Foods 2025, 14, 397 h ps://doi.o g/10.3390/ oods14030397
A icle
Enzyma ic Deg ada ion o Och a oxin A: The Role o
Ul a-Pu e Wa e
Joana San os
1
, Cons ança Oli ei a
1,2
, Filipe Teixei a
2
, A mando Venâncio
1,3
and Ca la Sil a
1,3,
*
1
Cen e o Biological Enginee ing, Uni e si y o Minho, Campus de Gual a , 4710-057 B aga, Po ugal;
joanasan os587@ho mail.com (J.S.); [email protected] (C.O.); a [email protected] (A.V.)
2
Cen e o Chemis y, Uni e si y o Minho, Campus o Gual a , 4710-057 B aga, Po ugal;
[email p o ec ed]
3
LABBELS—Associa e Labo a o y, 4710-057 B aga, 4800-058 Guima ães, Po ugal
* Co espondence: [email p o ec ed]o.p
Abs ac : Och a oxin A (OTA) is a oxic myco oxin, making i s emo al om ood
essen ial o public heal h. This s udy examines OTA deg ada ion by po cine panc ea ic
lipase (PPL) in ul a-pu e wa e e sus buffe sys ems h ough in i o assays and
molecula modeling. The esul s show ha PPL ully deg ades OTA in ul a-pu e wa e
wi hin 7 h a 44 °C, whe eas only pa ial deg ada ion occu s in phospha e buffe . A e 4
h, PPL in wa e deg ades 91% o OTA, compa ed o only 12% in buffe . The enzyme’s
hal -li e is longe in wa e (~4 h 4 min) han in phospha e buffe (~2 h 30 min), sugges ing
be e s abili y in wa e . O he buffe s, including ace a e, ci a e, and bo a e, con i med
highe deg ada ion efficiency in low-conduc i i y, acidic en i onmen s simila o ul a-
pu e wa e . Addi ionally, using he model compound p-ni ophenyl oc anoa e (p-NPO),
i was ound ha p-NPO deg ades as e in buffe , likely due o a sal ing-ou effec .
Molecula modeling and ci cula dich oism analysis indica e ha PPL’s seconda y
s uc u e in wa e p omo es an ideal con o ma ion o OTA binding. This s udy sugges s
ul a-pu e wa e as a g eene , sus ainable op ion o educing myco oxins in ood, wi h
b oad indus ial applica ions.
Keywo ds: ul apu e wa e ; enzyma ic deg ada ion; och a oxin A; lipase; ood
de oxi ica ion
1. In oduc ion
Myco oxins a e oxic seconda y me aboli es p oduced by ungi ha , when inges ed,
inhaled, o abso bed h ough he skin, can cause acu e and ch onic heal h effec s in
humans and animals [1,2]. The global p esence o myco oxins in ood and eed has a
signi ican impac on e e yone’s heal h, esul ing in billions o dolla s in annual economic
losses [3]. The myco oxins o mos conce n include a la oxins, och a oxins, zea alenone,
umonisins, ci inin, pa ulin, ni alenol, deoxyni alenol, and e go alkaloids [1].
Och a oxins a e highly oxic myco oxins, wi h och a oxin A (OTA) being he mos
oxic and commonly ound membe o he och a oxin amily. OTA is one o he op i e
myco oxins ha a e highly egula ed by he law due o i s neph o oxic, geno oxic,
cy o oxic, e a ogenic, mu agenic, and immuno oxic effec s [4]. Fu he mo e, he
In e na ional Agency o Resea ch on Cance (IARC) ca ego izes OTA as G oup 2B,
indica ing ha i is possibly ca cinogenic o humans [1].
Academic Edi o : Thie y Nogue
and Yihan Liu
Recei ed: 12 Decembe 2024
Re ised: 3 Janua y 2025
Accep ed: 24 Janua y 2025
Published: 25 Janua y 2025
Ci a ion: San os, J.; Oli ei a, C.;
Teixei a, F.; Venâncio, A.; Sil a, C.
Enzyma ic Deg ada ion o
Och a oxin A: The Role o
Ul a-Pu e Wa e . Foods 2025, 14,
397. h ps://doi.o g/10.3390/
oods14030397
Copy igh : © 2025 by he au ho s.
Submi ed o possible open access
publica ion unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/license
s/by/4.0/).
Foods 2025, 14, 397 2 o 17
Focusing on con ol s a egies o mi iga e OTA con amina ion in ood is c ucial. I
has only been in he las six decades, ollowing he iden i ica ion o a la oxins, ha he
impo ance o mi iga ing myco oxins o he well-being o humans and animals has been
ecognized [5–9]. Biological ans o ma ion is ega ded as he mos p omising bu
challenging me hod o dec ease he accumula ion o myco oxins, as p e- and pos -ha es
s a egies in ol ing chemical o physical emo al a e no sufficien ly effec i e [3,10–12].
