Cocoa pod husk, a new source of hydrolase enzymes for preparation of cross-linked enzyme aggregate

Extraction of hydrolase from CPH

Optimum pH and buffer concentration are important factors for the extraction of protein.
The optimum pH for the highest protein activity is different in various samples. The
highest lipase activity from olive fruit has been extracted at pH 5 (Panzanaro et
al. 2010]). In prior investigations, the optimal pH values for protein extraction from Mung
beans and Red beans were 9 (Wang et al. 2011]) and 8.8 (Liu et al. 2011]), respectively. The optimal conditions for protein extraction from red pepper seeds
and the highest enzyme activity were achieved at pH 8.8 (Firatligil-Durmus and Evranuz
2010]). These variations indicate that the activity is more sensitive to pH range. In general,
the enzyme activity increases when the pH of the medium is higher than 7.0 (Lv et
al. 2008]). Temperature is another crucial factor during the extraction in order to maintain
proteins’ shape, activity, and stability. To minimize protein denaturation as well
as the autolysis of enzymes, the sample must be kept at (0–4 °C) during the process.

Following the Bradford assay, the protein concentration of the crude enzyme mixture
is 358 µg/ml. It was observed that the colour of the assay was stable blue and in
gradient based on the concentration of BSA in the preparation of the standard curve.
The dark blue sample indicated the presence of active proteins and that the reaction
has occurred between the protein and dye.

Figure 1 shows the screening of hydrolase from CPH and each the enzymatic assay performed.
The highest enzyme activity was obtained from Protease (60.5 U/ml). The enzyme activity
for amylase, fructosyltransferase, mannanase, lipase, glucosidase, and glucanase were
conducted at (56.7, 9.04, 8.1, 5, 0.4, 0.3 U/ml), respectively. Among these hydrolases,
lipase was chosen for the next steps of the experiments due to its high efficiency
in various industrial applications.

Fig. 1. Measuring the activity of various hydrolases from CPH

Optimization of CPH extraction

Lipase production, increased by several folds, show that experimental designs based
on statistics is important in optimizing the medium. According to (Wu et al. 2007]), this method has several advantages such as establishing the effects of the factors
as well as building a system model that has less experimental requirements. Following
the OFAT experiments, optimal conditions for the three important factors (concentration,
pH, and ratio) were obtained using Face Centered Central Composite Design (FCCCD)
under RSM. Table 2 shows the experimental and predicted values of lipase activity acquired from the
regression equation of 20 combinations in each run. From the results, the highest
amount of lipase produced from CPH (11.43 U/ml) was observed in run 9 under 7 % ratio
of CPH and 50 mM sodium phosphate buffer at pH 8. The lowest was observed in run 14
(4.931 U/ml), where 5 % CPH is used with 100 mM buffer at pH 7. This proves that the
lipase production is better with the design matrix of FCCCD compared to OFAT and screening.
The results reported a 2.5-fold increase in the lipase production by using RSM.

A second order regression equation revealed the dependence of the lipase activity
from CPH on the medium constituents. The parameters of the equation have been obtained
by multiple regression and the variables that were screened were expressed in terms
of second-order polynomial equation as follows:

where response (Y) is the activity of lipase. A, B, and C represent the buffer pH,
buffer concentration, and ratio of CPH, respectively.

The analysis of variance (ANOVA) was conducted to verify the adequacy of the model
and Fisher’s statistical analysis was used to test it. The results are recorded in
Table 2. The F value of 20.55 and p value of 0.0001 from this model provides a significant inference. A p value less
than 0.05 is considered significant. The efficiency of the model is shown with a higher
value of R
²
(0.9487) and adjusted R
²
(0.9025). The R
²
value should be maintained within the range of 0–1.0. As the value gets closer to
1.0, the model is considered to be a better fit (Reddy et al. 2008]). The signal to noise ratio is measured by adequate precision, where a ratio greater
than 4 is considered a requirement for desirable models. The ability of the model
to navigate the design space is indicated by the adequate precision value of 17.618
for lipase activity.

Table 3 lists coefficient values of the regression equation. The significance of each coefficient
are determined using the p values, which is also an indication of the strength of interaction between each variable
that is independent; as the p values get smaller, the significance of the corresponding coefficients get bigger.
All the three linear coefficients (A, B, and C), interaction term AB (pH and concentration),
and all the three quadratic coefficients (A
²
, B
²
and C
²
), were found to be significant with (p  0.05) and affects the overall production
remarkably.

