Immobilization of neutral protease from Bacillus subtilis for regioselective hydrolysis of acetylated nucleosides: Application to capecitabine synthesis

Article abstract of DOI:10.3390/molecules21121621

This paper describes the immobilization of the neutral protease from Bacillus subtilis and its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave a poorly stable derivative that released almost 50% of the immobilized protein under the required reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was predicted in a 3D structure model of B. subtilis protease N built in silico by using the structure of Staphylococcus aureus metalloproteinase as the template. The immobilized enzyme shown a high regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative was obtained and successfully used in the development of efficient preparative bioprocesses for the hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in high yield.

Full text of DOI:10.3390/molecules21121621

molecules
Article
Immobilization of Neutral Protease from
Bacillus subtilis for Regioselective Hydrolysis of
Acetylated Nucleosides: Application to
Capecitabine Synthesis
Teodora Bavaro 1, Giulia Cattaneo 1, Immacolata Serra 2, Ilaria Benucci 3, Massimo Pregnolato 1
and Marco Terreni 1,
*
1
Department of Drug Sciences, University of Pavia, via Taramelli 12, I-27100 Pavia, Italy;
teodora.bavaro@unipv.it (T.B.); giulia.cattaneo01@universitadipavia.it (G.C.);
massimo.pregnolato@unipv.it (M.P.)
Department of Food, Environmental and Nutritional Sciences—DeFENS, University of Milano,
via L. Mangiagalli 25, I-20133 Milano, Italy; immacolata.serra@unimi.it
Department for Innovation in Biological, Agro-food and Forest Systems, University of Tuscia,
via S. Camillo de Lellis, I-01100 Viterbo, Italy; ilaria.be@unitus.it
2
3
*
Correspondence: marco.terreni@unipv.it; Tel.: +39-0382-987-265; Fax: +39-0382-422-975
Academic Editor: Roberto Fernàndez-Lafuente
Received: 10 September 2016; Accepted: 21 November 2016; Published: 25 November 2016
Abstract: This paper describes the immobilization of the neutral protease from Bacillus subtilis and its
application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful
for the preparation of anticancer products. Regarding the immobilization study, different results
have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave
a poorly stable derivative that released almost 50% of the immobilized protein under the required
reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic
carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained
enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was
predicted in a 3D structure model of B. subtilis protease N built in silico by using the structure of
Staphylococcus aureus metalloproteinase as the template. The immobilized enzyme shown a high
regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative
was obtained and successfully used in the development of efficient preparative bioprocesses for the
hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in
high yield.
Keywords: protease N; immobilization; nucleosides; regioselective hydrolysis; anticancer products;
capecitabine
1. Introduction
Proteases are hydrolytic enzymes that catalyze the cleavage of proteins. Proteolytic enzymes of
bacteria, fungi and viruses are largely studied due to their relevance in biotechnology for industrial
applications. Examples of important applications of neutral and alkaline proteases from Bacillus sp.
have been reported in fermentation and detergent industry [1]; and acid proteases of Aspergillus sp.
are used in the food industry for the production of cheese [2].
Most of the biotransformations used in food chemistry conveniently employ neutral proteases in
soluble form. The use of crude enzyme preparations as catalysts in fine chemical and pharmaceutical
processes is however affected by the stability limitations that characterize native enzymes, which could
Molecules 2016, 21, 1621; doi:10.3390/molecules21121621
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1621
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cause product contamination, raising problems of product quality and safety [
3
], especially in case of
drugs for parenteral use, or in the case of several anticancer and antiviral nucleosides.
These drawbacks may be overcome by immobilization of the biocatalyst on a solid carrier; in
fact, the binding with a matrix may increase the stability and the catalytic efficiency of the enzyme [4].
Moreover, the solid biocatalysts can be better manipulated than free enzymes. Immobilized enzymes
can be recovered and re-used at the end of the reaction, avoiding contamination of the final product
with residual proteins. However, immobilization could provide different outcomes depending on
the strategy used [5]. For example, immobilization of enzymes normally provides derivatives with
better stability, but in many cases a reduction of the enzyme activity is observed (caused by the
distortion of the enzyme due to interaction with the support and by diffusional problems). In some
instances, the activity may also be enhanced upon immobilization, such as in the case of lipases
adsorbed on hydrophobic supports via interfacial activation. However, enzyme adsorption (by ionic
or hydrophobic interaction) on solid carriers can be poorly stable and the protein can be released
under the non-physiological conditions (pH, temperature or presence of co-solvents) required in
preparative processes. On the contrary, covalent immobilization could ensure a suitable stability of the
enzyme derivative completely avoiding protein release, with the exception of multimeric enzymes
that require an additional post immobilization stabilization for preventing subunit dissociation [6].
In fact, in the case of multimeric enzymes, covalent immobilization would not be suitable to avoid the
enzyme inactivation and product contamination. In this case, only one or two subunits can be directly
immobilized by covalent attachment on the solid carrier while the others remain free to dissociate.
In this case a post-immobilization cross-linking is required for the stabilization of the active multimeric
structure of the enzyme, and to avoid the release of the protein from the solid biocatalyst.
Finally, it should be also considered that the catalytic performance of the biocatalyst can be
strongly affected by immobilization, depending on the carrier used (nature of the solid matrix and
binding chemistry) and the nature of the enzyme [6,7]. Consequently, the selection of the carrier and
immobilization condition should be carefully addressed considering physical-chemical phenomena,
such as substrate partition and diffusion, that could greatly affect the performance of the immobilized
enzyme [
or chemical modification of previously immobilized enzymes can be developed for improving the
properties of immobilized enzymes [ ]. In particular, the micro-environment around the immobilized
8]. In addition, different strategies, including the chemical modification of soluble enzymes
9
enzyme as well as any changes induced in its 3D structure by the covalent immobilization process
could be the main factors influencing the catalytic properties of immobilized enzymes [10].
Our research group has long studied the enzymatic synthesis of antitumoral and antiviral
modified nucleosides. Immobilized lipases have been successfully employed in the regioselective
hydrolysis of a large number of peracetylated building blocks [11,12]; however, to obtain an efficient
biocatalyst in terms of activity and selectivity, the immobilization of lipases needs to be performed by
hydrophobic adsorption [13]. This immobilization presents problems concerning the stability of the
enzyme derivative, including the desorption of the enzyme from the carrier in the reactions performed
in the presence of co-solvents (as required for the solubilisation of high amounts of substrate).
Enzymes immobilized by covalent binding demonstrated to be viable catalysts in a wide
variety of biotransformations, in particular for food and pharmaceutical applications [14,15].
Covalent immobilization can be particularly attractive to improve enzyme stability and to avoid
product contamination with residual protein, however, covalent immobilization of lipases provides
biocatalysts with very poor activity as a consequence of the reduced flexibility of the 3D structure that
prevents the lid to be open for the lipase activation.
Lipases, proteases and esterases have been used as catalysts for the regioselective deprotection of
nucleosides, for example, the neutral protease from B. subtilis (protease N) was reported to catalyse,
in aqueous medium, the selective hydrolysis of the primary position of peracetylated pyrimidine
5-substituted deoxyribonucleosides to give 30-O-acyl counterparts [16]. Furthermore, tri-O-acetylated
ester of purine and pyrimidine ribonucleosides were selectively converted by using soluble proteases

