Production of Anti-dementia Acetylcholinesterase Inhibitors from the Wild Yeasts Saccharomyces cerevisiae WJSL0113 and Wickerhamomyces anomalus JSF0128

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RESEARCH ARTCICLE

Production of Anti-dementia

Acetylcholinesterase Inhibitors from the Wild Yeasts Saccharomyces cerevisiae WJSL0113 and Wickerhamomyces anomalus JSF0128

Ji-Yoon Kim, Sang-Yeop Lee, Sang-Min Han, Jong-Soo Lee^*

Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 35345, Korea

*Corresponding author: [email protected]

ABSTRACT

In this paper, the screening of potent acetylcholinesterase (AChE) inhibitor - producing yeasts from wild yeasts and the condition for the production of anti-dementia AChE inhibitors are described. Among one hundred and seven non-pathogenic wild yeast strains from the waters and soils of three main rivers in Daejeon metropolitan city and midstream of Yeongsangang river in Sangju, sporogenous Saccharomyces cerevisiae WJSL0113 and asporogenous Wickerhamomyces anomalus JSF0128 were selected as useful strains for the production of potent AChE inhibitors. The AChE inhibitors of S. cerevisiae WJSL0113 and W. anomalus JSF0128 had a maximum yield when they were incubated in yeast extract-peptone-dextrose media (pH 6.0 in S. cerevisiae WJSL0113 and pH 5.0 in W. anomalus JSF0128) for 18 hr at 30℃, respectively.

Keywords: Acetylcholinesterase inhibitor, Anti-dementia, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Wild yeasts

INTRODUCTION

Recently, ^dementia^diseases^havesignificantlyincreasedbecauseoftheincreaseinelderlyoversixty- fiveyearsofageinKorea. ^Some^formsôf^dementiaâre^caused^byâ^lackôfneurotransmitters.

Acetylcholine (ÂCh) îs âneurotransmitterintheperipheral nervoussystemand centralnerve system, ândâ^shortageôf^cholineⁱⁿ^the^brain^has^beenâssociated^withÂlzheimer'^s^disease. ÂChîs converted intocholineand acetateby acetylcholinesterase (ÊC.³.¹.¹.⁷, ÂChE) [¹, ²]. ^Therefore, AChEisakeyenzymeinthepathophysiologyofdementia, ând^some^drugs^thatînhibitÂChEâre usedinthetreatmentofthatdisease [¹] ^becauseît^reduces^theâctivityôfcholinergicneurons. ÂChE inhibitors (anticholinesteraseinhibitors) ^reduce^the^rateât^whichÂChîs^broken^downând^hence increasetheconcentrationofAChinthebrain.

SeveralAChE inhibitorsasanti-^dementiaâgents ^have^beenêxtractedând characterized from OPEN ACCESS

https://doi.org/10.4489/KJM.20180049

Accepted: November 27, 2018 Revised: November 27, 2018 Received: October 30, 2018 Kor. J. Mycol. 2018 December, 46(4): 447-457 pISSN : 0253-651X

eISSN : 2383-5249

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variousplantsincludingUmbilicariaesculenta [³], ^Camellia^sinensis [⁴], ^Securinegasuffruticosa [⁵], Onosmahispida [⁶], ^Huperzia^serrate [⁷], ^Juglans^regia [⁸] ând^Coix^lacryma-^jobi [⁹], êtc. ^However, commercialAChEinhibitorssuchasGalantamine, Rivastigmine, ^Donepezil, ^Tacrineând^Memantine havebeenonlyapprovedbytheFDAasadrugtherapyfordementia [¹, ²]. ^Theyâlso^have^some^side effectsincludingnauseaandanorexia. ^Therefore, ^researchôn^thedevelopmentofnewanti-^dementia agentswithhighefficacyandnosideeffectsisnecessary.

Theproductionofusefulmetabolitesofyeasthaveledtotheiruseinthefieldofbiotechnology [¹⁰]. ^Yeastsâreâlsoôneôf^the^most^widelyûsed^modelôrganisms^for^geneticsând^cell^biology. Yeastshavelongbeenusedforthepreparationofalcoholicbeveragesand soysauces [¹⁰], êtc. Recently, ^yeasts^have^receivedâttention^because^they^have^variousphysiologicalfunctionalitiessuch asanti-^gout, ânti-hypertensive, ânti-^diabetes, ^GABAând^soôn [¹¹-¹⁵]. ^However, ^littleinformationon bioactivecompoundsfromyeastsissufficientlyavailable. ^Therefore, îtîs^necessary^to^study^the developmentofnewbioactivecompoundsfromnon-^pathogenic, ^wild^yeasts.

Forthedevelopmentofanewanti-^dementiaâgent^from^non-^pathogenic, ^wild^yeasts, ^screeningând characterizationofpotentanti-^dementiaÂChEînhibitor-^producing^wild^yeasts^wereinvestigatedand studiedfortheconditionstoproduceAChEinhibitorsfromtheselectedwildyeasts.

MATERIALS AND METHODS

Microorganisms and chemicals

Onehundredsevennon-^pathogenic, ^wild^yeasts^which^wereîsolated^from^the^watersând^soilsôf Daejeoncheon, Yudeungcheon and Gapcheon in Daejeon metropolitan city and midstream of YeongsangangriverinSangju, ^Korea ⁱⁿ²⁰¹⁷ât^the^Lab. ôfBiotechnology, ^PaichaiÛniversity, Korea [¹⁶] ^wereûsed^for^the^screeningôf^potentÂChEînhibitor-^producing^yeasts.

