Aspergillus niger Lipase: Heterologous Expression in Pichia pastoris, Molecular

Modeling Prediction and the Importance of the Hinge Domains at Both Sides
of the Lid Domain to Interfacial Activation
Zhengyu Shu
Engineering Research Center of Industrial Microbiology, Ministry of Education, Fujian Normal University,
Fuzhou Fujian 350108, P.R. China
College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan Hubei 430074, P.R. China
Mojie Duan, Jiangke Yang, Li Xu, and Yunjun Yan
College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan Hubei 430074, P.R. China

DOI 10.1021/bp.147
Published online February 26, 2009 in Wiley InterScience (www.interscience.wiley.com).

Aspergillus niger lipase (ANL) is an important biocatalyst in the food processing industry.
However, there is no report of its detailed three-dimensional structure because of difficulties
in crystallization. In this article, based on experimental data and bioinformational analysis
results, the structural features of ANL were simulated. Firstly, two recombinant ANLs
expressed in Pichia pastoris were purified to homogeneity and their corresponding second-
ary structure compositions were determined by circular dichroism spectra. Secondly, the pri-
mary structure, the secondary structure and the three-dimensional structure of ANL were
modeled by comparison with homologous lipases with known three-dimensional structures
using the BioEdit software, lipase engineering database (http://www.led.uni-stuttgart.de/),
PSIPRED server and SwissModel server. The predicted molecular structure of ANL pre-
sented typical features of the a/b hydrolase fold including positioning of the putative cata-
lytic triad residues and the GXSXG signature motif. Comparison of the predicted three-
dimensional structure of ANL with the X-ray three-dimensional structure of A. niger feruloyl
esterase showed that the functional difference of interfacial activation between lipase and
esterase was concerned with the difference in position of the lid. Our three-dimensional
model of ANL helps to modify lipase structure by protein engineering, which will further
expand the scope of application of ANL. V C 2009 American Institute of Chemical Engineers

Biotechnol. Prog., 25: 409–416, 2009

Keywords: Aspergillus niger, lipase, heterologous expression, Pichia pastoris, molecular
model, interfacial activation

Introduction et al. (1993) compared the open conformation of Candida

rugosa lipase with the closed conformation of the homolo-
Microbial lipases (triacylglycerol acylhydrolases, EC gous lipase from Geotrichum candidum and concluded that
3.1.1.3) catalyze the hydrolysis and the synthesis of esters the largest structural differences between them occurs in the
formed from glycerol and long-chain fatty acids. Apart from vicinity of the active site. Three loops in this region differ
their natural substrates, lipases catalyze the chemoselective, significantly in conformation and the interfacial activation of
the enantioselective and the regioselective hydrolysis and the lipase is likely to be associated with conformational rear-
synthesis of a broad range of non-natural esters, and are rangements of these loops.5 The active center of the lipase is
widely used in industry.1 Lipase activity is greatly increased completely buried beneath a short amphiphilic helical seg-
at the oil-water interface, a phenomenon known as interfacial ment (lid or flap), which prevents access of substrate. Upon
activation.2 With the development of industrial applications, binding of the lipase to oil-water interface, the active site
the relationship between structure and function of lipase has becomes accessible and a hydrophobic area is exposed by a
attracted particular attention. Three-dimensional structures of structural rearrangement, involving the movement of the
more than 28 microbial lipases have been solved and share a lid.6–8
common motif known as the a/b hydrolase fold.3,4
Both lipases and esterases (carboxylesterase, EC 3.1.1.1)
Elucidation of the three-dimensional structures of lipases are members of the a/b-hydrolase fold family with a com-
provides an explanation for interfacial activation. Grochulski mon architecture composed of a specific sequence of a heli-
ces and b sheets. The active site of lipases and esterases
Correspondence concerning this article should be addressed to Y. J. consists of a Ser-His-Asp/Glu catalytic triad.4 The active site
Yan at yanyunjun@tom.com and Z. Y. Shu at shuzhengyu@gmail.com. hydrolyses ester bonds, accepts a broad range of non-natural