Molecules 2016, 21, 1621
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0
into corresponding nucleosides bearing only a free hydroxyl group in C-5 position; in all cases, a very
high regioselectivity was observed [17,18].
B. subtilis secretes at least six extracellular proteases: the neutral proteases A and B, the alkaline
protease (subtilisin), extracellular protease, metalloprotease and bacillopeptidase F. Among them,
subtilisin and neutral protease A are considered the major proteases since their activity accounts for 20%
and 70%, respectively, of total proteolytic activity in the culture medium [19]. Since the composition
of the crude preparation named protease N provided by Amano Enzyme and used in the present
work, is not clearly defined, we assumed that it mainly consists of neutral protease A (or protease N).
This enzyme is one of the most active casein-hydrolysing enzymes reported to date, and it belongs
to metalloproteinase class [20,21]. Protease N is the product of nrpE gene and, in the mature form, it
consists of a polypeptide chain with a molecular weight of approximately 33 kDa [22]. Amino acid
sequence is known [19] while 3D structure of this enzyme has not been resolved yet, since the structure
determination of Bacillus neutral proteases has been hampered by their tendency to autolysis. A large
number of papers describe covalent immobilization of hydrolases such as proteases and esterases, for
example, trypsin and α-chymotrypsin were recently immobilized on aldehyde- activated carriers by
covalent attachment with very good results [23]; on the contrary, only few reports describe the use of
immobilized protease N; among them the use of protease N immobilized by adsorption on Celite, as
catalyst for peptide bond formation [24].
In the present work, we describe the enzymatic deacetylation of a set of fully acetylated
nucleosides 1–11, including two fluorinated compounds 7 and 8, precursors of capecitabine [25],
an important chemotherapeutic agent largely used for the treatment of several cancer forms, including
the advanced stage of colon cancer, as well as breast and ovary cancers [26].
All the reactions have been studied by using different hydrolases (esterases, lipases and proteases).
The results of this screening show that the highest activity and regioselectivity toward almost all of the
substrates considered was obtained with the commercial neutral protease from B. subtilis. Therefore, a
study for the covalent immobilization of this enzyme by using different carriers and immobilization
protocols was performed in order to obtain a derivative with suitable activity and stability useful for
developing preparative processes. In particular, the covalent immobilization of this enzyme has been
investigated using two commercial carriers characterised by hydrophobic (Eupergit® C and Sepabeads
EC-EP) and hydrophilic (agarose) surfaces, pre-activated with epoxy groups and cyanogen bromide
(CNBr) respectively. Immobilization can be easily performed directly by reacting these supports with
the enzyme at neutral pH, but in this case, a limited number of bonds can be obtained between the
carrier and the reactive groups of the protein [27].
Agarose activated with aldehyde groups (glyoxyl) has been also investigated. In this case, the
carrier should be activated before the use, but immobilization is performed at high pH to get the
immobilization of the enzyme that must be evenly distributed in the first place via several points [28].
Accordingly, this approach allows a multipoint interaction of the enzyme with the carrier that ensure
a greater stabilization of the enzyme [15]. Finally, immobilization of Protease N by cross-linking
(mediated by glutaraldehyde) has been investigated in order to obtain the immobilization of the
protein far from the carrier surface (avoiding protein distortion and the influence of the nature of
the carrier). Glutaraldehyde is a reagent frequently used with this goal in order to design efficient
biocatalysts [29].
The effects of the different immobilization procedures on the activity, stability and selectivity
of this protease have been evaluated. In particular, the catalytic performance of different biocatalyst
preparations was compared with those of the soluble (not immobilized) enzyme in the hydrolysis
of peracetylated uridine (
derivatives useful as precursors for different modified nucleosides [11
1
) and cytidine (
5
) (Scheme 1) with the aim to prepare 50-monodeacylated
12]. In order to optimize the
,
experimental conditions, the immobilized enzyme was tested under different temperature and pH
conditions, as well as by using organic co-solvents to ensure the complete solubilisation of high
concentration of substrates and products.

