Biochemical Engineering Journal 112 (2016) 161169

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

High level expression of a recombinant xylanase by Pichia pastoris

cultured in a bioreactor with methanol as the sole carbon source:
Purication and biochemical characterization of the enzyme
Maribel Cayetano-Cruz a , Ara Itzel Prez de los Santos a , Yolanda Garca-Huante a ,
Alejandro Santiago-Hernndez a , Patricia Pavn-Orozco b , Victor Eric Lpez y Lpez c ,
Mara Eugenia Hidalgo-Lara a,
a

Departamento de Biotecnologa y Bioingeniera, CINVESTAV-IPN, Mxico D.F., Mexico

Facultad de Ciencias Qumicas, Universidad Veracruzana, Veracruz, Mexico
c
Centro de Investigacin en Biotecnologa Aplicada del Instituto Politcnico Nacional, Mexico
b

a r t i c l e

i n f o

Article history:
Received 23 November 2015
Received in revised form 14 April 2016
Accepted 16 April 2016
Available online 19 April 2016
Keywords:
Pichia pastoris
Methanol
Fed-batch culture
Enzyme production
Purication
Recombinant DNA

a b s t r a c t
The xylanase gene xyn11A from Cellulomonas uda was expressed in Pichia pastoris under the control of an
inducible promoter AOX1. The recombinant xylanase was named Xyn11AAOX1 . The P. pastoris clone (C9)
showing the highest xylanase activity was selected to evaluate the production of Xyn11AAOX1 in liquid
cultures in a bioreactor. The culture was carried out by fed-batch fermentation using two strategies, onestage method using methanol, and two-stage method using glucose and methanol as carbon sources.
Interestingly, after 48 h of fermentation using one-stage method, a dry cell weight of 34 g/L and total protein concentration of 1.16 g/L were obtained, where Xyn11AAOX1 was the major enzyme secreted into the
culture medium. Xyn11AAOX1 was puried from the culture supernatant of P. pastoris/pPICZB xyn11A
and showed an estimated molecular mass of 45 kDa. The optimal temperature and pH were 50 C and 6.5,
respectively. The KM and Vmax values were 4.5 mg/mL and 5000 U/mg protein, respectively. This is the
rst report on cultivating P. pastoris with methanol as the sole carbon source in a minimal salt medium in
which the recombinant enzyme was obtained as the major enzyme secreted into the culture supernatant
within a short fermentation time.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Xylan is one of the major components of hemicellulose and
is the second most abundant renewable resource after cellulose. It is a complex polysaccharide composed of a main chain
consisting of xylose residues connected via -1,4-glycosidic linkages [1]. Given the complexity of this compound, its degradation
requires the synergistic action of several xylanolytic enzymes
including endo-xylanases, -xylosidases, -glucuronidases, arabinofuranosidases, and esterases. Endo--1,4-xylanase is the
most important enzyme in this process [2].
Endoxylanases act on the main chain of xylan by hydrolyzing the
internal (1 4) linkages between molecules of xylose, leading to
a mixture of different sizes of xylooligosaccharides [3]. Endoxylanases have important applications in industry because of their
enormous potential to transform lignocellulosic materials and they

Corresponding author.
E-mail address: ehidalgo@cinvestav.mx (M.E. Hidalgo-Lara).
http://dx.doi.org/10.1016/j.bej.2016.04.014
1369-703X/ 2016 Elsevier B.V. All rights reserved.

are widely used as raw materials in a large number of industrial processes. Currently, the major industrial application of xylanases is in
the paper industry [4]. The treatment of pulp with xylanases prior
to chemical bleaching provides signicant economic and environmental benets [5].
Although numerous studies have examined xylanases from various sources, it is still important to develop new methods for
improving the production of xylanase because of its potential for
industrial applications, such as in biobleaching and the animal feed
industry [1]. Heterologous expression systems may be useful for
obtaining increased amounts of xylanase. In eukaryotic systems,
Pichia pastoris has been successfully used to produce large amounts
of different enzymes. This organism is a methylotrophic yeast capable of producing signicant quantities of recombinant protein [6].
In the absence of glucose, this yeast can use methanol as the sole
carbon source because of its highly regulated methanol metabolism
pathway [7]. The alcohol oxidase enzyme (AOX1) is responsible for
the rst methanol oxidation reaction in P. pastoris; its synthesis is
regulated by the AOX1 promoter, which has been broadly used in
the expression of recombinant proteins in this microorganism [8,9].

