Available via license: CC BY 4.0
Content may be subject to copyright.
Citation: Guerineau, F. Properties of
Human Gastric Lipase Produced by
Plant Roots. Life 2022,12, 1249.
https://doi.org/10.3390/
life12081249
Academic Editor: Jianfeng Xu
Received: 15 July 2022
Accepted: 15 August 2022
Published: 16 August 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the author.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
life
Article
Properties of Human Gastric Lipase Produced by Plant Roots
François Guerineau
BioEcoAgro Research Unit, Universitéde Picardie Jules Verne, 33 Rue St Leu, 80039 Amiens, France;
francois.guerineau@u-picardie.fr
Abstract:
The properties of recombinant human gastric lipase produced in Arabidopsis thaliana roots
have been investigated with the goal of determining the potential of the enzyme. This enzyme is
stably bound to roots and can be extracted using a buffer at pH 2.2. This enzyme retains over 75% of
its activity after two weeks at room temperature when stored in a pH 2.2 buffer. Some of this activity
loss was due to the adsorption of the enzyme to the surface of the container. There was no loss of
lipase activity in dehydrated roots stored at room temperature for 27 months. The half-life of the
enzyme was approximately 15 min when stored in solution at 60
◦
C whereas dried roots retained 90%
lipase activity after one hour at 80
◦
C.
In vitro
binding assays using different root cell wall extracts
suggested that the lipase was bound to pectin in the roots. Lipase released from the root powder
hydrolyzed tributyrin. The high stability of the recombinant human gastric lipase makes this enzyme
a good candidate to be tested as a catalyst, whether in solution or bound to roots.
Keywords: Arabidopsis; hairy roots; lipase; protein production
1. Introduction
Lipases (EC 3.1.1.3) are enzymes of the hydrolase family that: (i) act on triacyl glyc-
erides as part of the catabolic process required for fatty acid absorption and (ii) exhibit
esterase activity. The two main lipases involved in digestion in humans are produced by
pancreas and stomach cells [
1
]. Unlike pancreatic lipase, gastric lipase (GL) acts at acidic
pH and does not require a colipase for activity [
2
]. Gastric lipase is a soluble enzyme found
at the interface of lipid droplets and aqueous solutions [
3
]. The hydrophobic active center
inside the enzyme structure is covered by a lid that opens upon contact of the enzyme with
a substrate [4].
The low pH tolerance of gastric lipase makes it a candidate for enzyme supplemen-
tation therapies for pancreatitis or cystic fibrosis patients [
5
]. Lipase supplementation is
currently done using enzymes extracted from livestock. A human recombinant enzyme
would be an interesting alternative to lipases of animal origin [
6
]. Lipases, especially of
microbial origin, have been used as biocatalysts in numerous biosynthetic reactions [
7
].
They can act in various solvents or ionic liquids to produce innovative molecules of thera-
peutic interest [
8
]. Human gastric lipase may also be used as a catalyst in esterification or
transesterification reactions.
Plants are now considered valuable hosts for the production of heterologous pro-
teins [
9
]. As a consequence of the ban on transgenic crops in some parts of the world,
in vitro
plant systems have been developed for the production of heterologous proteins
with the first approved plant-produced protein for therapeutic use being glucocerebrosi-
dase, produced by transgenic carrot cells grown in bioreactors [
10
]. Hairy roots are another
in vitro
system that can be used for the production of heterologous proteins [
11
]. Hairy
roots emerge following infection of plant tissue by the bacteria Rhizobium rhizogenes. Hairy
roots have higher stability than alternative cell culture systems, likely because they can
grow without the need to add phytohormones which can induce genetic
instability [12–14]
.
The Brassicaceae plant family can be used as an alternative to tobacco species for the pro-
duction of heterologous proteins [
15
–
17
]. In particular, Arabidopsis thaliana hairy roots have
Life 2022,12, 1249. https://doi.org/10.3390/life12081249 https://www.mdpi.com/journal/life
Life 2022,12, 1249 2 of 9
been established for the production of human gastric lipase [
18
]. Although the enzyme,
in this system, was targeted for secretion, it did not diffuse from the hairy roots to the
culture medium and had to be extracted from the roots, which suggested some interactions
between the enzyme and the roots producing it. In this work, I further investigated the
interaction of gastric lipase with the roots. Given that the stability of an enzyme produced
in a heterologous host was sometimes found to be lower than that of the native enzyme [
19
],
I investigated if this was similarly the case in recombinant human gastric lipase produced
by Arabidopsis thaliana hairy roots. This investigation paves the way for using this re-
combinant human gastric lipase enzyme as a biocatalyst or for lipase supplementation in
certain patients.
