Microparticle-based strategy for controlled release of substrate for the biocatalytic preparation of l -homophenylalanine

Article abstract of DOI:10.1021/cs4011919

Substrate inhibition is a universal challenge in biocatalytic process development. Herein, a controlled release of substrate from the microparticles was introduced and demonstrated to tackle this issue to increase the biocatalytic efficiency. Using phenylalanine dehydrogenase catalyzed production of l-homophenylalanine as a model reaction, and substrate-loaded microparticles were prepared and used as a reservoir to load a high amount of substrate and to control the release rate into the reaction media. Consequently, highly efficient biocatalysis could be sustainably achieved in the complex reaction system through constantly lowering the substrate concentration.

Full text of DOI:10.1021/cs4011919

Letter
pubs.acs.org/acscatalysis
Microparticle-Based Strategy for Controlled Release of Substrate for
the Biocatalytic Preparation of L‑Homophenylalanine
Jielin Zhang, Shanshan Tao, Baojie Zhang, Xuri Wu, and Yijun Chen*
State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, 24 Tongjia Street,
Nanjing, Jiangsu Province 210009, China
S
* Supporting Information
ABSTRACT: Substrate inhibition is a universal challenge in
biocatalytic process development. Herein, a controlled release
of substrate from the microparticles was introduced and
demonstrated to tackle this issue to increase the biocatalytic
efficiency. Using phenylalanine dehydrogenase catalyzed
production of L-homophenylalanine as a model reaction, and
substrate-loaded microparticles were prepared and used as a
reservoir to load a high amount of substrate and to control the
release rate into the reaction media. Consequently, highly
efficient biocatalysis could be sustainably achieved in the complex reaction system through constantly lowering the substrate
concentration.
KEYWORDS: biocatalysis, controlled release, microparticle, substrate inhibition, L-homophenylalanine
The concept of controlled release has been widely used in
the pharmaceutical settings. For example, controlled release of
drugs can sustainably give stable plasma drug concentration to
increase efficacy, reduce toxicity, and improve patient
compliance.15,16 The controlled release systems are typically
mixtures of a polymer and a drug so that the release behavior of
the drug could be controlled or predesigned on the basis of
diffusion, dissolution, osmosis, erosion, and other mecha-
nisms.17,18 Recently, micro- or nanoparticle-based drug delivery
systems have attracted much attention owning to their small
size, accurate control, and potential for easy preparation and
design.19 Therefore, we proposed that constantly controlling
the released substrate concentration at a relatively low level by
micro- or nanoparticle delivery systems could effectively avoid
substrate inhibition and consequently increase the volumetric
productivity for biocatalytic processes. Moreover, for the micro-
or nanoparticles encapsulating organic compounds, particularly
those with a predominant release mechanism of diffusion, the
substrate release is typically driven or hindered by the
conversion rate of the enzymes, serving as a demand-dependent
delivery system.14 This provides benefits to avoid substrate
inhibition for complex bioconversions involving multiple
enzymes and substrates. It is very difficult to decide adequate
substrate concentration and the supply speed because of
complicated and unpredictable kinetic behaviors during the
reaction. In another aspect, the solid−liquid biphasic system
could also prevent an undesirable increase in the volume from
batch-fed processes for poorly water-soluble or highly toxic
iocatalysis has been an important route in the production
Bof enantiomerically pure compounds because of its
inherent enantioselectivity, mild conditions, and potential to
reduce total synthetic steps;1,2 however, biocatalysts are usually
inhibited to reduce activity or even inactivated by high substrate
concentration.3,4 Because substrate concentration is commonly
a decisive factor to achieve high volumetric productivity and the
scalability of an efficient process, substrate inhibition has
become a universal issue for nearly all biocatalytic processes
and their industrial usefulness.
To address this important issue, a variety of approaches have
been explored, such as fed-batch addition of substrate,5,6
development of aqueous−organic biphasic systems,7 utilization
of resins in the reaction system,8,9 and use of ionic liquids as
reaction media10,11 and neat organic solvent.12,13 Most of these
approaches are designed to control substrate concentration in
the reaction below the critical inhibition level, which could thus
possibly diminish or reduce substrate inhibition and enable
sustainable transformation.14 However, these approaches have
certain limitations to efficiently increasing the volumetric
productivity. For example, batch-fed addition of substrate, the
most commonly used manner, usually causes an unavoidable
increase in the reaction volume and is less effective when
substrate inhibition is very serious.7,14 Organic solvents are
associated with the issues on safety and volatility that are
unsuitable for environmentally friendly processes.14 In addition,
the inclusion of resins in solid−liquid bioconversion usually
requires extensive screening and testing, which may also
hamper the stability of biocatalysts.14 Thus, it is necessary to
exploit a novel, green, and effective strategy for the substrate
supply to robustly increase the efficiency of biocatalysis.