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In bioreactor cultures, P. pastoris can grow to high cell densities (100 g/L) in minimum salt medium [10,11]. A common strategy
for achieving high cell densities involves three stages in fed batch
cultures. The rst stage corresponds to a batch culture with glucose or glycerol as the carbon source. Secondly, fed-batch control
is initiated by feeding concentrated glucose and glycerol until a certain cell density is reached. The third stage consists of an induction
step during which methanol is added to induce heterologous protein production. The duration of the induction period is longer than
that of biomass generation, resulting in a total fermentation time
of approximately 100 h [12,13].
We previously reported the cloning and expression of the C
xyn11A xylanase gene from Cellulomonas avigena in Escherichia
coli; however, most of the protein was found in the insoluble fraction of the bacterial cell lysate in inclusion bodies [14]. The aim
of this work was to extracellularly overexpress Xyn11A xylanase
from C. uda CDBB-1960 in P. pastoris under fed-batch fermentation
in minimal salt medium using methanol as the sole carbon source.
Further, the recombinant enzyme was puried and characterized.
2. Materials and methods
2.1. Chemicals
The DNA gel extraction kit and restriction enzymes were
obtained from Qiagen (Hilden, Germany). Birchwood xylan, 3,5dinitrosalysilic acid, and phenylmethylsulfonyl uoride (PMSF)
were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). SDSPAGE markers were obtained from Bio-Rad (Hercules, CA, USA).
All other chemicals were of analytical grade. Xylo- oligosaccharide standards (xylose, xylobiose, xylotetrose and xylohexose) were
purchased from Megazyme (Wicklow, Ireland).
2.2. Molecular techniques
DNA manipulations were performed using standard methods
[15]. P. pastoris was manipulated as described in the manual of the
EasySelectTM Pichia Expression Kit (Invitrogen, Carlsbad, CA, USA).
2.3. Strains, plasmids, and mediums
C. avigena CDBB-531 strain was identied as Cellulomonas uda
CDBB-1960 by taxonomic analysis based on 16S rDNA, and this
strain was deposited in the National Collection of Microbial Cultures, CINVESTAV (Mexico City, Mxico). E. coli JM109 (Invitrogen)
was used for high-level production of plasmid DNA, and P. pastoris X-33 (Invitrogen) was used for xylanase expression. The vector
pPICZB was supplied by Invitrogen. E. coli JM109 was grown at
37 C and 150 rpm on Luria-Bertani (LB) medium containing 25 g
Zeocin/mL to select clones transformed with the pPICZB vector.
P. pastoris was grown in a shaking ask at 28 C in rich medium
containing 1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% (w/v) yeast nitrogen base,
4 105 % (w/v) biotin, and 1% (w/v) glucose or 0.5% (v/v) methanol
(BMMY) for induction. For strain maintenance, YPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v)
dextrose was used.
2.4. Subcloning of xylanase gene
The C xyn11A gene was subcloned into pPICZaB vector as
follow. Plasmid DNA from E. coli XL1-Blue MRF carrying the C
xyn11A gene [14] was used as a template to amplify the coding
region. The coding region of the C xyn11A gene (GenBank accession number AM182259.2) was amplied by PCR using forward
primer (5 -CTGCAGCGGTGACGTCCAACCAG-3 ) and reverse primer

(5 -TCTAGACCGCAGAACGCGCTGGGCGTCCA-3 ) into the restriction

sites XbaI and PstI (underlined), respectively. PCR was performed
using HotStar HiFidelity DNA Polymerase (Qiagen) under the following conditions: one cycle for 5 min at 95 C, followed by 30
cycles for 1 min at 94 C, 1 min at 59 C, 1.2 min at 72 C; and one
cycle for 10 min at 72 C in a Gene-Cycler (Bio-Rad). The PCR products were puried and directionally cloned in the pPICZB vector
in the XbaI and PstI sites, generating the construct pPICZaB-xyn11A,
which was transferred into E. coli JM109 by heat shock. E. coli JM109
cells harboring the construct pPICZaB-xyn11A were cultivated in LB
medium at 37 C supplemented with 25 g Zeocin/mL. A transformant E. coli JM109/pPICZB-xyn11A clone was selected on an LB
agar plate containing Zeocin (100 g/mL) and analyzed by restriction proling and DNA sequencing.
2.5. Expression in pastoris
For expression in P. pastoris, 10 g of the pPICZB-xyn11A construct was linearized with SacI and transferred into P. pastoris X-33
by electroporation (4.5 kV/cm, 25 mF and 400 W for 11 ms) using
(EasyjecT Optima, Equibio Ltd., New York, NY, USA). pPICZB without the insert was used as a negative control. Transformed clones
were selected based on their ability to grow on YPD agar plates
containing 100, 200, 500, or 1000 g Zeocin/mL.
2.6. Selection of recombinant clones
Candidate clones were cultured at 30 C for 8 h on BMMY
medium agar plates containing 0.5% (v/v) methanol and 0.2% (w/v)
xylan. Positive clones were selected for expression of the xylanase
activity, which was visualized using Congo Red staining [16].
2.7. Xylanase and protein assay
A 20 L supernatant aliquot was added to 980 L citratephosphate buffer (50 mM, pH 6.5) containing 0.2% (w/v) birchwood
xylan. The xylanase activity was determined from the amount of
reducing sugars released during incubation at 50 C. The reducing sugar was measured by the dinitrosalicylic acid method using
xylose as a standard [17]. One unit of activity (U) is dened as
1 mol of xylose released per minute under the assay conditions.
The protein concentration was measured using the Lowry method
[18], with bovine serum albumin as a standard. All tests were carried out in triplicate and the average values were recorded.
2.8. Fed-batch culture for enzyme production in bioreactor
Fermentations were carried out in a 7 L bioreactor (BIOSTAT
Aplus, Sartorius, Goettingen, Germany). For propagation, P. pastoris
was cultured in YPD medium. The propagation culture for the reactor was carried out in 200 mL of YPD medium inoculated with 2%
inoculum in a 1 L Erlenmeyer ask incubated at 28 C with shaking
at 200 rpm for 24 h. The medium was then centrifuged at 8000 rpm
for 10 min and the pellet was resuspended in 300 mL of minimum
salt medium (MSM) to inoculate the bioreactor.
For fermentation, the basal media MSM [19] containing the
trace element solution PTM1 (Amresco, Solon, OH, USA) was used.
The MSM contained the following: 4 g/L KH2 PO4 , 4 g/L (NH4 )2 SO4 ,
0.38 g/L CaCl2 , 18.2 g/L K2 SO4 , 9.4 g/L MgSO4 7H2 O, 1 mL PTM1, and
2.5 g/L methanol or 40 g/L glucose. The trace element solution
PTM1 contained the following: 5.99 g/L CuSO4 5H2 O, 8 mL 10 NaI
stock solution, 3 g/L MnSO4 H2 O, 0.20 g/L Na2 MoO4 2H2 O, 0.50 g/L
CoCl2 6H2 O, 20.04 g/L ZnCl2 5H2 O, 65.05 g/L FeSO4 7H2 O,800 L
100 H3 BO3 stock solution,19.2 mL 96.2% H2 SO4 , and 0.40 g/L
biotin.