2. Materials and Methods
2.1. Materials
The Arabidopsis thaliana hairy root line used in this work was the GL28 line de-
scribed in [
18
]. It was obtained by hypocotyl transformation of the Arabidopsis sgs3-12
co-suppression mutant line with the Rhizobium rhizogenes 15834 strain containing the cDNA
encoding the mature human gastric lipase, fused to a plant signal peptide-encoding se-
quence, under control of the 2
×
35 S cauliflower mosaic virus promoter. The lipase
substrates 4-methylumbelliferyloleate (MUO) (ref. 75164) and tributyrin (ref. W222305)
were from Sigma-Aldrich, St-Quentin-Fallavier, France.
2.2. Root Culture and Storage
Hairy roots were grown at 21
◦
C on an orbital shaker at 60 RPM in 55 mm diameter
Petri dishes in Gamborg B5 medium containing sucrose at 5% and 2,4-D at 0.5 mg/L. They
were harvested after 18 to 21 days, briefly washed in distilled water, and freeze-dried
overnight. The dehydrated roots were kept in Eppendorf tubes having their lids punctured.
The tubes were placed in closed containers containing silica gel and stored in the dark at
room temperature.
2.3. Lipase Extraction and Assay
Lipase was extracted by grinding 5 to 10 mg of dry roots in 200 to 400
µ
L of lipase
extraction buffer (LEB: 0.1 M glycine-NaOH pH 2.2, 0.15 M NaCl). The mixture was
incubated for 15 min at 37
◦
C in an Eppendorf Thermomixer at 1000 RPM and then
centrifuged at 13,000
×
gfor 2 min. Fluorometric assays were done on the supernatants
diluted 1/100 or 1/200 in LEB in Eppendorf Protein LoBind
®
tubes. As previous work has
established linearity of lipase activity on tributyrin and esterase activity on MUO [18], for
convenience, the fluorometric assay using MUO was used to quantify hGL activity. The
assays were done in MUO substrate solution (10 mM acetate buffer pH 5, 0.15 M NaCl,
7 ppm Triton X-100, 0.15 mM MUO) as described before [
18
]. The assays were done in
duplicates. The activity was expressed in
µ
moles of 4-methylumbelliferone (MU) produced
per min per ml of undiluted extract or per mg of roots. It was then converted to units/mg
of roots, knowing that one unit of enzyme catalyzed 0.219
µ
moles of MUO per min under
our assay conditions [
18
]. For the reaction on tributyrin, 100
µ
L of tributyrin was emulsified
in 10 mL of 10 mM acetate buffer pH 5, 0.15 M NaCl, 1% molten agarose and the mixture
was poured into a 90 mm diameter Petri dish. After setting, root powder was sprinkled on
the plate. Photographs were taken after 1, 4, and 24 h.
2.4. Lipase Stability and Tube Binding Assays
Lipase was extracted from different batches of dry roots. Extracts were diluted
1/200 either in 50 mM glycine buffer at pH 2.2, 0.15 M NaCl or in 50 mM acetate buffer at
pH 5, 0.15 M NaCl. The diluted extracts were kept in Protein LoBind
®
Eppendorf tubes
at room temperature and assayed for lipase activity after 1, 3, 6, 10, and 14 days. After
14 days
, the solutions were transferred into new tubes and the lipase activity was assayed
24 h
later. The activities in stored extracts were expressed as a percentage of activity related
Life 2022,12, 1249 3 of 9
to freshly diluted extracts. To test for lipase binding to tubes, 100
µ
L of lipase diluted 1/200
in one or the other above buffer were placed in Protein LoBind
®
Eppendorf tubes for
24 h
.
The solution was removed and the tubes were rinsed with 100
µ
L of buffer of the same
composition as the one used to dilute the extracts. One hundred
µ
L of MUO substrate
solution was placed for one min in the empty tubes. One hundred
µ
L of stop solution was
added and the fluorescence of the MU (4-methylumbelliferone) was measured, indicating
the activity of lipase bound to the tubes.