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2. Results and Discussion
2.1. Screening of Different Hydrolases in the Enzymatic Deacylation of Peracetylated Nucleosides
For a preliminary screening different hydrolyses have been compared in the deacylation of
peracetylated nucleosides. The enzymes were used after a simple covalent immobilization on an epoxy
carrier (Eupergit® C). The crude extract of Aspergillus niger contains several esterases and lipases.
In this case, the esterase fraction was immobilized on Eupergit® C after a preliminary removal of the
lipases by adsorption on hydrophobic carriers, according to the previously reported procedure [30].
Other biocatalysts were obtained by direct immobilization of the commercial enzyme preparation
(see Materials and Methods section).
In all cases the immobilization yield (% of activity recovered after complete immobilization
of the enzyme) was higher than 20% (results not shown), with the exception of the protease N
from Bacillus subtilis that provided lower yields (the residual activity was about 20% of the total
activity immobilized). The screening was performed with different peracetylated substrates including
pyrimidine derivatives (Scheme 1) such as uridine (
thymidine ( ), cytidine ( ), N-acetyl-cytidine (
), N4-pentyloxycarbonyl-5-fluorocytidine (
and 5-fluorocytidine ( ), as well as some adenosine derivatives including adenosine (
arabinosyladenine (10) (Scheme 2) and ribavirin (11) (Scheme 3).
1
), arabinosyluracil (
2
), 20-deoxyuridine (
3),
7),
9),
4
5
6
8
X
X
X
R
R
R
N
N
N
N
O
N
O
N
O
R1
O
R1
AcO
AcO
+
R1
HO
O
O
OAc R2
OH R2
OAc R2
R1 = H
1a
2a
3a
4a
5a
6a
7a
8a
1
2
3
4
5
6
7
8
R = H
R = H
R = H
R = CH3
R = H
R = H
R = F
R = F
R
2 = OAc X = OH
R1 = OAc
R1 = H
R2 = H
R2 = H
X = OH
X = OH
X = OH
3b
4b
1 = H
R
2 = H
R
R1 = H
R1 = H
R1 = H
R1 = H
R2 = OAc X = NH2
R2 = OAc X = NHAc
R2 = OAc X = NHCOO(CH2)4CH3
R2 = OAc
X = NH2
Scheme 1. Enzymatic hydrolysis of substrates 1–8.
NH2
N
NH2
N
N
N
N
N
N
N
R3
AcO
R
R
O
O
R2 R1
OAc R1
R1 = OAc
R1 = H
R1, R2 = OAc R3 = OH
R1, R3 = OAc R2 = OH
9 R = H
10 R = OAc
9a R = H
10b R = OAc
Scheme 2. Enzymatic hydrolysis of substrates 9 and 10.
O
O
N
NHAc
N
NHAc
N
N
N
N
HO
AcO
O
O
OAc OAc
OAc
OAc
11
11a
Scheme 3. Enzymatic hydrolysis of substrate 11.

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In the enzymatic hydrolysis of peracetylated pyrimidine ribonucleosides
1
–
9
, protease N exhibited
a marked preference in terms of activity and regioselectivity toward the 50-position affording the
monodeprotected compounds in very good yield (64%–93%), and with a comparable biotransformation
rate (Table 1).
Table 1. Enzymatic hydrolysis of peracetylated pyrimidine nucleosides 1–11.
Products (Yield %)
Vh 1
Enzyme
t (h)
Conversion (%)
Substrate
1
0
0
5 -OH
3 -OH
Protease N
Esterase PPL
Esterase ANL
Acylase
22
96
5
0.52
0.02
3.13
0.66
100
79
98
1a (93)
1a (52)
1a (20)
1a (56)
n.i.
n.i.
n.i.
n.i.
4
97
Protease N
Esterase PPL
Esterase ANL
Acylase
48
120
5
0.04
0.02
5.66
0.10
48
47
99
97
2a (34)
2a (28)
2a (35)
2a (14)
n.i.
n.i.
n.i.
n.i.
2
3
4
5
6
24
Protease N
Esterase PPL
Esterase ANL
Acylase
48
48
5
0.10
0.01
4.46
0.68
93
28
99
98
3a (64)
3a (23)
3a (44)
3a (49)
3b (13)
3b (5)
3b (2)
3b (5)
5
Protease N
Esterase PPL
Esterase ANL
Acylase
24
48
3
0.71
0.36
2.07
0.07
98
91
92
92
4a (74)
4a (41)
4a (42)
4a (68)
4b (6)
4b (8)
4b (10)
4b (9)
5
Protease N
Esterase PPL
Esterase ANL
Acylase
48
48
5
0.16
0.90
5.10
0.89
94
88
98
97
5a (84)
5a (51)
5a (13)
5a (49)
n.i.
n.i.
n.i.
n.i.
3
Protease N
Esterase PPL
Esterase ANL
Acylase
48
48
3
0.19
0.09
6.14
0.35
82
99
99
94
6a (77)
6a (54)
6a (22)
6a (73)
n.i.
n.i.
n.i.
n.i.
7
Protease N
Esterase PPL
Esterase ANL
Acylase
24
24
5
18
3
0.10
0.15
4.30
0.18
3.3
97
92
94
90
98
7a (92)
7a (48)
7a (66)
7a (78)
7a (39)
n.i.
n.i.
n.i.
n.i.
n.i.
7
CRL
Protease N
CRL
30
30
0.18
0.12
99
93
8a (89)
8a (80)
n.i.
n.i.
8
9
Protease N
Esterase PPL
Esterase ANL
Acylase
48
48
3
0.12
0.03
4.42
1.11
93
72
97
99
9a (80)
9a (72)
9a (21)
9a (42)
n.i.
n.i.
n.i.
n.i.
3
Protease N
Esterase PPL
Esterase ANL
Acylase
24
24
5
0.09
0.04
3.61
0.77
55
36
99
99
n.i.
n.i.
n.i.
n.i.
10b (42)
10b (34)
10b (57)
10b (12)
10
11
4
Protease N
Esterase PPL
Esterase ANL
Acylase
48
48
4
0.05
0.10
3.16
0.76
62
58
98
11a (31)
11a (31)
11a (26)
11a (3)
n.i.
n.i.
n.i.
n.i.
4
100
Experimental conditions: 10% CH3CN in 25 mM KH2PO4 buffer pH 7, immobilized enzyme 50 IU, reaction
volume 2.5 mL, (substrate): 10 mM, r.t., n.i.: not isolated, 1 µmol·min/IU.
The only exception was 20,30-O-acetylarabinosyluracil (2a) synthesized in only 34% yield after
48 h. The yields were much lower compared with those previously reported for Candida rugose lipase
(CRL), the highest performing enzyme in terms of activity and selectivity toward the 50-position of
pyrimidine nucleosides reported to date [11].