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163

Fig. 1. Selection of P. pastoris/pPICZB-xyn11A clones by growth on (a) YPD medium with Zeocin (100 g/mL) and (b) BMMY medium containing 0.5% methanol and 0.2%
(w/v) xylan. Stained with 1% (w/v) Congo red and faded with 1 M NaCl. Detection of xylanolytic activity by formation of hydrolysis halos on agar-xylan. Negative control is
shown in black circle. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

The operation conditions were: 3.5 L working volume, 28 1 C,

pH 7.0, 600 rpm initial stirrer speed, and 1 vvm initial aeration. The
pH was maintained automatically by the addition of NH4 OH (30%)
and H3 PO4 (8.5%). In order to maintain the dissolved oxygen (DO)
above 20%, the speed and aeration were controlled between 600
and 1000 rpm and 11.5 vvm, respectively.
For the two-stage method, fermentation was initiated with a
batch culture stage using a glucose concentration of 40 g/L. Following consumption of glucose, the methanol-fed batch phase was
initiated. For the one-stage method, fermentation was initiated
with a batch culture stage using an initial methanol concentration
of 2.5 g/L. After consumption of methanol during the batch phase,
indicated by an increase of the DO concentration (data not shown),
the methanol-fed batch phase was initiated. For both methods, the
feed solution for the fed-batch stage fermentation contained a mixture of 12 mL of PTM1 solution and 1000 mL of 100% methanol (the
nal methanol concentration was 98.8%). The methanol solution
was fed shot-wise in order to maintain a methanol concentration
of 0.5% in the reactor. Antifoam was used as a foam suppressor
and was added as necessary (Antifoam 204, Sigma-Aldrich). During
fermentation, samples were withdrawn periodically to determine
the methanol concentration, biomass, total protein, and xylanolytic
activity. The biomass was determined by measuring the optical
density at 600 nm and the dry cell weight. The methanol concentration was monitored by using a biochemical analyzer (YSI 2700
Select, YSI, Yellow Springs, OH, USA).
2.9. Xylanase purication
Recombinant Xyn11AAOX1 was puried from the culture supernatant of P. pastoris cells harboring the pPICZaB-xyn11A construct
by anion exchange chromatography. The cell-free supernatant was
dialyzed in buffer A (50 mM TrisHCl, 25 mM KCl, 0.1 mM PMSF, pH
7.5). The dialysate was loaded in a UNOsphere Q (Bio-Rad) column
that was pre-equilibrated with buffer A. The absorbed protein was
eluted from the column over a linear gradient of KCl (25 mM1 M) in
buffer A at a ow rate of 2.5 mL/min, and 2.0-mL fractions were collected. Fractions with xylanase activity were pooled and analyzed
by 10% SDS-PAGE.
2.10. SDS-PAGE analysis and enzyme deglycosylation
Protein analyses were carried out via 10% SDS-PAGE using
the Mini-PROTEAN III System (Bio-Rad). Proteins in the gel were
stained with Coomassie Brilliant Blue R-250 and the molecular weight (MW) was estimated with reference to broad-range
molecular weight protein standards (Bio-Rad). Periodic acid-Schiff

staining was used to detect carbohydrates in the protein as

described by Dubray and Bezard [20]. For enzyme deglycosylation,
puried Xyn11AAOX1 xylanase was incubated in a boiling water
bath for 15 min and then treated with Endoglycosidase H (EndoH) (Roche Diagnostic GmbH, Mannheim, Germany) according to
the manufacturers instructions.

2.11. Zymogram analysis

Zymogram analysis was carried out as previously described [21],
with some modications as follows. Briey, protein samples were
separated in a 10% polyacrylamide gel co-polymerized with 1%
Remazol Brilliant Blue linked to xylan (RBB-xylan), under semidenaturing conditions. Protein samples were resuspended in SDS
sample buffer without 2--mercaptoethanol, and the samples were
incubated at 37 C for 30 min. After electrophoresis, the gels were
incubated at 50 C for 20 min in 50 mM citrate-phosphate buffer at
pH 6.5 to reveal xylanase activity.