2.5. Cell Wall Polysaccharide Extraction
Alcohol-insoluble matter (AIM) was prepared using a method described in [
20
]. Two
hundred mg of freeze-dried untransformed roots were ground in 2 mL of 70% ethanol. The
suspension was vortexed for 20 s and centrifuged at 13,000
×
gfor 30 s. The pellet was
further extracted twice with 2 mL of 70% ethanol, twice with 100% ethanol, twice with
chloroform/methanol (vol/vol), and once with acetone. The dry pellet was AIM. For pectin
removal, AIM was extracted twice with 20 mM Tris-HCl pH 6.8, 50 mM EDTA for
15 min
in an Eppendorf Thermomixer set at 60
◦
C and 1000 RPM, and then twice with
50 mM
Na
2
CO
3
under the same conditions. The pectin-deprived AIM pellet was then washed in
1 mL of acetone and dried at 65 ◦C.
2.6. In Vitro Lipase Binding Assays
Lipase was extracted from GL28 roots using LEB as described above (40 µL LEB/mg
roots). The proteins were precipitated by 70% ammonium sulfate for 5 min at room
temperature. After centrifugation for 5 min at 13,000
×
g, the pellets were dissolved in
the same volume of 10 mM acetate buffer, pH 5. Two hundred
µ
L of the solution was
incubated for 15 min with 5 mg of either root powder or AIM or pectin-deprived AIM in
an Eppendorf Thermomixer set at 30
◦
C and 900 RPM. The suspension was centrifuged
at 13,000
×
gfor
2 min
. The supernatants were collected for the quantification of unbound
lipase. The pellets were washed once with 10 mM acetate buffer pH 5 and resuspended
in 200
µ
L of LEB for lipase extraction for 15 min in an Eppendorf Thermomixer set at
37 ◦C
and 1000 RPM. After centrifugation at 13,000
×
gfor 2 min, the lipase activity in the
supernatant was assayed, indicating the amount of bound lipase.
2.7. Statistical Analysis
For each experiment, n indicates the number of lipase extractions done on dry roots.
Two enzyme assays were done on each extract. The two values were averaged and the
mean activities in the different extracts were calculated. The confidence intervals of the
means (CI; p= 0.95) were calculated on vassarstats.net and indicated as error bars on the
graphs. The number of replicates (n) is given for each experiment in the figures or after the
mean values and CI in the text.
3. Results
3.1. Extraction of Lipase from Roots
Dehydrated roots were ground in various extraction buffers and the lipase activities in
the extracts were measured. As found before [
18
], much less lipase was released in acetate
buffer at pH 5 than in glycine buffer at pH 2.2 (LEB). However, the addition of 1 M NaCl
or of 0.2 M CaCl
2
to acetate buffer resulted in the release of 2.7 and 8 times more lipase
activity than by acetate alone, respectively (Figure 1).
Life 2022,12, 1249 4 of 9
Figure 1.
Release of gastric lipase from dry root powder in different solutions. Ac is 10 mM acetate
buffer. Gly is 0.1 M glycine buffer. Bars are confidence intervals (0.95; n= 5).
3.2. Stability of Lipase in Solution at Room Temperature
A time course of lipase activity in solution at pH 2.2 or at pH 5 kept at room temper-
ature was performed. These pHs were chosen because they are the pH of the extraction
buffer and of the assay buffer, respectively. Whereas extracts at pH 2.2 retained approxi-
mately 75% of lipase activity after 14 days, those at pH 5 lost over 50% activity over the
same time span (Figure 2). Transferring the diluted solutions of lipase to new tubes on
day 14 resulted in a sharp drop of lipase activity after 24 h, especially at pH 5. To test for
adsorption on tubes, lipase diluted in solutions at pH 2.2 or at pH 5 were placed in tubes
for 24 h and removed. The tubes were then rinsed, and the activity of lipase adsorbed onto
the tube was measured. The lipase activity per tube was 0.095
±
0.012 mU (0.95 CI, n= 6)
or 1.5 ±0.39 mU (0.95 CI, n= 6) for lipase in buffer at pH 2.2 or at pH 5, respectively.
Figure 2.
Time course of gastric lipase activity in extracts kept at 20
◦
C, at pH 2.2 (circles) or at
pH 5
(triangles). The extracts were placed in new tubes on day 14. Bars are confidence intervals (0.95;
n= 6).