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Interestingly, the selectivity of protease N in the hydrolysis of pyrimidine deoxyribonucleosides
and was different from those previously reported for CRL [11]; in fact, it was reported that CRL
gave a preferential hydrolysis in 30-position while protease N gives products bearing a free hydroxyl
group in position 50 (3a and 4a) with 74% and 64% yields, respectively [11]. For substrates
and
, protease N shown similar regioselectivity and afforded similar yields of the products 5a and 6a
3
4
5
6
compared with CRL.
Protease N was also successfully tested in the hydrolysis of substrates
7 and 8 to give products 7a
and 8a (in about 90% yield), two new intermediates for the preparation of capecitabine (Scheme 4) [25].
X
N
X
N
NHCOO(CH2)4CH3
N
F
F
F
N
N
O
O
N
O
Protease N
AcO
HO
CH3
O
O
O
OAc
OAc OAc
OH OH
OAc
7
8
X = NHCOO(CH2)4CH3
X = NH2
7a X = NHCOO(CH2)4CH3
8a X = NH2
Capecitabine
Scheme 4. General scheme of capecitabine synthesis.
The hydrolysis of the substrates
CRL [11]. In the case of substrate the results were comparable with those obtained by protease N,
while with the substrate the CRL gave a low regioselectivity. In the hydrolysis of substrate , the yield
(80%) of the 50-deprotected product 9a upon treatment with protease N resulted much higher if
7 and 8 was also tested with previously reported immobilized
8
7
9
compared with those reported for CRL (about 60% of yield) [11]. In the hydrolysis of substrate 10
the protease from B. subtilis shown the same regioselectivity compared with CRL, but in this case,
the yield of product 10b resulted lower than the yield previously reported with the lipase. Generally,
a lower regioselectivity than with protease N was observed for esterase from A. niger and acylase from
A. melleus under the same experimental conditions. These enzymes, in fact, gave nearly complete
hydrolysis in a shorter reaction time for almost all the substrates tested, if compared with protease N,
but the yields of monohydrolysed products were much lower compared with protease N. The esterase
from PPL extract shown poor activity and regioselectivity toward the 50-position of the peracetylated
pyrimidine nucleosides.
,
Finally, the protease N was also tested on peracetylated ribavirin 11, an example of a base-modified
ribonucleoside used for the treatment of hepatitis C infection [31]. Also in this case, the hydrolysis only
0
occurs in position C-5 in the sugar moiety (31% of 11a in 48 h), while the hydrolysis of the amide group
the heterocycle was not observed (Scheme 3). All the results are summarized in Table 1. Products 1a
as well as 10b and 11a obtained in the reaction catalysed by protease N, were identified by comparison
with analytical standards previously fully characterized by 1H and COSY NMR [11
12 25]. Furthermore,
–9a
,
,
product 3b and 4b, were not isolated and identified by HPLC analysis of the reaction mixture.
2.2. Covalent Immobilization of Protease N
Protease N provided the best results in terms of activity and selectivity in the hydrolysis of the
different substrates tested. A better characterization of the derivative obtained by immobilization
on epoxy-activated Eupergit C® was performed and this enzyme preparation was compared with
other derivatives obtained by immobilization on different carriers. Therefore, the effect induced by the
nature of the different carriers (hydrophobic or hydrophilic) and by the use of different immobilization
chemistry was evaluated measuring the final activity and stability of the enzyme derivatives prepared.
First, the stability of native protease N was studied under the experimental conditions required
for the different protocols of immobilization considered in this study (Figure S1). In all cases, activity
of the soluble enzyme was completely retained after 5 h; after this period, the activity decreased to a
different extent depending on the operational conditions. The enzyme is not completely stable under

Molecules 2016, 21, 1621
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the immobilization conditions used in the case of hydrophobic epoxy carriers such as Eupergit® C
and Sepabeads EC-EP. In fact, this immobilization procedure requires 24 h because a first hydrophobic
adsorption is induced by using high concentration buffers (at pH 8). Once the enzyme is adsorbed,
a covalent interaction occurs between lysine amino groups and the epoxy groups of the carrier [32].
Under these conditions, the enzyme lost more than 50% of its initial activity after 24 h (Figure S1).
Accordingly, the final activity obtained after immobilization on epoxy-carriers ranged from 15% to
20% (Table 2). In addition, about 50% of protease N was released from both these catalysts (Figure 1)
indicating that not all the loaded protein is covalently linked to the carriers. Therefore, the stability of
these enzyme derivatives resulted very low, comparable to the free enzyme (Figure 2). This evidence
could explain the low activity and the consequently the long reaction time in the hydrolysis of the
different substrates. In order to obtain an active and stable biocatalyst, the covalent attachment of
protease N on agarose (a hydrophilic carrier) was investigated using different activations, including
activated agarose (glyoxyl-agarose and glutaraldehyde-agarose).
Table 2. Covalent immobilization of protease N.
Loading 1 mg
Protein/g Support
Imm. Protein 2
(%) (SD)
Activity 3
Yield 4
(%) (SD)
Support
Activation
(IU·g−1) (SD)
Eupergit®
Sepabeads EC-EP
Glyoxyl agarose
C
Epoxy
Epoxy
Aldehyde
Aldehyde
Isocyanate
Isocyanate
10
10
10
10
10
50
62 (8)
58 (11)
90 (6)
98 (4)
96 (3)
65 (5)
1 (0.5)
20 (10)
16 (8)
32 (6)
22 (4)
24 (6)
31 (2)
0.8 (0.4)
1.6 (0.3)
1.1 (0.2)
1.2 (0.3)
7.8 (0.4)
Agarose glutaraldehyde
Agarose CNBr
Agarose CNBr
1 Loaded activity (IU −1): 5 IU for a loading of 10 mg/g, 25 IU for a loading of 50 mg/g (IU =
g µmol of substrate
·
hydrolyzed per min), 2 Measured by Bradford assay, 3 The activity of immobilized protease N is expressed as
IU·g−1 of support, 4 (Activity of immobilized protease N/loaded activity) × 100, SD = standard deviation.
Figure 1. Desorption of protease N in the experimental conditions used for the hydrolysis of different
peracetylated nucleosides.
100
80
60
40
20
0
0
20
40
60
80
100
120
Time (h)
Native
Eupergit C
Agarose CNBr
Sepabeads EC-EP
Agarose glutaraldehyde
Figure 2. Stability of protease N derivatives in 30% v/v of acetonitrile. Experimental conditions:
30% CH3CN in 25 mM phosphate buffer pH 7, immobilized protease N 250 mg, volume 1 mL, r.t.