2.12. Biochemical characterization of Xyn11AAOX1

The effect of pH on the enzymatic activity of Xyn11AAOX1 was
determined at pH values from 4 to 8 using 50 mM citrate-phosphate
buffer (pH 47) and 100 mM phosphate buffer (pH 78) with 0.2%
(w/v) xylan, at 50 C. The effect of temperature on the enzymatic
activity of Xyn11AAOX1 was evaluated in 0.2% (w/v) xylan solution
prepared in 50 mM citrate-phosphate buffer, pH 6.5, under different temperature (3075 C) conditions.
The thermal stability of recombinant xylanase was determined
by measuring the residual activity of the enzyme that had been
exposed to three different temperatures: 40 C, 50 C, and 60 C
in 50 mM citrate-phosphate buffer (pH 6.5) until the half-life was
reached. Aliquots of xylanase were removed at different time intervals and assayed to determine the enzymatic activity as described
above. The xylanase activity prior to the pre-incubation was considered to be 100%.
The kinetic parameters KM and Vmax were determined under the
optimum temperature and pH conditions while varying the xylan
concentration from 1 to 10 mg/mL, and were calculated graphically
using a Lineweaver-Burk plot.
The effect of metal ions (HgCl2 , CuCl2 , LiCl, ZnSO4 , CaCl2 , MgCl2 ,
FeSO4 , NiCl2 , NaCl, MnCl2 ) and the chelating agent EDTA, at respective nal concentrations of 1 and 5 mM, on the enzyme activity were
independently evaluated by determining the xylanolytic activity
under standard assay conditions.

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M. Cayetano-Cruz et al. / Biochemical Engineering Journal 112 (2016) 161169

Fig. 2. Growth (triangle), xylanolytic activity (circles), and methanol concentration (dotted line) of Pichia pastoris/pPICZB-xyn11A. (a) Two-stage method and (b) One-stage
method. Growth was monitored by cell dry weight, xylanolytic activity by DNS method, and methanol concentration with a biochemical analyzer YSI 2700.

2.13. Thin-layer chromatography (TLC) assay

Xylan degradation products were analyzed by TLC. A total of 7 U
of Xyn11AAOX1 was incubated at 40 C in 150 L of 1% (w/v) birchwood xylan prepared in 50 mM citrate-phosphate buffer, pH 6.5.
Aliquots were withdrawn at different times. A total of 3 L of each
sample and 2 L of the xylo-oligomer standard (xylose, xylobiose,
xylotetrose, and xylohexose) was spotted on silica gel (Merck KGaA,
Darmstadt, Germany). The hydrolysis products were separated in
a solvent system consisting of ethanol (30% v/v), butanol (50% v/v),
and H2 O (20% v/v). The plate was then sprayed with sulfuric acid
(15% v/v) and heated for 40 min at 80 C in an oven to visualize the
xylo-oligomers.
3. Results
3.1. Expression of xyn11A gene in pastoris
The coding region of the xyn11A gene, which encodes for the
mature Xyn11A, was cloned into the pPICZB expression vector to generate the plasmid pPICZB-xyn11A. The construct was
then transferred to P. pastoris by electroporation and transformed
clones were selected based on their ability to grow on YPD agar
plates containing 100, 200, 500, and 1000 g Zeocin/mL (Fig. 1a).
P. pastoris/pPICZB-xyn11A clones that grew in 1000 g Zeocin/mL
were selected for further analysis. Candidate P. pastoris/pPICZBxyn11A clones were cultured at 28 C for 8 h on BMMY medium agar

plates containing 0.5% methanol and 0.2% xylan. Positive clones

were selected for expression of the xylanase activity and visualized by Congo red staining. A positive clone, C9, which showed the
highest xylanolytic activity was chosen for further analysis (Fig. 1b).
3.2. Fed-batch culture for production of Xyn11AAOX1 in bioreactor
The production of extracellular Xyn11AAOX1 was evaluated
using P. pastoris cells harboring the construct pPICZB-xyn11A by
fed-batch culture, using the traditional two-stage method and a
one-stage method using methanol as the sole carbon source. The
two-stage method consists of a batch culture stage with glucose
followed by a methanol-fed batch phase. The one-stage method
consists of a batch and fed-batch stages using methanol as the sole
carbon source. The temperature and pH conditions were maintained at 28 C and 7, respectively. The speed and air ux were
varied in order to maintain the dissolved oxygen above 20%.
For the two-stage method, a well-known fermentation strategy
was employed consisting of one stage to generate biomass with
glucose as a carbon source and a second stage using methanol as
the sole carbon source, for the production of the Xyn11AAOX1 . Batch
stage with glucose lasted approximately 22 h, at this moment the
percentage of dissolved oxygen started to increase due to glucose
exhaustion in the medium. Once glucose was exhausted, the rst
methanol pulse was implemented in order to begin with the induction stage and xylanase expression. Methanol solution was fed
shot-wise in order to maintain the methanol concentration below

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165

Table 1
Summary of purication of xylanase Xyn11AAOX1 .