3.3. Long-Term Storage of Lipase in Roots
Extracts from roots that had been freeze-dried the day before had a lipase activity
of 6.59
±
0.26 U/mg roots (0.95 CI, n= 10). Extracts from roots that were kept in silica
gel-containing pots for 27 months had a lipase activity of 6.63
±
0.3 U/mg roots (0.95 CI,
Life 2022,12, 1249 5 of 9
n= 10
). Similar lipase activity was observed in extracts from dry roots that were stored for
shorter amounts of time (Table S1).
3.4. Temperature Stability of Lipase
To investigate whether the association of gastric lipase to roots translated into higher
temperature stability, the stability at 60
◦
C of lipase in solution or in dehydrated roots was
compared. The half-life of lipase in lipase extraction buffer at 60
◦
C was approximately
15 min
whereas there was no loss of lipase activity extracted from dry roots kept at 60
◦
C
for one hour (Figure 3). Incubating lipase in extraction buffer for 5 min at 80
◦
C resulted in
a total loss of lipase activity (data not shown). In contrast, the activity of lipase extracted
from dry roots incubated at 80
◦
C for one hour was 92.7
±
9.4% (0.95 CI, n= 4) of that of
lipase extracted from the control roots kept at room temperature. Similarly, 56.4
±
17.1%
(0.95 CI, n= 4) activity could be recovered from roots incubated for one hour at 100
◦
C.
To assess the relative contributions of the association with roots or of the dehydration
in the thermal stability of dry root-associated lipase, dehydrated roots were ground and
hydrated in 20 mM acetate buffer pH 5, 0.15 M NaCl. The mixes were incubated either
at room temperature or at 60
◦
C for 30 min. The lipase was then extracted from the roots
and assayed for lipase activity. The activity extracted from heat-treated hydrated roots
was 33.4
±
5.4% (0.95 CI, n= 4) of the activity extracted from hydrated roots kept at
room temperature.
Figure 3.
Stability at 60
◦
C of gastric lipase in solution in lipase extraction buffer (circles) or in dry
roots (triangles). Bars are confidence intervals (0.95; n= 4).
3.5. In Vitro Binding Assays
To investigate the interaction of human gastric lipase with plant cell wall polysaccha-
rides,
in vitro
binding assays were undertaken. Lipase in 10 mM acetate buffer at
pH 5
was
incubated with either untransformed root powder, cell wall powder, or cell wall powder
from which pectin had been extracted. The unbound and bound fractions of lipase were
extracted and assayed as indicated in the materials and methods section above. The binding
of lipase to each powder was expressed as a percentage of bound lipase activity related to
the total lipase activity. The binding of hGL was much more extensive on root powder and
cell wall powder than on pectin-deprived cell wall powder (Figure 4).
Life 2022,12, 1249 6 of 9
Figure 4. In vitro
binding of gastric lipase to root powder (RP), cell wall powder (CW), or pectin-
deprived cell wall powder (PDCW). Bars are confidence intervals (0.95, nindicated on graph).
3.6. Enzyme Release from Root Powder
To see whether gastric lipase could be released from root powder to hydrolyze a sub-
strate, root powder was sprinkled on a tributyrin-containing agarose plate. Untransformed
root powder was used as a control. The formation of a clear halo around each grain of
transgenic root indicated tributyrin hydrolysis, with the size of the halos increasing with
time (Figure 5).
Figure 5.
Degradation of tributyrin by gastric lipase diffusing from transgenic root powder on an
agarose plate. (
a
) after 1 h; (
b
) after 4 h; (
c
) after 24 h. In each plate: left, untransformed root powder;
right, transgenic root powder.
4. Discussion
Previous work has shown that unlike EGFP (enhanced green fluorescent protein), the
human gastric lipase targeted for secretion failed to diffuse from transgenic hairy roots
into the culture medium [
18
]. The enzyme, which had likely accumulated in the cell walls,
had to be extracted from the roots. Similarly, gastric lipase produced in Pichia pastoris also
remained in the cell wall [
21
]. The efficiency of the extraction of human gastric lipase from
hairy roots was shown to be pH-dependent, with markedly more lipase being released at
pH 2.2 than at pH 5 [
18
]. Here, the effect of salts on lipase release was investigated. The
addition of 1 M NaCl or of 0.2 M CaCl
2
enhanced the release of lipase from root powder at
pH 5 (Figure 1). The effect of the 0.2 M CaCl
2
solution on lipase release was found to be
three times higher than that of the 1 M NaCl solution, although the ionic strength of the
CaCl
2
solution was lower than that of the NaCl solution. This result suggests a specific
action of calcium ions. In plant cell walls, calcium ions interact with pectin by forming
bridges between homogalacturonan chains [
22
]. The extensive effect of CaCl
2
on lipase
release from roots suggests involvement of pectin in lipase retention in the cell wall at pH 5.