The deg ada ion o OTA by isola ed o c ude enzymes is an effec i e me hod o
de oxi ying ood [11,13–18]. The use o isola ed enzymes is a highly efficien app oach o
speci ically a ge ing OTA. This me hod is en i onmen ally iendly and does no in ol e
he use o ha m ul chemicals. Hyd olases, mo e speci ically lipases, a e he enzymes mos
used in his p ocess o mi iga ion [10]. Lipases [EC 3.1.1.3] a e a subclass o es e ases,
isola ed om a ious species as mic oo ganisms, plan s, and animal issues, ha can
ca alyze a a ie y o eac ions including hyd olysis, alcoholysis, acidolysis,
anses e i ica ion, and es e i ica ion.
In bio ans o ma ion eac ions, po cine panc ea ic lipase (PPL) is an effec i e enzyme
due o i s cos effec i eness compa ed o o he comme cially a ailable mic obial and
animal lipases [19]. PPL is a p o ein o 50 kDa wi h 448 amino acid esidues. In 1996,
He moso e al. esol ed he c ys alline s uc u e o he PPL–colipase complex in he open
s a e (PDB: 1ETH) [20]. The PPL p o ein consis s o wo dis inc domains, an α/β- old N-
e minal domain ( esidues: 1–336), e med domain 1, and a β-sandwich C- e minal
domain ( esidues: 337–448) e e ed o as domain 2. The esidues Se 153, Asp177, and
His264 cons i u e he ac i e si e o he enzyme, which is shielded by a lid egion ex ending
om Cys238 o Cys262. The ac i i y o he enzyme is modula ed by con o ma ional
changes in he icini y o he lid [21].
In biphasic sys em in e aces, he lid opens due o con o ma ional changes,
p oducing he “open s uc u e” o he lipases; his p ocess is known as in e acial
ac i a ion. In an open con o ma ion, subs a es can access he ac i e si e and unde go
con e sion. Howe e , when he lid closes, he lipase becomes inac i e by shielding he
ac i e si e om he en i onmen , hus p e en ing subs a e access [22]. In pu e aqueous
en i onmen s, he lid emains p edominan ly closed, whe eas in he p esence o a
hyd ophobic laye , i s ays open.
Hanque and P abhu [23] p oposed ha he PPL in i s closed con o ma ion is s able
in wa e , whe eas he open con o ma ion is signi ican ly less s able and ends o ansi ion
owa ds he closed s a e. The au ho s also epo ed ha despi e signi ican luc ua ions in
he closed con o ma ion o PPL, ha ha e been obse ed in he p esence o wa e , i was
no possible o induce he opening mo emen o he lid. In he open con o ma ion, he
luc ua ions a e smalle , and he lid mo es owa d he closed con o ma ional s a e, as i
he s uc u e we e eezing in a mo e pola en i onmen . The e o e, he eac ion
en i onmen mus be ca e ully conside ed when using lipases. Rega ding he enzyma ic
deg ada ion o OTA, mos s udies p esen deg ada ion in phospha e-buffe ed solu ions
[13], o in cul u e media [14,24–26]; howe e , hese s udies did no epo on he
explo a ion o he sol en effec on enzyma ic ac i i y.
San os e al. ha e p e iously demons a ed ha PPL has he abili y o deg ade 43%
o OTA and comple ely deg ade i s non-chlo ina ed de i a i e, och a oxin B, in 9 h, using
phospha e buffe (100 mM, pH 7.5) as eac ion medium [13]. Ab unhosa e al. had
p e iously pe o med OTA deg ada ion using panc ea in enzyme and p o ease unde he
same buffe ed condi ions [11].
The die a y in ake o phospha e and he le el o phospha e in he bloods eam a e
signi ican conce ns o bo h indi iduals wi h kidney disease and he gene al popula ion.
Recen esea ch indica es ha phospha e addi i es in ood can ad e sely affec
indi iduals wi h no mal kidney unc ion. O e he pas decade, hype phospha emia has
Foods 2025, 14, 397 3 o 17
eme ged as a s ong p edic o o mo ali y in ad anced ch onic kidney disease [27]. In
2009, a g oundb eaking s udy i s es ablished a connec ion be ween phospha e
concen a ions and ca dio ascula disease in he gene al popula ion [28]. Since hen,
moun ing e idence has ein o ced his link, showing ha ele a ed se um phospha e
le els, e en i hey emain wi hin he no mal ange, co ela e wi h ad e se ca dio ascula
ou comes, including co ona y a e y calci ica ion, conges i e hea ailu e, and mo ali y
[29]. The p esence o phospha e addi i es in ood is a signi ican conce n, and hei
po en ial impac on heal h may be mo e subs an ial han p e iously es ima ed.