C 2009 American Institute of Chemical Engineers

V 409
410 Biotechnol. Prog., 2009, Vol. 25, No. 2

substrates, and exhibit excellent stereoselectivity. Most li- 30 , italics indicate the encoding sequence for His-tag) and
pases display the unique feature of interfacial activation, the reverse primer R1 (50 CGGCGGCCGCTTATAGCAGG-
whereas the esterase not. CACTCGGAAATC 30 ) incorporated the EcoRI and Not I
Aspergillus niger lipase displays positional selectivity to- restriction sites (underlined), respectively. The PCR amplifi-
ward the one- and three- positions of the glycerol moiety,9 cation protocol consisted of a 5 min denaturation at 94
C,
and is generally regarded as safe by the Food and Drug followed by 25 cycles of denaturation at 94
C for 1 min,
Administration of the United States of America, which annealing at 56
C for 1 min, extension at 72
C for 1 min,
makes it a conventional food additive in the food processing and a final hold for an extra 5 min at 72
C. The PCR prod-
industry. Recently, A. niger lipase (ANL) has also been used uct was digested with EcoRI and Not I, and then ligated into
as a detergent additive, and used in the acetylation of cellu- the EcoRI, Not I -digested pPIC9K plasmid. The resulting
lose, the degradation of ochratoxin A and asymmetric or- plasmid, pPIC9K-lipanl, was then transformed into E. coli
ganic synthesis.10–13 Much research on ANL has been DH5a, and the sequence of the ANL encoding region was
concentrated on its production, purification, characterization, confirmed by DNA sequencing.
immobilization, and application. However, there is no report The pPIC9K-lipanl plasmid and the pPIC9K plasmid were
of its three-dimensional structure. linearized with Sac I and then introduced into P. pastoris
In a previous work, a novel acid-resistant and thermo- GS115 by the lithium chloride transformation method. Indi-
stable lipase from A. niger F044 was purified, biochemically vidual colonies were picked up and screened on the Geneti-
characterized and the corresponding gene was cloned (Gen- cin-YPD plates and then on olive oil-emulsion YPD plates.
Bank access No. DQ647700) and expressed in Escherichia The best lipase-producing clone was chosen for further
coli in our laboratory.14,15 The amino acid sequence of ANL, induction expression.
deduced from the cDNA, was 50% and 36% identical to that
of Thermomyces lanuginosus lipase and A. niger feruloyl
esterase A, respectively. Although the lipase gene from A. Expression and purification of the recombinant lipase
niger was over-expressed in E. coli, the recombinant lipase
The P. pastoris recombinant strains were grown in 50 mL
protein accumulated in the cells in an insoluble form as
YPD media (1% yeast extract, 2% peptone, 2% dextrose) in
inclusion bodies. In this article, the lipase gene from A. niger
a 250 mL baffled flask at 28
C at 250 rpm until the culture
has been functionally expressed in Pichia pastoris. The
reached OD600 ¼ 2.0–6.0. The cells were harvested by cen-
recombinant lipase has been secreted directly into the culture
trifugation at room temperature and resuspended to an OD600
media and purified to homogeneity. From bioinformatics
of 1.0 in YPD media to induce expression. The culture was
data, the structural features of ANL have been predicted,
cultivated at 28
C at 250 rpm, with added methanol to a
which will may help ANL protein engineering to improve its
final concentration of 1% (v/v) every 24 h.
properties and then to further extend the scope of its indus-
trial application. A structural comparison between ANLs and After 96 h of induction expression, the supernatant was
the homologous esterases brought clues to elucidate their collected by centrifugation at 12,000 rpm for 10 min and
functional difference on interfacial activation. concentrated by lyophilization. The recombinant ANL,
tagged with an amino-terminal 6His, was purified on a Ni-
NTA agarose column followed by Sephadex G-75 gel-filtra-
Materials and Methods tion chromatography.
Strains and plasmids
A. niger F044 was isolated and identified in our labora-
Lipase activity assay
tory. E. coli DH5a was used as the cloning host and P. pas-
toris GS115 (Novagen) was used as the heterologous Lipase activity was quantitatively determined by an alkali
expression host. Plasmid pPIC9K (Novagen) was used as titration method using olive oil as substrate.16 The reaction
gene expression vector. was carried out in 50 mM Tris-HCl buffer (pH 7.5) for 10
min at 45
C. One unit of lipase activity was defined as the
amount of lipase necessary to liberate 1 lmol fatty acid
The primary structure elucidation of ANL from olive oil per min under the standard assay conditions.
The homologous amino acid sequence, which has more
than 30% identity to that of ANL and codes a protein with
known three-dimensional structure, was selected and aligned Secondary structure compositions of ANL
with the amino acid sequence of ANL in BioEdit software.
Some important amino acid residues, including the oxyanion The secondary structure compositions of ANL were deter-
hole residues and the active site residues, were identified mined using the JASCO J-810 spectropolarimeter. The deter-
in lipase engineering database (LED, http://www.led. mination parameters were as follows: 0.25-s response, 200
uni-stuttgart.de/). nm/min scanning speed, six accumulations, 0.01 cm cell
length, 0.5 nm band width. The wavelength ranged from 260
to 170 nm and the concentration of the recombinant proteins
Construction of the expression plasmid and ANL I and ANL II were 0.18 and 0.16 mg/mL, respectively.
transformation of P. pastoris Based on computer analysis using CCA program, the sec-
The DNA fragment encoding the mature A. niger F044 ondary structure compositions of the recombinant lipase
lipase (designated ANL, without the signal peptide sequence) were calculated from circular dichroism spectra.17
was amplified by PCR using the cDNA from A. niger F044 Secondary structure compositions of the recombinant ANL
as the template. The forward primer F1 (50 GCGAATTC- from P. pastoris was also predicted using the PSIPRED pro-
CATCACCATCACCATCACAGTGTCTCGACTTCCACGTTGG tein structure prediction server.18
Biotechnol. Prog., 2009, Vol. 25, No. 2 411

