Effect of alkylamine on activity and stability of immobilized angiotensin converting enzyme

Article abstract of DOI:10.1016/j.catcom.2012.03.008

Angiotensin converting enzyme (ACE) was immobilized on chitosan microspheres with glutaraldehyde as cross-linking reagent. The activity and stability of immobilized ACE (I-ACE) were improved by further modification with alkylamines. The modification conditions were optimized. The characteristics of I-ACE and modified I-ACE (MI-ACE) including activity, kinetic parameters, optimal pH and temperature, stability and reusability were investigated. After modification, I-ACE activity was increased, K_m decreased from 4.33 mM to 2.61 mM, optimal pH value was altered, pH stability, storage stability and thermal stability were improved, and optimal temperature did not change.

Full text of DOI:10.1016/j.catcom.2012.03.008

Catalysis Communications 24 (2012) 16–19
Contents lists available at SciVerse ScienceDirect
Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Short Communication
Effect of alkylamine on activity and stability of immobilized angiotensin
converting enzyme
Qingqing Tian a, Naiyuan Zhou a,b, Weixiu Zeng a, Yingying Chen a, Shuang Wu a, Wei Duan a,
a
Bo Chen a, , Shouzhuo Yao
⁎
a
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research Ministry of Education, Hunan Normal University, Changsha 410081, China
China National Center for Biotechnology Development, Beijing 100036, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 January 2012
Received in revised form 29 February 2012
Accepted 7 March 2012
Available online 15 March 2012
Angiotensin converting enzyme (ACE) was immobilized on chitosan microspheres with glutaraldehyde as
cross-linking reagent. The activity and stability of immobilized ACE (I-ACE) were improved by further mod-
ification with alkylamines. The modification conditions were optimized. The characteristics of I-ACE and
modified I-ACE (MI-ACE) including activity, kinetic parameters, optimal pH and temperature, stability and re-
usability were investigated. After modification, I-ACE activity was increased, Km decreased from 4.33 mM to
2.61 mM, optimal pH value was altered, pH stability, storage stability and thermal stability were improved,
and optimal temperature did not change.
Keywords:
Angiotensin converting enzyme
Modification
Alkylamine
© 2012 Elsevier B.V. All rights reserved.
Activity
Stability
1. Introduction
In the present study, water-soluble alkylamines (methylamine,
diethylamine, triethylamine and n-butylamine) were used to modify
Angiotensin converting enzyme (ACE) is a regulatory factor in the
rennin–angiotensin–aldosterone system, which plays an important
role in the regulation of blood pressure [1,2]. Immobilization was usual-
ly employed to improve ACE stability and reusability since the purifica-
tion of ACE is difficult and its activity loses fast. Generally, ACE was
immobilized via the bonding between primary amino groups in enzyme
and aldehyde-functionalized carriers to form Schiff bases [3–7].
Comparing to the immobilized enzyme, the reactive groups in
carriers are in large excess. These excess groups could further react
with enzyme to gradually induce the change of its conformation to
deactivate it [8,9], and they might cause non-specific adsorption and
give a false binding activity when the immobilized enzyme was used
as affinity absorbent [10,11]. A modification aiming at quenching
these excess reactive groups in carriers has been proved to be effective
in mitigating the loss of the activity during storage or use and enhancing
the stability [12–15]. For ACE immobilized on aldehyde-functionalized
chitosan microspheres (AFCM), aldehyde groups are the quenching tar-
gets. NaHB4 [16], ethanolamine [17], amino acids [12,17], biomacromole-
cules and polymers [18] have been used for quenching aldehyde groups.
However, in view of minimizing the possible non-specific adsorption, the
optimal quenching agents should be as simple as possible.
immobilized ACE (I-ACE) by quenching the excess aldehyde groups
in carriers to regulate I-ACE activity and stability. After modification
under optimum condition, the characteristics of I-ACE and the modi-
fied I-ACE (MI-ACE) including activity, kinetic properties, optimal pH
and temperature, stability and reusability were investigated.
2. Materials and methods
2.1. Materials
Chitosan (deacetylation 90%) was produced by China National
Medicines Corporation Ltd. (Shanghai, China). N-Hippuryl-His-Leu
hydrate (HHL) was purchased from Sigma. All other chemicals were
of analytical grade without further purification.
2.2. Enzyme preparation
The ACE was prepared according to Ref. [19]. Pig lung was purchased
from a local slaughterhouse (Changsha, China). Tissue was diced and
homogenized in 10-fold volumes of ice-water containing 0.3% Triton
X-100, then centrifuged at 2000 rpm for 20 min. The supernatant was
fractionated with (NH4)2SO4 between 1.6 and 2.6 M. Then the precipi-
tate was dissolved and dialyzed against 0.1 M borate buffer with pH
8.3 (including 0.3 M NaCl, buffer A). The activity of the prepared ACE ex-
tract was 0.3531 U/mL (4 mM HHL).
⁎
Corresponding author. Tel.: +86 731 88865515; fax: +86 731 88872046.
E-mail address: dr-chenpo@vip.sina.com (B. Chen).
1566-7367/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.catcom.2012.03.008

