Vol.1, No.3, 185-190 (2009)
doi:10.4236/ns.2009.13024
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/NS/
Natural Science
Preparation of lignin derivatives and their application as
protease adsorbents
Run Fang1,2, Yin Lin1 Xian-Su Cheng*1
1College of Material Science and Engineering, Fuzhou University, Fuzhou, China
2Department of Chemistry and Chemical Engineering, Minjiang University, Fuzhou, China; Corresponding author:
Received 1 September 2009; revised 25 September 2009; accepted 27 September 2009.
ABSTRACT
Synthesis of two lignin derivatives, lignophenol
and lignin-aminophenol, were presented in this
article. The chemical structure and the func-
tional groups of lignin derivatives were charac-
terized through FT-IR analysis. The immobiliza-
tion of three proteases (papain, trypsin and
pepsin) on lignin and lignin derivatives was
carried out using adsorption technique. The
influence of contact time and pH on the enzyme
adsorption by different adsorbents was inves-
tigated. Furthermore, enzyme activity recovery
was also evaluated. Results showed that lignin
and lignin derivatives could adsorb proteases
effectively and the adsorption capacity of lingo-
phenol and lignin-aminophenol was higher than
that of pure lignin. Meanwhile, the activity re-
covery of papain and pepsin immobilized on
lignin derivatives was very high. This pheno-
menon suggested that there is a supramole-
cular interaction between enzymes and lignin
derivatives which do not inhibit enzyme activity.
Therefore, lignophenol and lignin-aminophenol
are both promising adsorbents for enzyme im-
mobilization under acid and neutral conditions.
Keywords: Lignin Derivative; Papain; Trypsin;
Pepsin; Adsorption
1. INTRODUCTION
Papain, trypsin and pepsin are all of proteases found in
plant and animal cells that break down proteins or pep-
tides by catalyzing the hydrolysis of peptide bonds.
These enzymes, after being immobilized, offer several
advantages over their free form equivalent. Examples
include better stability, possible reuse, greater sensitivity
and greater reproducibility of effectiveness. The immo-
bilization technology of enzyme has been playing an
important role in biological industry, medicine and
clinical diagnosis, chemical analysis, environmental pro-
tecttion and energy exploitation. Apart from recycling
the biocatalyst, immobilization yields further advantages,
such as the easy removal of the biocatalyst from the re-
action mixture and thus, simplified product purification.
Different immobilization techniques have been devel-
oped including covalent coupling [1,2], enzyme cross-
linking molecules [3], adsorption on a carrier [4] and the
encapsulation in polymeric gels or membranes [5,6].
Support material is a key factor in enzyme immobili-
zation and considerable attention has been paid to the
searching for ideal support materials, which may give
the best combination of high remaining activity, low cost
and friendly to human health and environment. Lignin
(EH-lignin) isolated from the residue of enzymatically
hydrolyzed cornstalks (a by-product of fuel ethanol in-
dustry) is a novel organosolv lignin developed in recent
years [7]. Making use of this biomaterial will not only
enhance economic benefits of bioindustry but also di-
minish potential environmental pollutions.
Lignin is a totally renewable aromatic polymer noted
for its versatility and applicability in a variety of uses.
Since they are safe to human consumption and environ-
ment, some lignin derivatives can even been used as
food additives which has been permitted by EPA and
FDA. Due to the unique isolation procedures, the content
of polar functional groups, such as carbonyl, hydroxyl
and phenolic hydroxyl, is very high in EH-lignin. This
characteristic makes it possible to utilize EH-lignin, after
mild modification, as enzyme immobilization supports.
Furthermore, the crosslink structure of modified lignin is
helpful in improving the chemical and structure stability
of support materials. Enzymes immobilized on lignin
derivatives are advantageous because of the capability of
transferring active compounds to heterogeneous reac-
tions and the easy separation of them from the reaction
mixture subsequently.
The adsorption of endotoxin and bromelain on lignin
and lignin derivatives has been reported in our previous
works [8,9]. It has been found that lignin derivatives,

R. Fang et al. / Natural Science 1 (2009) 185-190
SciRes Copyright © 2009 http://www.scirp.org/journal/NS/Openly accessible at
186
prepared through phenol or amino modification, are
ideal supports for enzyme immobilization with good
adsorption capacity and high remaining activity. In this
work, we present the synthesis of two lignin derivatives
(lignophenol and lignin-aminophenol) and their applica-
tion as protease adsorbents. The immobilization of three
proteases (papain, trypsin and pepsin) on EH-lignin and
lignin derivatives was evaluated by measuring the en-
zyme adsorption capacity and the activity recovery. The
influence of contact time and pH on the enzyme adsorp-
tion was discussed in detail.