Conside ing his, he op imal solu ion o deg ading myco oxins may be he use o he
g eenes sol en —ul a-pu e wa e . Wa e is non- oxic o bo h heal h and he
en i onmen , and i s abundan , enewable a ailabili y makes i an economically and
ecologically iable choice [27]. The physical p ope ies o wa e , and hei changes wi h
empe a u e and p essu e, enable a wide ange o eac ions in his pola sol en han
ini ially expec ed [28].
Gi en ha he majo ood c ops wo ldwide a e p one o myco oxins, i is impe a i e
o de elop sa e me hods o elimina ing hese oxins om he ood supply. In ecen
yea s, he e has been signi ican p og ess in using g een sol en s o myco oxin
deg ada ion, as hey can be used di ec ly in ood wi hou comp omising i s quali y.
To ou knowledge, no s udies ha e ye been published on he use o ul a-pu e wa e
o OTA deg ada ion. Despi e e idence o low PPL ac i i y in wa e , his s udy aims o
cla i y, h ough in i o assays and molecula modeling, how ul a-pu e wa e enhances
OTA deg ada ion compa ed o buffe solu ions. This po en ially g oundb eaking
app oach offe s a simple ye inno a i e me hod wi h nume ous u u e applica ions in
ood p ocessing. Al hough PPL can be con amina ed by aces o ypsin, which impa s
a bi e as e, and o he impu i ies like animal ho mones, he enzyme emains highly
p omising [30].
2. Ma e ials and Me hods
2.1. Ma e ials
Lipase om po cine panc eas (Type II, ≥125 U/mg) (PPL), och a oxin A (OTA),
och a oxin alpha (OTα), di-po assium hyd ogen o hophospha e, and po assium
dihyd ogen o hophospha e we e pu chased om Sigma-Ald ich (Po o, Po ugal).
Ace oni ile (HPLC g ade), ace ic acid, and sy inge il e s PTFE memb ane we e
pu chased om The mo Scien i ic (Po o, Po ugal). The ul apu e wa e was ob ained
om a Wa e 75 il e wi h a pH o 5.6 and conduc i i y o 0.001 mS/cm. The mic opla e
eade used o e alua e he enzyma ic ac i i y was he Syne gy H1 Mul i-Mode Reade
om BioTek (USA). The LC-MS is a The mo Scien i ic™ Vanquish™ Flex UHPLC sys em
coupled o a The mo Scien i ic™ O bi ap Explo is™ 120 high- esolu ion accu a e mass
spec ome e (The mo Fishe Scien i ic, B emen, Ge many). The ins umen con ol and
da a p ocessing we e ca ied ou by Xcalibu 4.5 so wa e (The mo Scien i ic).
2.2. Enzyme Ac i i y
The speci ic enzyme ac i i y o lipases was de e mined using p-ni ophenyl
oc anoa e (p-NPO) as subs a e. The s anda d assay was pe o med a 37 °C in a inal
olume o 4 mL con aining he subs a e (6 mM), he enzyme, and he assay buffe
(K2HPO4 buffe , pH 7.8, 50 mM). The eac ion was ini ia ed by he addi ion o he enzyme
and s opped wi h he addi ion o ace one. The hyd olysis o p-NPO was obse ed by
measu ing he o ma ion o p-ni ophenol using wo me hods. The o ma ion o he
p oduc was measu ed a a wa eleng h o 400 nm using spec opho ome y [31]. The
p oduc was also quan i ied using LC-MS acco ding o he me hod p esen ed below. One
Foods 2025, 14, 397 4 o 17
uni o enzyme ac i i y is de ined as he amoun o enzyme ha ca alyzes he p oduc ion
o 1 µmol p-ni ophenol om he ini ial subs a e pe minu e.
2.3. Enzyma ic Deg ada ion o Och a oxin A
OTA deg ada ion assays we e conduc ed using lipase om po cine panc eas in he
p esence o pu e wa e and diffe en buffe s (phospha e buffe 100 mM, pH 7.5; phospha e
buffe 50 mM, pH 7.5; bo a e buffe 100 mM, pH 7.5; ace a e buffe 100 mM, pH 5; ci a e
buffe 100 mM, pH 6). The p ocedu e in ol ed incuba ing he enzyme (10 mg/mL) in 1
mL o sol en con aining myco oxin (10 µg/mL), a diffe en empe a u es (21, 30, 37, 44,
50, 57, 60 °C) o diffe en pe iods o incuba ion (15 and 30 min; 1, 2, 4, 6, 7 and 10 h). The
eac ion samples we e dilu ed in he LC-MS mobile phase, il e ed h ough PTFE sy inge
il e s (13 mm diame e , 0.2 µm po e size), and analyzed by LC-MS. Addi ionally, a con-
ol assay wi hou enzyme was p epa ed and subjec ed o he same p o ocol o each p o-
cedu e.
