Intestinal Lipase Characterization in Common Snook (Centropomus undecimalis) Juveniles 2022 Bartolo Concha-Frías.pdf


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fishes
Article
IntestinalLipaseCharacterizationinCommonSnook
(Centropomusundecimalis)Juveniles
BartoloConcha-Frías1,MarthaGabrielaGaxiola-Cortes2,FannyJanetDelaCruz-Alvarado3,
LuisDanielJimenezMartinez1,EmyrSaulPeña-Marin3,4,MarciaAngélicaOliva-Arriagada5,
JoeLuisArias-Moscoso6,*andCarlosAlfonsoAlvarez-González3,*
1LaboratoriodeBiologíaMolecular,DAMJ-UJAT,CarreteraEstatalLibreVillahermosa-ComalcalcoKm.
27+000s/nRancheríaRiberaAlta,JalpadeMéndez86205,Mexico;******@(.-F.);
******@(.)
2UnidadMultidisciplinariadeDocenciaeInvestigación,FacultaddeCiencias,UNAM,PuertodeAbrigos/n,
Sisal97356,Mexico;******@
3LaboratoriodeFisiologíaenRecursosAcuáticos,DACBIOL-UniversidadJuárezAutónomadeTabasco,
CarreteraVillahermosaCá,Villahermosa86139,Mexico;******@(.-A.);
******@(.-M.)
4ConsejoNacionaldeCienciayTecnología,,éditoConstructor,
Juárez,CiudaddeMéxico03940,Mexico
5LaboratoriodePeces,DepartamentodeAcuicultura,FacultaddeCienciasdelMar,UniversidadCatólicade
Chile,CampusGuayacánLarrondo1281,Coquimbo1780000,Chile;******@
6DepartmentofEngineering,TechnologicalNationalofMexico,TechnologicalInstituteoftheYaquiValley,
Bacum85276,Mexico
Citation:Concha-Frías,B.;*Correspondence:******@(.-M.);alvarez_******@(.-G.);
Gaxiola-Cortes,.;DelaTel.:+52-9933581500()(.-G.)
Cruz-Alvarado,.;Jimenez
Martinez,.;Peña-Marin,.;Abstract:Thecommonsnook(Centropomusundecimalis)isaeuryhalineflshwithhighcommercial
Oliva-Arriagada,.;demandintheMexicansoutheast,Caribbean,,someaspectsofits
Arias-Moscoso,.;digestivephysiologyarestillunknown,,the
Alvarez-González,

LipaseCharacterizationinCommon
Snook(Centropomusundecimalis)digestivelipase’soptimaltemperatureis35C,beingstablebetween25and35C,andshows
,7,,
/presentedbyOrlistat(%),EbelactoneA(%),EbelactoneB(%),SDS1%(%),%
flshes7030107(%),%(%).OrlistatandEbelactoneAandBcompletelyinhibitedthelipase
bandinthezymogram,
AcademicEditor:Francisco
.

Received:23March2022Keywords:digestivelipase;inhibitors;temperature;pH;Centropomidae
Accepted:6May2022
Published:10May2022
Publisher’sNote:MDPIstaysneutral