Received: December 15, 2013
Revised: April 10, 2014
Published: April 16, 2014
© 2014 American Chemical Society
1584
dx.doi.org/10.1021/cs4011919 | ACS Catal. 2014, 4, 1584−1587

ACS Catalysis
Letter
substrates. Therefore, controlled release of substrate could be
an effective solution for the prevention of substrate inhibition
and the increase of biocatalytic efficiency, regardless of the
complexity of the reaction system.
To test our hypothesis, an appropriate reaction system and a
polymer material used for the encapsulation of substrate are
needed. First, the reductive amination of sodium 2-oxo-4-
phenylbutanoic acid (OPBA-Na) to L-homophenylalanine (L-
HPA) catalyzed by genetically modified phenylalanine dehy-
drogenase (mPDH) was chosen as a model reaction20 (Scheme
1) on the basis of the following factors: (a) the conversion
by a remarkable decrease in the particle size (Supporting
Information Table S3), which is in accordance with previous
reports.23,24 The loading efficiency, calculated as the ratio of the
substrate amount in the microparticles versus total particle
amount, increased with the input of OPBA-Na. When the
polymer/substrate ratio was 4:1, a loading efficiency of 17.48%
was achieved to give the most significant sustainable release
(Figure 1), which can deliver enough substrate into the reaction
media.25,26
Scheme 1. Reductive Amination of Sodium 2-Oxo-4-
phenylbutanoic Acid (OPBA-Na) to L-Homophenylalanine
(L-HPA) by Modified Phenylalanine Dehydrogenase
a
(mPDH) from Thermoactinomyces intermedius
a
Pichia pastoris formate dehydrogenase (FDH) coexpressed with
mPDH was coupled for the regeneration of NADH using HCOOH as
a cosubstrate.
Figure 1. Release of OPBA-Na from the microparticles with different
Eudragit RS100/substrate ratios. Data are presented as average values
of three independent experiments, and error bars represent standard
deviations. The release experiments were performed in 0.1 M
potassium phosphate buffer (pH 8.0) containing 4 M NaCl at 25
requires cofactor NADH, which is regenerated by coupling
formate dehydrogenase (FDH) and its substrate formate; (b)
the reaction contains multiple enzymes and substrates including
mPDH, FDH, NADH, OPBA-Na, ammonia and formate; (c) at
25 °C and pH 8.0, the biocatalytic process seriously suffered
from substrate inhibition, in which the initial velocity greatly
declined when substrate concentration was above 150 mM, and
the reaction was completely inhibited when OPBA-Na was over
250 mM (Supporting Information Table S1); (d) the reaction
kinetics are unable to fit into any current models for accurate
determination of the kinetic parameters; (e) our previous
efforts on a batch-fed process resulted in a significant increase
in the reaction volume, and the reaction was limited to only
eight cycles;20 and (f) product inhibition is not an issue because
L-HPA precipitates out during the reaction and the byproduct is
a gaseous CO2.21 Meanwhile, the use of nanoparticles as a
vehicle for substrate supply was not appropriate because the
amount of OPBA-Na loaded in the nanoparticles was too low
to achieve meaningful productivity and recovery rate, and the
release profiles of nanoparticles were unsatisfactory (data not
shown). Consequently, microparticles were the ultimate choice
for our study.
Next, a suitable polymer to prepare microparticles was
screened by examining the encapsulation rate and the release
profile of the substrate. As shown in Supporting Information
Table S2, Eudragit RS100, an acrylic polymer with quaternary
ammonium groups,22,23 exhibited desirable characteristics for
use as a material to load OPBA-Na compared to other
polymers tested. To prepare OPBA-Na loading microparticles,
the o/o emulsion solvent vaporization method was used to
obtain the microparticles with irregular shapes and rough and
condensed surface (Supporting Information Figure S1).
