Solid-surface activated recombinant: Rhizopous oryzae lipase expressed in Pichia pastoris and chemically modified variants as efficient catalysts in the synthesis of hydroxy monodeprotected glycals

Article abstract of DOI:10.1039/c7cy00255f

Lipase of Rhizopus oryze expressed in Pichia pastoris (ROL_pp) was selectively immobilized on octyl-Sepharose, fixing the open and active conformations. This enzyme was compared to the commercial one (ROL_sigma) and a unique enzyme of 32 kDa was selectively adsorbed in both cases. Small differences in the N-terminal peptide sequence of both lipases seem to be involved in the enzyme fixing on the solid support, affecting the active site structure. This phenomenon resulted in a strong difference in catalytic properties between immobilized enzymes, with ROL_pp being the most active, specific and regioselective heterogeneous biocatalyst in the hydrolysis of lactal hexaacetate. Immobilized ROL_pp was 8 times more active, and more specific than immobilized ROL_sigma, with excellent regioselectivity (monodeprotection in 3-OH, >99% yield). Solid-phase chemical modification of the N-terminus of immobilized ROL_pp was attempted because of the moderate results obtained in the hydrolysis of glucal triacetate. Different biomolecules were introduced and the enzyme catalytic properties in this reaction were assessed. The modification of ROL_pp with a polycarboxylated peptide (pA) improved the activity, specificity and regioselectivity of the enzyme, producing mainly the 3-OH monodeprotected glucal. The presence of acetonitrile 3% (v/v) in the reaction medium negatively affected ROL_pp, being completely unspecific, whereas ROL_pp modified with p1 conserved the specificity and regioselectivity shown in fully aqueous medium. The presence of dioxane improved the specificity and varied the regiopreference of the immobilized lipase (from C-3 to C-6 and C-4 monohydrolyzed products). The posterior modification with pA improved the specificity and the regio-preference of ROL_pp towards C-6 monohydrolyzed product.

Full text of DOI:10.1039/c7cy00255f

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Solid-surface activated recombinant Rhizopous
oryzae lipase expressed in Pichia pastoris and
chemically modified variants as efficient catalysts
in the synthesis of hydroxy monodeprotected
glycals
Cite this: DOI: 10.1039/c7cy00255f
Marco Filice,ab Marta Molina,a M. Dolors Benaiges,c Olga Abian,defg
a
Francisco Valeroc and Jose M. Palomo
*
Lipase of Rhizopus oryze expressed in Pichia pastoris (ROLpp) was selectively immobilized on octyl-
Sepharose, fixing the open and active conformations. This enzyme was compared to the commercial one
(ROLsigma) and a unique enzyme of 32 kDa was selectively adsorbed in both cases. Small differences in the
N-terminal peptide sequence of both lipases seem to be involved in the enzyme fixing on the solid sup-
port, affecting the active site structure. This phenomenon resulted in a strong difference in catalytic prop-
erties between immobilized enzymes, with ROLpp being the most active, specific and regioselective hetero-
geneous biocatalyst in the hydrolysis of lactal hexaacetate. Immobilized ROLpp was 8 times more active,
and more specific than immobilized ROLsigma, with excellent regioselectivity (monodeprotection in 3-OH,
>99% yield). Solid-phase chemical modification of the N-terminus of immobilized ROLpp was attempted
because of the moderate results obtained in the hydrolysis of glucal triacetate. Different biomolecules were
introduced and the enzyme catalytic properties in this reaction were assessed. The modification of ROLpp
with a polycarboxylated peptide (pA) improved the activity, specificity and regioselectivity of the enzyme,
producing mainly the 3-OH monodeprotected glucal. The presence of acetonitrile 3% (v/v) in the reaction
medium negatively affected ROLpp, being completely unspecific, whereas ROLpp modified with p1 con-
served the specificity and regioselectivity shown in fully aqueous medium. The presence of dioxane im-
proved the specificity and varied the regiopreference of the immobilized lipase (from C-3 to C-6 and C-4
monohydrolyzed products). The posterior modification with pA improved the specificity and the regio-
preference of ROLpp towards C-6 monohydrolyzed product.
Received 10th February 2017,
Accepted 28th March 2017
DOI: 10.1039/c7cy00255f
rsc.li/catalysis
bohydrate chemistry, with tremendous versatility in the prep-
aration of different biologically relevant molecules such as
Introduction
Glycals, pyranose derivatives containing an unsaturated bond,
are quite useful in organic chemistry and in particular in car-
carbohydrate-based vaccines.1–3
In the application of these molecules to the architecture
of a final active structure it is necessary to consider adequate
strategies to obtain a regioselectively free unique hydroxyl
group in the molecules without affecting the stability of the
double bond. Indeed, many chemical deprotection–protec-
tion approaches modify this double bond.4 In this way the
use of biocatalytic strategies has been recognized as one of
the most successful methods to obtain this kind of building
block without affecting the double bond. This green process
permits the achievement of these molecules in a highly spe-
cific and regioselective way.5 However, the stability and the
production of the enzyme must be taken into account in the
final process for practical industrial implementation.
a Department of Biocatalysis, Institute of Catalysis (CSIC), c/marie curie 2,
Cantoblanco Campus UAM, Madrid, 28049, Spain.