2. EXPERIMENTAL
2.1. Materials
EH-lignin was isolated from the residue of enzymati-
cally hydrolyzed cornstalks and purified in laboratory
according to the procedures described in literature [7].
Papain, trypsin and pepsin as well as 4-aminophenol and
4-cresol were purchased from Sinopharm Chemical Re-
agent Co., Ltd, China. All other reagents were of ana-
lytical grade.
2.2. Preparation of Reagents
The papain, trypsin and pepsin solutions with different
pH were prepared by dissolving a certain amount of sol-
ute in 0.1mol/l phosphate buffer, 0.05mol/l Tris-HCl
buffer and 0.05mol/l lactic acid and sodium lactate
buffer respectively. Thermo Orion 828 (Orion, U.S.) pH
meter was employed in determining pH values of the
solutions.
2.3. Preparation of Lignophenol and
Lignin-Aminophenol
Lignophenol was prepared by modifying EH-lignin in a
two-phase system composed of p-cresol and 72% sulfu-
ric acid. The preparation procedures and the characteris-
tics of lignophenol were introduced in our previous
works [10].
Synthesis of 4-aminophenol modified EH-lignin (lig-
nin-aminophenol) was carried out in a jacketed reactor
flask equipped with a stirrer and a reflex condenser.
0.5mol/l 4-aminophenol solution was prepared by add-
ing 3g 4-aminophenol into 55ml distilled water in the
reactor and dissolved at 80. 5g EH℃-lignin and 35ml
glyoxal was then added into this solution with stirring.
After 1h reaction at 80, the precipitates were filtered, ℃
washed with distilled water and ethanol repeatedly and
then dried in a vacuum oven at 50 until a constant ℃
weight was obtained. Spectrum 2000 FT-IR spectrometer
(Perkinelmer, U.S.) was employed in FT-IR analysis of
the derivatives.
2.4. Adsorption of Enzyme on Lignin and
Lignin Derivatives
50mg support material (EH-lignin, lignophenol and lig-
nin-aminophenol) was incubated with 5ml enzyme solu-
tion in a shaker at 25 for a period of time, the initial ℃
enzyme concentration C0 was varied depending on ex-
periments. At the end of this period, the supernatant was
separated by centrifuging at 3000 rev/min and then di-
luted with buffer to 25mL. Meanwhile, the precipitate
was collected and vacuum dried for enzyme activity as-
say. The amount of immobilized enzyme was determined
by measuring the concentration of the free enzyme in the
supernatant before and after adsorption with Cary50
UV/VIS spectrophotometer (Varian, U.S.). Reference
samples were prepared according to identical procedures
described above by adding 5mL buffer instead of en-
zyme solutions.
2.5. Enzyme Activity Assay
The activities of free enzymes were determined by
measuring the tyrosine amount produced by enzyme-
catalyzed hydrolysis of casein according to Chinese drug
standards: papain [11]. One unit of enzyme activity is
defined as the amount of enzyme that produces 1μg ty-
rosine from casein per min at 40. The conce℃ntration of
tyrosine was determined at 275nm using UV/VIS spec-
trophotometer. The activity of enzymes immobilized on
EH-lignin and its derivatives was measured using the
same method as above, except that the enzyme solutions
were replaced by a given amount of immobilized en-
zymes.
3. RESULTS AND DISCUSSIONS
3.1. Characteristics of Lignin and Lignin
Derivatives
EH-lignin is a kind of organosolv lignin isolated from the
residue of enzymatically hydrolyzed cornstalks. Com-
pared with traditional lignin derivatives, such as lignosul-
fonate, EH-lignin possesses some valuable characteristics
such as high content of functional groups, less impurities
and narrow molecular weight distribution [12].
In the 4-cresol concentrated acid system, lignin frac-
tions contact with acid for a short time may give reactive
carbocations (α C
+), which are stabilized quickly by 4-
cresol and results in the formation of diphenyl-methane
type materials (lignophenol) presented in Figure 1. Pre-
liminary studies show that lignophenol is a kind of
cross-linked polymer with lots of phenolic hydroxyl
groups [10].