To maximize the loading efficiency and obtain a desired
release profile, different ratios between the polymer and OPBA-
Na were compared. As the percentage of the substrate
increased, the particle yield slightly changed, accompanyied
■
○
△
▼
°C, 250 rpm. Ratio of 4:1; ratio of 2:1; ratio of 1:1; ratio of
1:2.
As an ammonio methacrylate polymer, Eudragit RS100 is
insoluble but pH-independently permeable in water, and
compounds encapsulated in this material could be released by
diffusion.27 In this study, the release rate by a higher polymer/
drug ratio was significantly slower than that by lower ones as a
result of the possibility of an increased diffusion barrier (Figure
1). In particular, <60% of substrate was released in 6 h for ratios
of 4:1 and 2:1, suggesting their great potential for controlled
release of the substrate during the biocatalytic reaction.
Meanwhile, the release profile revealed a biphasic behavior
consisting of a retained phase in the beginning and a declined
phase after the release reached maximum. Because OPBA-Na is
stable in aqueous solution at pH 8 for 3 days (data not shown),
substrate decomposition in the declined phase was not likely to
occur. Meanwhile, adding NaCl into the reaction media could
increase the amount of OPBA-Na to release (Supporting
Information Table S4), indicating possible roles of charge
interactions between OPBA and the polymer. On this basis, the
release process should operate as a dynamic combination of
diffusion and absorption. When most of the substrate
encapsulated in the polymer diffused to the media, the
adsorption played a predominant role to give a downward
release profile. Similar adsorption was also observed for the
product L-HPA and the enzymes. Therefore, NaCl at optimal 4
M concentration was added in the release and reaction media in
the following experiments to disrupt the adsorption while
maintaining enzyme activity (Supporting Information Table
S4). Although NaCl is gentle to most enzymes, some enzymes
may have limited tolerance to such a high concentration.
Fortunately, in this case, a high concentration of NaCl did not
appear to have a negative impact on either enzyme.
1585
dx.doi.org/10.1021/cs4011919 | ACS Catal. 2014, 4, 1584−1587

ACS Catalysis
Letter
Subsequently, we employed the microparticles with poly-
mer/substrate ratios of 2:1 and 4:1 to take part in the model
reaction at a substrate concentration of 250 mM. Theoretically,
on the basis of Figure 1, substrate inhibition could be avoided
when the substrate concentration was controlled lower than the
critical inhibition concentration of 150 mM. Surprisingly, the
bioconversion was only <30% after 12 h, in which the actual
substrate concentration was >200 mM to cause substrate
inhibition (data not shown). This accelerated release in the
reaction was most likely from the shifted reaction equilibrium,14
or the dissociation of the substrate from the particles in the
presence of complicated reaction components.
Given that the particle size could greatly affect the release
rate, it is desirable to increase the particle size to achieve
controlled slower release. Hence, sieves were used to screen
particles for larger size and slower release (Supporting
Information Figure S2).23,25 After comparison, the micro-
particles with size over 250 μm were proven to be suitable, with
which 20% substrate was released in 12 h (Figure 2a) while
loading efficiency remained constant (17.48%). Then these
particles were used to supply 250 mM OPBA-Na (1.43 g
microparticles) for the reductive amination. This is a
concentration that is impossible for conventional batch reaction
by the same enzymes and cofactors (see Supporting
Information and Table S1). This time, 85% of OPBA-Na was
released out and successively transformed to L-HPA in 12 h
(Figure 2b). This was a remarkable increase compared with 3%
conversion by conventional batch reaction with severe substrate
inhibition. Through the process, the microparticles swelled a
little but remained intact. After collecting and drying, the
microparticles were intact and well-separated with shapes and
sizes that were similar to those at the beginning. However,
white precipitates of L-HPA were observed on the surface of the
microparticles. Thus, another 2 M NaCl was added into the
resulting solution for further dissociation of L-HPA. Meanwhile,
the pH was adjusted to 11 for dissolving L-HPA, and the
remaining microparticles were removed via filtration. The
product was finally recovered from the filtrate by adjusting the
pH back to neutral to obtain a recovery yield of 68.8%. This
slightly lower yield than previous batch-fed processes with the
same enzymes could be due to the strong binding of L-HPA to
the particles. Then, the pH value for dissolving L-HPA was
increased to 14 to further dissolve strongly bound product, and
the final yield was increased to 72.4%, higher, than previous
batch-fed processes.20 This continuous transformation for 12 h
suggested that both mPDH and FDH maintained stable
catalytic activity in the presence of the microparticles and
NaCl. In addition, the substrate concentration was constantly
controlled under the critical inhibition concentration (150
mM). Collectively, the initial reaction velocity increased 18-
fold, suggesting that Eudragit RS100 microparticles could avoid
substrate inhibition and increase overall catalytic efficiency.