2.4. LC-MS Me hod
De ec ion and iden i ica ion o he compounds was pe o med using a The mo Sci-
en i ic™ Vanquish™ Flex UHPLC (The mo Fishe Scien i ic, B emen, Ge many) sys em
coupled o a The mo Scien i ic™ O bi ap Explo is™ 120 high- esolu ion accu a e mass
spec ome e (The mo Fishe Scien i ic, B emen, Ge many). The ins umen con ol and
da a p ocessing we e ca ied ou by Xcalibu 4.5 so wa e (The mo Fishe Scien i ic). The
UHPLC column u ilized was a YMC-T ia C18 column (150 × 2.1 mm i.d., 3 µm pa icle
size) p o ec ed wi h a gua d column o he same ma e ial (2.1 mm i.d.). The mobile phases
consis ed o A (wa e wi h 0.1% o ace ic acid) and B (ace oni ile wi h 0.1% o ace ic acid).
The low a e was 0.35 mL/min using he ollowing linea g adien scheme ( in min; %
A): 0 min, 70%; 3 min, 40%; 10 min, 70%; and o al un ime was 15 min. The column
empe a u e was 35 °C, he au osample empe a u e was se a 15 °C, and he injec ion
olume was 10 µL. Ion sou ce (The mo Scien i ic™ Op aMax™ NG ion sou ce) was
equipped wi h a hea ed elec osp ay ioniza ion (HESI) p obe. The ex e nal mass calib a-
ion o he Q-O bi ap was pe o med once a week o ensu e a wo king mass accu acy <
3 ppm. The op imized HESI empe a u e was se a 350 °C, he capilla y empe a u e a
325 °C, and he elec osp ay ol age a 3.5 kV and 2.5 kV o posi i e and nega i e modes,
espec i ely. Shea h and auxilia y gas we e 50 and 10, espec i ely. All quali a i e da a in
his s udy we e acqui ed using he SIM and MS2 mode. In he las mode, he samples we e
agmen ed wi h a no malized collision ene gy o 30% o ob ain p oduc ion spec a.
2.4.1. Moni o ing o OTA Deg ada ion
The enzyma ic eac ion was moni o ed by he disappea ance o OTA, de ec ed by
LC-MS, and con i med by he appea ance o OTα, one o he deg ada ion p oduc s. The
iden i ica ion o he peaks co esponding o OTA and OTα was alida ed by compa ison
wi h a s anda d solu ion and by he MS2 spec um o OTA, which can be ound in he
Suppo ing in o ma ion (Figu e S1). OTA quan i ica ion was pe o med using a calib a-
ion cu e spanning he ange o 0.98–500 µg/mL. The calcula ed limi s o de ec ion (LOD)
and quan i ica ion (LOQ) we e 0.8 µg/mL and 2.4 µg/mL, espec i ely.
2.5. Ci cula Dich oism
The seconda y s uc u e o PPL was s udied by ci cula dich oism spec oscopy, us-
ing a Jasco J-1500 spec opola ime e , equipped wi h a empe a u e con olle se a 37 °C.
The enzyme concen a ion was se a 5 µM, dissol ed in he buffe solu ions unde s udy
and in pu e wa e . The baseline was eco ded using hese sol en s and sub ac ed o he
enzyme spec a. The spec a we e eco ded o be 190–240 nm a a scan speed o 20 nm/min
Foods 2025, 14, 397 5 o 17
and bandwid h o 1 nm. The pa hleng h cell was 1 mm. The inal spec a we e ob ained
by he a e age o h ee scans o each sample. The da a analysis o ci cula dich oism was
conduc ed using Dich oWeb (h p://dich oweb.c ys .bbk.ac.uk, accessed on 19 Ap il
2024), an online ool specialized in de e mining he seconda y s uc u e o p o eins om
ci cula dich oism spec a.
2.6. Compu a ional S udies
The model used o he compu a ional s udies was buil om he X- ay c ys allo-
g aphic s uc u e o PPL (PDB:1ETH; esolu ion: 2.8 Å) [20], which was ob ained om he
RCSB P o ein Da a Bank (h ps://www. csb.o g/, accessed on 9 Ma ch 2024). C ys alliza-
ion wa e molecules we e emo ed, as well as 2-me cap oe hanol and (hyd oxye h-
yloxy) i (e hyloxy)oc ane molecules ound in he c ys allized s uc u e. Hyd ogen a oms
we e hen added using he Open Babel so wa e package e sion 2.4.1, a ge ing a pH o
7.