Figure 1. Alignment of the amino acid sequence of A. niger lipase (ANL) with those of homologous proteins of T. lanuginosus lipase
(TLL, NCBI accession No. O59952 and PDB accession No. 1DT5), P. camembetri mono- and diacylglycerol lipase (PCMDL,
NCBI accession No. P25234 and PDB accession No. 1TIA), R. miehei lipase (RML, NCBI accession No. P19515 and PDB
accession No. 3tgl) and A. niger feruloyl esterase A (ANE, NCBI accession No. O42807 and PDB accession No. 1uwc).
Underlining indicates the conserved Gly-X-Ser-X-Gly motif in lipase and esterase. Close triangles indicate the serine, aspar-
tic acid, and histidine residues, which are presumed to form the catalytic triad. The putative oxyanion hole residues are
indicated by closed circles. Cylinders are represented as a helices and arrows as b strands. The positions of a specific amino
acid residue were ascertained by LED (Lipase Engineering Database, http://www.led.uni-stuttgart.de/).

Homology modeling and evaluation ular model was evaluated using root mean square deviation
The amino-acid sequence of ANL shares 50% identity (RMSD)21 and Ramachandran Plot22 and checked using
with that of T. lanuginosus lipase (TLL). The atomic coordi- ProSA-web service.23 The optimized molecular model was
nates of TLL in their open conformation19 were obtained visualized using VMD1.8.6 molecular graphics viewer
from the Protein Data Bank database. 1DT5 was selected as software.24
template and the three-dimensional structure model of ANL
was built using SWISS-MODEL server.20 The initial molec- Three dimensional structural superposition
ular model was subject to energy minimization using Swiss- The atomic coordinates of A. niger feruloyl esterase,
PdbViewer and the geometric quality of the resulting molec- 1UWC,25 were obtained from the Protein Data Bank. The
412 Biotechnol. Prog., 2009, Vol. 25, No. 2

crystal structure of A. niger feruloyl esterase was superposed

over the crystal structure of TLL (PDB accession No. 1DT5)
and the predicted molecular model of ANL. Comparison
their three-dimensional structures was carried out using the
combinatorial extension method (http://cl.sdsc.edu/).26 The
resulting PDB files were visualized using RASMOL molecu-
lar visualization software.