Crude extract
Anion exchange chromatography

Total activity (U)

Total protein (mg)

Specic activity (U/mg)

Yield (%)

Fold purication

91.58
60.40

1.78
0.50

51.45
120.80

100
65.95

1
2.35

analyzed by 10% SDS-PAGE. Two major bands with an estimated

MW of 45 kDa (Fig. 3a) were found to have xylanolytic activity, as
revealed by zymogram performed under semi-denaturing conditions, using 1% RBB-xylan as the substrate (Fig. 3b).
3.4. Xylanase purication
Xyn11AAOX1 was puried from the culture supernatant by anion
exchange chromatography as a single peak with a yield of 65.95%
and specic activity of 120.80 U/mg. The results of the procedure
are summarized in Table 1. Additionally, 10% SDS-PAGE analysis of
the puried protein showed two major bands with an estimated
MW of approximately 45 kDa, which was 7 kDa higher than the
expected value for the mature form of Xyn11AAOX1 (38 kDa) (Fig. 4a,
lanes 35).
Fig. 3. (a) SDS-PAGE analysis of expressed proteins in culture supernatant in bioreactor with methanol (0.5%) during fermentation. Lane (1) MW protein standards,
(27) Supernatant at 10, 18, 30, 44, 46, 48, respectively, (b) Zymogram analysis of
xylanase activity.

5 g/L during the fed-batch stage. At the end of the fed-batch culture, a dry cell weight of 19.5 g/L and total protein of 1.15 g/L were
achieved (Fig. 2a). Maximum xylanolytic activity of 57 U/mL was
detected at 33 h of culture.
In the one-stage method, with methanol as the sole carbon
source, after 26 h of batch operation and when 7 g/L dry cell weight
was reached, an increase in the dissolved oxygen was observed,
indicating the depletion of methanol in the culture medium. At
that time, methanol solution was fed shot-wise in order to maintain the methanol concentration below 5 g/L during the fed-batch
stage. A total of 587 mL of methanol solution was added during
the fed-batch stage. After 48 h of fermentation, the dry cell weight,
total protein, and xylanolytic activity were 34.1 g/L, 1.16 g/L, and
161.5 U/mL, respectively (Fig. 2b). Findings here indicated that
using the one-stage method, higher volumetric xylanolytic activity
and biomass were obtained after 48 h of fermentation compared
to that observed following the traditional two-stage method. We
thus decided to carry out the purication and biochemical characterization of Xyn11A AOX1 from the supernatant obtained from the
fermentation using methanol as the sole carbon source.
3.3. Extracellular expression of Xyn11A in pastoris
To evaluate the extracellular expression of Xyn11A in P. pastoris,
samples of the culture supernatant from the one-stage fermentation of P. pastoris, removed at different fermentation times, were

3.5. Deglycosylation of Xyn11AAOX1

To assess the presence of O- and N-linked carbohydrate moieties, the puried Xyn11AAOX1 was analyzed by 10% SDS-PAGE and
stained with periodic acid-Schiff reagent. A positive reaction was
detected (Fig. 4b), indicating that Xyn11AAOX1 was a glycoprotein.
Additionally, the glycosylated nature of Xyn11AAOX1 was conrmed
by deglycosylation by Endo-H treatment. 10% SDS-PAGE analysis of
the deglycosylated forms of Xyn11AAOX1 showed two bands with
an estimated MW of 38 kDa, whereas Xyn11AAOX1 showed two
bands with an estimated MW of 45 kDa (Fig. 4c). Given that EndoH cleaves N-linked glycans but not O-linked glycans, the puried
Xyn11AAOX1 was treated with Protein Deglycosylation Mix; however, the same pattern as in Fig. 4c was observed by 10% SDS-PAGE
(data not shown).
3.6. Biochemical characterization of recombinant xylanase
Xyn11AAOX1
The puried recombinant xylanase Xyn11A from C. uda
expressed in P. pastoris was biochemically characterized to determine the effect of pH, temperature, metallic ions, and substrate
concentration on the xylanolytic activity of the enzyme using birchwood xylan as the substrate.
The inuence of pH on the xylan hydrolysis ability of
Xyn11AAOX1 was determined at pH values ranging from 4 to 8
at 50 C. Xyn11AAOX1 displayed the highest activity at pH 6.5 and
exhibited more than 50% of the maximal activity over a broad pH
interval from 5 to 7.5 (Fig. 5a).
The effect of temperature on the enzymatic activity of
Xyn11AAOX1 was assayed over a wide range of temperatures from

Table 2
Biochemical properties of Xyn11A expressed in E. coli. and Xyn11AAOX1.
Biochemical characters

Xyn11A (C. uda)

Xyn11AAOX1

Optimum temperature ( C)
Optimum pH
KM (mg/mL)
Vmax (U/mg)
Temperature stabilityHalf-lives (t1/2 )
Reference

55
6.5
0.98
5.7
35 C 15.8 ha 45 C 6.1 ha 55 C 1.1 ha
[14]

50
6.5
4.5
5000
40 C 35 ha 50 C 27 minb 60 C 1.5 minb
This work

a
b

Theoretical half-life using Ln A = Ln A0 ki t.

Experimental half-life data.

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M. Cayetano-Cruz et al. / Biochemical Engineering Journal 112 (2016) 161169

Fig. 4. SDS-PAGE analysis (a) Xylanase purication. Lane (1) MW protein standards, (2) Dialyzed supernatant (35) Puried recombinant xylanase. (b) Periodic acid-Schiff
stain and (c) Deglycosylation analysis. Lane (1) Xyn11AAOX and (2) Xyn11AAOX after treatment with Endo H.