Finally, to demonstrate the feasibility of the present strategy,
a further increase in the substrate concentration and reaction
scale was performed. In a 50 mL reaction system, OPBA-Na in
the microparticles was increased to 500 mM (100 g/L, 28.7 g
microparticles). After 33 h, the conversion was 80% (Figure
2c). Over the course of the reaction, the substrate sustainably
released from the microparticles and was immediately trans-
formed to L-HPA with >99% ee. In particular, the substrate
concentration was well controlled under the inhibition level
throughout the reaction. Total 400 mM (71.6 g/L) L-HPA was
produced, and the space−time productivity reached 51.7 g L−1
Figure 2. Reductive amination of OPBA-Na with the microparticles. A
polymer/substrate ratio of 4:1 and particle size >250 μm were used.
Data are presented as average values of three independent experi-
ments, and error bars are standard deviations. (a) Release profile of
●
OPBA-Na from the microparticles. , Dissolution behavior of OPBA-
□
Na; , Eudragit RS100 microparticles with particle size >250 μm. (b)
Time course of reductive amination of OPBA-Na using the
microparticles to load 250 mM substrate in 5 mL system. Red bar,
percentage of conversion with the microparticles; black bar, percentage
○
of conversion of conventional batch substrate addition; , actual
substrate concentration; dashed line, critical inhibition concentration.
(c) Time course of reductive amination of OPBA-Na using the
microparticles to load 500 mM substrate in the 50 mL system, and
finally, 400 mM L-HPA was formed. Red bar, percentage of conversion
with the microparticles; black bar, percentage of conversion of
■
conventional batch substrate addition (less than 1%). , actual
substrate concentration; dashed line, critical inhibition concentration.
1586
dx.doi.org/10.1021/cs4011919 | ACS Catal. 2014, 4, 1584−1587

ACS Catalysis
Letter
day−1. To the best of our knowledge, this is equal to the highest
substrate concentration ever reported for this biocatalytic
reaction.20,28 Moreover, the final reaction volume was increased
less than 2-fold over the beginning, significantly lower than that
of the batch-fed reaction (an over 5-fold increase), showing the
particular advantages for substrates with low aqueous solubility
in a very limited operational room, which is quite common for
the preparation of a large number of pharmaceuticals or
intermediates.14 Therefore, by applying a microparticle-assisted
substrate supply, the substrate inhibition in biocatalysis could
be completely eliminated to robustly enhance the biocatalytic
efficiency. In addition, the potential reuse of the polymer and
the advancement in developing less expensive materials for
more efficient encapsulation can further facilitate the
implementation of the present strategy. Although the present
study was intended only to deal with the issues associated with
substrate inhibition in biocatalytic processes, an effective
solution to simultaneously address both substrate and product
inhibitions would be more desirable and beneficial because
these inhibitions typically coexist to affect the catalytic
efficiency in most biocatalytic reactions.
In summary, we have demonstrated the concept of a
microparticle-based strategy to control substrate release for
efficient biocatalysis. Using a Eudragit RS100 microparticle
system, a high substrate concentration was achieved for mPDH-
catalyzed reductive amination of OPBA-Na. Because the
microparticle encapsulation technique can be applied to various
types of chemicals for controlled release, the present study
provides a general solution for efficient, environmentally
friendly biocatalytic processes for the preparation of valuable
chemicals.
(5) Li, Y.; Li, Y. Y.; Mi, Z. Y.; Li, D. S.; Tang, Y. J. Bioresour. Technol.
2009, 100, 2271−2277.
(6) Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.;
Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J.;
Devine, P. N.; Huisman, G. W.; Hughes, G. J. Science 2010, 329, 305−
309.
(7) Schmolzer, K.; Madje, K.; Nidetzky, B.; Kratzer, R. Biotechnol.
̈
̈
Bioeng. 2012, 108, 216−221.
(8) Brenna, E.; Gatti, F. G.; Monti, D.; Parmeggiania, F.; Sacchetti, A.
Chem. Commun. 2012, 48, 79−81.
(9) Truppo, M. D.; Rozzell, J. D.; Turner, N. J. Org. Process Res. Dev.
2010, 14, 234−237.