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Glyoxyl- and glutaraldehyde agarose possess the same aldehyde functionalization, but in
the second case, the reactive groups are placed far from the carrier surface by introduction of a
spacer. Glyoxyl activation is directly obtained by oxidation of diols groups on the carrier surface,
while the glutaraldehyde carrier is prepared through the reductive amination of the glyoxyl-groups
(with ethylenediamine), followed by reaction of the obtained amino groups with glutaraldehyde [33].
In both cases, the immobilization was carried out at high pH values (pH 10) to ensure that the
aldehyde groups efficiently react with the amino groups of the enzyme to form the imino double
bonds. Imino bonds are reduced with sodium borohydride to obtain irreversible covalent attachment
through stable C-N bonds [33].
At a high pH value the amino groups of the enzyme are very reactive and the immobilization
is driven by the formation of the first imino double bonds (correlated to the reactivity of the
lysines). However, under these conditions, the formation of additional bonds is possible, and a
multipoint interaction can be achieved [32]. Immobilization on aldehyde-activated carriers can be
also performed at neutral pH but, in this case, a lower interaction degree between the enzyme and
the solid matrix would be obtained. Agarose activated with cyanogen bromide (CNBr) groups
has been also investigated. This carrier is characterized by the presence of isocyanate groups that
directly react with amino groups of the protein. In this case, immobilization efficiently occurs under
mild pH conditions [27] forming enzyme derivatives with few interactions between the protein and
the carrier (normally the terminal amino group is the only reactive site). The immobilization of
protease N on agarose gave similar results regardless of the activation of the carrier (Table 2) and
the conditions used, generally, the percentage of expressed activity after immobilization was higher
than that achieved on epoxy-hydrophobic carriers. In particular, when glyoxyl agarose was used
the immobilization yield (32%) was higher than those obtained by activation with glutaraldehyde
(22%). In both cases, the enzyme was completely immobilized and for immobilization at neutral pH
on glutaraldehyde-activated carrier the results were similar to those obtained at pH 10 (results not
shown). Similarly, immobilization on CNBr agarose was performed at neutral pH, providing a final
immobilization yield around 25%–30% (Table 2). No relevant differences in the final activity expressed
were observed when the protein loading was increased from 10 to 50 mg/g of carrier.
The immobilization on activated agarose carriers is completed in few hours (see Materials and
Methods) and under these conditions, the free enzyme is completely stable (Figure S1). Consequently,
the loss of activity observed after immobilization of protease N on agarose, (independently on the
experimental conditions and activation of the carrier), seems not correlated to the 3D distortion of
the protein structure associated to a high number of covalent bonds with the support. Moreover,
the enzyme desorption studies on the differently immobilized protease N shown, in all cases, that
enzyme was completely retained by the matrixes (Figure 1). Accordingly, these enzyme derivatives
shown a much higher stability than both the free enzyme and the epoxy-carriers derivatives (Figure 2).
The stability of the different preparations of protease N was evaluated in presence of buffer/acetonitrile
30% (v/v) (Figure 2).
As in preparative processes co-solvents could be used to ensure the solubility of substrates, we
tested the stability of the derivatives also in the presence of co-solvents [11,12,30]. All the agarose
derivatives shown a remarkable stability regardless the activation of the carrier. The retained activity
in all cases was complete after 120 h, while the native enzyme and the epoxy-carrier derivatives were
rapidly inactivated.
2.3. In Silico Evaluation of the 3D Structure of Protease N
In order to explain the results obtained after covalent immobilization of protease N on the
different carriers, the hypothetical localization of lysines on the enzyme surface was predicted in
a 3D structure model of B. subtilis protease N, obtained in silico by using the bioinformatics tools
HH-pred [34]; and Modeller [35]. Briefly, HH-pred, starting from a protein query sequence, produces a

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list of closest homologs. The 3D structural model is then calculated by the MODELLER software from
HHpred alignments.
In the case of protease N, the amino acid sequence of neutral protease A from B. subtilis [21
]
was used as query, and the closest homolog, with 52% of sequence identity (Figure S2), turned out to
be Staphylococcus aureus metalloproteinase, which was subsequently used to build the 3D structure
model (Figure 3). Since it is not known if protease N exists as a single monomer or as a homodimer,
two hypothesis can be considered.
Figure 3. Front (panel
A) and back side (panel B) in the 3D structure of neutral protease A
(protease N) from B. subtilis built in silico using the structure of Staphylococcus aureus metalloproteinase
as the template.
Considering the structure of monomeric protease N, the poor stability of the biocatalyst obtained
by immobilization on epoxy carrier of the enzyme can be explained. The simulation of the structure of
this enzyme (Figure 3) shown a large side of its surface (around the active site) completely lacking in
lysines, differently from the S. aureus metalloproteinase used as homolog for the structure prediction
and reported in the Supplementary Materials (Figure S3).
During the immobilization on hydrophobic epoxy carrier, the enzyme is very likely adsorbed
through this planar area of the enzyme surface. However, after adsorption, the formation of covalent
bonds is difficult or almost impossible to occur since the absence of reactive lysines in this side of the
protein surface and, consequently, a large surface of the enzyme will be only adsorbed on the carrier,
but not covalently linked. On the contrary, the orientation of the enzyme during immobilization on
aldehyde or isocyanate activated supports is driven by the formation of the first bonds with the most
reactive residues on the protein surface. For this reason, the preliminary adsorption is not necessary
and, consequently, the enzyme can be differently oriented with respect to hydrophobic epoxy carriers.
Also in the case of a homodimer, immobilization on epoxy hydrophobic carriers will not be suitable
to ensure a covalent interaction of the monomers with the support maintaining the intact dimeric
active structure of the enzyme. In fact, one protein chain would be linked to the carrier while the other
would be free to dissociate thus releasing the 50% of the protein, as observed after immobilization of
protease N on epoxy carriers.
Considering what we observed for the monomeric protease, we expected better results for the
covalent immobilization of a dimeric enzyme due to the reticular structure of this solid matrix that
would ensure the link of both protein chains thus retaining the 3D structure. The possible distortion of
the “active” conformation of the dimers could determine a loss of activity after immobilization.
2.4. Optimization of Enzymatic Hydrolysis of 1 and 5
The enzyme preparations obtained with protease N immobilized on agarose activated with
glutaraldehyde and agarose CNBr were used as biocatalysts in the hydrolysis of the peracetylated
nucleosides
1 and 5 (Scheme 1 and Table 3). The behaviour of these enzyme derivatives was compared
with that of the soluble enzyme. The enzymatic hydrolysis was carried out at room temperature in
phosphate buffer (pH 7) containing 10% (v/v) of acetonitrile.