Life 2022,12, 1249 7 of 9
To investigate the interaction of lipase with cell wall components, gastric lipase was
added to either root powder or crude cell wall extracts or cell wall extracts from which
soluble pectin had been removed. The binding of lipase to root powder and to cell wall
powder was very effective whereas it was markedly lower on cell wall powder from which
pectin had been extracted (Figure 4). This strengthened the hypothesis of lipase interaction
with pectin in root cell walls. The negative charges of pectin at pH 5 might be responsible
for the association with lipase, expected to be positively charged below its pI of 6.9. In
contrast, at pH 2.2, homogalacturonans are expected not to be charged, which would
explain the release of lipase at this pH. Similarly, the addition of CaCl
2
might trigger lipase
release at pH 5 by competing for the negative charges of pectin.
The time course analysis of lipase activity in solution at pH 2.2 revealed a slow decrease
down to approximately 75% of the initial activity after 14 days (Figure 2). There was more
activity loss at pH 5, down to approximately 45%. Although this could indicate higher
stability of lipase at pH 2.2 than at pH 5, the following observation points towards another
mechanism. The transfer of lipase solutions into new tubes after 14 days resulted in a sharp
drop in activity after 24 h. This strongly suggested that lipase adsorption on tubes was
occurring. This hypothesis was confirmed by a tube-binding assay that revealed lipase
activity on the surface of the tubes. The binding was 15 times more extensive at pH 5 than at
pH 2.2. The adsorption of various lipases on polypropylene beads has been reported, with
the immobilization causing a loss of enzyme activity in some cases [
23
]. The propensity of
gastric lipase to adsorb on surfaces has to be taken into account when using this enzyme.
Human gastric lipase was found to be more stable in gastric juice than when puri-
fied [
24
]. It was found that the half-life of purified gastric lipase was only 25 min at pH 2
and pH 3 and 101 min at pH 5 whereas it was over 24 h in gastric juice at pH 3 and pH 5
and 510 min at pH 2. The stability of recombinant lipase extracted from plant roots here
was higher than that of native lipase in gastric fluid. Some plant co-extracted proteins
or other molecules may have a stabilizing effect on lipase. Alternatively, higher stability
might result from differences in the protein structures of the native and the recombinant
enzymes, due to differences in the glycan structures deposited on proteins by animal and
plant cells [
16
]. In any case, the higher stability of the recombinant enzyme extracted from
plant roots is a valuable feature for its use as a biocatalyst.
The effect of the association of lipase with root cell wall on the lipase thermal tolerance
was evaluated. The half-life of lipase in solution in LEB at 60
◦
C was approximately
15 min
(Figure 3). In contrast, the half-life of lipase in dry roots at 100
◦
C was over an
hour, revealing much higher temperature stability of the lipase in dehydrated roots than
in solution. When hydrated roots were incubated at 60
◦
C for 30 min, the lipase activity
dropped by 67% whereas lipase in solution under similar treatment lost 80% of its activity
(Figure 3). The thermal tolerance of lipase in dehydrated roots is therefore mainly due
to dehydration.
The protective effect of dehydration was also confirmed by long-term stability mea-
surements. It has been previously shown that approximately 17% of lipase activity was
lost in roots kept at room temperature for two months [
18
]. In that experiment, no silica
gel was used for the storage of the roots, which resulted in the roots being exposed to
atmospheric moisture. Here, the storage of roots in the presence of silica gel ensured more
extensive dehydration and long-term preservation of lipase activity. There was no loss
of extractable lipase activity in dehydrated roots over a period of 27 months. Although
freeze-drying is a well-established method of tissue or protein preservation, long-term
studies are lacking [
25
]. In order to maintain the activity of proteins upon freeze-drying,
a protecting agent must often be added to them [
26
]. The high stability of human gastric
lipase in lyophilized plant roots revealed a high tolerance of the enzyme to dehydration.