E-mail: josempalomo@icp.csic.es; Fax: +34 91 5854760; Tel: +34 91 5854768
b Spanish National Research Centre for Cardiovascular Disease (CNIC),
Biomedical Research Networking Center for Respiratory Diseases (CIBERES), c/
Melchor Fernandez Almagro 3, 28029 Madrid, Spain
c Departament d'Enginyeria Química, Biológica i Ambiental, EE, Universitat
Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
d Instituto Aragonés de Ciencias de la Salud (IACS), Zaragoza, 50009, Spain
e Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Units
IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, Zaragoza, 50018,
Spain
f Aragon Institute for Health Research (IIS Aragon), Zaragoza, 50009, Spain
g Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades
Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
In this way, the use of the methylotrophic yeast, Pichia
pastoris, as a cell factory for the expression of recombinant
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proteins has become increasingly popular in recent times
and a substantial workhorse for biotechnology.6
In particular, P. pastoris used as a host for the production
of Rhizopus oryzae lipase (ROL) offers advantages compared
to other hosts such as high productivity, the capacity to grow
in a minimal medium at high cell densities, low levels of
endogenous protein secretion and the ability to efficiently
secrete heterologous protein.7,8
ROL is widely used in industrial applications10 and re-
cently this heterologous ROL version from P. pastoris has
been successfully described as an interesting biocatalytic tool
in the preparation of asymmetric compounds, cholesterol de-
rivatives and biofuels.9–13
Nevertheless, the production of this lipase in P. pastoris
(ROLpp) generates small differences in the N-terminal se-
quences14 compared with the same lipase expressed in E. coli
(ROLsigma).15 The structure of closed conformation of the li-
pase from R. niveus (RNL)16 – which presents an identity with
ROL of over 95% – is shown in Fig. 1. In general the
N-terminal sequences of RNL, ROLpp and ROLsigma show a
highly hydrophobic character (more than 10 of the last 28
aminoacids present hydrophobic residues)16 and part of
these sequences is stabilized by a near β-sheet (Fig. 1).
The N-terminal analysis showed some differences between
the recombinant and native lipases, especially in a tetra-
Fig. 2 SDS-PAGE of the immobilized form of ROL in octyl-Sepharose.
Lane 1. LMW marker. Lane 2. Soluble recombinant ROLpp. Lane 3.
Supernatant after immobilization of ROLpp on octyl-Sepharose. Lane 4.
Octyl-Sepharose-ROLpp immobilized preparation. Lane 5. Soluble
commercial ROLsigma. Lane 6. Supernatant after immobilization of
ROLsigma on octyl-Sepharose. Lane 7. Octyl-Sepharose-ROLsigma
immobilized preparation.
peptide sequence; the Glu-Ala-Glu-Phe sequence in ROLpp
(where two aminoacids correspond to the final sequence of
the alpha-factor of Saccaharomyces, and the next two corre-
spond to the restriction site where ROLpp was cloned)14 ver-
sus the Thr-Asn-Ser-Ala sequence in ROLsigma (aminoacids
from the prosequence of ROL) (Fig. 1). Indeed, the
Fig. 1 A. Surface lipase structure in closed conformation. B. Cartoon structure of Rhizopus lipase. The protein structure corresponds to Rhizopus
niveus lipase (>95% homology with ROL, identical N-terminus sequence with ROLsigma) obtained from the Protein Data Bank (pdb code: 1LGY) and
the pictures were created using Pymol v. 0.99. Lid oligopeptide (blue), hydrophobic residues (red), serine active site (green). C. N-terminal se-
quences of ROLpp and ROLsigma. The underlined aminoacids correspond to the N-terminus sequence in the protein structure in A and B.
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Fig. 3 Characterization of ROL lipases. A) Far-UV CD spectra; B) near-UV CD; C) fluorescent spectra. ROLpp (black line), ROLsigma (red line).
Fig. 4 Characterization of ROL lipases in the presence of Triton X-100. A) Fluorescent spectra of ROLpp. B) Fluorescent spectra of ROLsigma. C)
Near-UV CD spectra of ROLpp. D) Near-UV CD of ROLsigma. Without Triton (black line), with Triton (red line).
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Fig. 5 A. Surface lipase structure in open conformation. B. Lipase in open conformation adsorbed onto a hydrophobic surface. The protein
structure corresponds to Rhizopus delemar lipase (>95% homology with ROL, identical N-terminus sequence with ROLsigma) obtained from the
Protein Data Bank (pdb code: 1TIC) and the pictures were created using Pymol v. 0.99. Lid oligopeptide (blue), hydrophobic residues (red), serine
active site (green), N-terminus sequence (orange).
Table 1 Biocatalytic hydrolysis of 1
Biocatalyst
Specific activitya
Time (h)
Conversion (%)
Yield of 2 (%)
Others
Octyl-Sepharose-ROLsigma
Octyl-Sepharose-ROLpp
1.83 0.1
13.6 0.6
72
23
100
100
48
>99
52
—
a
The initial rate in μmol × mgprot−1 × min−1 × 10−3. It was calculated at 10–30% conversion.
Table 2 Hydrolysis of 3 catalyzed by different butyl-Sepabeads-ROLpp preparations at pH 5 and 25 °C
Biocatalyst
Specific activitya
Time (h)
Conversion (%)
Yield of 4 (%)
Yield of 5 (%)
Yield of 6 (%)
Others
Butyl-Sepabeads-ROLsigma
Butyl-Sepabeads-ROLpp
Butyl-Sepabeads-ROLpp-pA
Butyl-Sepabeads-ROLpp-pB
Butyl-Sepabeads-ROLpp-pC
Butyl-Sepabeads-ROLpp-pD
10.83 0.86
11.12 0.55
16.99 0.85
13.33 0.52
13.01 0.60
17.49 0.80
28
28
24
24
24
24
95
95
73
93
84
91
29
23
46
6
24
11
8
5
5
5
7
8
2
6
2
7
7
8
56
61
20
75
46
64
a
−1
The initial rate in μmol × mgprot × min−1 × 10−3. It was calculated at 10–20% conversion. Ac-Cys-Asp-Asp-Asp-Asp-Asp-COOH (pA), Hippuryl-
Arg-OH (pB), dipeptide Boc-Ala-Gly-OH (pC) and polygalacturonic acid (pD).
N-terminus in ROLpp is slightly more hydrophobic than that
in ROLsigma (Fig. 1C).