Lignin-aminophenol was prepared by modifying EH-
lignin with 4-aminophenol, glyoxal act as cross-linking
agent. By introducing amino and phenolic hydro- xyl
groups into EH-lignin, the enzyme adsorption capacity

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187
Openly accessible at
and the molecular structure of lignin- aminophenol were
of great difference to that of native EH-lignin. FT-IR
spectra of EH-lignin, lignophenol and lignin-aminophenol
are presented in Figure 2. It can be found in the spec-
trum of lignin-aminophenol that, compared with EH-
lignin, the relative intensity of the adsorption bands at
3200-3600cm-1 (assigned to –OH and –NH2 stretching
vibration) and 1637cm-1 (assigned to N–H bending vi-
bration) increase significantly while the intensity of
other bands remain nearly unchanged. Similar pheno-
menon can be observed in the spectrum of lignophenol
at 3200-3600cm-1 (assigned to –OH stretching vibration).
Therefore, it can be tell from FT-IR analysis that the
content of –OH on lignophenol and the content of –OH
and –NH2 on lignin-aminophenol have been greatly en-
hanced after modification.
3.2 Adsorption of Enzymes on Lignin and
Lignin Derivatives
The influence of contact time (t) and initial pH of en-
zyme solution on the adsorption amount of different en-
zymes on EH-lignin, lignophenol and lignin- aminophe-
nol were studied in this paragraph.
For initial pH studies, the papain solutions were pre-
pared with phosphate buffer, pH vary form 5.0 to 8.0;
the trypsin solutions were prepared with Tris-HCl buffer,
pH vary form 7.0 to 9.0 and the pepsin solutions were
prepared with lactic acid and sodium lactate buffer, pH
form 2.5 to 5.0. The initial concentration of enzyme was
C0=5.0 mg/ml and the contact time t=50 min. For
OH
OCH3
Lignin
α
+
CH3
OH
72%H2SO4
OH
OCH3
Lignin
CH3
OH
Figure 1. Molecular structure of lignophenol.
c
b
a
Wavenumber (cm-1)
40003000200010004000 3500 3000 2500 20001500 1000500
Figure 2. IR spectra of (a) EH-lignin; (b) ligno-
phenol; (c) lignin-aminophenol.
contact time studies, the papain solutions were prepared
with phosphate buffer pH 8.0, the trypsin solutions were
prepared with 0.05mol/L Tris-HCl buffer pH 9.0 and the
pepsin solutions were prepared with 0.05mol/L lactic
acid and sodium lactate buffer pH 3.0. The initial con-
centration of enzyme was 3.0 mg/ml. All the adsorption
experiments were performed at 25 according to pr℃o-
cedures mentioned in 2.5.
3.2.1. Effect of pH
The influence of pH on the adsorption amount of papain,
trypsin and pepsin on lignin and lignin derivatives is
presented in Figure 3. Figure 3a shows the papain ad-
sorption by different support materials at pH 5.0, 6.0, 7.0
and 8.0. In these cases, the adsorption of papain in-
creased with increasing pH. The maximum adsorption
amount 265 mg.g-1 was reached at pH 8.0 by lig-
nin-aminophenol and 193 mg.g-1 at pH 8.0 by lignophe-
nol. Similarly, the adsorption amount of trypsin on three
adsorbents (Figure 3b) increased with the rising of pH
until equilibrium was reached at pH 8.0-9.0. The maxi-
mum adsorption amount 233 mg.g-1 was reached at pH
9.0 by lignin-aminophenol and 171 mg.g-1 at pH 9.0 by
lignophenol. The variation of adsorption amount of pep-
sin (Figure 3c) was a little different from that of papain
and trypsin. With increasing pH, three peaks of adsorp-
tion amount on different adsorbents appeared at pH 3.0
simultaneously. Further increase of pH led to the de-
crease of enzyme adsorption amount. The largest ad-
sorption amount was 361 mg.g-1 by lignin-aminophenol
and 344 mg.g-1 by lignophenol.
It has been reported that the isoelectric point of papain
is pH 8.6 [13]. Meanwhile, we noticed that the adsorp-
tion amount of papain on three adsorbents increased with
the rising of pH value, especially when it was close to
the isoelectric point of papain. Similarly, the adsorption
amount of trypsin and pepsin reached the maxima when
the pH values were closed to their isoelectric point, i.e.
about 8.2 and 2.8 respectively [14,15].
This phenomenon may be related to induce polarization
that the variation of pH leads to a corresponding change in
the relative abundance of positive and negative sites on
the adsorbents and enzymes, thus modulating the strength
of the electrostatic interactions between them.
3.2.2. Effect of Contact Time
The influence of contact time on the adsorption amount
of papain, trypsin and pepsin on three different adsorb-
ents is shown in Figure 4.