Molecula Dynamics (MD) simula ions we e hen ca ied ou o explo e he s uc-
u al elaxa ion o PPL unde pu e wa e and PBS. To p epa e he simula ion box o he
s uc u al elaxa ion in pu e wa e , he s uc u e o PPL was placed a he cen e o a 10.5
× 11.5 × 17.5 nm box, accompanied by 2 chlo ide ions o balancing he o e all cha ge o
PPL. A o al o 51,700 wa e molecules we e hen added o he simula ion box using he
Packmol so wa e package 20.14.3 [32]. A simila p o ocol was used o c ea e he ini ial
s a e o PPL in PBS, by adding 170 po assium ions, 69 monohyd ogenphospha e ions and
25 dihyd ogenphospha e ions. The Ambe o ce ield was used h oughou he MD p o-
ocol, using he ff14SB pa ame e s [33] o he p o ein s uc u e, GLYCAM-06j [34] o he
glycosyla ed si es o PPL, he wa e TIP3P model [35], and he GAFF2 [36] pa ame e s o
he emaining species, excep he long- ange pa ame e s o po assium and chlo ide
which we e collec ed om Joung and Chea ham [37]. All calcula ions we e ca ied ou
using he LAMMPS so wa e [38], e sion da ed om he 21s o No embe o 2023. The
same MD p o ocol was applied o he wo sys ems: i s he s ain caused by he manipu-
la ion o he PPL s uc u e was elaxed using he hessian- ee new on minimiza ion
me hod. A e minimiza ion, he ini ial eloci ies o he sol en a oms we e c ea ed om
a andom dis ibu ion a ge ing an ini ial empe a u e o 150 K, and he sol en was al-
lowed o hea (wi h he enzyme s uc u e ozen) o 300 K in 1 ns, unde NVT condi ions
using he Nosé–Hoo e he mos a and a ime s ep o 0.5 s. This allowed he sol en mol-
ecules o pe mea e e en ual acancies in he PPL s uc u e and p e en i s collapse. The
eloci ies o he enzyme a oms we e gene a ed a ge ing an ini ial empe a u e o 150 K,
and he sys em was allowed o hea o 300 K du ing 5 ns. Finally, he sys em was equili-
b a ed a 300 K using he NVT condi ions desc ibed abo e o 5 ns, ollowed by a 20 ns
p oduc ion un. The s a is ics on he s uc u e o PPL in each sol en we e collec ed om
he las 5 ns o his p oduc ion un, he ajec o y along which was sampled a a a e o 1
ame pe 0.01 ns. The affini y o OTA and p-NPO o pu e wa e and PBS was accessed
by simula ing one solu e molecule in NVT condi ions in a 10 × 10 × 20 nm box di ided in
wo isola ed egions along he zz axis, wi h pe iodic bounda y condi ions along he xx and
yy di ec ions. The lowe pa o he box was illed wi h pu e wa e o he a ge densi y o
0.0033 Å⁻3, whe eas he uppe hal was illed wi h a p e-equilib a ed PBS solu ion model.
The solu e molecule was placed a he cen e o he uppe hal and allowed o equilib a e
o 1 ns, a e which a p oduc ion un o 10 ns was ca ied ou . The solu e molecule was
hen alchemically ansla ed o he lowe hal o he box (occupied by pu e wa e ), allowed
o equilib a e, and subjec ed o a new p oduc ion un.
Finally, he affini y o p-NPO and OTA o PPL was e alua ed o he ini ial (c ys al-
line) s uc u e, as well as o he las eco ded s uc u e o PPL in bo h wa e and PBS.
This was ca ied ou using a s anda d docking p o ocol [39]. The docking s udies we e
Foods 2025, 14, 397 6 o 17
p epa ed using he MGLTools 1.5.7 so wa e sui e [40] and he binding mode sea ch and
anking we e ca ied ou using Au oDock Vina, e sion 1.2.5 [41,42]. The con o ma ional
sea ch was es ic ed o a box con aining he pu a i e ac i e si es o PPL, which is com-
p ised by he amino acid esidues Se 156, Asp177, and His264 in chains A and C. A g id
spacing o 1 Å was used, oge he wi h an exhaus i eness se ing o 24, and collec ing he
30 lowes ene gy binding modes wi hin 5 kcal.mol−1 o he lowes ene gy mode.