30 C to 75 C at pH 6.5. The enzyme displayed optimal activity at

50 C and exhibited more than 50% of the maximal activity over
a broad range of temperatures from 30 to 60 C (Fig. 5b). A dramatic reduction to 40% of the maximal activity of Xyn11AAOX1 was
observed at temperatures ranging from 65 to 75 C.
The thermal stability of Xyn11AAOX1 was evaluated by preincubation of the puried enzyme at 40 C, 50 C, and 60 C at pH 6.5.
The puried xylanase Xyn11AAOX1 was negatively affected at 60 C
(t1/2 of 1.5 min), but at 40 C the enzyme retained more than 65% of
the maximal activity after 20 h of incubation and had a half-life of
35 h (Table 2).
The hydrolytic activity of Xyn11AAOX1 toward birchwood xylan
was evaluated by variation of the substrate concentration from 1
to 10 mg/mL in the reaction mixture. A graph of the initial reaction
rate versus the substrate concentration showed that the enzyme
obeyed Michaelis-Menten kinetics and had a KM of 4.5 mg/mL and
Vmax of 5000 U/mg (R2 = 0.98) (Supplementary data).
It has been reported that metal ions generally act as inhibitors
or activators of enzyme activity. To study the effect of various
metal ions and the chelating agent EDTA on the enzymatic activity,
xylanase Xyn11AAOX1 was exposed to 10 different metal ions (Hg2+ ,
Cu2+ , Li+ , Zn2+ , Ca2+ , Mg2+ , Fe2+ , Ni2+ , Na+ , Mn2+ ) at nal concentrations of 1 mM, and 5 mM each. Table 3 shows the effect of each
of the metal ions (1 mM) and the chelating agent EDTA (1 mM) on
the xylanolytic activity. No difference was observed between the
results obtained at the two different concentrations. No metal ion
was found to activate xylanolytic activity. The addition of mercury
ions drastically inhibited the xylanase activity of Xyn11AAOX1 .

Fig. 6. TLC analysis of the products after hydrolysis of birchwood xylan by

Xyn11AAOX xylanase. Xylo-oligomer standards: xylohexose (X6), xylotetraose (X4),
xylobiose (X2) and xylose (X1), lane (18) different reaction times for hydrolysis (0,
3, 6, 9, 24, 48, 72, 96 h) in citrate-phosphate buffer, pH 6.5 a 40 C, respectively.

3.7. Analysis of hydrolysis products by TLC

Birchwood xylan degradation was monitored by TLC analysis.
The main products of xylan hydrolysis were xylobiose and xylohexose, with a smaller amount of xylotriosa, as shown in Fig. 6.
After 96 h, no xylose was detected.

Fig. 5. pH and temperature proles of the puried xylanase. (a) Xyn11AAOX was incubated at pH ranging from 4 to 7 in citrate phosphate buffer (50 mM) or phosphate buffer
(100 mM) from 6.5 to 8 with 0.2% xylan at 50 C for 4 min, (b) Xyn11AAOX was incubated at temperatures ranging from 30 to 75 C for 4 min in citrate phosphate buffer 50 mM,
pH 6.5 and xylan 0.2%. The values are from three parallel determinations.

M. Cayetano-Cruz et al. / Biochemical Engineering Journal 112 (2016) 161169

Table 3
Inuence of metal ions and EDTA on xylanolytic activity.
Metal ion/EDTA (1 mM)

Relative activity (%)

No addition
HgCl2
CuCl2
LiCl
ZnSO4
CaCl2
MgCl2
FeSO4
NiCl2
NaCl
MnCl2
EDTA

100.00 5.68
ND
97.14 2.67
105.25 0.96
110.10 5.23
101.23 3.27
104.91 2.45
97.27 4.53
101.43 2.16
100.82 5.39
65.55 4.01
82.38 4.37

ND: no activity was detected.

4. Discussion
Different enzymes have been expressed in P. pastoris under
the control of the AOX1 promoter, and the levels of expression
ranged between 0.004 g/L and 2.5 g/L [22,23]. It has been reported
that the expression level for a recombinant protein produced in
P. pastoris appears to be decided by the inherent protein properties. Since these factors are interdependent, the strategies for
achieving high-level protein expression have generally involved
improving the operational strategies, including the medium composition, implementation of different growth stages with different
carbon sources, or using a mixture of different carbon sources [24].
Such strategies, with different growth stages, are based on avoiding strong repression of the AOX1 promoter by glucose [6]. It has
been reported that repression of the AOX1 promoter can decrease
the production of recombinant protein [10,25]. Implementation
of strategies involving different growth stages has also allowed
maximum biomass production of the order of 100 g/L, and recombinant protein production of the order of milligrams; however, the
required fermentation time was between 100 and 150 h [10,11].
The production of Xyn11AAOX1 was successfully carried out
by fed-batch fermentation using a traditional two-stage method,
using glucose and methanol as carbon sources [6,11,26]. In order
to shorten the fermentation-time, a one-stage method, using
methanol as the sole carbon source, was implemented. Interestingly, after 48 h of fermentation, the xylanolytic activity (2.8-fold)
and the biomass (1.7-fold) obtained using the one-stage method
was higher than those observed following a conventional twostage method (Fig. 2). Although, the total protein was comparable
using both methods of fermentation, electrophoretic analysis of the
protein prole in the culture supernatant from the one-stage fermentation showed that Xyn11AAOX1 was the main protein secreted
into the culture medium; whereas, in the culture supernatant
from the two-stage method (Supplementary data), several bands
of about the same intensity of Xyn11AAOX1 were observed, which
correspond to different proteins secreted into the medium. Hence,
the one-stage method with methanol as the sole carbon source may
be and effective way of promoting protein synthesis under the control of AOX1 promoter, rather than other proteins from the host P.
pastoris.
Mellitzer et al. [22] reported the expression of one optimized
codon TlXynA xylanase from Thermomyces lanuginosus in P. pastoris under the AOX1 promoter; the yeast was fed-batch cultured
by using two stage fermentation with glycerol and methanol as
carbon sources. In this study, a yield of 1.2 g/L of TlXynA with a
specic activity of 115 U/mg was obtained after 90 h of fermentation. These data are similar to our ndings, where a yield of
1.16 g/L of Xyn11AAOX1 and a specic activity of 120.80 U/mg were
obtained; however, the fermentation-time was shortened to 48 h
when methanol was used as the sole carbon source.