Tributyrin was hydrolyzed around root powder specked on an agarose plate (Figure 5),
which indicated that some gastric lipase was released from the roots and diffused onto
the agarose to hydrolyze the substrate. The slow release of lipase at pH 5 initiated the
production of butyric acid, which decreased the pH around the roots, causing the release of
Life 2022,12, 1249 8 of 9
more enzymes. This induction mechanism likely explains the diffusion of the enzyme on
the agar plate, indicated by the enlargement of the halos around the specks, over 24 h.
5. Conclusions
This work has revealed interesting properties of a plant-produced human gastric
lipase, such as high stability at room temperature and high tolerance to dehydration, that
could be leveraged in the design of supplement formulations for the treatment of certain
patients. The propensity of gastric lipase to bind pectin or to adsorb on surfaces suggests
that the immobilization of the enzyme may be easy. The stability of the enzyme favors its
use as a biocatalyst in solvents or in ionic liquids. The strong association of lipase with roots
provided a slow-release form of the enzyme that can be utilized in biochemical reactions,
whether in aqueous or non-aqueous solvents. The ease of production of this extremophilic
human enzyme by plant roots enables the production of structural variants of the enzyme
that will improve its properties and its biosynthesis capacity.
Supplementary Materials:
The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/life12081249/s1, Table S1: Lipase activity extracted from roots kept at room
temperature for various amounts of time.
Funding:
This research was funded by MESRI (Ministère de l’Enseignement Supérieur, de la
Recherche et de l’Innovation), grant number EA3900 and by the SFR (Structure Fédérative de
Recherche) Condorcet, LIPACT project.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or Supplementary Materials.
Acknowledgments:
I am very grateful to Catherine Sarazin for helpful discussions and critical
reading of the manuscript.
Conflicts of Interest: The author declares no conflict of interest.
References
1.
Roussel, A.; Canaan, S.; Egloff, M.P.; Rivière, M.; Dupuis, L.; Verger, R.; Cambillau, C. Crystal structure of human gastric lipase
and model of lysosomal acid lipase, two lipolytic enzymes of medical interest. J. Biol. Chem.
1999
,274, 16995–17002. [CrossRef]
[PubMed]
2.
Chahinian, H.; Snabe, T.; Attias, C.; Fojan, P.; Petersen, S.B.; Carrière, F. How gastric lipase, an interfacial enzyme with a
Ser-His-Asp catalytic triad, acts optimally at acidic pH. Biochemistry 2006,45, 993–1001. [CrossRef] [PubMed]
3.
Miled, N.; Canaan, S.; Dupuis, L.; Roussel, A.; Rivière, M.; Carrière, F.; de Caro, A.; Cambillau, C.; Verger, R. Digestive lipases:
From three-dimensional structure to physiology. Biochimie 2000,82, 973–986. [CrossRef]
4. Canaan, S.; Roussel, A.; Verger, R.; Cambillau, C. Gastric lipase: Crystal structure and activity. Biochim. Biophys. Acta 1999,1441,
197–204. [CrossRef]
5.
Fieker, A.; Philpott, J.; Armand, M. Enzyme replacement therapy for pancreatic insufficiency: Present and future. Clin. Exp.
Gastroenterol. 2011,4, 55–73.
6.
Aloulou, A.; Carrière, F. Gastric lipase: An extremophilic interfacial enzyme with medical applications. Cell. Mol. Life Sci.
2008
,
65, 851–854. [CrossRef]
7.
De Godoy Daiha, K.; Angeli, R.; de Oliveira, S.D.; Almeida, R.V. Are lipases still important biocatalysts? A study of scientific
publications and patents for technological forecasting. PLoS ONE 2015,10, e0131624.
8.
Elgharbawy, A.A.; Riyadi, F.A.; Alam, M.Z.; Moniruzzaman, M. Ionic liquids as a potential solvent for lipase-catalysed reactions:
A review. J. Mol. Liq. 2018,251, 150–166. [CrossRef]
9.
Schillberg, S.; Raven, N.; Spiegel, H.; Rasche, S.; Buntru, M. Critical analysis of the commercial potential of plants for the
production of recombinant proteins. Front. Plant Sci. 2019,10, 720. [CrossRef]
10.
Tekoah, Y.; Shulman, A.; Kizhner, T.; Ruderfer, I.; Fux, L.; Nataf, Y.; Bartfeld, D.; Ariel, T.; Gingis-Velitski, S.; Hanania, U.; et al.