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Table 3. Enzymatic screening of peracetylated nucleosides (1, 5) by immobilized protease N.
0
Product (Yield %) 5 -OH
Substrate
Support
t (h)
Vh
Conversion (%)
1
1
1
5
5
5
Native enzyme
Agarose CNBr
Agarose glutaraldehyde
Native enzyme
Agarose CNBr
Agarose glutaraldehyde
22
15
24
48
48
0.52
0.14
0.14
0.16
0.05
0.06
100
99
81
92
94
88
1a (93)
1a (94)
1a (69)
5a (84)
5a (66)
5a (54)
216
Experimental conditions: 10% CH3CN in 25 mM phosphate buffer pH 7, immobilized protease N 2 IU, reaction
volume 10 mL, substrate: 10 mM, r.t.
In the hydrolysis of substrate
1 the best results were obtained by using protease N immobilized on
agarose CNBr that afforded 1a in about 94% yield, similarly to what observed with the soluble enzyme.
When agarose glutaraldehyde derivative was used as biocatalyst in the hydrolysis of substrate 1,
the yield of 1a diminished (Table 3).
In the enzymatic hydrolysis of peracetylated cytidine 5
, the higher yield of 50-monodeprotected
compound (5a) was obtained with the protease N immobilized on agarose CNBr, compared with
the glutaraldehyde derivative. In fact, 5a was achieved in 66% yield in 48 h using the agarose CNBr
derivative, but regioselectivity and yields are lower with respect to the native enzyme (Table 3).
The hydrolysis of peracetylated nucleosides
1 and 5 was investigated under different reaction
conditions, studying the influence of parameters such as organic co-solvent, temperature or pH.
In particular, methanol, acetone and acetonitrile (10% v/v), were considered as co-solvents.
The temperature has been found to play a pivotal role in controlling the selectivity of the
hydrolysis of peracetylated uridine 1. In fact, as reported in Table 4, when the temperature decreases,
the regioselectivity and the yield markedly increased, although a reduction of activity and a consequent
increase of the reaction time required for complete hydrolysis of the substrates was observed. In fact,
◦
performing the hydrolysis at 4 C and pH 7, the mono-hydrolysed product 1a was obtained with
90% yield in 144 h. When peracetylated cytidine
5 was used as substrate, a decrease in the yield of
5a was observed increasing the pH (Table 4). The yield of 5a at pH 8.5 was lower with respect to
pH 7 (50% instead of 66% yield). The catalytic performance of protease N versus compound 5a was
not affected by temperature (4 ◦C or at 25 ◦C) and no difference was observed in the yields obtained.
Finally, the best co-solvent resulted acetonitrile, whereas methanol or acetone decreased the yields of
all products.
Table 4. Enzymatic hydrolysis of peracetylated nucleosides 1 and 5 catalysed by agarose CNBr-protease N.
0
Substrate
Solvent
pH Temperature
t (h)
Vh 1
Conversion (%)
Product (Yield %), 5 -OH
◦
1
1
1
1
1
1
5
5
5
5
5
5
MeOH
Acetone
CH3CN
CH3CN
CH3CN
CH3CN
MeOH
Acetone
CH3CN
CH3CN
CH3CN
CH3CN
pH 7, 25 C
7
23
15
144
216
27
48
120
48
0.12
0.09
0.14
0.02
0.01
0.08
0.07
0.02
0.09
0.01
0.005
0.02
96
99
99
99
95
97
88
85
94
82
92
93
1a (74)
1a (82)
1a (94)
1a (90)
1a (82)
1a (72)
5a (42)
5a (58)
5a (66)
5a (63)
5a (62)
5a (50)
◦
pH 7, 25 C
◦
pH 7, 25 C
◦
pH 7, 4 C
◦
pH 5.5, 25 C
◦
pH 8.5, 25 C
◦
pH 7, 25 C
◦
pH 7, 25 C
◦
pH 7, 25 C
◦
pH 7, 4 C
360
192
120
◦
◦
pH 5.5, 25 C
pH 8.5, 25 C
Experimental conditions: 10% of solvent in 25 mM phosphate buffer, immobilized lipase 2 IU, reaction volume
1.25 mL, (substrate): 10 mM, 1 µmol·min/IU.
The development of a preparative process revealed a poor productivity by increasing the
concentration of peracetylated uridine
1 (Table 5). The yield of 1a progressively diminished from 94%

Molecules 2016, 21, 1621
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to 79% when substrate concentration was increased up to 40 mM. On the contrary, the yield of 5a
increased from 66% to 85% when concentration was increased from 10 to 40 mM (Table 5).
Table 5. Hydrolysis of substrates 1 and 5 at different concentration.
0
Substrate
Conc. (mM)
t (h)
Vh 1
Conversion (%) (SD)
Product (Yield %) 5 -OH (SD)
1
1
1
5
5
10
20
40
10
40
24
48
72
48
72
0.48
0.30
0.28
0.09
-
94 (5)
92 (4)
90 (8)
94 (3)
88 (9)
1a (94) (5)
1a (86) (6)
1a (79) (10)
5a (66) (7)
5a (85) (6)
Experimental conditions: 10% CH3CN in 25 mM phosphate buffer pH 7, agarose CNBr-protease N 6 IU, reaction
volume 5 mL, r.t., SD = standard deviation.
3. Materials and Methods
3.1. General Procedures
The crude extracts of lipases from Porcine pancreas (PPL) and Aspergillus niger (ANL) were
purchased from Sigma-Aldrich (Milano, Italy). The crude extracts of protease N from Bacillus subtilis
and acylase Aspergillus melleus were kindly donated by Amano Enzyme Europe (Chipping Norton,
UK). Uridine was kindly donated by Adorkem Technology s.p.a. (Costa Volpino, Bergamo, Italy);
arabinosyluracil, 20-deoxyuridine, adenosine, arabinosyl adenine and peracetylated ribavirin were
kindly donated by Pro.bio.sint s.r.l. (Varese, Italy). Protease N PRD0950504N was a gift from
Amano Enzyme (Tokyo, Japan). Eupergit® C was kindly donated from Rohmpharma Rohm GmbH
(Darmstadt, Germany). Sepabeads EC-EP and EC-HG was a gift from Resindion (Binasco, Milano, Italy).
Bradford reagent, cyanogen bromide activated-Sepharose® 4B, Nα-Benzoyl-L-arginine ethyl ester
hydrochloride (BAEE), Bovine Serum Albumin (BSA), agarose, cytidine and thymidine were purchased
from Sigma-Aldrich.
Chromatographic purifications were performed on silica gel (Merck 60, Munich, Germany,
40–63 µm) with the solvent system indicated; TLC analyses were run on silica plates (Merck 60 F254)
and visualized by UV light (254 nm). HPLC analyses were run with a L-7100 HPLC (Merck Hitachi,
Tokyo, Japan) equipped with Merck Hitachi D-7000 HPLC Multi HSM Manager, a L-7400 detector,
an L-7300 oven and a C18 column (Kromasil, 250 mm
×
4.6 mm, 5 µm particles) The analysis were
conducted at 1 mL
·
min−1. UV wavelength and the percentage of organic modifier are selected
according to the maximum of absorption of the desired compound. The pH during the enzymatic
hydrolyses was kept constant by automatic titration and the enzymatic activities were measured by
using a pH-stat 718 Stat Tritino (Metrohm, Herisau, Switzerland). Peracetylated nucleosides
were synthesized using well-known previously reported methods [11]. Compound was prepared
in according to the literature [25]. Products 1a 9a as well as 10b and 11a obtained in the reaction
catalysed by protease N, were identified by comparison with analytical standards previously fully
characterized by 1H and COSY NMR [11
12 25]. Furthermore, product 3b and 4b, were not isolated
1–6 and
8
,9
7
–
,
,
and identified by HPLC analysis of the reaction mixture.
3.2. Enzyme Immobilization
Enzyme immobilization on different supports was realized applying the respective immobilization
procedure (see below), the enzymatic activity was monitored during the immobilization by enzymatic
standard assay and the amount of immobilized enzyme was assessed by measuring the enzyme
concentration before and after immobilization (Bradford method). The enzymatic preparation was
washed with deionized water and kept at 4 ◦C whenever not in use.