3. Resul s and Discussion
Ul a-pu e wa e was employed he ein as he eac ion medium o he enzyma ic
deg ada ion o OTA by PPL. This app oach seeks o e ine he p e iously desc ibed
me hod, enhancing i s sui abili y o applica ion in ood o eed.
He e, he op imal condi ions iden i ied in p e ious s udies we e ep oduced, and he
ole o wa e in OTA deg ada ion by PPL was in es iga ed, compa ing he esul s wi h
deg ada ion in he p esence o a buffe (phospha e buffe ). P e ious esea ch has shown
ha PPL exhibi s i s highes ac i i y in alkaline media, wi h a pH ange om 7.3 o 9 and
an op imal empe a u e o 35–45 °C [19]. Speci ically, Milek ound ha a pH 7.5, he op-
imal empe a u e o oli e oil hyd olysis was 36 °C [43]. Addi ionally, selec i e hyd oly-
sis o wo medicinal seed oils was success ully ca ied ou a pH 8.0 [44]. Al hough PPL is
an alkaline lipase, i has also been used in neu al o mode a ely acidic condi ions (pH 6.5–
7.0). Lei e al. in es iga ed he ca aly ic ac i i y o PPL in he hyd olysis o an oli e oil
emulsion a pH 6.9 [45]. While i s op imal ca aly ic ac i i y occu s in an alkaline en i on-
men , PPL demons a es g ea e s abili y in neu al o mode a ely acidic media.
3.1. Enzyma ic-Assis ed Deg ada ion o OTA in Pu e Wa e
To elucida e he ac i i y o PPL in ul a-pu e wa e and phospha e buffe (100 mM,
pH 7.5) o OTA deg ada ion, an ini ial sc eening was ca ied ou unde p e iously es ab-
lished condi ions a 37 °C o 10 and 24 h [13].
A e 10 h in pu e wa e , he deg ada ion o he myco oxin was comple e, unlike wha
happens in phospha e buffe , whe e PPL deg ades only 21% o OTA. A e an incuba ion
pe iod o 24 h a he same empe a u e, only 40% o he myco oxin was deg aded in he
buffe , compa ed o 100% deg ada ion in pu e wa e . Al hough he li e a u e sugges s
ha PPL in wa e is in i s closed o m, du ing OTA deg ada ion, his enzyme appea s o
ha e ac i i y in wa e , con a y o expec a ions.
To e alua e he op imal empe a u e o PPL, es s we e conduc ed a diffe en em-
pe a u es o e a pe iod o 2 h in bo h wa e and buffe . The s udy explo ed empe a u es
abo e and below he e e ence alue o 37 °C, offe ing a wide ange o da a poin s o
iden i y he op imal condi ions o maximizing enzyma ic ac i i y and OTA deg ada ion
efficiency. The incuba ion ime o 2 h was chosen based on e idence showing he mos
signi ican diffe ences in deg ada ion pe cen ages. The s udy e ealed ha he highes
OTA deg ada ion du ing his pe iod occu ed a 50 °C o bo h condi ions, a he han a
37 °C (Figu e 1a). Gi en he sligh diffe ence be ween 50 and 44 °C, he la e was p e e ed
due o i s lowe ene gy consump ion du ing incuba ion.
The expe imen was ep oduced a 44 °C in pu e wa e o de e mine he ime e-
qui ed o PPL o achie e comple e deg ada ion o OTA. I was obse ed ha a e 7 h,
OTA was 100% deg aded a 44 °C, compa ed o he 10 h equi ed a 37 °C. Examining
Figu e 1b, apid deg ada ion is e iden up o he second hou o hyd olysis a 44 °C, a e
which he p ocess decele a es. This slowdown can be a ibu ed o he dec easing a aila-
bili y o subs a e as he eac ion p og esses o o losses in ac i i y.
Foods 2025, 14, 397 7 o 17
Figu e 1. E alua ion o he op imal condi ions o OTA hyd olysis by PPL in wo diffe en media.
The eac ion was moni o ed a diffe en (a) empe a u es (21, 30, 37, 44, 50, 57, and 60 °C) and (b)
incuba ion imes (15 and 30 min; 1, 2, 4, 6, 7, and 10 h). The op imal empe a u e o OTA hyd olysis
was 44 °C in wa e and buffe . The incuba ion ime equi ed o he comple e hyd olysis o OTA
was 7 h in pu e wa e (44 °C).
Based on p elimina y op imiza ion ials, a maximum eac ion ime o 4 h a 44 °C
was es ablished o he s udy o enzyma ic ac i i y o assess mo e e iden diffe ences be-
ween he eac ion media. The esul s depic ed in Figu e 2 highligh a no able diffe ence
in he deg ada ion a e o OTA when compa ing pu e wa e and phospha e buffe o e
diffe en ime in e als. In pu e wa e , OTA deg ada ion by PPL eached 18% in jus 15
min and 91% in 4 h, unde sco ing he sys em’s efficiency in his sol en wi h a no ably
apid deg ada ion a e. Con e sely, in he phospha e buffe , OTA deg ada ion was sig-
ni ican ly slowe , wi h only 12% deg aded a e 4 h o eac ion.
