Download Enzymatic synthesis of sialic acid derivative by immobilized lipase

Bioresource Technology 102 (2011) 10136–10138
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Bioresource Technology
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Short Communication
Enzymatic synthesis of sialic acid derivative by immobilized lipase
from Candida antarctica
Chi-Min Chau a,b, Kuan-Ju Liu c,⇑, Chun-Hung Lin d
a
School of Applied Chemistry, Chung Shan Medical University, Taichung 40201, Taiwan, ROC
Department of Medical Research, Chung Shan Medical University Hospital, Taichung 40201, Taiwan, ROC
c
Department of Food Science, National Penghu University of Science and Technology, Makung City, Penghu 88046, Taiwan, ROC
d
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, ROC
b
a r t i c l e
i n f o
Article history:
Received 9 June 2011
Received in revised form 23 July 2011
Accepted 25 July 2011
Available online 28 July 2011
Keywords:
Sialic acid methyl ester monononanoate
N-acetyl neuraminic acid methyl ester
Organic solvent
Esterification
Lipase
a b s t r a c t
The effects of important reaction parameters on the enhancement of sialic acid derivative lipophilic properties through the lipase-catalyzed esterification of N-acetyl neuraminic acid methyl ester are investigated in this study. It is found that the lipase Novozym 435 from Candida antarctica is particularly
useful in the preparation of sialic acid methyl ester monononanoate (SAMEMN). The optimum temperature for the SAMEMN synthesis reaction using Novozym 435 is 60 °C, and nonanoic anhydride is found to
be the best substrate among all acyl donors. The Novozym 435-catalyzed esterification of N-acetyl neuraminic acid methyl ester gave a maximum yield of 87.7% after 6 h in acetonitrile at 60 °C. Because the
novel method developed is simple, yet effective, it could potentially be used industrially for the production of sialic acid derivatives.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Sialic acids (N-acetylneuraminic acids, Neu5Ac) are negatively
charged 9-carbon sugars predominantly found in vertebrates, a
few higher invertebrates, and certain types of bacteria (Schauer,
2000). In vertebrates, sialic acids are primarily expressed at the
outermost ends of the carbohydrate structures on cell surface glycoproteins and glycolipids or as homopolymers of N-acetylneuraminic acid in a-2,8 linkages in mammalian brain tissue (Angata
and Varki, 2002). Sialic acid-containing structures in eukaryotic
systems play important roles in various physiological and pathological processes, including cell–cell adhesion, viral infection, and
cell growth regulation (Schauer, 2000; Angata and Varki, 2002).
Therefore, sialic acid and its derivatives have broad applications
in health food and the pharmaceutical industry (Roy et al., 1992).
In the investigation of the receptor recognition of sialic acid for rational drug development, structurally modified sialic acids are
invaluable tools for understanding the important biological and
physiological properties of sialylated structures.
Esterification may be a suitable method for increasing the lipophilicity and stability of N-acetyl neuraminic acid methyl ester,
because the ester residue is well-characterized as a nontoxic carrier
moiety with a high affinity for cell membranes and great
⇑ Corresponding author. Tel.: +886 6 926 4115x3803; fax: +886 6 926 0259.
E-mail address: [email protected] (K.-J. Liu).
hydrophobicity to prevent degradation. However, the traditional
chemical synthesis of sugar or polyol esters requires acidic and metal catalysts at high pressures and temperatures (Ress and Linhardt,
2004; Yang et al., 2011), resulting, in most cases, in complex mixtures of monoester and di- or triester isomers and numerous byproducts (Zhou et al., 2011). Enzymatic reactions in nonaqueous
media by lipases have become increasingly valuable tools for generating sialic acid ester derivatives. By employing enzymatic technology, reactive processes can be carried out under mild
conditions with a broad range of substrates in an environmentally
friendly manner. Thus, this has become one of the most practical
and efficient methods for the production of complex sialates and
their derivatives. In the present work, it is found that sialic acid
methyl ester monononanoate (SAMEMN) can be efficiently synthesized from N-acetyl neuraminic acid methyl ester and nonanoic
anhydride using Novozym 435 in acetonitrile. The influences of several parameters on the esterification reaction are investigated.