Some reports have demonstrated differences between
these two ROL enzymes in terms of specific activity with
long-chain esters, although all these studies were performed
with the lipase in soluble form.14 However, lipases in solu-
tion are in a certain equilibrium between a closed and an in-
active conformation, where the active site is secluded by an
oligopeptide chain (lid), (main) and an open and active con-
formation, where the lid is shifted, making the active site ac-
cessible, (minor). This heterogeneity of lipase solution means
that enzyme concentration strongly affects the final activity
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Fig. 6 Activity remaining of the different butyl-Sepabeads-ROL preparations in the presence of solvent. Relative activity values were calculated
considering the activity of the non-modified biocatalyst (nm) 100%. A) Activity of the butyl-Sepabeads-ROL after modification. B) Activity of the dif-
ferent immobilized ROL catalysts in the presence of co-solvent (v/v). Acetonitrile (10%) (blue), acetonitrile (20%) (red), dioxane (20%) (green). Activ-
ity was determined using the pNPB activity assay. Ac-Cys-Asp-Asp-Asp-Asp-Asp-COOH (pA), Hippuryl-Arg-OH (pB), dipeptide Boc-Ala-Gly-OH
(pC) and polygalacturonic acid (pD).
Table 3 Stability of the different butyl-Sepabeads-ROLpp preparations in the presence of co-solvent (v/v). The biocatalysts were incubated in the sol-
vent solution and the activity was measured after 3 h
Relative activitya (%)
Biocatalyst
10% (v/v) acetonitrile
20% (v/v) acetonitrile
20% (v/v) dioxane
Butyl-Sepabeads-ROLpp
Butyl-Sepabeads-ROLpp-pA
Butyl-Sepabeads-ROLpp-pB
Butyl-Sepabeads-ROLpp-pC
Butyl-Sepabeads-ROLpp-pD
56 2.8
42 2.5
41 2.0
56 2.6
25 1.6
100 6.0
20 1.3
83 4.1
60 3.0
30 1.8
60 2.4
100 4.0
40 1.6
60 2.9
40 1.5
a
The relative activity is determined considering 100% the activity of the biocatalyst dissolved in phosphate buffer 25 mM pH 7. Ac-Cys-Asp-Asp-
Asp-Asp-Asp-COOH (pA), Hippuryl-Arg-OH (pB), dipeptide Boc-Ala-Gly-OH (pC) and polygalacturonic acid (pD).
Table 4 Hydrolysis of 3 catalyzed by different butyl-Sepabeads-ROLpp preparations at pH 5 and 25 °C in the presence of 3% (v/v) acetonitrile
Biocatalyst
Specific activitya
Time (h)
Conversion (%)
Yield of 4 (%)
Yield of 5 (%)
Yield of 6 (%)
Others
Butyl-Sepabeads-ROLpp
Butyl-Sepabeads-ROLpp-pA
Butyl-Sepabeads-ROLpp-pB
Butyl-Sepabeads-ROLpp-pC
Butyl-Sepabeads-ROLpp-pD
14.66 0.88
1.66 0.1
4.10 0.23
4.16 0.20
3.83 0.20
48
72
48
48
48
99
85
100
100
100
1
48
0
0
1
1
0
0
1
2
1
3
0
0
0
96
34
100
99
97
a
−1
The initial rate in μmol × mgprot × min−1 × 10−3. It was calculated at 10–20% conversion. Ac-Cys-Asp-Asp-Asp-Asp-Asp-COOH (pA), Hippuryl-
Arg-OH (pB), dipeptide Boc-Ala-Gly-OH (pC) and polygalacturonic acid (pD).
values.17 Therefore the application of immobilization strate-
gies where a unique conformation of lipase can be present
on a solid-phase is quite important for a real comparison.
For the regioselective deprotection of carbohydrates using
lipases, the application of enzymes in open conformation has
been demonstrated to be mandatory for successful results.5
Many different examples demonstrate that lipases adsorbed
by hydrophobic interactions result in a fixed open and more
stable conformation.18–21 Indeed, in the case of carbohydrate
reactions, the best catalysts using lipases have been an
immobilized form where the lipase is adsorbed onto a hydro-
phobic support material.5
In this work we analyzed the effect of fixing the open con-
formation of both ROL lipases, by selective adsorption on a
hydrophobic matrix. The effect on the catalytic properties
and especially their application in the regioselective hydroly-
sis of different peracetylated glycals have been tested. Also
site-specific modifications of the ROLpp on the solid-phase
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Table 5 Hydrolysis of 3 catalyzed by different butyl-Sepabeads-ROLpp preparations at pH 5 and 25 °C in the presence of 3% (v/v) dioxane
Biocatalyst
Specific activitya
Time (h)
Conversion (%)
Yield of 4 (%)
Yield of 5 (%)
Yield of 6 (%)
Others
Butyl-Sepabeads-ROLpp
Butyl-Sepabeads-ROLpp-pA
Butyl-Sepabeads-ROLpp-pB
Butyl-Sepabeads-ROLpp-pC
Butyl-Sepabeads-ROLpp-pD
13 0.66
2.50 0.12
10 0.5
15 0.74
14 0.70
24
72
24
24
24
83
89
62
95
90
8
9
0
4
10
14
29
2
5
11
14
13
2
5
14
47
38
58
81
55
a
−1
The initial rate in μmol × mgprot × min−1 × 10−3. It was calculated at 10–20% conversion. Ac-Cys-Asp-Asp-Asp-Asp-Asp-COOH (pA), Hippuryl-
Arg-OH (pB), dipeptide Boc-Ala-Gly-OH (pC) and polygalacturonic acid (pD).
were performed by introducing different peptide sequences
in the N-terminal and their effect on catalytic properties was
studied.
were analyzed spectrophotometrically, measuring the incre-
ment in absorbance at 348 nm (∈ = 5.150 M−1 cm−1) pro-
duced by the release of p-nitrophenol (pNP) in the hydrolysis
of 0.4 mM of p-nitrophenol butryrate (pNPB) in 25 mM so-
dium phosphate buffer at pH 7 and 25 °C. To initialize the
reaction, 0.05–0.2 mL of lipase solution (blank or superna-
tant) or suspension was added to 2.5 mL of substrate solu-
tion. Enzymatic activity was determined as μmol of hydro-
lyzed pNPB per minute per mg of enzyme (IU) under the
conditions described above.