It can be seen in Figure 4a that the adsorption of pa-
pain by lignin and lignin derivatives was rapid in the
first 30 min and a contact time of only 1h was required
to attain the equilibrium adsorption. The adsorption
amounts of papain on lignophenol and lignin-aminophenol
increased up to the highest level i.e. 189 mg/g-1 at 50
min and 259 mg/g-1 respectively at 70 min respectively,
and then remained almost constant. Similar adsorption

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188
5.0 5.5 6.0 6.5 7.0 7.5 8.0
50
100
150
200
250
300
EH-lignin
lig nophe no l
lignin aminophenol
E
nzyme a
d
sorpt
i
on
(
mg.g
-1
)
pH
0 10203040506070
0
30
60
90
120
150
180
210
240
270
Enzyme adsorption (mg.g-
1
)
Contact Time
(
min
)
EH-lignin
lignophenol
lignin aminophenol
(a) (a)
0 10203040506070
0
40
80
120
160
200
240
Contact Time
(
min
)
E
nzyme a
d
sorpt
i
on
(
mg.g
-1
)
EH-lignin
lignophenol
lignin aminophenol
7.0 7.5 8.0 8.5 9.0
0
40
80
120
160
200
240
EH-lignin
lignophenol
lignin aminophenol
Enzyme adsorption (mg.g-1)
pH
(b) (b)
0 10203040506070
0
40
80
120
160
200
240
280
320
360
Enzyme adsorption (mg.g-
1
)
Contact Time (min)
EH-ligni n
lignophenol
lignin aminophenol
2.5 3.0 3.5 4.0 4.5 5.0
180
210
240
270
300
330
360
E
nzyme a
d
sorpt
i
on
(
mg. g
-1
)
pH
EH-lignin
li gnophenol
lignin aminophenol
(c)
(c)
Figure 4. Influence of contact time on the
adsorption amount of three proteases.
Co=3.0 mg/ml, T=25℃ (a) papain at pH 8;
(b) trypsin at pH 9; (c) pepsin at pH 3.
Figure 3. Influence of initial pH of enzyme
solution on the adsorption amount of three
proteases. Co=5.0 mg/ml, t=50min,T=25℃
(a) papain; (b) trypsin; (c) pepsin.
the higher adsorption capacity of lignin derivatives un-
behavior can be seen in Figure 4b. The adsorption of
trypsin reached a maximum value of 158 mg/g-1 by
lignophenol and 224 mg/g-1 by lignin-aminophenol at
50min. The adsorption of pepsin (Figure 4c) increased
steadily with extending contact time and the highest ad-
sorption amount was 211 mg/g-1 at 70min by lignophenol
and 351 mg/g-1 at 70min by lignin-aminophenol. In all
these studies, the sequence of enzyme adsorption amount
goes as follows: lignin-aminophenol > lignophenol >
EH-lignin.
der acid condition are both possible reasons.
Despite the variation of contact time and initial pH,
the sequence of enzyme adsorption amount of three
support materials goes unchanged as follows: lig-
nin-aminophenol > lignophenol > EH-lignin. Therefore,
it is clear that the enzyme adsorption capacity of lignin
derivatives increase significantly after modification. On
considering the chemical structure, as has been men-
tioned in 3.1, the content of phenolic hydroxyl groups
was increased in lignophenol and both of amino and
phenolic hydroxyl groups were enriched in lig-
nin-aminophenol after modification. These polar func-
tional groups, which may interact with amino and car-
oyl groups on enzymes, will favor the enzyme adsorp-
It can be found from Figure 3 and Figure 4 that the
adsorption amount of pepsin on lignin derivatives is
higher than that of papain and trypsin, which may indi-
cated stronger interactions between lignin derivatives
and pepsin. The features of their chemical structure and b
x
Openly accessible at

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189
Table1. The activities of three proteases before and after adsorption.
Proteases Adsorbent Before adsorption
/U·mg-1
After adsorption
/U·mg-1
Activity recovery
/％
EH-lignin 1.68×103 0.19×103 11.3
lignophenol 1.16×103 0.65×103 55.7
Papain
lignin-aminophenol 0.96×103 0.51×103 53.1
EH-lignin 6.98×103 0.52×103 7.5
lignophenol 1.37×104 0.13×104 9.6
Trypsin
lignin-aminophenol 1.42×104 0.31×104 21.5
EH-lignin 5.15×103 0.65×103 12.6
lignophenol 6.62×103 5.00×103 75.5 Pepsin
lignin-aminophenol 5.20×103 3.65×103 70.3
tion through the formation of hydrogen bonding [16,17].
Furthermore, due to their hydrophilic characteristic, the
introduction of amino and hydroxyl groups into support
materials will lead to a better contact between lignin
derivatives and free enzymes suspended in aqueous so-
lution and thus, a larger quantity of enzymes become
linked.