Table 3. Analysis of variance of quadratic model for lipase production

Literature has documented several fold increases in the production of lipase using
RSM (Aybast?er and Demir 2010]; Dwevedi and Kayastha 2009]; Khoramnia et al. 2010]; Muralidhar et al. 2001]; Ruchi et al. 2008]). For example, a 20 % increase in the production of lipase was achieved under optimal
conditions following the use of RSM (He and Tan 2006]).

Sodium dodecyl sulphate SDS-PAGE was used to obtain the protein profile and to estimate
the molecular weight of the lipase extracted from the CPH. The molecular weight of
the lipase has been estimated 40 kDa in rice bran (Bhardwaj et al. 2001]), 24 kDa in buckwheat seed (Suzuki et al. 2004]), and 55 kDa in rapeseed (Sammour 2005]). These studies reveal that enzymes have different molecular weights depending on
their sources. In silver staining, more than one band is resulted from the SDS-PAGE
analysis, which indicates that the extract of the CPH contains many proteins of different
sizes. Since the sample was only precipitated, all the proteins’ bands were clearly
shown and possibly one of them belongs to lipase (Fig. 2). Additionally, the thickness of the band indicates the abundance or the intensity
level of the protein.

Fig. 2. SDS-PAGE of lipase extracted from CPH. Lane 1 molecular weight marker, Lane 2 molecular weight of lipase

Study of operating conditions of extraction of CLEA-lipase from CPH (OFAT analysis)

In this study, the individual effect of three selected parameters (concentration of
ammonium sulphate, glutaraldehyde, and BSA) were investigated to determine the highest
enzyme activity. Production of CLEA-lipase from CPH was carried out in 15 ml falcon
tubes. The content of the tube was made up of 0.5 ml crude lipase from CPH, (10–50 %
w/v) saturated ammonium sulphate, (40–80 mM) glutaraldehyde and (0–0.37 mM) BSA and
all reactions were allowed to proceed for 17 h.

Among the tested values, 30 % (w/v) ammonium sulphate, 70 mM glutaraldehyde, and 0.23 mM
BSA were found to be optimum values for the production of CLEA-lipase. Figure 3 shows the formation of CLEA with the addition of BSA as an additive.

Fig. 3. Schematic picture of CLEA preparation with addition of BSA as additive

The final particle size of CLEA depends on the type and amount of the precipitant,
stirring rate, concentration of glutaraldehyde, and protein concentration (Garcia-Galan
et al. 2011]; Yu et al. 2006]). In order to capture the enzyme activity in the final product of CLEA, the entire
free enzyme should be precipitated to a significant level. Addition of organic solvents,
salts, non-ionic polymers or acids can aggregate and precipitate the enzyme. Among
different precipitants, ammonium sulphate has worked the best for precipitating all
the free enzyme activity (Talekar et al. 2012]; Yu et al. 2013]). The amount of ammonium sulphate plays a significant role in controlling the enzyme
activity and particle size formation. It was noted that the maximum activity of the
insoluble cross-linked enzyme was detected when most of the protein has been precipitated
out of the solution and vice versa (Yu et al. 2006]).

The production of CLEA-lipase from CPH was amplified with the increase of ammonium
sulphate concentration. The observed increase in the production of CLEA-lipase was
between 10 and 30 % (w/v) ammonium sulphate. However, further increase in the concentration
rather decreased the production (Fig. 4). High concentrations of ammonium sulphate can decrease the enzyme activity and reduce
the protein residues in the supernatant. This may result in too little precipitant
to form the aggregates and indicates that additional free lipase was forming insoluble
enzyme aggregates. High ammonium sulphate concentration may lead to protein denaturation
that is responsible for the loss of activity (Wang et al. 2011]).

Fig. 4. Effect of different concentration of ammonium sulphate (10–50 %w/v) on CLEA-lipase
activity from CPH

The substrate concentration, below the requirement for the enzyme saturation, allows
the enzyme environment to partition the substrate toward the enzyme. This results
an increase in the enzyme activity.