Figu e 2. PPL enzyma ic ac i i y in pu e wa e and in phospha e buffe (100 mM, pH 7.5) e alua ed
as OTA deg ada ion o 4 h o incuba ion a 44 °C.
The s udy sugges s ha he enzyme’s s abili y and efficiency a e in luenced by he
sol en . To p o e his, he s abili y o PPL in pu e wa e and phospha e buffe was e al-
ua ed by incuba ing he enzyme in hese sol en s a 44 °C o e a ime ange (0.25 o 120
h). Enzyme ac i i y was measu ed using he model subs a e p-ni ophenyl oc anoa e,
wi h quan i ica ion o he p oduc p-ni ophenol pe o med using LC-MS echnique.
The decay cons an (K), which ep esen s he enzyme inac i a ion a e, is signi i-
can ly highe in he buffe (0.27) compa ed o wa e (0.14), as shown in Table 1. This in-
c eased inac i a ion a e sugges s ha , in he buffe en i onmen , he enzyme loses i s
ca aly ic ac i i y mo e apidly. This diffe ence is u he suppo ed by he hal -li e alues.
The hal -li e is sho e in he buffe (~2 h3 0 min) compa ed o wa e (~4 h 4 min),
% deg ada ion
(b)
(a)
% deg ada ion
Foods 2025, 14, 397 8 o 17
indica ing educed con o ma ional and unc ional s abili y o he enzyme in he buffe .
These alues we e calcula ed using G aphPad P ism 9.0 so wa e, allowing o p ecise
da a analysis.
Table 1. Hal -li e decay pa ame e s o PPL enzyma ic ac i i y in ul a-pu e wa e and phospha e
buffe (100 mM, pH 7.5), calcula ed using G aphPad P ism.
Ul a-Pu e Wa e Phospha e Buffe
Decay cons an (K) 0.14 ± 0.06 0.27 ± 0.11
Hal -li e (h) 4.74 ± 1.8 2.56 ± 0.98
Ini ial ac i i y (Y0) 1.02 ± 0.09 1.20 ± 0.12
R2 0.90 0.87
These esul s sugges g ea e enzyme s abili y in pu e wa e compa ed o phospha e
buffe . The diffe ence in s abili y may be a ibu ed o ionic in e ac ions ha can des abi-
lize p o eins h ough con o ma ional changes in hei s uc u e. Such s uc u al changes
can inc ease he enzyme’s suscep ibili y o dena u a ion o agg ega ion, esul ing in a
apid loss o ca aly ic ac i i y. Pu e wa e , despi e no p o iding he same buffe ing ca-
paci y as phospha e buffe , appea s o allow o g ea e s uc u al s abili y o e a longe
pe iod.
3.2. The Effec o pH and Conduc i i y on OTA Deg ada ion
To unde s and he efficiency o he enzyme in wa e , expe imen s we e conduc ed
using wa e and diffe en buffe s, wi h a ia ions in buffe ype, pH, and ionic concen a-
ion, a 44 °C (Figu e 3). As shown in he p e ious esul s, he enzyme appea s o be mo e
efficien in deg ading OTA when in wa e . Ul a-pu e wa e ideally has a pH o 7 a 25 °C,
which cha ac e izes i as neu al. Howe e , in p ac ice, measu ing he pH o pu e wa e
can be challenging due o i s low elec ical conduc i i y and high sensi i i y o en i on-
men al con amina ion. Small amoun s o ca bon dioxide om he ai can dissol e in wa-
e , o ming ca bonic acid (H2CO3), which can sligh ly lowe he pH o alues a ound 5.5
o 6.5. The e o e, pu e wa e has a sligh ly acidic pH and low conduc i i y.
The hyd olysis o OTA by PPL in phospha e buffe a pH 7.5 (100 mM) showed a
much slowe and less efficien pe o mance, possibly due o he phospha e concen a ion.
Reducing he phospha e concen a ion o 50 Mm did no signi ican ly imp o e he deg a-
da ion, indica ing ha he p esence o phospha e, ega dless o concen a ion, may be de -
imen al o he enzyma ic ac i i y (Figu e 3).