3.3. Remaining Activity Analysis
The remaining activities of immobilized enzymes were
evaluated and the results were compared to that of free
enzymes. Since we have already investigated the influ-
ence of adsorption time and pH value on enzyme adsor-
ption, the optimum conditions mentioned in 3.2.1 were
used in the activity recovery experiments. The activities
of free and immobilized enzymes were determined fol-
lowing the method mentioned in 2.6.
Table 1 shows the activities of papain, trypsin and
pepsin before and after adsorption by lignin and lignin
derivatives. It can be seen in Table 1 that the activities
recovery of all three proteases immobilized on ligno-
phenol and lignin-aminophenol is higher than that of
proteases adsorbed by native EH-lignin. Compared with
EH-lignin, the larger enzyme adsorption capacity of lin-
gophenol and lignin-aminophenol is an important factor
that leads to higher activity recovery which has been
discussed in 3.2. Another factor, the interaction between
enzymes and support materials, also makes a contribu-
tion to this outcome. Enzyme immobilization can causes
changes in the tertiary structure of the protein which in
turn may influence the activity under specific conditions.
Therefore, the activity decrease after immobilization can
be explained by the intermolecular interaction between
enzymes and support materials that change the confor-
mation of the enzymes. The high activity recovery of
papain and pepsin adsorbed on lignophenol and lig-
nin-aminophenol indicates a supra-molecular interaction
between enzymes and lignin derivatives which has little
side-effect on the activity of the enzymes. Modified EH-
lignin is a high molecular composed of phenylpropane
skeleton as the hydrophobic group and amino, hydroxyl
and carboxyl as the hydrophilic groups. Therefore, the
supramolecular interaction may be a combination of hy-
drogen bonding and hydro- phobic interaction [18].
Another phenomenon that deserves our attention is the
significant difference of the activity recovery between
three enzymes adsorbed on the same adsorbent. Despite
the variation of support materials, the sequence of activ-
ity recovery of three enzymes goes unchanged as follows:
pepsin > papain > trypsin. The variation of adsorption
amount of three enzymes is a factor that will affect the
activity recovery. However, as was shown in 3.2, the
adsorption amount of different enzymes on the same
support material do not differ a great deal, which means
that other influencing factors should also be taken into
account.
From the standpoint of the physical characteristics,
this phenomenon may be ascribed to the influence of pH
on the dissolution of lignin and lignin derivatives in
aqueous solutions. It has been pointed out in some stud-
ies that as the pH of the solution increases, the dissolva-
bility of lignin and lignin derivatives will increase si-
multaneously [19]. It was also found in our research that
when the buffer turned to alkaline, the dissolvability of
lignin and lignin derivatives increased rapidly. The ad-
sorption of pepsin, papain and trypsin was carried out
under the optimum conditions at pH 3.0, 8.0 and 9.0,
respectively. The dissolution of support materials under
alkaline condition decreased the stability and the yield of
immobilized enzymes, which in turn reduced the activity
recovery of trypsin remarkably. These analyses indicate
that lignin and lignin derivatives are more suitable for
papain and pepsin immobilization under acid or neutral
conditions.
4. CONCLUSIONS
The preparation and characteristics of two lignin deriva-
tives, lignophenol and lignin-aminophenol, was pre-
sented in this article. Compared with native lignin, the
content of amino and phenolic hydroxyl groups was
greatly enhanced after modification.
The results received from adsorption experiments
show that three proteases (papain, trypsin and pepsin)
can be adsorbed by lignin and lignin derivatives effec-
tively. The adsorption capacity was affected by contact

R. Fang et al. / Natural Science 1 (2009) 185-190
SciRes Copyright © 2009 http://www.scirp.org/journal/NS/
190
Openly accessible at
time and pH depending on the feature of enzymes. De-
spite the variation of enzymes, the sequence of enzyme
adsorption capacity goes as follows: lignin-aminophenol
> lignophenol > EH-lignin, which is attributed to the
enrichment of polar functional groups, such as –OH and
–NH2, in lignin derivatives after modification.
The activity recovery of pepsin and papain immobi-
lized on lignin derivatives under acid and neutral condi-
tions was very high which indicates a combination of
hydrogen bonding and hydrophobic interaction between
enzymes and lignin derivatives. These intermolecular
interactions greatly enhance enzyme adsorption and
hardly inhibit enzyme activity. Therefore, lignophenol
and lignin-aminophenol are promising support materials
for enzyme immobilization under acid and neutral con-
ditions.
5. ACKNOWLEDGEMENT
This paper has received support from the fund of State Key Laboratory
of Guangzhou Institute of Chemistry, Chinese Academy of Science
(LCLC-2004-158）.
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