The level of glutaraldehyde concentration for maximal lipase activity was determined
by testing different levels of glutaraldehyde with the concentration of 40–80 mM.
The highest lipase activity of 5.23 U (Fig. 5) was observed at approximately 70 mM concentration of glutaraldehyde. In general,
the reaction of glutaraldehyde molecule with amine residues of the lipase surface
can impact (positively/negatively) the lipase activity (Guauque et al. 2014]).

Fig. 5. Effect of different concentration of glutaraldehyde (40–80 mM) on CLEA- lipase activity
from CPH

The concentration of glutaraldehyde has significant effects on the enzyme activity.
In the presence of low amounts of cross-linkers, the enzyme molecule will leach into
water, because the immobilized enzyme is still flexible and unstable. An excessive
amount of GA can decrease the enzyme activity due to loss of the minimum flexibility
needed for the activity (Sheldon 2011]). Zhu and Sun (2012]) reported that this reaction could be due to more multi-point chemical bonds between
enzyme molecules and membrane surface at a high GA concentration.

The internal aldehyde group of glutaraldehyde attached with primary ?-amino group
from lysine and the result of this reaction is the Schiff bases (Barbosa et al. 2014]).

Sometimes CLEA may not be effective due to low enzyme Lys residue, since the cross
linking involves reaction of amino groups of Lys residues at the external surface
of the enzyme (Talekar et al. 2012]). In order to enhance the activity of enzyme, a second protein like BSA with a large
amount of Lys can be used for the preparation of CLEA. The addition of BSA can prevent
the formation of clusters in the excess concentration of glutaraldehyde, which leads
to the limitation of mass transfer. Moreover, the addition of BSA provides lysine
residues and makes positive impact by creating bonds with glutaraldehyde to prevent
the denaturing of the targeted protein. However, the BSA decreases the volumetric
loading of the target enzyme, since the inert protein occupies a portion of the volume
(Barbosa et al. 2014]; Rodrigues et al. 2014]).

Figure 6 shows the effect of different concentrations of BSA as additives (0–0.37 mM) on the
CLEA-lipase activity. A rise in the CLEA-lipase production was seen at levels ranging
from 0 to 0.23 (mM) BSA, where a decrease in the production was observed with subsequent
increase in concentrations. This may be due to leaching of extra protein in the media.

Fig. 6. Effect of different concentration of BSA (0–0.37 mM) on CLEA-lipase activity from
CPH

The addition of 10 mg BSA in the formation of CLEA of aminoacylase increased the activity
to 82 %, which was only 24 % before the addition (Dong et al. 2010]).

The lower activity of CLEA lipase of CPH (9 U) compared to the crude enzyme (11.26
U/ml) may be due to the hydrophobic and rigid structure of the enzyme after cross
linking. Therefore, it is hard for the enzyme to accept a large substrate like pNPP. The immobilization of the enzyme can block the active site of the enzyme (Iyer and
Ananthanarayan 2008]; Mateo et al. 2007]; Sheldon 2007]) and may also increase the diffusion problems (Gottifredi and Gonzo 2005]). These issues have led to the reduction of enzyme activity after the immobilization.
The enzyme can denature and decrease in activity after solubilising in the immobilization
medium.

After the formation of CLEA, the excess glutaraldehyde was removed by repeatedly washing
in water to avoid any modification of protein by the free glutaraldehyde. However,
the already reacted glutaraldehyde may continue the cross linking process with the
protein even after the washing steps.

Stability of CLEA-lipase against hydrophilic organic solvents

In our previous work, the CLEA-lipase from CPH was characterized and the results indicated
that the immobilized lipase has superior stability in response to different ranges
of temperature (25–60 °C) and pH (5–10) in comparison to free form of the enzyme (Khanahmadi
et al. 2015]). In this experiment, the stability of CLEA-lipase against hydrophilic organic solvents
(methanol, dioxane and acetone) was studied. The optimum temperatures for enzymatic
reaction of immobilized and free lipase were found to be 60 and 45 °C, respectively.
Organic solvents are known to have detrimental effect on the enzyme action. Based
on Fig. 7a, the stability of both forms of lipase were low in the presence of methanol. However,
CLEA was relatively more stable against methanol at a higher level. In the presence
of 60 % (v/v) methanol, the free lipase retained only 4 % of its initial activity,
whereas for CLEA, it was 29 %. When solvent concentrations of both dioxane (Fig. 7b) and acetone (Fig. 7c) were increased up to 100 % (v/v), a gradual reduction in the activity was observed
for both enzyme forms comparatively within a range (0–100 %, v/v). CLEA exhibited
a higher retention in activity than the free enzyme. A similar trend of stability
was too observed in the other study (Xu et al. 2011]).