Q. Tian et al. / Catalysis Communications 24 (2012) 16–19
17
2.3. Preparation of chitosan microspheres and immobilization of ACE
3. Results and discussion
The AFCM was prepared according to Ref. [6]. More details were
provided in Supporting information. AFCM and ACE extract (g/3 mL)
were mixed and oscillated at room temperature for 1 h, then stored
at 4 °C overnight. Finally, I-ACE was washed with distilled water
and buffer A until no protein could be detected. The prepared I-ACE
activity was 0.1245 U/g (4 mM HHL).
3.1. Modification of I-ACE with alkylamines
I-ACE was first modified by different kinds of alkylamine solutions,
NaHB4 and ethanolamine. As shown in Fig. 1A, except NaHB4 and
triethylamine, all modifiers showed activation on I-ACE. And the acti-
vation effects were conformed to the level of difficulty of the reaction
with aldehyde group. Ethanolamine, methylamine and n-butylamine
exhibited activation in a broad concentration range (140–6720 mM,
prepared with water, pH 8.3). At the optimum condition (modified
by 2240 mM methylamine), MI-ACE activity was 2 folds higher than
I-ACE (Fig. 1B).
2.4. Enzyme assay
The activities of free ACE and I-ACE were analyzed by using HHL
(4 mM, prepared with buffer A) as substrate according to Ref. [20].
An assay mixture consisted of 0.1 g I-ACE and 400 μL HHL or 10 μL
free ACE and 200 μL HHL was incubated at 37 °C for 30 min, then
100 μL acetonitrile was added to terminate reaction. The product hip-
puric acid was determined by RP-HPLC (Shimadzu, Tokyo, Japan) on a
Johnson Spherigel C18 column (200 mm×4.6 mm, 5 μm, Dalian, China).
The mobile phase was 0.1% H3PO4 in 25% acetonitrile–water with a
flow-rate of 1 mL/min. The effluent was monitored with an UV detector
at 228 nm. A unit (U) of enzyme activity was defined as the amount of
enzyme releasing 1 μmol of hippuric acid from HHL per min under the
assay conditions.
For I-ACE immobilized on AFCM activated with glutaraldehyde
of different concentrations (0.6%, 0.9%, 1.25%, 1.75%, 2.5% and 5%),
2.5. Modification of I-ACE
A series of varied modifier solutions with different concentrations
(140–6720 mM, prepared with water) and pH (5.3–11.3, adjusted by
HCl) were first prepared. For modification, I-ACE was added to modifier
solutions (g/10 mL) under shaking at 25 °C for 3 h, and then washed
with distilled water and buffer A till no alkylamine left.
2.6. Effect of pH and temperature on the activity of free ACE, I-ACE and
MI-ACE
Activity was determined at 37 °C under varied pHs (5.3–10.3) or
at different temperatures (25–60 °C) under pH 8.3 for 30 min to in-
vestigate the effect of pH and temperature on the activity of free
ACE, I-ACE and MI-ACE.
2.7. Stability and reusability
pH stabilities and thermal stabilities of I-ACE and MI-ACE were
studied by measuring the residual activities of enzyme after preincu-
bation in buffer A with different pHs (5.3–11.3) at 25 °C or in buffer A
at different temperatures (40–60 °C) for 3 h, respectively. For storage
stability experiments, I-ACE and MI-ACE were stored in buffer A at 4 °C,
and enzyme activity was measured periodically. The reusability was de-
termined by repeated determination of their activities (n=10).
2.8. Kinetic analysis
The kinetic properties of free ACE, I-ACE and MI-ACE were deter-
mined using different HHL concentrations (0.4–4 mM). The kinetic
constants Km and Vmax were calculated from the Lineweaver–Burk
plots. All the experiments in the paper were carried out in triplicate.
Table 1
The enzyme kinetic parameters of free ACE, I-ACE and MI-ACE.
Km (mM)
Vmax (mol/L/min)
Fig. 1. (A) Effect of different modifiers on the activity of I-ACE. The concentrations of all
the modifiers were 280 mM. (B) Effect of concentrations on the activity of I-ACE. (C)
Effect of modification on the activity of I-ACE immobilized on AFCM which was activat-
ed by different concentrations of glutaraldehyde.
Free ACE
I-ACE
MI-ACE
1.63
4.33
2.61
0.52
0.26
0.66