Experimental section
General
Ac-Cys-Asp-Asp-Asp-Asp-Asp-COOH (pA) was purchased from
Isogen (Netherlands). Lipase from Rhizopus oryzae (ROLsigma),
per-O-acetylated lactal (1), per-O-acetylated glucal (3), N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride
(EDC), Hippuryl-Arg-OH (pB), Boc-Ala-Gly-OH (pC), poly-
galacturonic acid (25–50 kDa) (pD) and p-nitrophenyl butyrate
(pNPB) were from Sigma Chem. Co (St. Louis, USA). Octyl-
Sepharose 4BCL fast flow was from GE heathcare (Uppsala,
Sweden). TLC analyses were run on silica plates (Merck 60
Immobilization of lipases
Two grams of ROL powder (containing 5 mg total protein per
gram) was dissolved in 60 mL of sodium phosphate buffer
pH 7.
F
254). HPLC analyses were performed using an HPLC-spectra
P400 (Thermo Separation products). The column was a
Kromasil-C18 (250–ϕ4.6 mm and mm) from Analisis
Octyl-Sepharose support was added to the enzyme solu-
tion; 1 g in 30 mL of ROL solution. The other 30 mL of ROL
solution was added to 1 g of butyl-Sepabeads. The reactions
were incubated at 25 °C for 4 h. After this time, enzymatic ac-
tivity of the suspension and supernatant were determined
using the pNPB enzymatic assay to determine the immobili-
zation yield and remaining enzymatic activity in the
immobilized preparation. After immobilization, the enzyme
derivative was recovered by filtration under vacuum, washed
with abundant distilled water and conserved dry in the
fridge. In all cases more than 95% of enzymatic activity was
immobilized on the support and the immobilized enzyme
conserved the initial activity.
5
Vinicos (Tomelloso, Spain). Analyses were run at 25 °C using
an L-7300 column oven at a flow of 1 mL min−1 and UV de-
tector L-7400. NMR spectra were recorded using a Varian
Mercury 500 MHz spectrometer and calibrated in accordance
with solvent standard peaks.
Production of Rhizopus oryzae lipase in Pichia pastoris
Rhizopus oryzae lipase (ROL) was produced by a mixed sub-
strate fed-batch cultivation of a recombinant P. pastoris strain
using methanol as an inductor.22 The culture broth was
centrifuged and microfiltered to remove the biomass. The
supernatant was concentrated by ultrafiltration with
a
N-terminal chemical modification of ROL immobilized on
butyl-Sepabeads
Centrasette® Pall Filtron system (New York, USA) equipped
with an Omega membrane with a 10 kDa cut-off, and subse-
quently dialyzed against 10 mM Tris-HCl buffer pH 7.5 and
thereafter lyophilized.23
Compounds pA–pD (5 equiv.) were respectively dissolved in
0.5 mL of acetonitrile and added to 10 mL of distilled water
pH 4.8 containing EDC (10 mM). After 30 min, one gram of
the ROL immobilized on butyl-Sepabeads (butyl-Sepabeads-
ROL) was added to the solution and the mixture was incu-
bated for 18 h. Then, the suspension was filtrated, washed
with abundant distilled water and stored in the fridge.
Enzymatic activity assay (pNPB assay)
In order to follow the immobilization process, the activities
of the soluble lipases and their immobilized preparations
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Stability of the different biocatalysts in the presence of co-
solvent
tion was complete, the reaction mixture was filtered by vac-
uum on a sintered glass filter and the solution was saturated
with NaCl and extracted with ethyl acetate. After evaporation
of the solvent under reduced pressure, the crude product was
purified by flash chromatography and determined by 1H-NMR.
1H-NMR (500 MHz, CDCl3) of 4. δ (ppm): 6.42 (dd, J = 6.1
Hz, 1H, H-1), 4.95 (dd, J = 6.2, 2. Hz, 1H, H-4), 4.84 (dd, J = 6.2,
3.2, 2.7 Hz, 1H, H-2), 4.43 (ddd, J = 6.7, 5.3 Hz, 1H, H-5), 4.22–
4.38 (m, 2H, H-6A, H-6B), 4.20–4.11 (dd, J = 6.2, 2.2 Hz, 1H,
H-3), 2.55 (bs, 1H, OH), 2.16 (s, 3H, CH3), 2.11 (s, 3H, CH3).
1H-NMR (500 MHz, CDCl3) of 5. δ (ppm): 6.49 (dd, J = 6.1
Hz, 1H, H-1), 5.41–5.50 (m, 1H, H-3), 5.22 (dd, J = 9.0, 6.5 Hz,
1H, H-4), 4.81 (dd, J = 5.9, 2.8 Hz, 1H, H-2), 3.98–4.09 (m, 1H,
H-5), 3.66–3.86 (m, 2H, H-6A, H-6B), 2.07–2.13 (2s, 6H, 2CH3).
1H-NMR (500 MHz, CDCl3) of 6. δ (ppm): 6.45 (dd, J = 6.2,
1.5 Hz, 1H, H-1), 5.31 (ddd, J = 7, 2.4 Hz, 1H, H-3), 4.76 (dd, J
= 3.6, 2.6 Hz, 1H, H-2), 4.38–4.57 (dd, J = 14.7, 7 Hz, 2H, H-
6A, H-6B), 4.01 (m, J = 2.6 Hz, 1H, H-5), 3.84 (bt, J = 9.6 Hz,
1H, H-4), 3.54 (bs, 1H, OH), 2.14–2.16 (s, 6H, 2CH3).
The different unmodified and modified butyl-Sepabeads-ROL
biocatalysts were incubated in phosphate buffer solution with
different amounts of co-solvent (v/v) (dioxane or acetonitrile).
The activity of the immobilized enzyme was evaluated over
time using the pNPP assay. At least triplicates of each assay
were made.