167

Because of the lack of reports on bioreactor cultivations expressing xylanases in P. pastoris, the present ndings were compared
with those obtained for the heterologous expression of xylanases
in P. pastoris cultured in a shake ask, or to those observed for P.
pastoris expressing other enzymes under the AOX1 promoter, cultured in a bioreactor. Notably, the yield of 1.16 g/L of Xyn11AAOX1
is 8-fold higher than that reported for a xylanase from T. lanuginosus IOC-4145 (148 mg/L obtained in 96 h of culture) [27], and
6-fold higher than that reported for a xylanase from T. lanuginosus
195 (26 U/mL, obtained in 120 h of culture) [28], both cultured in a
shake ask. Regarding the expression of other enzymes in P. pastoris
at the bioreactor level, the expression of a human calreticulin (CRT)
under the AOX1 promoter following a four-step growth protocol,
consisting of a glycerol batch, a glycerol fed-batch, and transition
phase and methanol fed-batch phase has been reported. Notably,
in the afore mentioned work, the authors were able to obtain
a CRT concentration and dry cell weight of 1.6 g/L and 85.9 g/L,
respectively; however, these results were achieved after 143 h of
fermentation time [29]. Interestingly, in the present work, a concentration of 1.16 g/L of Xyn11AAOX1 was obtained from 34.1 g/L of
dry cell weight after 48 h of fermentation time, which corresponds
to less than half of the yeast biomass (85.9 g/L) reached by Ciplys
et al. [29], where Xyn11AAOX1 was the main enzyme secreted into
the culture medium. Hence, we are currently undertaking studies
to optimize the biomass yield with the aim to further improve the
protein concentration without increasing the fermentation time.
The ndings presented herein suggest that the utilization of
methanol as the only carbon source, in both batch and fed-batch
mode, may affect the P. pastoris culture in two ways. First, improving the synthesis of Xyn11AAOX1 in P. pastoris by controlling the
AOX1 promoter, thus resulting in Xyn11AAOX1 being the main
enzyme secreted into the culture medium; this in turn reduces the
operations required in downstream processing. Second, decreasing
the fermentation time due to time phase transition between carbon
sources is not required in this strategy; therefore, the global cost of
process production can be reduced.
Since Xyn11AAOX1 was the major enzyme secreted into the
culture medium using the one-stage method, protein purication required only one step of anion exchange chromatography,
especially because P. pastoris exports only low levels of endogenous proteins to the medium [6]. Analysis of puried Xyn11AAOX1
using 10% SDS-PAGE showed two major bands with an estimated
MW of approximately 45 kDa, and with xylanolytic activity as
determined based on zymogram analysis. The expected MW for
Xyn11AAOX1 is 38 kDa, that accounts for the open reading frame of
the xyn11A gene (999 bp), encoding a polypeptide of 332 amino
acids residues with a calculated MW of 35.1 kDa [14], plus the epitope myc and the Histidine tag added by the pPICZB vector. The
7 kDa difference could be attributed to glycosylation of Xyn11AAOX1
during its expression in P. pastoris, corresponding to a glycosylation content of 18%; further, it has been reported that recombinant
proteins secreted by P. pastoris show a certain degree of glycosylation [6]. The Endo-H treatment of Xyn11AAOX1 (two bands of
approx. 45 kDa) yielded a deglycosylated form of the enzyme (two
bands of approx. 38 kDa) thus conrming the glycosylated nature of
Xyn11AAOX1 . The presence of two bands before and after EndoH and
Protein Deglycosylation Mix treatment of Xyn11AAOX1 is possibly
due to slight differences in the glycosylation pattern of Xyn11AAOX1 ,
that were not completely removed by the treatment with these
enzymes or to the presence of other post-translational modications carried out by P. pastoris [6,9,13], that were no investigated in
this study.
Xyn11AAOX1 displayed optimal pH at 6.5 (Table 2), similar to
Xyn11A expressed in E. coli [14] and several xylanases obtained
from different microorganisms that have been reported to have
optimal function at pH 6.5, such as in Paenibacillus sp. DG-22

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M. Cayetano-Cruz et al. / Biochemical Engineering Journal 112 (2016) 161169