The eac ion in a bo a e buffe a pH 7.5 and 100 mM showed imp o emen com-
pa ed o he eac ion in phospha e buffe . This sugges s ha he ype o ion in he buffe ,
in addi ion o low conduc i i y, signi ican ly in luences enzyma ic ac i i y. Bo a e may
in e e e less wi h he enzyme o subs a e s uc u e, esul ing in highe ac i i y, hough
i s ill does no ma ch he efficiency obse ed in pu e wa e . When using buffe s wi h pH
le els o 5 and 6, he enzyme’s pe o mance a pH 5 was be e han a pH 6. Howe e ,
nei he condi ion was as effec i e as pu e wa e .
The esul s indica e ha buffe s, especially hose wi h high conduc i i y, seem o de-
c ease enzyma ic ac i i y, sugges ing sensi i i y o he enzyme o hese ions and/o a
change in he o m o OTA ha educes i s a ailabili y as a subs a e. Ionic in e e ence
can comp omise subs a e a ailabili y o he enzyme by o ming complexes be ween he
p esen ions and OTA, as well as di ec in e ac ions wi h he enzyme s uc u e, affec ing
i s con o ma ion, including he lid con o ma ion, and consequen ly, i s ca aly ic ac i i y.
Simila ly, he pH can al e he ioniza ion o OTA molecules, affec ing hei a ailabili y as
a subs a e o he enzyme. Unde he pH condi ions desc ibed as op imal in he li e a u e,
Foods 2025, 14, 397 9 o 17
he ionized o m o OTA may no be ideal o binding o ecogni ion by he enzyme, hus
educing deg ada ion efficiency.
Figu e 3. The hyd olysis o OTA by PPL in diffe en buffe s and ul a-pu e wa e a 44 °C is ep e-
sen ed by ba s. The conduc i i y in mS/cm o each solu ion unde s udy is ep esen ed by a black
do abo e each ba .
A pH 7.5, he ca boxylic g oup (-COOH) o OTA ends o be p edominan ly in he
ionized o m (-COO−), while he phenolic g oup (-OH) emains neu al. This ioniza ion
inc eases he molecule’s pola i y, as dep o ona ed g oups ( o ming anions) end o be
mo e pola . This occu s because he loss o a p o on inc eases he elec onega i i y o he
unc ional g oup’s a om, esul ing in a mo e pola bond. In ul a-pu e wa e , a a mo e
acidic pH (~5–6), bo h g oups may be p edominan ly in he p o ona ed o m, lea ing he
molecule wi h i s no mal less-pola cha ac e . Since wa e has a high dielec ic cons an
and high pola i y, he hyd ophobic effec may occu , which e lec s he clus e ing o non-
pola molecules o unc ional g oups o educe he pola /nonpola in e acial a ea. This
could lead o he concen a ion o OTA molecules and make hem mo e a ailable o e-
ac ion.
In pu e wa e , he enzyme exhibi ed i s bes pe o mance, indica ing ha low con-
duc i i y and acidic pH a e ideal condi ions o enzyma ic ac ion.
3.3. Seconda y S uc u e o PPL—Ci cula Dich oism
The analysis o he seconda y s uc u e o PPL as pe o med by ci cula dich oism
(CD) spec a in he a ious sol en s s udied is p esen ed in Figu e 4. Pe o ming CD spec-
a in ci a e buffe was no easible due o a apid ol age inc ease ha would exceed he
maximum ecommended alue. The esul s (Table 2) e ealed signi ican a ia ions in he
seconda y s uc u e composi ion o he enzyme ac oss diffe en sol en s. The esul s in-
dica e ha he enzyme exhibi s a highe numbe o helical s uc u es (helices 1 and 2)
when in wa e (8.68% in o al) compa ed o 100 mM phospha e buffe (6.8% in o al). Ad-
di ionally, he quan i ies o be a shee s (s and 1 and s and 2) a e simila in bo h condi-
ions, wi h a sligh dec ease in buffe (36.4% in wa e s. 37.2% in buffe ). The amoun o
u ns and uno de ed s uc u es also emained ela i ely cons an be ween he wo condi-
ions, displaying simila alues (22.1% and 32.9% in wa e s. 22.7% and 33% in buffe ).
PPL in bo a e buffe exhibi s a beha io al pa e n like ha obse ed in pu e wa e (9.4%
in o al o helices 1 and 2). Ace a e buffe showed he lowes con en o alpha helices (5.6%
in o al), along wi h he highes amoun o be a shee s (39.3%). These diffe ences sugges
ha he aqueous en i onmen a o s he o ma ion o helical s uc u es in he enzyme,
while some buffe solu ions seem o educe he s abili y o hese helical s uc u es, possi-
bly leading o a mo e diso de ed con o ma ion. The highe p esence o alpha helices in
% deg ada ion
mS/cm
Foods 2025, 14, 397 16 o 17
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