Biocatalytic deprotection of per-O-acetylated lactal (1)
1 (0.01 mmol) was added to a 2 mL solution of phosphate
buffer (25 mM) with acetonitrile (20%) at pH 5, 25 °C and
the reaction was initialized by adding 0.4 g of biocatalyst.
The hydrolytic reaction was carried out under mechanical
stirring, and the pH value was controlled by automatic titra-
tion. At least triplicates of each assay were made. Hydrolysis
reactions were followed by TLC. TLC conditions were hexane :
AcOEt 5 : 5 v/v, Rf2: 0.25, Rf1: 0.56. The reaction was also
followed by HPLC, where the eluent was an isocratic mixture
of 40% acetonitrile in 10 mM ammonium phosphate buffer
at pH 3.8, UV detection at 220 nm and a flow rate of 1 mL
min−1. Under these conditions, the retention times were: 1:
18.50 min, 2: 11.01 min.24
HPLC determination: retention times of the different
products in HPLC were: Rt3 = 24.6 min, Rt4 = 6.3 min, Rt5
6.6 min, Rt6 = 8.1 min.
=
Circular dichroism
The reaction was scaled up using 2 g of biocatalyst in the
hydrolysis of 0.05 mmol (24 mg) of 1 in 15 mL volume
(containing 20% ACN) at pH 5. When full conversion was
achieved, the reaction mixture was filtrated by vacuum on a
sintered glass filter. The catalyst was washed with distilled
water (3 times) and stored at 4 °C. The aqueous solution was
filtrated and saturated with NaCl and then extracted with
ethyl acetate (3 × 15 mL). The collected organic layers were
dried over anhydrous Na2SO4 which was then removed by fil-
tration and concentrated under vacuum, producing a white
Circular dichroism (CD) spectra of the different lipases were
recorded in a Chirascan spectropolarimeter (Applied Photo-
physics) at 25 ( 1) °C. Near-UV spectra were recorded at wave-
lengths between 250 and 310 nm in a 1 cm path-length cu-
vette and far-UV spectra were recorded at wavelengths
between 200 and 260 nm in a 0.1 cm path-length cuvette.
Protein concentrations were 20 and 10 μ, respectively, in
phosphate buffered saline, pH 7.2 (PBS; bioMerieux).
1
solid (2, 21 mg, 90%) which was identified by H-, 13C-NMR
Fluorescence spectroscopy
1
and 2D-COSY. H-NMR (500 MHz, CDCl3) of 2. δ (ppm): 6.33
Fluorescence measurements were performed in a Varian Cary
Eclipse fluorescence spectrophotometer (Agilent Technolo-
gies) monitoring the intrinsic tryptophan fluorescence in 2
μM of different ROL variants and peptide conjugate solu-
tions, using an excitation wavelength of 280 nm, with excita-
tion and emission bandwidths of 5 nm, and recording fluo-
rescence emission spectra between 300 and 400 nm. All
spectroscopic measurements were made in water.
(d, 1H, J = 5.90 Hz, H-1), 5.38 (d, 1H, J = 3.39 Hz, H-4′), 5.25 (t,
1H, J = 8.37 Hz, H-2′), 5.00 (dd, 1H, H-3′), 4.76 (dd, 1H, H-2),
4.59 (d, 1H, J = 8.0 Hz, H-1′), 4.43 (m, 1H, H-3), 4.20–4.10 (m,
2H, H-6a, 6b), 4.13–4.07 (m, 2H, H-6′a, 6′b), 4.08 (m, 1H, H-
5′), 3.98 (m, 1H, H-5), 3.63 (m, 1H, H-4), 2.15–2.09 (5s, 15H, 5
× CH3). 13C-NMR (300 MHz,CDCl3). δ (ppm): 170.58, 170.03,
169.60, 143.75, 102.66, 102.19, 81.96, 73.77, 71.51, 70.82,
68.70, 68.26, 66.98, 62.58, 62.10, 20.79, 20.59, 20.55, 20.45.
Results and discussion
Biocatalytic deprotection of per-O-acetylated glucal (3)
Characterization and adsorption of Rhizopus oryzae lipase
(ROL)
Per-O-acetylated glucal (3) (1.5 mM) was dissolved in 1 mL of
25 mM sodium phosphate buffer at pH 4.8, containing in
some cases 3% of co-solvent (v/v). 0.1 g of butyl-Sepabeads-
ROL preparation was added. The reaction was followed by
HPLC analysis. At least triplicates of each assay were made.
Analyses were run at 25 °C using an L-7300 column oven and
UV detector L-7400 at 220 nm. The mobile phase was an iso-
cratic mixture of 20% acetonitrile–80% water (containing
0.01% TFA); flow rate 1.0 mL min−1. For the product charac-
terization the reaction was scaled up 5 times. When the reac-
The two lipases, ROLpp and ROLsigma, were adsorbed on com-
mercial octyl-Sepharose at 25 °C for 3 h. In both cases, the li-
pase immobilization yield was excellent (>95%). This immo-
bilization method, using low ionic strength during the
incubation, permits the specific immobilization of lipases in a
crude mixture, e.g. containing esterases.25 Thus, the SDS-PAGE
analysis of the crude, supernatant and suspension of the differ-
ent immobilized proteins revealed that ROLpp presents three
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bands, one around 34 kDa, another slightly more intense at 36
kDa and another around 70 kDa (Fig. 2, lane 2). In the case of
ROLsigma, 3 bands also appeared, the main one at 40 kDa and
another two slighter bands at 34 kDa and around 97 kDa, re-
spectively (Fig. 2, lane 5). These results showed different pro-
teins contained in the crude extract of both ROL lipases. How-
ever, in both cases only a single strong band at 34 kDa was
observed in the immobilized form (Fig. 2, lane 4 and 7). The
other bands were not adsorbed on octyl-Sepharose under these
conditions, which could indicate that those proteins are not li-
pases, (e.g. the 40 kDa which has been identified as esterase).14
Thus, the two immobilized ROL preparations can be
completely comparable for an evaluation of whether the varia-
tion in the N-terminal sequence has any effect on the catalytic
properties of the enzyme.