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3.2.1. Immobilization of Esterase Fraction Contained in the Crude Extract of Aspergillus niger Lipase
The crude extract of ANL (1.6 g), containing three lipase fractions (31, 43 and 65 kDa),
was dissolved in 22 mL of 10 mm phosphate buffer pH 7. The solution was stirred at room temperature
for 1 h. Octyl agarose (4 g), previously washed with the same buffer, was added to the enzyme solution
and the suspension was kept under stirring for 1.5 h at 4 ◦C. The octyl agarose derivative was then
filtered and washed with distilled water. To the solution was then added octadecyl Sepabeads (2 g),
previously washed with the same buffer, and the suspension was kept under stirring for 1.5 h at
◦
4 C. The enzyme derivative, containing a lipase fraction (31 kDa), was then filtered and washed with
distilled water. The supernatant, showing esterase activity, was diluted 1:1 with a solution of 2 M
phosphate buffer at pH 7.5. Sepabeads EC-EP (4 g), previously washed with the same buffer, was
added and the suspension was stirred at room temperature for 24 h. The enzyme derivative was then
filtered and washed with distilled water.
3.2.2. Immobilization of Esterase PPL and Acylase on Eupergit® C
The crude extract (0.5 g) were dissolved in potassium phosphate buffer 1 M pH 8.0 (12.6 mL).
After, 1 g of support, previously washed with water and potassium phosphate buffer, was added
thereto. The suspension was kept under stirring for 24 h at room temperature and then filtered.
The enzymatic preparation was washed with deionized water and kept at 4 ◦C whenever not in use.
3.2.3. Immobilization of Protease N on Eupergit® C and Sepabeads EC-EP
25 IU of protease N crude extract (0.5 g) were dissolved in potassium phosphate buffer 1 M pH 8.0
(12.6 mL). After, 1 g of support, previously washed with water and potassium phosphate buffer,
was added thereto. The suspension was kept under stirring for 24 h at room temperature and then
filtered. The enzymatic preparation was washed with deionized water and kept at 4 ◦C whenever not
in use.
3.2.4. Immobilization of Protease N on Glyoxyl Agarose and Agarose Glutaraldehyde
The supports were previously activated with the respective procedure and then submitted to
enzyme immobilization. The glyoxyl-agarose is obtained by reaction of agarose gel in reductive-basic
environment (NaOH 1.7 M, with NaBH4 28.4 mg/mL, 4 ◦C) with glycidol overnight, then oxidized
with NaIO4 for 2 h and washed.
The agarose glutaraldehyde support was activated as previously reported [34], briefly the
aldehyde-agarose (17.5 g) is aminated with ethylenediamine (EDA, 2 M pH 10.00) for 2 h and reduced
with NaBH4 (1 g, 2 h). The EDA activated agarose was then suspended in 0.2 M phosphate buffer pH 7
(3.4 mL) and a solution of 25% (v/v) glutaraldehyde (5.1 mL) was added. The mixture was kept under
stirring for 16 h at room temperature in the darkness.
Immobilization on aldehyde activated carriers was performed slightly modifying the procedure
previously reported [36,37]. The aldehyde-agarose gel (1.4 mL), glyoxyl agarose or agarose
gluteraldehyde, were suspended in 50 mM carbonate buffer at pH 10.05. After the addition of
25 IU of protease N enzyme extract (0.5 g), the suspension (14 mL) was kept under mechanical stirring
during 2.5 h. The chemical reduction of Schiff bases was carried out by adding to the mixture 14 mg of
NaBH4 (1 mg/mL) over 30 min. The immobilized enzyme was then filtered and washed with 10 mM
potassium phosphate buffer pH 5.0.
3.2.5. Immobilization of Protease N on Cyanogen Bromide-Activated Sepharose® 4B
25 IU of protease N (0.5 g of crude extract) were dissolved in 12.6 mL of immobilization buffer
(0.1 M NaHCO3, 0.5 M NaCl pH 8.5). The support (1 g) was previously activated in 200 mL of
1 mM HCl solution for 15 min, after that it was filtered and conditioned with immobilization buffer and
added to the enzymatic solution (final volume 14 mL). The suspension was kept at room temperature

