zhi-Xiang QIN, Cui ZHAO, Bei LI, Zhong-Ying LIAO and Feng LIU for their
technical assistance.
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