publishedmapsandinstitutionalaffll-Thecommonsnook(Centropomusundecimalis,Bloch,1792)isacarnivorousmarine
,partoftheCaribbean,and
SouthAmerica[1,2],Thissituationhasbeenaccentuatedbypopulationgrowthandthe
projectionsmadebytheFAOfor2030(FAO,2020),anditisthereforenecessarytointegrate
,studiesondigestivephysiologyfocusingonthe
Copyright:©[3,4]toimprovethe
LicenseeMDPI,Basel,,withanincreaseingrowthandlowercosts[5–8].
ThisarticleisanopenaccessarticleExogenouslipasesfromthemicrobiotaofflshsuchasrainbowtrout(Oncorhynchusmykiss)
distributedunderthetermsand
havebeenusedand,inmanycases,didnotfunctionasfoodadditives[9],sothelackof
conditionsoftheCreativeCommons
knowledgeaboutthedigestivephysiologyofflshisalimitationfortheformulationof
Attribution(CCBY)license(https://
[10].,somestudieshavebeencarriedoutontheimportance
/).
Fishes2022,7,:///flshes7030107:.
Fishes2022,7,1072of11
ofdigestiveenzymessuchastheactivityandearlyexpressionofproteases,lipases,and -
amylase[11,12].However,lipasesrequirespecialattentionsincetheyareessentialenzymes
ofthedigestivesystem,wherecatalyticactivitycanbeevaluatedbyfast,reliable,speciflc,
selective,andsensitiveanalyticalmethods[13].
Typically,carnivorousflshfeedpreywithhighlipidcontent;consequently,itiscon-
sideredthattheyrequiremoresigniflcantlipaseactivitycomparedtootheromnivorous
species[7,14].AccordingtoRefs.[3,8],lipidsareessentialinthestructureofcellmembranes
bymaintainingflexibilityandpermeability,inthestorageofenergyandfattyacids,andin
participatingincellsignaling[15,16].
Carboxylesterlipase(CEL)areagroupofwater-solublecarboxylicesterhydrolase
enzymesthatmovebetweenthecellsofanorganism[17]andcarryoutthehydrolysisof
triglyceridestomonoglyceridestoreleasefattyacids[18,19]bybreakinglipidbonds,acting
betweentheaqueousandorganicphases[13,20,21].Theyareclassifledintotwogroups:
(1)Esterases(),whichperformtheexcisionoflow-molecular-weightfattyacids
andhavesolubilizingcapacity,actingonsimpleesterbondsbycatalyzingtheruptureof
esterbondsofvitamins,phospholipids,triglycerides(TGS),
activeinwater-insolublelipidsubstrates;(2)True()or
bile-salt-dependentlipases,thedominantlipaseinflsh[22,23],whicharesynthesizedinthe
pancreas,requiringbilesaltsfortheirproperfunctioninginconcentrationsbetween25mM
and250mM,suchassodiumtaurocholate(C26H44NNaO7S),sodiumtaurodeoxycholate
(C26H44NNaO6S),andcholicacid(C24H40O5),producedand/orrecycledintheliver
in60to95%asaproductofimmunoglobulindegradation,lipidssuchascholesterol
andsteroids,whichareessentialforthesolubilization,hydrolysis,andadsorptionof
lipidsthroughenterocytesandpreservingtheirdenaturationandconsiderablyincreasing
theiractivityatpH8[24–27].Lipaseisauniquepolypeptidechainthatfoldsintoa
largeN-terminaldomainbelongingtothefold / -hydrolaseandasmallerC-terminal
domaincontainingacatalytictriadofserine,asparticacid,andhistidinethatisanalogous
toserine,thusfavoringtheadsorptionofthesubstrateontheintestinalwallsofflsh,
allowingthemtoactonpoorlysolublesubstrates[28].Theyarecurrentlyusedinthefood,
pharmaceutical,detergent,biotensive,
reasons,flshisbeingstudiedasafundamentalsourceoflipaseproductiontoimprove
industrialprocesses[29,30].
Lipaseactivityanditscharacterizationhavebeenreportedinseveralflshspecies
suchasYellowflntuna(Thunnusalbacares),Longtailtuna(Thunnustonggol),Skipjacktuna
(Katsuwonuspelamis)[4],Siameseflghtingflsh(Bettasplendens)[14],Europeanperch(Perca
fluviatilis)andArcticchar(Salvelinusalpinus)[7],andMozambiquetilapia(Oreochromis
mossambicus)[8].However,,thistypeofstudyhasnotbeencarriedout;
consequently,ourmainobjectiveistocharacterizebile-salt-activatedlipasebybiochemical
,anditisherethatthey
exerttheircatalyticactivity.


Fishwerehandledincompliancewiththestandardsforthegoodwelfarepractices
oflaboratoryanimalsfromtheNormaMexicanaNOM-062-ZOO-1999delaSecretaríade
Agricultura,Gandara,DesarrolloRural,PescayAlimentación.

Forthisstudy,atotalof50wildjuveniles()were
capturedinthemonthofJulyfromthenaturalenvironment,withconicalmosquitonets
15mlong3mhigh,inArroyoVerde,Comalcalco,
transferredincontainerswithconstantaerationtotheLaboratoriodeFisiologíaenRecursos
AcuáticosbytheDivisiónAcadémicadeCienciasBiológicasoftheUniversidadJuárez
AutónomadeTabasco,México,andfedwithtilapia(Oreochromisniloticus)flngerlings,in:.
Fishes2022,7,1073of11
circularplasticpondsat28C,-222(tricaine
methanesulfonate),theywerestarvedfor48h.