Molecules 2016, 21, 1621
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under stirring for 2.5 h and then filtered. the immobilization requires a quenching passage realized for
2 h at 4 ◦C under stirring in 40 mL of 0.1 M TRIS-HCl buffer pH 8.0.
3.3. Activity Assay
To a solution of Nα-benzoyl-L-arginine ethyl ester hydrochloride (BAEE, 17 mg) in 25 mM
phosphate buffer pH 7 (final volume = 5 mL, (BAEE) = 10 mM), the enzyme was added (10 mg of
protease N enzymatic extract or 10–50 mg of immobilized enzyme). The reaction was kept under
mechanical stirring at room temperature. At fixed times, aliquots of the reaction mixture were
withdrawn (20 and 30 min), filtered by centrifugation (10 kDa MWCO centrifugal filter devices,
12,000 rpm, 22 ◦C, 10 min) to remove the protein and analyzed by HPLC using the method below
reported for the separation of the product, Nα-benzoyl-L-arginine acid (BAA), from the unreacted
substrate Scheme 1). Analytical method: A: 10 mM potassium phosphate buffer pH 4.2 (75%, v/v),
B: 9:1 CH3CN/H2O (25%, v/v) isocratic elution,
λ
223 nm, Rt
= 3.1 min, Rt
= 7.0 min.
BAEE
BAA
The enzyme activity was calculated as IU, that corresponds to the amount of enzyme that converts
1 µmoL of BAEE into BAA per minute.
3.4. Enzyme Stability
The enzyme stability was evaluated in phosphate buffer (25 mM, pH 7.0) in presence of 30% (v/v)
◦
of CH3CN during 5 days period at 25 C. The samples were periodically withdrawn and their activities
were measured with the activity assay described elsewhere.
3.5. Evaluation of the Attachment between the Enzyme and the Support
The different enzyme derivatives were incubated with buffered solutions containing acetonitrile
10% (v/v) at pH 7 for 24 h. After that, the release of the protein in the supernatant was determined
according to Bradford assay [38] using bovine serum albumin as standard.
3.6. Enzymatic Hydrolysis of Peracetylated Nucleosides 1–10
A solution of a peracetylated nucleoside (10–40 mM) in acetonitrile 10% (v/v) was added to a
solution of potassium phosphate buffer (pH 7, 25 mM). The pH was adjusted to 7.0 and the appropriate
amount of immobilized protease N was added. The suspension was maintained under mechanical
stirring at room temperature until the maximum hydrolysis of the substrate. During the reaction the
pH was kept constant by automatic titration (Metrohm 718 STAT Tritino). Samples of the reaction
mixture were analyzed at different times by TLC and HPLC. Finally, the enzyme was filtered off and
washed with deionized water and a solution of acetonitrile (10%), and the filtrate was concentrated
under reduced pressure and extracted with ethyl acetate (3
×
20 mL). The collected organic phases
were dried with anhydrous Na2SO4, filtered, and dried under vacuum. The residue, when necessary,
was further purified by silica gel column chromatography (CH2Cl2 100% to CH2Cl2/MeOH, 97:3) to
afford the different deprotected nucleosides 1a–9a, 3b, 4b and 10b.
3.7. Semi-preparative Method
Hydrolysis reaction was purified by using an HP-1100 pump (Agilent, Palo Alto, CA, USA),
equipped with a manual Rheodyne sample valve (1.4 mL loop) and connected to octadecyl silica
columns (LichroCart C18, 250 mm
The system was connected to an HPLC ChemStation (Revision A.04.01). The analytical method was
adapted to semi-preparative purpose: the flow rate was enhanced to 4 mL
min−1 and the proper
×
10 mm, 10 µm) and a single wavelength UV-detector (λ 260 nm).
·
volatile buffer have been chosen. Mobile phases were A: 100% 10 mM ammonium acetate buffer where
pH was adjusted to 4.2 adding acetic acid, B: 9:1 CH3CN/H2O and the purification was realized with
30% of B in isocratic mode and peaks were collected separately. Identity and purity of each fraction

Molecules 2016, 21, 1621
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was identified by injection on the analytical system. After the identification, the solution corresponding
to the desired product was lyophilised.
4. Conclusions
In this work, the protease N from B. subtilis has been conveniently immobilized on agarose
(hydrophilic carrier) by covalent attachment independently from the activation of the carrier,
maintaining about the 30% of initial activity and producing solid biocatalysts completely stable
in presence of co-solvent for the regioselective hydrolysis of acetylated nucleosides. Differently, the
immobilization on an hydrophobic acrylic carrier activated with epoxy groups leads to instable
biocatalysts because part of the enzyme is released from the support under the reaction conditions.
The enzyme leakage seems related to the fact that part of the protein is not covalently linked to the
carrier, but it is only adsorbed in its monomeric or dimeric form. This adsorption seems mediated by a
large area of the protein surface lacking in lysines (the amino acid reactive in the covalent interaction).
Protease N gave similar or better results compared with immobilized CRL, the best catalyst
reported to be a useful catalyst for the hydrolysis of peracetylated nucleosides (substrates 1, 5, 6 and 9),
to obtain products with a free hydroxyl group in position C-50. For this reason, the biocatalysts have
been tested also in the hydrolysis of two derivatives of the fluorinated cytosine in order to obtain
products 7a and 8a, useful as building blocks for the synthesis of Capecitabine and, in both cases,
immobilized protease N provided better results compared with CRL. Interestingly, the selectivity of
protease N in the hydrolysis of deoxyribonucleosides
previously reported for CRL allowing the hydrolysis in position C-50 instead of C-30.
Finally, in the hydrolysis of arabinosylnucleosides (substrates and 10), the protease N maintain
3 and 4 was opposite compared with the results
2
the same regioselectivity previously observed with CRL, but afforded much lower yields of products 2a
and 10b compared with this lipase.
In conclusion, the protease N covalently immobilized on agarose can be preferred to the
immobilized CRL for the preparation of acetylated ribonucleosides with only one free hydroxyl
group in position C-50, as useful building blocks for the synthesis of monophosphate nucleotides or
50-deoxyribonucleosides such as Capecitabine. The use of agarose-CNBr can be conveniently used for
the preparation of a solid biocatalyst suitable to be used in preparative reactions. Accordingly, with
the protease N derivative acetylated nucleosides bearing only one free hydroxyl group have been
prepared in 80%–85% yield and up to 14 g/L of concentration.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
12/1621/s1.
Acknowledgments: The authors kindly acknowledge Andrea Mattevi (Department of Biology and Biotechnology
Lazzaro Spallanzani University of Pavia) for the helpful suggestion for the in silico evaluation of the 3D structure
of protease N.
Author Contributions: Teodora Bavaro participated in enzyme immobilization and preparation of the manuscript;
Giulia Cattaneo participated in the enzymatic hydrolysis of some substrates; Immacolata Serra participated in
the in silico evaluation of the 3D structure of protease N; Ilaria Benucci participated in the immobilization of
protease N; Massimo Pregnolato and Marco Terreni participated in the preparation and revision of the manuscript.
All co-authors participated equally and substantially to the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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