[30], Saccharopolyspora pathumthaniensis S582 [31], and Sphingobacterium sp. TN19 [32]. Sukhumsirichart et al. [33] reported the
purication of xylanase rXynSW3 from Streptomyces sp. SWU10,
where the optimal activity was demonstrated at pH 5.56.5.
Xyn11AAOX1 displayed optimal activity at a temperature of
50 C, which was 5 C lower than that reported for Xyn11A
expressed in E. coli [14] (Table 2). Xylanase enzymes from other
microorganisms exhibited an optimal activity at 50 C, such as
that from Penicillum purpurogenum [34], Aspergillus sojae [35],
Geotrichum candidum 3C [36], and Trichoderma reesei Rut C-30 [37].
Regarding the thermostability, at 60 C, the half-life of
Xyn11AAOX1 (t1/2 of 1.5 min) was shorter than those of xylanases
PhX20 (t1/2 of 10 min) from Pseudozyma hubeiensis [38] and Xyn2
(t1/2 of 25 min) from Trichoderma reesei [39]. At 50 C, the thermal stability of Xyn11AAOX1 (t1/2 of 26.8 min) was comparable to
that reported for xylanase XynII (t1/2 of 28 min) from the fungus
Aspergillus usamii [40], but lower than that of xylanase Xyn21 from
T. reesei, which retained above 80% of the original activity after
30 min [39]. At 40 C, the half-life of Xyn11AAOX1 exceeded the
reported value of 24 h for a xylanase from Bacillus sp. NTU-06 [41].
The KM and Vmax values of Xyn11AAOX1 were higher than those
reported for Xyn11A expressed in E. coli (KM of 0.98 mg/mL and
Vmax of 5.7 U/mg) [14] in 4.6-fold and 877-fold, respectively. Findings here may be attributed to the carbohydrate component (18%)
added to the enzyme during the heterologous expression in P. pastoris. Changes in the kinetic parameters due to glycosylation has
been previously reported for the native invertase INV3a-N of Candida guillermondii MpIIIa [42], recombinant invertases D-INVAAOX1
and D-INVBAOX1 expressed in P. pastoris [43], the non-glycosylated
invertase from Candida utilis [44] and the human glutaminyl cyclase
expressed in P. pastoris [26].
The KM value of Xyn11AAOX1 was lower than those reported
in previous studies. Sajjad et al. [45] reported two recombinant
xylanases with KM values of 15.4 and 33.3 mg/mL and Li et al.
[46] reported a cellulose-free xylanase with a KM of 5.4 mg/mL. In
contrast, the Vmax value of Xyn11AAOX (5000 U/mg) was 2.9-fold
higher than those reported for xylanase xyl I (Vmax = 1679 U/mg)
from Aspergillus caespitosus [47], but lower than that reported for a
xylanase (10571 U/mg) from Penicillium sp. CGMCC 1669 [48].
Concerning the effect of metal ions and EDTA on the activity
of Xyn11AAOX , no metal ion was found to activate the xylanolytic
activity, but the inhibition of Xyn11AAOX by the addition of mercury ions suggested the existence of thiol groups at the active site
of the enzymes [49]. A 95% decrease in the xylanolytic activity was
observed when Hg2+ was added to Xyn11NX from Nesterenkonia
xinjiangensis CCTCC AA001025 at a nal concentration of 1 mM
[50], and complete inhibition of XylG from Streptomyces thermocarboxydus HY-15x was observed when Hg2+ was added at a nal
concentration of 1 mM [51]. The Mn2+ ion (1 mM) decreased the
xylanolytic activity of Xyn11AAOX by 34.45%. Xylanase from Bacillus
stearothermophilus T-6 was inhibited by 38% in the presence of Mn2+
(1 mM) [52], whereas the xylanase KRICT PX1 from Paenibacillus
sp. HPL-001 was completely inhibited by Mn2+ at a concentration
of 5 mM [53]. The xylanolytic activity of the recombinant enzyme
was decreased by 18.62% following the addition of EDTA, suggesting that a few metals are required for enzymatic activity. Similarly,
slight inhibition of the xylanase activity of Aspergillus versicolor [54]
and S. pathumthaniensis S582 was observed [31]. However, strong
inhibition (64%) of xylanase KRICT PX1 [53] was reported in the
presence of 5 mM EDTA. The behavior of enzymes in the presence
of metal ions is variable and still cannot be predicted [55].
Based on TLC analysis, Xyn11AAOX1 is an endo-type xylanase
without -xylosidase activity since xylose was not observed as
a product. This result supports the previous classication of this
species into the family 11 of glycoside hydrolases, since it has been
reported that xylanases from family GH11 do not hydrolyze xylo-

biose [56]. Lee et al. [57] reported that a xylanase from Laetiporus
sulphureus without -xylosidase activity and xylobiose and xylotetraose generated hydrolytic products from birchwood xylan.
5. Conclusion
We identied a single positive clone of P. pastoris that could
extracellularly express the endo-1,4--xylanase Xyn11AAOX with
a high degree of purity into the culture supernatant in a bioreactor. Additionally, we used methanol as the sole carbon source to
reduce the number of fermentation steps. Using this strategy, the
fermentation time was shortened to 48 h and high biomass and
recombinant xylanase production were achieved. Additionally, we
used minimal salt medium to grow the yeast, which can be used
to reduce costs in industrial application. Overall, we could produce the recombinant xylanase with a good balance among the
simple medium used, methanol as a sole carbon source, a short
fermentation time and the amount of xylanase produced.
Acknowledgements
Research was funded by the Departamento de Biotecnologa y
Bioingeniera, CINVESTAV-IPN, Mxico. M. Cayetano-Cruz received
a PhD scholarship from CONACYT, Mxico.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2016.04.014.
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