Results and Discussion

Amino acid sequence analysis of ANL
The amino acid sequence of (ANL, NCBI accession No.
ABG37906), deduced from the cDNA, was aligned with
those of filamentous fungi lipases and an esterase with
known three-dimensional structure, namely (TLL, NCBI
accession No. O59952 and PDB accession No. 1DT5),
Penicillium camemberti mono- and diacylglycerol lipase Figure 2. SDS-PAGE analysis of the recombinant A. niger
lipase from P. pastoris GS115 transformants. Lane
(PCMDL, NCBI accession No. P25234 and PDB accession M: protein markers (in kDa); Lane 1: the purified
No. 1TIA), Rhizomucor miehei lipase [(RML), NCBI acces- ANL I; Lane 2: the purified ANL II; Lane 3: the
sion No. P19515 and PDB accession No. 3tgl] and A. niger recombinant ANL purified only by Ni-NTA agarose
feruloyl esterase A (ANE, NCBI accession No. O42807 and column; Lane 4: the concentrated culture superna-
tant of P. pastoris GS115 transformant integrated
PDB accession No. 1uwc). The amino-acid sequence of with A. niger lipase gene; Lane 5: the concentrated
ANL shares 50, 42, 34, and 36% identity with those of TLL, culture supernatant of P. pastoris GS115 transform-
PCMDL, RML, ANE, respectively. As can be observed in ant integrated with the linearized plasmid pPIC9K.
Figure 1, the amino acid residues forming the catalytic triad The purified recombinant ANL (Lane 3) corre-
in filamentous fungi lipases and esterase are completely con- sponded to the over-striking band in Lane 4.
served, and the serine residue, one of the catalytic triad, is
also located in a highly conserved pentapeptide motif.27
Combination of the three-dimensional structural information
of TLL, 19 ANE, 25 PCMDL, 28 and RML29 with the LED acid sequence of the ANL from His258, one of the catalytic
(http://www.led.uni-stuttgart.de/),3 we conclude that (1) the triad, to carboxylic terminal; (2) The His-tag at the amino-
catalytic triad of ANL is formed by Ser146, Asp201, and terminal of the recombinant ANL designed for affinity chro-
His258; (2) the oxyanion hole of ANL consist of Ser83 and matography was intact; (3) Bioinformatics analysis to
Leu147. the amino acid sequence of ANL also showed that there
were three potential N-glycosylation sites, including Asn32-
ValThrCys, Asn123LeuThrSer, and Asn242SerThrAla, res-
Expression and purification of ANL pectively. The predicted molecular weight of ANL was 31.6
After 96 h of induction expression of the transformant, the kDa (not including the signal peptide), which suggests that
lipase hydrolysis activity of the culture supernatant was 15.5 there exists some post-translational modification of the
U/mL. The expression level of ANL gene in P. pastoris recombinant ANL. Moreover, the purified recombinant li-
GS115 was lower than that of other fungi lipase genes,30–32 pases gave a positive periodic acid-schiff staining reaction,
which might be correlated with codon usage bias. The nucle- which shows that the recombinant lipases were modified by
otide sequence of the ANL gene did not have sequence iden- glycosylation (data not shown).
tity to any known lipase genes except for another ANL gene The specific activity of the native ANL is much higher
(GenBank access No. DQ680030) detected from GenBank than that of the recombinant ANLs. The glycosylation modi-
by the BLAST nucleotide sequence alignment (data not fication of the native ANL was also observed (data not
shown), while the amino acid sequence of ANL had obvious shown). A more in-depth investigation is required to explore
homology to that of other filamentous fungi lipases (Figure the effect of glycosylation modification on the specific activ-
1). With further analysis of the encode sequence, there found ity of ANL. It’s presumed that the site difference of the
three rare codons of P. pastoris in the ANL gene, which are glycosylation modification, the extent difference of the gly-
Arg75 (CGG), Arg195 (CGC), and Arg205 (CGG). cosylation modification, the conformation difference of the
After purification on a Ni-NTA agarose column and by modified ANL, and (or) the obstacle of the oligosugar
Sephadex G-75 gel-filtration chromatography, two recombi- branch to the substrate combination, etc. could have impact
nant ANLs, named ANL I and ANL II, were isolated from on the specific activity of ANL.
the culture supernatant. Their specific activity of the ANL I
and ANL II were 49 U/mg and 77 U/mg, respectively (Fig-
ure 2, lane1 and lane2) and was lower than that of the native Three dimensional molecular model building of ANL
ANL (1943.75 U/mg).14 The relative molecular mass of the As the amino-acid sequence of ANL shares 50% sequence
purified recombinant ANL was 35–40 kDa (Figure 2, lane3), identity with that of TLL solved at 2.4 Å resolution,19 it is
which was the same as that of the native ANL.14 The forms expected that most features of our comparative model are
of recombinant ANL I and ANL II seem to depend on the correctly predicted within a RMS deviation of 0.1 Å.33 There
difference in the degree of glycosylation. Firstly, the struc- are seven crystal structure data of T. lanuginosa lipase in the
ture of the recombinant lipase was intact because (1) no PDB database, including 1DT3, 1DTE, 1DT5, 1DU4, 1EIN,
potential protease cleavage site was identified in the amino 1GT6, and 1TIB, respectively. 1DT3, 1DTE, 1DU4, and
Biotechnol. Prog., 2009, Vol. 25, No. 2 413