2. Methods
2.1. Chemicals and enzymes
The following 5 commercial lipases were used: Pseudomonas
cepacia lipase (Amano PS) and Candida rugosa lipase (Amano AY)
were purchased from Amano International Enzyme Co. (Nagoya,
Japan); Rhizomucor miehei lipase (Lipozyme RM IM) and Candida
0960-8524/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.07.093
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C.-M. Chau et al. / Bioresource Technology 102 (2011) 10136–10138
antarctica (Novozym 435), from Novo Nordisk Inc. (Danbury, CT,
USA); and C. rugosa type VII lipase, from Sigma Chemical Co. (St.
Louis, MO, USA). Lipozyme RM IM and Novozym 435 were immobilized enzymes. N-Acetyl neuraminic acid methyl ester and nonanoic anhydride were the products of Sigma Chemical Co.
2.2. Optimization of SAMEMN synthesis in organic solvent
SAMEMN was synthesized in a 10-ml reaction vessel via the
esterification of N-acetyl neuraminic acid methyl ester (20 mM)
with nonanoic anhydride (60 mM) in acetonitrile (Liu and Shaw,
1995). The reaction mixture (1.5 ml) contained 50 mg of biocatalyst. Reactions were performed at 60 °C with orbital shaking
(200 rpm). The reaction was stopped by removing the enzyme
through filtration. The transformation products were analyzed by
high-performance liquid chromatography (HPLC) after removing
the reaction solvent by vacuum evaporation. Experiments were
conducted to study which factors, such as enzyme source, reaction
time (6–48 h), reaction temperature (30–60 °C), acyl donor type,
and reaction solvent, affected the yield of SAMEMN. The effects
of several parameters on SAMEMN yield were studied keeping all
other conditions constant.
2.3. Product and lipase activity analysis
The analysis and quantization of SAMEMN were performed on a
Hitachi L-7100 HPLC system equipped with a UV–VIS detector and
a system controller. Samples (5 ll) were run using the gradient
mode with a 15-min gradient from solvent A (CH3CN:H20 = 26:74)
to solvent B (CH3CN = 100) on a reverse-phase Mightysil RP-18GP
column (250 4.6 mm ID, 5 lm, particle size) at 40 °C. The flow
rate was adjusted to 1 ml/min, and peaks were monitored at
200 nm. Percent molar yields were determined using the standard
curve of SAMEMN. Triple samples were each analyzed twice. Lipase-specific activity was measured according to the method described by Rúa et al. (1993) using p-nitrophenyl butyzate as the
substrate. One unit of enzyme was defined as the amount of enzyme that released 1 lmol of p-nitrophenol per minute.
2.4. Statistical analysis
A variance analysis of the results was carried out using the General Linear Model Procedure from the SAS Statistical Software, Version 6.11 (1995). Lipase source, reaction time, temperature, acyl
donor, and organic solvent were each tested in triplicate. Multiple
comparisons of means were carried out by Duncan’s multiple
range test at p < 0.05.
3. Results and discussion
3.1. Screening of biocatalysts
The esterification of N-acetyl neuraminic acid methyl ester has
previously been carried out with lipases from various sources, in
either free or immobilized form; however, because of the relative
low stability of free lipases against high pressure and temperature
(Knez and Habulin, 2002), immobilized lipases have become the focus of current research and industrial applications. Three commercial free lipases (Lipases AY, PS, and Candida rugosa lipase Type VII)
and 2 immobilized lipases (Novozym 435 and Lipozyme RM IM)
were tested in acetonitrile at 60 °C to compare their effects on the
esterification producing SAMEMN. As shown in Table 1, 36.3 U/
mg protein of immobilized C. antarctica lipase, Novozym 435,
showed remarkable catalysis and specificity in the enzymatic synthesis of SAMEMN. When synthesis was catalyzed by Lipozyme
Table 1
Sialic acid methyl ester monononanoate formation by lipase from different sources.