In order to know if some structural effect can be observed,
both enzymes were desorbed from the support after adding 1%
Triton X-100 and the soluble pure enzymes were concentrated
by ultracentrifugation using an Amicon Ultra filter 10 kDa.
Experiments of far circular dichroism (CD) to study possi-
ble modifications in the secondary structure and near CD
and fluorescence assays to analyze the tertiary structure were
performed (Fig. 3). According to the far CD spectrum, the
content of α helix secondary structure in the enzymes was
the same, which suggested that no significant differences in
that were observed (Fig. 3A). Additionally, small differences
in intensity were observed in the near CD spectrum but not
in shape and the fluorescence spectra of both enzymes were
identical (Fig. 3C). These results suggested that this small
variation in the N-terminal seems not to be significant
enough to structurally affect the whole enzyme.
sis of lactal hexaacetate 1 at pH 5 and 25 °C. Although the
structural results did not show clear differences, a tremen-
dous difference in catalytic properties was observed for this
particular reaction. ROLpp immobilized on octyl-Sepharose
was 8-fold more active than the immobilized ROLsigma
(Table 1). Furthermore, octyl-Sepharose-ROLpp showed excel-
lent specificity and regioselectivity, producing exclusively the
3-OH monohydrolyzed 2 in >99% yield, whereas immobilized
ROLsigma showed moderate specificity (Table 1). Thus ROL
showed a similar regio-preference in the monodeacetylation
to other lipases,5,27,28 although this result using immobilized
ROLpp is the best in terms of activity and excellent regio-
selectivity. This excellent result supports the theory by which
a simple change in some aminoacids in the N-terminal se-
quence of the enzyme expressed by Pichia pastoris generates
a more active and specific enzyme in this complex biocata-
lytic reaction.
In this way in order to expand the study, the ROL catalysts
were evaluated in the hydrolysis of glucal triacetate (3). To
this end, ROL enzymes were immobilized on butyl-
Sepabeads, a specific resin which does not suffer swelling,
which can thus be used both in aqueous and in organic me-
dia (where Sepharose collapsed29).
In the hydrolysis of 3, ROLsigma was slightly better in spec-
ificity and selectivity than ROLpp (Table 2). The butyl-
Sepabeads-ROLpp preparation showed low specificity in
monodeprotection and also low regioselectivity, producing
3-OH (4), 6-OH (5), and 4-OH (6) monohydrolyzed products,
with moderate yields for 4 (23%) (Table 2).
Therefore, considering these moderate results for butyl-
Sepabeads-ROLpp, a new strategy for improving the specificity
and regioselectivity of the enzyme in the hydrolysis of 3 was
attempted.
CD and fluorescent analyses of ROLpp and ROLsigma in the
presence of detergent were performed (Fig. 4).
In the fluorescence spectrum there was a displacement in
the maximum of 28 nm (from 330 to 302 nm), identical for
both lipases (Fig. 4A and B). This data showed the conforma-
tional change from mainly closed (without detergent) to open
conformation (in the presence of detergent) of the enzyme,
which is similar in both cases.
After the clear differences in catalytic performance of both
ROL enzymes in the hydrolysis of lactal due to the tetra-
peptide difference in the protein sequence (higher anionic
with hydrophobic degree residues in ROLpp) as a key role, a
site-specific chemical modification in the N-terminus of
immobilized ROLpp was attempted.
Near CD spectra also showed a change in the tertiary
structure with the presence of detergent in both cases; some
deviations were observed in the range of 310–285 nm in a
comparison between both enzymes.
The N-terminus of the protein seems to be also involved
in the interaction of the protein with a hydrophobic surface
(28-mer peptide sequence with 50% hydrophobic groups),16
considering the main hydrophobic pocket, the lid and sur-
rounding areas in the open conformation (Fig. 5). Further-
more, the N-terminal peptide of ROL is also interacting with
a loop (Asp232-Phe242) bearing the catalytic aspartic acid,
which could be influencing the final catalytic properties
when this lipase is fixed on a hydrophobic interface.25,26
Different biomolecules were selected for incorporation
into the protein, considering a balance of hydrophilic/hydro-
phobic. For that purpose, a polycarboxylated peptide Ac-Cys-
Asp-Asp-Asp-Asp-Asp-COOH (pA), which was recently demon-
strated to greatly alter the properties of a genetically modified
lipase,30 was used. Also Hippuryl-arginine (pB) which pre-
sents a hydrophobic N-protecting group with a positively
charged character (opposite to pA), dipeptide Boc-Ala-Gly-OH
(pC) (introducing hydrophobicity) and finally a polysaccha-
ride, polygalacturonic acid (pD), a hydrophylic and very car-
boxylated structure.
The effect of the modification on the enzyme activity was
evaluated (Fig. 6). The modification using molecules
containing carboxylic groups (pA, pC and pD) improved the
enzyme activity from 2 to 4 fold, whereas the introduction of
N-terminal arginine protected by a hydrophobic group (pB),
decreased by half the activity of the immobilized enzyme
Application of immobilized ROL preparations
The octyl-Sepharose immobilized forms of ROLpp and
ROLsigma were used as catalysts in the regioselective hydroly-
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(Fig. 6A). The activities analyzed in the presence of different
solvents and concentration results are shown in Fig. 6B. The
butyl-Sepabeads-ROLpp underwent an increase in activity es-
pecially in the presence of dioxane. Concerning the modified
butyl-Sepabeads-ROLpp a high hyper-activation of enzyme ac-
tivity is observed with pB modification in the presence of di-
oxane (around 8 fold) and also with acetonitrile (4 fold),
whereas the other modified ROLpp biocatalysts just conserved
the activity, or suffered strongly decreasing values such as
was the case of modification with pD.
tration, a solvent percentage high enough to conserve the
enzyme stability during the reaction times.