(10) Park, S.; Viklund, F.; Hult, K.; Kazlauskas, R. J. Green Chem.
2003, 5, 715−719.
(11) van Rantwijk, F.; Sheldon, R. A. Chem. Rev. 2007, 107, 2757−
2785.
(12) Klibanov, A. M. Nature 2001, 409, 241−246.
(13) Muttia, F. G.; Kroutila, W. Adv. Synth. Catal. 2012, 354, 3409−
3413.
(14) Kim, P. Y.; Pollard, D. J.; Woodley, J. M. Biotechnol. Prog. 2007,
23, 74−82.
(15) Parker, R. S.; Doyle, F. J., 3rd Adv. Drug Delivery Rev. 2001, 48,
211−228.
(16) Grassi, M.; Grassi, G. Curr. Drug Delivery 2005, 2, 97−116.
(17) Fredenberg, S.; Wahlgren, M.; Reslow, M.; Axelsson, A. Int. J.
Pharm. 2011, 415, 34−52.
(18) Feng, K.; Wang, S.; Ma, H.; Chen, Y. J. Pharm. Pharmacol. 2013,
65, 1757−1764.
(19) Singh, M. N.; Hemant, K. S. Y.; Ram, M.; Shivakumar, H. G. Res.
Pharm. Sci. 2010, 5, 65−77.
(20) Zhang, J.; Zhu, T.; Wu, X.; Chen, Y. Appl. Microbiol. Biotechnol.
2013, 97, 8487−8494.
(21) Cho, B. K.; Seo, J. H.; Kang, T. W.; Kim, B. G. Biotechnol.
Bioeng. 2003, 83, 226−234.
(22) Wu, P. C.; Huang, Y. B.; Chang, J. S.; Tsai, M. J.; Tsai, Y. H. Eur.
J. Pharm. Sci. 2003, 19, 115−118.
ASSOCIATED CONTENT
* Supporting Information
■
(23) Trapani, A.; Laquintana, V.; Denora, N.; Lopedota, A.;
Cutrignelli, A.; Franco, M.; Trapani, G.; Liso, G. Eur. J. Pharm. Sci.
2007, 30, 64−74.
S
Details concerning materials, preparation, and characterization
of microparticles and experimental procedures of the
biocatalytic reactions. This material is available free of charge
via the Internet at http://pubs.acs.org/.
(24) Jain, V.; Singh, R. Acta Poloniae Pharmaceutica. Drug Research
2010, 67, 407−415.
(25) Haznedar, S.; Dortunc,̧ B. Int. J. Pharmacol. 2004, 269, 131−140.
(26) Lamprecht, A.; Yamamoto, H.; Takeuchi, H.; Kawashima, Y. J.
Controlled Release 2003, 90, 313−321.
AUTHOR INFORMATION
Corresponding Author
*E-mail: yjchen@cpu.edu.cn.
■
(27) Rowe, R. C.; Sheskey, P. J.; Quinn, M. E. Handbook of
Pharmaceutical Excipients, 6th ed.; Rowe, R. C., Sheskey, P. J., Quinn,
M. E., Eds.; Pharmaceutical Press: London, 2009; p 525−533.
(28) Ahmad, A. L.; Oh, P. C.; Abd Shukor, S. R. Biochem. Eng. J.
2010, 52, 296−300.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the “‘111’” Project
from the Ministry of Education of China, and the State
Administration of Foreign Experts Affairs of China (No. 111-2-
07), National Key Project of Science and Technology
(2013ZX09402103), the Research Project of th State Key
Laboratory of Natural Medicines, China Pharmaceutical
University (SKLNMZZCX201301), and the Priority Academic
Program Development of Jiangsu Higher Education Institu-
tions.
REFERENCES
■
(1) Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.;
Moore, J. C.; Robins, K. Nature 2012, 485, 185−194.
(2) Hall, M.; Bommarius, A. S. Chem. Rev. 2011, 111, 4088−4110.
(3) Shen, N. D.; Ni, Y.; Ma, H. M.; Wang, L. J.; Li, C. X.; Zheng, G.
W.; Zhang, J.; Xu, J. H. Org. Lett. 2012, 14, 1982−1985.
(4) Shin, J. S.; Kim, B. G. Biotechnol. Bioeng. 2010, 106, 541−552.
1587
dx.doi.org/10.1021/cs4011919 | ACS Catal. 2014, 4, 1584−1587