Source
Trade name
Yield of
SAMEMN (%)*
Specific activity (U/mg
protein)
Candida
antarctica
Candida rugosa
Candida rugosa
type VII
Pseudomonas
cepacia
Rhizomucor
miehei
Novozym
435
Amano AY
Sigma
87.7 ± 0.8c
36.3
0.0 ± 0.0
0.0 ± 0.0a
1.9
82.0
Amano PS
0.0 ± 0.0a
111.0
Lipozyme
RM IM
25.0 ± 0.3b
34.0
a
Experimental conditions: 50 mg of crude lipase powder, 20 mM N-acetyl neuraminic acid methyl ester, and 60 mM nonanoic anhydride; the suspension was shaken
at 60 °C and 200 rpm for 6 h.
*
Triplicate data from separate experiments are expressed as mean ± SEM. Mean
values in the same column with different letters are significantly different
(p < 0.05).
RM IM, the yield after 6 h was 62.7% lower than that after synthesis
catalyzed by Novozym 435. Higher activity of Novozym 435 was
also found in the kinetic resolution of secondary alcohols in monoether-functionalized ionic liquids (Zhou et al., 2011). Thus, Novozym
435 was observed to efficiently catalyze the esterification of N-acetyl neuraminic acid methyl ester with nonanoic anhydride and appeared to be suitable for SAMEMN production.
3.2. Effects of reaction time and temperature on SAMEMN production
A time course was produced to monitor reaction progress and
possibly minimize process costs. After 6 h of incubation with Novozym 435 at 2% (w/w of reactants), a yield of 87.7% was observed
for SAMEMN. The amount of product increased with increasing
reaction time up to 6 h and then declined (12–48 h) significantly
because of a possible reverse hydrolysis reaction (Mutua and Akoh,
1993). Similar results have been reported for the synthesis of
methyl acrylate where the maximum ester yield was obtained in
6 h by the hydrogel-bound lipase of Pseudomonas aeruginosa
MTCC-4713 and gradually declined thereafter (Kanwar et al.,
2007). In addition, Yang et al. (2011) reported that the quality of
poly(oleic diacid-co-glycerol) was high when the reaction catalyzed by Novozym 435 in a vacuum (10 mmHg) system for 6 h.
Therefore, after considering these findings, a reaction time of 6 h
appeared to be optimal and was used in the following experiments.
In lipase-catalyzed reactions, temperature significantly influences both initial reaction rate and enzyme stability. In most cases,
the reaction rate increases with temperature while the stability of
enzymes declines (Ward et al., 1997; Foresti and Ferreira, 2007).
In order to investigate the effect of temperature on the activity of
Novozym 435 in acetonitrile, 4 different temperatures were employed, ranging from 30 to 60 °C. Fig. 1 (Supplementary data) shows
the effect of temperature on the catalytic activity of Novozym 435.
Nag (1988) have demonstrated that high temperatures can change
the conformation of enzymes that can alter the free energy of the
system, potentially affecting substrate binding capacity and reducing the yield of the reaction. Because Novozym 435 is quite thermostable, it was possible to run the reaction at temperatures as high as
60 °C, which allowed a maximum yield of 87.7% SAMEMN to be obtained. This result is in agreement with results obtained by De
Diego et al. (2011) in the production of biodiesel. Thus, 60 °C was
chosen as an optimal temperature for further reactions.
3.3. Selection of acyl donor
Acyl donors with different chain lengths (nonanoic acid, nonanoic anhydride, methyl nonanoate, and trinonanoin) were used
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C.-M. Chau et al. / Bioresource Technology 102 (2011) 10136–10138
Table 2
Effect of organic solvents on the Novozym 435-catalyzed sialic acid methyl ester
monononanoate at 60 °C.