In the presence of 3% (v/v) of acetonitrile, most butyl-
Sepabeads-ROLpp preparations suffered a strong decrease in
activity and specificity (Table 4). The butyl-Sepabeads-ROLpp
modified with pA was unique in that it conserved the speci-
ficity and regioselectivity from that without co-solvent.
When 3% (v/v) dioxane was in the reaction medium, an
improvement in specificity for butyl-Sepabeads-ROLpp and
some modified preparations was observed (Table 5). How-
ever, the most significant change was the enantiopreference
of the enzyme: now the three different monohydrolyzed prod-
ucts were formed, in higher amounts for 5 (6-OH) and 6
(4-OH) instead of 4 (3-OH). The best result was obtained with
butyl-Sepabeads-ROLpp-pA, which showed the best specificity
and regioselectivity towards 5 (Table 5). The other modified
catalysts showed the same regioselectivity but worse specific-
ity than butyl-Sepabeads-ROLpp (Table 5).
These new biocatalysts were tested in the hydrolysis of 3
at pH 5 and 25 °C (Table 2).
Under these conditions, the butyl-Sepabeads-ROLpp-pA
showed better activity (2 fold) and much better specificity
and regioselectivity than the non-modified catalyst. This bio-
catalyst permitted us to obtain mainly 3-OH product (46%
yield from 73% conversion). The enzyme modification with
the other carboxylated molecules (pC and pD) improved the
activity but not significantly the specificity. Interestingly, the
butyl-Sepabeads-ROLpp-pB worsened the enzyme specificity,
also decreasing the regioselectivity (Table 2).
The specificity and regioselectivity achieved by the differ-
ent immobilized ROL biocatalysts were maintained during all
the reaction times.
Therefore, the site-specific chemical modification of
ROLpp with pA, a tetracarboxylated peptide, seems to be the
best catalyst for this monodeprotection reaction. Without a
solvent, this catalyst showed the best specificity, stability and
regioselectivity in the production of 4. However, acetonitrile
addition did not improve the enzyme catalytic properties for
this reaction, whereas the presence of dioxane had an inter-
esting effect in modulating the regioselectivity of the enzyme,
and in particular for this modified ROL, producing mainly
the 5 (6-OH) in a ratio of 2.23 against 6 (4-OH).
Stability of the new butyl-Sepabeads-ROLpp biocatalysts
The different modified ROLpp biocatalysts were incubated for
3 h in the presence of different co-solvent concentrations and
the remaining activity was calculated in the pNPB assay
(Table 3). In the presence of 10% (v/v) acetonitrile, around
half the activity value was conserved in the non-modified
ROL preparation, whereas the chemically modified catalyst
showed similar or less activity after this time. More inhibi-
tion was observed after incubation in the presence of 20%
acetonitrile. In this case the butyl-Sepabeads-ROL-pB con-
served higher activity than the non-modified one, whereas
the other modifications did not improve the result. As we
previously observed in Fig. 6 acetonitrile in general inhibited
enzyme activity more than dioxane. Indeed, in the presence
of 20% (v/v) dioxane, butyl-Sepabeads-ROL conserved up to
60% activity in 3 h of incubation. Surprisingly, the modifica-
tion with pA maintained the full initial activity of the enzyme
(Table 2). In the other biocatalysts the modification did not
help the stability of the enzyme.
Conclusions
The expression of R. oryzae lipase in P. pastoris generates
small changes in the N-terminal sequence of the protein
compared with the commercial version, which seem to be
structurally not significant although strongly affecting the li-
pase catalytic properties. The adsorption onto a hydrophobic
solid support permitted a real comparison of both enzymes,
because a unique enzyme is fixed in an active form (open
conformation) in both cases. The N-terminal sequence with a
hydrophobic nature is involved in fixing the enzyme on the
solid support which is also related to modifications in the ac-
tive site structure. This resulted in a strong difference in ac-
tivity and specificity between immobilized enzymes, with
ROL from P. pastoris being the most active, specific and
regioselective catalyst in the hydrolysis of fully acetylated di-
saccharide lactal.
In a second approach, chemical modification of the
N-terminus of ROLpp was attempted. Different biomolecules
were introduced at this position on the immobilized enzyme,
obtaining excellent results by modification with a poly-
carbxylated peptide, increasing the activity, specificity and
regiseolectivity of the enzyme in the hydrolysis of glucal, es-
pecially important when a co-solvent was used. Therefore, the
Thus, it was clear that the modification performed on the
N terminus of ROLpp showed an important effect on activity
and stability.
Hydrolysis of 3 catalyzed by butyl-Sepabeads-ROLpp
biocatalysts in the presence of co-solvent
In order to alter the catalytic properties of the immobilized en-
zyme, the hydrolytic reaction of 3 catalyzed by butyl-Sepabeads-
ROLpp biocatalysts was performed in aqueous media
containing 3% (v/v) of different co-solvents (Tables 4 and 5).
This relatively low co-solvent concentration was demonstrated
to be enough to alter the enzyme structure to improve the speci-
ficity and regioselectivity,31 and considering the previous re-
sults of immobilized ROL stability at higher co-solvent concen-
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Catalysis Science & Technology
directly immobilized enzyme or site-specific modified ones
could be excellent heterogeneous biocatalysts for application
in the improved preparation of glycosyl building blocks.
12 S. H. Duarte, G. L. del Peso Hernández, A. Canet, M. D.
Benaiges, F. Maugeri and F. Valero, Bioresour. Technol.,
2015, 183, 175–180.
13 J. Rodrigues, A. Canet, I. Rivera, N. M. Osorio, G. Sandoval,
F. Valero and S. Ferreira-Dias, Bioresour. Technol., 2015, 213,
88–95.