18
Q. Tian et al. / Catalysis Communications 24 (2012) 16–19
although the activities and the contents of the excess aldehyde groups
in each kind of I-ACE were different, the increments of activity for the
six kinds of I-ACE were almost the same (Fig. 1C). It could be concluded
that the increment of activity mainly depended on the concentration of
the modifier.
The activity improvement is supposed to result from the altered
microenvironment around the I-ACE due to the quenching of residual
aldehyde groups in carriers. The conformation change of I-ACE induced
by excess aldehyde groups could partly reinstate after quenching,
thereby enzyme activity was reinstated. Notice that the IC50 of captopril
to MI-ACE (1.1625 μM) was higher than that to I-ACE (0.3372 μM), indi-
cating an increase of the active enzyme after modification. Besides, I-
ACE might also be activated by the unfolding–refolding action of dena-
turants via converting its rigid structure into a flexible structure. The
flexible structure was hardly recovered after removing methylamine
because of the improved microenvironment for enzyme, and thus the
I-ACE activation was sustained. Such phenomenon was opposite to
some reports about the disappeared activation of enzyme after the re-
moval of denaturants [21,22]. By further increasing methylamine con-
centrations, I-ACE suffered denaturation and the activity was decreased.
3.2. Influence of pH and salt concentration on modification
The activity of I-ACE was an indicator for investigating the effect of
the pHs (5.3–11.3, adjusted by HCl) of methylamine solutions
(2240 mM prepared with water) on modification. The best activity
was obtained at pH 10.3 (Fig. 2). Besides, the modification effect re-
duced when using buffer A as solvent for methylamine, which was
caused by the increment of ionic strength.
Fig. 3. Effect of pH and temperature on the activity of free ACE, I-ACE and MI-ACE.
H-bond with water, which would affect the enzyme active center.
Without aldehyde groups, the I-ACE was less susceptible to hydrogen
or hydroxide ions. Thus the optimal pH of MI-ACE shifted. The optimal
temperature was 40 °C for free ACE (Fig. 3), while, I-ACE and MI-ACE
had a wide temperature endurance range (40–50 °C) with the same
optimal temperature of 40 °C.
3.3. The kinetic parameters of free ACE, I-ACE and MI-ACE
The increase in Km and decrease in Vmax of the enzyme had been
immobilized, thus suggesting that the I-ACE had a lower affinity for
substrate than that of free ACE, which was caused by the loss of en-
zyme flexibility and the increase of diffusional resistance. While, the de-
crease in Km and increase in Vmax of the MI-ACE indicated that the
modification improved the affinity between immobilized enzyme and
substrate (Table 1).
3.5. Stability and reusability
Stability is an important parameter to evaluate immobilized en-
zyme. As presented in Fig. 4, MI-ACE showed a good stability in a
wider pH range (5.3–11.3) than I-ACE (5.3–8.3), which was mainly
due to the inert surface of MI-ACE and the newly introduced pendent
groups were insensitive to the pH change. MI-ACE showed better
thermal stability than I-ACE at 40 °C. After preincubating at 40 °C for
3 h, I-ACE and MI-ACE lost 52% and 32% activities, respectively. But
after preincubating at 50 °C for 3 h, the activities of both lost as
much as 90%. Moreover, MI-ACE showed better storage stability
than I-ACE. MI-ACE retained 75.9% activity 30 days later while I-ACE
retained 52.3%. After 120 days, only 8.9% activity was left for I-ACE,
but still 22.4% for MI-ACE. Furthermore, they had high repeated utili-
zation after 10 cycles of operation with 78.6% and 82.4% of their orig-
inal activities for I-ACE and MI-ACE, respectively.
3.4. Effect of pH and temperature on enzyme activity
I-ACE and MI-ACE had a wider pH endurance range (7.3–9.3) than
free ACE (Fig. 3). The difference in the optimal pH for free ACE and I-
ACE of 8.3, and for MI-ACE of 9.3 was caused by the change of the
pendent groups, residual charges and the enzyme microenvironment
[23,24]. The residual aldehyde group is hydrophilic, it could form
Fig. 2. Effect of pH of the methylamine solution on modification. The concentration of
Fig. 4. pH stability of I-ACE and MI-ACE. Preincubated in buffer A (pH 5.3–11.3) at 25 °C
the methylamine solution was 2240 mM.
for 3 h.

Q. Tian et al. / Catalysis Communications 24 (2012) 16–19
19
3.6. Recovering of the activity of I-ACE
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Acknowledgments
This work was financially supported by the National “863” Research
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Science Research Foundation of Hunan Province (2010TT1001, 05k009)
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Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.catcom.2012.03.008.