Solvent
Log P
Yield of SAMEMN (%)*
n-Hexane
Isopropyl ether
Chloroform
t-Butanol
Tetrahydrofuran
Acetonitrile
3.50
2.20
2.00
0.80
0.49
0.33
0.0 ± 0.0a
0.0 ± 0.0a
0.0 ± 0.0a
0.0 ± 0.0a
0.0 ± 0.0a
87.7 ± 0.8b
Novozym 435 (50 mg) was added to a reaction mixture (1.5 ml) containing 20 mM
N-acetyl neuraminic acid methyl ester and 60 mM nonanoic anhydride. The reaction was carried out during 6 h in various organic solvent at 60 °C.
*
Triplicate data from separate experiments are expressed as mean ± SEM. Mean
values in the same column with different letters are significantly different
(p < 0.05).
at a 1:3 M ratio of N-acetyl neuraminic acid methyl ester:acyl donor in the presence of acetonitrile at 60 °C. SAMEMN was esterified
under optimized reaction conditions because of its great potential
for developing an intrinsic antioxidant applicable to health food
and the pharmaceutical industry. The influence of fatty acids, acid
anhydrides, fatty acid methyl esters, and triacylglycerols with different chain lengths as acyl donors on yield was investigated.
Nonanoic anhydride showed the best performance of all the nonanoyl samples in the esterification reaction, producing an 87.7%
yield (1.3-fold increase with respect to nonanoic acid), followed
by trinonanoin (79.8%), nonanoic acid (67.9%), and methyl nonanoate (46.5%). Liu et al. (1997) discovered similar trends in the SCCO2 system using different acyl donors for lipase-catalyzed interesterification. Romero et al. (2005) reported that acid anhydride
performed better than all other acyl donors, yielding 91% esterification in n-hexane at 313 K (a 4-fold increase with respect to acetic
acid). Nonanoic anhydride was confirmed to be the most suitable
acyl donor for the synthesis of SAMEMN.
3.4. Effect of different solvents
Sialic acid fatty acid esters consist of a long chain fatty acid,
which is soluble in organic solvents, and a sugar, which is insoluble
in most organic solvents. A suitable organic solvent had to be identified in which both substrates would dissolve and react as required. The effect of different organic solvents on the lipasecatalyzed synthesis of SAMEMN at 60 °C was studied (Table 2). It
was important to select an organic solvent should not affect lipase
activity and selectivity and has to be permitted for general use in
the manufacture of pharmaceuticals (Liu and Shaw, 1995). Esterification was performed in n-hexane, isopropyl ether, chloroform,
t-butanol, tetrahydrofuran, and acetonitrile. The highest yield,
namely, 87.7%, was obtained in acetonitrile after a 6 h reaction period at 60 °C. The final yield after a 6-h reaction period at 60 °C was
0% in all other solvents tested (Table 2). In this case, solvents with
log P > 3 values are not suitable because the solubility of the glycoside in this medium is very poor or null. These results are a good
indication that the nature of the solvent can significantly affect
the enzymatic synthesis of SAMEMN.
4. Conclusions
The lipase-catalyzed esterification of N-acetyl neuraminic acid
methyl ester with nonanoic anhydride in a small amount of organic
solvent–adjuvant was performed at atmospheric pressure. The
influence of different types of lipase on reaction rate was studied,
with the immobilized lipase Novozym 435 from C. antarctica giving
the best results. The highest conversion, 87.7% after 6 h of reaction,
was achieved in acetonitrile, which is biocompatible for the production of health foods. These results are of general interest for
developing industrial processes for the preparation of SAMEMN,
which is used in pharmaceuticals and in the synthesis of other sialic acid derivatives.
Acknowledgements
Financial support for this study from the National Science Council of the Republic of China under Grant (NSC 97-2313-B-346-002)
is greatly appreciated.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2011.07.093.
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