Figure 3. The overall three-dimensional structure of A. niger

lipase, as obtained by homology modeling. b strands
are represented as arrows and a helices as cylinders. Figure 5. Energy plot of the predicted model of A. niger
Ser146, Asp201, and His258 form the catalytic triad. lipase.
The oxyanion hole consists of Ser83 and Leu147.
The a-helix span between Leu86 to Asp92 formed
the lid. Table 1. Secondary Structure Distribution of A. niger Lipase
Structure Element ANL I ANL II ANL*
a-Helix 36.1 32.8 32.59
b-Strand 25.9 25.0 21.85
Turns 11.5 16.5
Other 26.5 25.7 45.56
* The value originated from structure prediction by PSIPRED.

Figure 4. Ramachandran plot of the predicted model of A.

niger lipase.

1TIB are the close conformation. 1GT6 is originated from T.

lanuginosa lipase mutant. Although both 1DT5 and 1EIN are
the open conformation, only the crystal structure of 1DT5
was obtained in the presence of nonionic detergents and was
selected as the model template.
After refinement, one of the satisfactory structures
obtained is shown in Figure 3. The Ramachandran plot of Figure 6. Ser146, Asp201, and His258 form the catalytic triad
within the range of H-bond interactions.
this predicted model of ANL indicated that angles were in
allowed regions (Figure 4). The RMSD between the pre-
dicted model and the X-ray of TLL (1DT5) is 0.1 Å. The idues energy distribution is below the zero baseline, consist-
z-score of the predicted model is 8.01, the value close to ent with the parameters of a native conformation (Figure 5).
that of the experimentally determined structure of TLL Judged from the experimental data, the predicted proportion
(8.88), and is in the range of native conformation. The res- of the secondary structure elements and the predicted three-
414 Biotechnol. Prog., 2009, Vol. 25, No. 2

Figure 7. Three-dimensional structure comparison between lipase and esterase. (A) Superimposition of the three-dimensional
structure of A. niger feruloyl esterase on that of A. niger lipase; (B) Superimposition of the three-dimensional structure of
A. niger feruloyl esterase on that of T. lanuginosa lipase.