14 M. Guillén, M. D. Benaiges and F. Valero, Comparison of the
biochemical properties of a recombinant lipase extract from
Rhizopus oryzae expressed in Pichia pastoris with a native
extract, Biochem. Eng. J., 2011, 54, 117–123.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
Acknowledgements
15 A. Sayaria, F. Frikhaa, N. Mileda, H. Mtibaaa, Y. B. Alia, R.
Vergerb and Y. Gargouria, FEBS Lett., 2005, 579, 976–982.
16 M. Kohno, J. Funatsu, B. Mikami, W. Kugimiya, T. Matsuo
and Y. Morita, J. Biochem., 1996, 120, 505–510.
17 J. M. Palomo, M. Fuentes, G. Fernández-Lorente, C. Mateo,
J. M. Guisan and R. Fernández-Lafuente, Biomacromolecules,
2003, 4, 1–6.
18 M. Mathesh, B. Luan, T. O. Akanbi, J. K. Weber, J. Liu, C. J.
Barrow, R. Zhou and W. Yang, ACS Catal., 2016, 6,
4760–4768.
19 S. Sarkar, K. Das and P. K. Das, Langmuir, 2016, 32,
3890–3900.
20 N. Willems, M. Lelimousin, H. Koldsø and M. S. P. Sansom,
Biochim. Biophys. Acta, Biomembr., 2017, 1859, 340–349.
21 M. Marciello, M. Filice and J. M. Palomo, Catal. Sci.
Technol., 2012, 2, 1531–1543.
22 C. Arnau, R. Ramón, C. Casas and F. Valero, Enzyme Microb.
Technol., 2010, 46, 494–500.
23 P. Ferrer, M. Alarcón, R. Ramón, M. D. Benaiges and F.
Valero, Biochem. Eng. J., 2009, 43, 271–277.
24 M. Filice, R. Vanna, M. Terreni, J. M. Guisan and J. M.
Palomo, Eur. J. Org. Chem., 2009, 20, 3327–3329.
25 A. Bastida, P. Sabuquillo, P. Armisen, R. Fernandez-
Lafuente, J. Huguet and J. M. Guisan, Biotechnol. Bioeng.,
1998, 58, 486–493.
The authors thank the Spanish National Research Council
(CSIC) and the Ramon Areces Foundation for financial sup-
port. This work was supported by Fondo de Investigaciones
Sanitarias [PI15/00663 to OA] Miguel Servet Program from
Instituto de Salud Carlos III [CPII13/0017 to OA] Diputacion
General de Aragon [Digestive Pathology Group B01 to OA] and
by the project CTQ2016-74959-R of the Spanish Ministry of
Economy and Competitiveness. The CNIC is supported by the
Spanish Ministry of Economy and Competitiveness (MINECO)
and the Pro CNIC Foundation and by Severo Ochoa Center of
Excellence (MINECO award SEV-2015-0505). We also thank
Spanish Ministry of Economy for research grant SAF2014-
59118-JIN co-funded by Fondo Europeo de Desarrollo Regio-
nal (FEDER).
References
1 R. R. Schmidt and Y. D. Vankar, Acc. Chem. Res., 2008, 41,
1059–1073.
2 S. Koch, D. Schollmeyer, H. Löwe and H. Kunz, Chem. – Eur.
J., 2013, 19, 7020–7041.
3 S. J. Danishefsky, Y.-K. Shue, M. N. Chang and C.-H. Wong,
Acc. Chem. Res., 2015, 48, 643–652.
4 S. J. Danishefsky and M. T. Bilodeau, Angew. Chem., Int. Ed.
Engl., 1996, 35, 1381–1419.
5 M. Filice, J. M. Guisan, M. Terreni and J. M. Palomo, Nat.
Protoc., 2012, 7, 1783–1796.
6 M. Ahmad, M. Hirz, H. Pichler and H. Schwab, Appl.
Microbiol. Biotechnol., 2014, 98, 5301–5317.
7 G. P. L. Cereghino, J. L. Cereghino, C. Ilgen and J. M. Cregg,
Curr. Opin. Biotechnol., 2002, 13, 329–332.
26 U. Derewenda, L. Swenson, Y. Wei, R. Green, P. M. Kobos, R.
Joerger, M. J. Haas and Z. S. Derewenda, J. Lipid Res.,
1994, 35, 524–534.
27 R. Alnoch, R. Rodrigues de Melo, J. M. Palomo, E. Maltempi
de Souza, N. Krieger and C. Mate, Catalysts, 2016, 6, 191,
DOI: 10.3390/catal6120191.
8 X. Ponte, J. L. Montesinos-Seguí and F. Valero, Process
Biochem., 2016, 51, 1954–1963.
28 T. Bavaro, P. Torres-Salas, D. Ubiali and M. Terreni, RSC
Adv., 2013, 3, 7355–7359.
9 A. V. De Paula, G. F. M. Nunes, J. D. L. Silva, H. F. De Castro
and J. C. Dos Santos, Appl. Biochem. Biotechnol., 2010, 160,
1146–1156.
29 M. Filice, O. Romero, A. Aires, J. M. Guisan, A. Rumbero
and J. M. Palomo, Adv. Synth. Catal., 2015, 357,
2687–2696.
10 P. G. Quintana, M. Guillén, M. Marciello, F. Valero, J. M.
Palomo and A. Baldessari, Eur. J. Org. Chem., 2012, 23,
4306–4312.
11 P. G. Quintana, A. Canet, M. Marciello, F. Valero, J. M.
Palomo and A. Baldessari, J. Mol. Catal. B: Enzym.,
2015, 118, 36–42.
30 O. Romero, M. Filice, B. de las Rivas, C. Carrasco-Lopez, J.
Klett, A. Morreale, J. A. Hermoso, J. M. Guisan, O. Abian and
J. M. Palomo, Chem. Commun., 2012, 72, 9053–9055.
31 C. Godoy, G. Fernandez-Lorente, B. de las Rivas, M. Filice,
J. M. Guisan and J. M. Palomo, J. Mol. Catal. B: Enzym.,
2011, 70, 144–148.
Catal. Sci. Technol.
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