Alltheflshweresacriflced,andtheirintestinesweredissectedundericeconditions
(4C).Theorganswereweighed,thentheorgansetwashomogenizedin50mMTris-HCl+
25mMCaCl,,inaratioof1:5,andcentrifugedat16,000gfor30minat4C.
2
Subsequently,thesupernatantwasremovedandstoredat20Cforsubsequentanalysis.
ActivitywasmeasuredusingthemethoddescribedbyVersawetal.[31],wherethe
reactionmixconsistedof20Lofenzymeextractin200Lof100mMsodiumtaurocholate
-
for5min,andthereactionwasstartedwith20Lof -naphthylcaprylate(20mM)for
30minat35,20Loffastbluewasaddedat100mMandincubatedfor5min
Loftrichloroaceticacid(TCA;
):1v/,theabsorbance

usingtheBradford[32]
ofspeciflcactivityofindividualextractswasdeterminedusingthefollowingequations:
(1)unitsmL1=[Dabsflnalreactionvolume(mL)][MECtime(min)extract
volume(mL)]and(2)mgproteinunits1=[unitspermL][mgofsolubleprotein]1,
whereDabs=Increaseinabsorbanceatagivenwavelength;Finalvolume=Finalvolumeof
thereaction(mL);MEC=Molarextinctioncoefflcient(MEC)calculatedfromtheregression
lineof2-naphthol();Time=incubationtimeofthecatalyzedreaction
(min);Extra-spectrumvolume=Volumeofmultienzymeextract(mL).Alltestswere
performedintriplicate.

Theeffectoftemperatureonlipaseactivitywasdeterminedwithuniversalbuffer[33]
atpH9intherangebetween25and65Candusingthetechniquepreviouslydescribed.
TheeffectofpHonlipaseactivitywasdeterminedwithuniversalbuffer[33],varying

wasdeterminedatdifferenttimes(30,60,90,and120minofpre-incubation),varying
temperatures(25,35,35,55C)andpH(2,3,4,5,6,7,8,9,10and11)comparedtoacontrol
withoutpre-incubationusingthetechniqueofVersawetal.[31].Alltestswereperformed
intriplicate.

Theeffectofinhibitorsonlipaseactivitywasdeterminedbyamulti-enzymaticextract
pre-,microbialEbelactoneA1mM,andmicrobialEbelactone
B1mM[34]andinincreasingamounts(,,and1%)ofsodiumlauryldoudecylsulfate
(SDS)usingthetechniqueproposedbyGörgünandAkpınar[15],comparedtoacontrol
withoutpre-incubation,usingthesametechniqueasVersawetal.[31],aspreviously
.

Electrophoresiswasperformedundernativeconditionsfollowingthetechniquepro-
posedbyDavis[35]on10%polyacrylamidegelsinbufferTris-.
Theelectrophoreticwasrunat80Vfor15minandthenincreasedto120Vfor2hat
4 -naphthylcaprylate(200mmolL1),wheregel
,fastbluesolution(100mmolL1)wasadded,and
itwasincubatedat25
inhibitorspreviouslymentionedwereused,whichwerepre-incubatedina1:1ratio(en-
zyme/inhibitor).(Markham,ON,Canada)
BM523andQuantityOne1-DAnalysisSoftwarefromBio-Rad(Hercules,CA,USA)(phos-:.
Fishes2022,7,xFORPEERREVIEW4of11
1-DAnalysisSoftwarefromBio-Rad(Hercules,CA,USA)(phosphorylase97kDa,bovine
serumalbumin66kDa,Ovalbumin45kDa,carbonicanhydrase29kDa,trypsinogen24
kDa,andSBTI20kDa)wereusedtocalculatethemolecularweightofthebandwithac-
Fishes2022,7,

phorylase97kDa,bovineserumalbumin66kDa,Ovalbumin45kDa,carbonicanhydraseNormalityandhomoscedasticitywerecorroboratedforthevaluesofenzymaticactivity>
29kDa,trypsinogen24kDa,andSBTI20kDa)wereusedtocalculatethemolecularweight
-wayanalys

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