Fig. 7. Comparison in the resistance of both the free lipase and its CLEA against hydrophilic
solvents. Free lipase and CLEA were incubated at 45 and 60 °C, respectively, for 30 min
in various buffer/solvent mixtures: methanol (a), dioxane (b), and acetone (c), and their residual activities were determined. A value of 100 % refers to the activity
obtained by the enzyme in the solvent-free buffer solution

There are several factors responsible for the effects of organic solvents. The organic
solvents mostly cause denaturation of proteins mainly due to the disruption of intra
non-covalent interactions. In other cases, organic solvents can act either as an inhibitor
or a substrate itself rendering competitive inhibition or substrate inhibition. Organic
solvents can interrupt intra-molecular hydrogen bonding that stabilizes the tertiary
structure of any proteins. Apart from that, the organic solvents change the physical
properties such as dielectric constant, polarity, and hydrophobicity. This change
affects the solvation of substrate and/or transition states, and hence interferes
with their binding to enzyme molecule (Mozhaev et al. 1989]).

High stability of immobilized lipase can be attributed to the structure of the immobilized
enzyme that becomes rigid because of the cross-linking. The immobilization is a useful
approach to protect the enzyme from the detrimental influence of organic solvents.
This is achieved by suppressing the tendency of the enzyme towards unfolding and is
accompanied by the loss of tertiary structure necessary for the activity.

The absence of water in the incubation solution can play a role, since increased stability
of enzymes under low moisture conditions has already been proven. It has been shown
that dehydration can exorbitantly retard the thermal inactivation of enzymes, which
preserves their conformational rigidity (Volkin et al. 1991]). Although rigid structure of CLEA maintains higher retention at higher concentration
of dioxane and acetone, the result was contradictory for pure methanol solvent known
to cause severe denaturation in various enzymes (Herskovits et al. 1970]).

Structural characterization of CLEA lipase from CPH by Field Emission Scanning Electron
Microscopy (FE-SEM)

The shape and size of CLEA particle is still relatively unknown. The final particle
size of CLEA depends on the rate of stirring, rate of precipitant addition, concentration
of protein, and precipitation time. CLEA has a small pore size that reduces the substrate
diffusion rate. Therefore, the proposed methods will allow adequate enzyme volumetric
activities even when the substrate is unsuitable for the enzyme. In the case of highly
suitable substrate, the enzyme activity will most likely overtake the substrate to
drop the substrate concentration inside the particle (and also a pH gradient if H
⁺
is consumed or produced by the reaction) (Garcia-Galan et al. 2011]). During the immobilization, enzyme solubility decreases in the surrounding medium.
While aggregation is slow, the extreme force applied on the structure of the enzyme
can cause denaturation. The enzyme structure can be retained if protein molecules
are found in time to surround the enzyme. The aggregation speed can be increased to
recover a poor enzyme activity. The molecule diameter is set by the surface tension
of the aggregate and the hydrophobicity of the surface will control the structures
of protein aggregates. As stated by the classical nucleation theory, the nucleus size
is controlled by the interaction of the free energy which is either increased by interfacial
surface formation or decreased in solid formation. The size of the primary particles
in the final aggregate is likely to be small and controlled by the ratio of nucleation
and growth.

Field emission scanning electron micrographs revealed that the CLEA from CPH has a
spherical appearance. With regard to CLEAs, Schoevaart et al. (2004]) reported that CLEAs have either a spherical appearance (Type 1) or a less-structured
form (Type 2). Therefore, the CLEA of CPH was the Type 1 aggregates (Fig. 8). The Type 1 aggregates can accommodate a thousand times more enzyme molecules than
the Type 2. Unlike the free protein, the enzyme molecules are packed in a small volume,
where mass-transport limitations will surely be expected, especially with fast reactions.
This effect is found to be small when the CLEA is dispersed in the solution at the
end of the cross-linking process.

Fig. 8. FE-SEM picture of CLEA-lipase from CPH