dimensional structure characterization of ANL correspond to ment results support the validity of the prediction model.
those of a/b hydrolase fold enzyme family4,27 when 1DT5 The differences of the secondary structure elements mea-
was used as structure template. sured in the two isoforms, ANL I, and ANL II, may be
The secondary structure elements of ANL, whether pre- attributed to the glycosylation modification.
dicted from the amino acid sequence of ANL by PSIPRED Similar to most basic features of microbial lipase, the
protein structure prediction server or determined from the model structure includes both a-helix and extended b-sheet
recombinant ANL I and ANL II by circular dichroism spec- secondary structures in the folded protein and the b-sheet is
tropolarimeter, consist of more than 30% a-helix and more in the core region surrounded with a-helix. Ser146, Asp201,
than 20% b-strand (Table 1). The proportion of the second- and His258, forming the catalytic triad, are adjacent to each
ary structure elements from ANL conforms to the structural other in the tertiary structure (Figure 3) and their separation
characteristics of the a/b hydrolase. The corresponding distances are within the range of H-bond interactions as
amino acid residue sequences of the different secondary shown in Figure 6. The active Ser146 residue occurs on
structure elements were shown in Figure 1. The measure- the hairpin turn, where a central b-sheet is converted to an
Biotechnol. Prog., 2009, Vol. 25, No. 2 415

Figure 8. Alignment of the amino acid sequence of the lid domain and the hinge domain at both sides of the lid. Boxes indicate the
a-helix and arrows indicate the b-sheet.

a-helix. An a helix fragment from Leu86 to Asp92, which is activation.36 There also existed a corresponding amino acid
known as a lid, is located in the top right corner of the residue, Ser84 at the lid’s left hinge in ANL molecular
active center. The oxyanion hole consisted of Ser83 and model. While in A. niger esterase molecular, the correspond-
Leu147, which donate their backbone amide protons to stabi- ing amino acid residue is Gly69, which has a more flexibility
lize the tetrahedral intermediate transiently formed in the hy- and destabilize the protein conformation. At the lid’s right
drolysis reaction, and Ser83 was neighbored to a conserved hinge of ANL molecular model, residues 97–99 form a b-
glycine residue (Figure 3). The distance predicted from the sheet component. Asp99 is substituted by Pro84 at its’ corre-
model between residues Cys22 and Cys268, Cys35 and sponding site of A. niger esterase. Due to the lack of a
Cys40, Cys104 and Cys107, respectively, allowed us to con- hydrogen atom on its nitrogen atom, Pro can’t form the cor-
nect them by three disulfide bridges (not shown in Figure 3). rect pattern of hydrogen bond and will destroy the b-sheet
component. Such amino acid residues as Gly69, Pro84, etc.
which are prone to form random coil component within the
Three dimensional structure comparison between lipase hinge regions at both sides of the specific a-helix will help
and esterase to make the a-helix far away the active site of A. niger
The canonical a/b hydrolase fold common to lipases and esterase.
esterases consists of a mostly parallel, eight-stranded b sheet From these results, it can be concluded that (1) the situa-
surrounded on both sides by a helices (with only the second tion of the specific a helix (named as lid in lipase) deter-
b strand antiparallel).4 It will help to understand the func- mines the functional difference of interfacial activation
tional difference of interfacial activation between the lipase between the lipase and the esterase; (2) the situation of the
and the esterase by structure comparison. specific a helix is determined by the conformation of the
An obvious structural difference is worth noting when the hinge regions at its both sides. These findings provide us
three-dimensional structure of ANL is superimposed on that some hints to design a lipase with a permanently open lid by
of A. niger feruloyl esterase. The lid domain of ANL is situ- optimizing amino acids within the hinge regions at both
ated in the top right corner of the active site while the corre- sides of the lid.
sponding a helix domain of A. niger feruloyl esterase is far
away from the active site (Figure 7A). This result can also Acknowledgment
be obtained by superimpose of the three-dimensional struc-
ture of T. lanuginosa lipase on that of A. niger feruloyl This work was supported by the National High-Technology
esterase (Figure 7B). A further analysis of the structural Project (‘‘863’’ Project) of P.R. China (No.2006AA020203,
components on both sides of the lid shows that there are two No.2007AA100703, No.2007AA05Z417) and the National
b-sheet components at the carboxyl side of the lid, while Natural Science Funds of P.R. China (No. 30870545).
there exist only random coil component at the corresponding
domain of A. niger feruloyl esterase (Figure 8).
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