Large-scale production of pharmaceutical proteins in plant cell culture-the protalix experience. Plant Biotechnol. J.
2015
,13,
1199–1208. [CrossRef]
11. Ono, N.N.; Tian, L. The multiplicity of hairy root cultures: Prolific possibilities. Plant Sci. 2011,180, 439–446. [CrossRef]
12.
Aird, E.L.N.; Hamill, J.D.; Rhodes, M.J.C. Cytogenetic analysis of hairy root cultures from a number of plant species transformed
by Agrobacterium rhizogenes.Plant Cell Tissue Organ Cult. 1988,15, 47–57. [CrossRef]
Life 2022,12, 1249 9 of 9
13.
Häkkinen, S.T.; Moyano, E.; Cusido, R.M.; Oksman-Caldentey, K.M. Exploring the metabolic stability of engineered hairy roots
after 16 years maintenance. Front. Plant Sci. 2016,7, 1486. [CrossRef]
14.
Sun, J.; Ma, L.; San, K.Y.; Peebles, C.A.M. Still stable after 11 years: A Catharanthus roseus hairy root line maintains inducible
expression of anthranilate synthase. Biotechnol. Prog. 2017,33, 66–69. [CrossRef]
15.
Huet, Y.; Ele Ekouna, J.P.; Caron, A.; Mezreb, K.; Boitel-Conti, M.; Guerineau, F. Production and secretion of a heterologous
protein by turnip hairy roots with superiority over tobacco hairy roots. Biotechnol. Lett. 2014,36, 181–190. [CrossRef]
16.
Ele Ekouna, J.P.; Boitel-Conti, M.; Lerouge, P.; Bardor, M.; Guerineau, F. Enhanced production of recombinant human gastric
lipase in turnip hairy roots. Plant Cell Tissue Organ Cult. 2017,131, 601–610. [CrossRef]
17.
Mai, N.T.P.; Boitel-Conti, M.; Guerineau, F. Arabidopsis thaliana hairy roots for the production of heterologous proteins. Plant Cell
Tissue Organ Cult. 2016,127, 489–496. [CrossRef]
18.
Guerineau, F.; Mai, N.T.P.; Boitel-Conti, M. Arabidopsis hairy roots producing high level of active human gastric lipase. Mol.
Biotechnol. 2020,62, 168–176. [CrossRef]
19. Rai, M.; Padh, H. Expession systems for production of heterologous proteins. Curr. Sci. 2001,80, 1121–1128.
20.
Fangel, J.U.; Jones, C.Y.; Ulvskov, P.; Harholt, J.; Willats, W.G.T. Analytical implications of different methods for preparing plant
cell wall material. Carbohydr. Polym. 2021,261, 117866. [CrossRef]
21.
Sams, L.; Amara, S.; Chakroun, A.; Coudre, S.; Paume, J.; Giallo, J.; Carrière, F. Constitutive expression of human gastric lipase
in Pichia pastoris and site-directed mutagenesis of key lid-stabilizing residues. BBA-Mol. Cell Biol. Lipids
2017
,1862, 1025–1034.
[CrossRef]
22.
Cao, L.; Lu, W.; Mata, A.; Nishinari, K.; Fang, Y. Egg-box model-based gelation of alginate and pectin: A review. Carbohydr. Polym.
2020,242, 116389. [CrossRef]
23.
Gitlesen, T.; Bauer, M.; Adlercreutz, P. Adsorption of lipase on polypropylene powder. Biochim. Biophys. Acta
1997
,1345, 188–196.
[CrossRef]
24.
Ville, E.; Carrière, F.; Renou, C.; Laugier, R. Gastric disorders physiological study of pH stability and sensitivity to pepsin of
human gastric lipase. Digestion 2002,65, 73–81. [CrossRef]
25.
Molnar, A.; Lakat, T.; Hosszu, A.; Szebeni, B.; Balogh, A.; Orfi, L.; Szabo, A.J.; Fekete, A.; Hodrea, J. Lyophilization and
homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques.
Amino Acids 2021,53, 917–928. [CrossRef]
26.
Roy, I.; Gupta, M.N. Freeze-drying of proteins: Some emerging concerns. Biotechnol. Appl. Biochem.
2004
,177, 165–177. [CrossRef]