Themedia usedinthisstudy, ^yeastêxtract-^peptone-^dextrose (^YPD), ^yeastêxtract-^maltêxtract (^YM) ând^potato-^dextrose (^PD) ^were^purchased^from^the^Becton^Dickinson (^Sparks, ^MD, ÛSA).

Unlessotherwisespecified, âll^chemicalsând^solvents^wereôfânalytical^grade.

AChE (recombinanthumanAChE, ^E.^C. ³.¹.¹.⁷), acetylthiocholinechlorideand 5,⁵'-^Dithiobis (²-nitrobenzoicacid, ^DTNB) ^were^purchased^from^the^Sigma^Chemical^Co. (^St. ^Louis, ^MO, ^USA).

TheVERSAmaxmicroplatereader (^Molecular^Devices, ^Sunnyvale, ^CA, ^USA) ^was^used^to^measure theAChEactivity.

Screening of potent AChE inhibitor-producing yeasts and its microbiological characterization

YeastswereinoculatedinYPDmedium (².⁰% ^peptone, ¹.⁰% ^yeastêxtractând².⁰% ^dextrose) ând culturedat30℃ ^for²⁴^hr. Âftercentrifugationat15,⁰⁰⁰×^g^for¹⁵^min, ^the^cells^wereôbtainedând suspendedand washedindistilled water. ^The^cells^wereresuspendedindistilledwaterandthen disrupted by glass bead. Âftercentrifugation at 15,⁰⁰⁰×^g (¹⁰ ^min, ⁴℃), ^the ÂChEînhibitory

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activitiesofthesupernatantweredeterminedtoselecttheAChEinhibitor - ^producing^yeast. Themorphologicalcharacteristicsoftheselectedstrainsincludingcellshapeandsizeandmodeof vegetativereproductionweredeterminedbythemicroscopyof24hroldculturesgrowninYPD brothat28℃ [¹⁷]. ^The^other^culturalcharacteristicswereinvestigatedaccordingtothemethodsofthe generalmicrobiologylabmanual [¹⁷].

Assay of the AChE inhibitory activity

TheAChEinhibitoryactivitywasmeasuredspectrophotometricallyusingthetechniqueofEllmanet al. [¹⁸]. Â^mixtureôf¹¹⁰^μLôfâssay^buffer (⁰.¹^M^sodium^phosphate, ^pH⁷.³), ³⁰^μLôfÂChE (⁰.⁸ U/^mL), ³⁰^μLôf^substrate (Acetylthiocholinechloride), ²⁰^μLôf⁵,⁵'-^Dithiobis (²-nitrobenzoicacid) (^DTNB), ând¹⁰^μLôf^sample^dissolvedⁱⁿ^theâssay^buffer^wasîncubated^for⁶⁰^minât³⁷℃. ^The reactionproduct5-^thio-²-nitrobenzoateproducedenzymaticallywasmeasuredat415nm [²].

Theinhibitionratiowasobtainedbythefollowingequation: Inhibition (%) = [¹-{(^S-^S0)/(^C-^C0)}]×¹⁰⁰

, ^where^C^was^the^radiationôf^the^control (ênzyme, âssay^buffer, ^DTNB, ând^substrate) âfter⁶⁰^min ofincubation; ^C0^was^the^radiationôf^the^controlât^time^zero; ^S^was^the^radiationôf^the^tested samples (ênzyme, ^sample^solution, ^DTNB, ând^substrate) âfter⁶⁰^minôfîncubation, ând^S0^was^the radiationofthetestedsamplesattimezero.

Alldataarethemeanofduplicatedexperiments. ^To^check^the^quenchingêffectôf^the^samples, ^the samplesolutionwasaddedtothereactionmixtureC, ândâny^reductionⁱⁿ^radiation^by^the^sample wastheninvestigated. ^TheÎC50valuewasdefinedastheconcentrationoftheAChEinhibitorthatis requiredtoinhibit50% ôf^theînhibitory (ÂChE) âctivity.

Condition for the production of AChE inhibitor

ThemediaandtheinitialpH, ^culturetemperatureandcultivationtimefortheproductionofAChE inhibitorsfromtheselectedstrainswereinvestigatedintherangeof15~⁴⁰℃, ^initial^pH⁴.⁰~⁹.⁰^and culturetime0~⁴⁸^hr^bydeterminingtheAChEinhibitoryactivityofeachcellfreeextract.

Assay of anti-dementia using PC12 cell

Constructionandcultivation oftherecombinantCT105cellline: ^PC12^cells (¹⁰³/^mL) ^that^were culturedinthe5% ^FBSând^penicillin/streptomycin-^containing^RPMI^medium^weretransferredtoa 6-^well^plateând^culturedôvernightât³⁷℃. ^The^culture^broth^consistedôfâ^mixtureôfÂ^solution (^mixtureôf²^μgôf^pCT105^plasmidând¹⁰⁰^μLôf^serum^free^medium, ^SFM) ând^B^solution (^mixture of10μLoflipofectintransfectionreagentand100μLofSFMincubatedtogetherfor45min) ^which wasreactedfor15minAfterthecellswereharvestedandwashedtwotimeswithPBS, ^they^were culturedin1.⁵^mLôf^SFM^mediumât³⁷℃ ^for⁶^hrⁱⁿâ^CO2incubator. ^Then, ⁵% ^RPMI^medium^was addedtothecellculturebrothandculturedovernight. ^The^cells^weresubculturedontofresh6-^well platescontaining450μg/^mL^G-⁴¹⁸, ândâ^single^clone^was^selectedâfter²^weeksôf^culturing.

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Inductionofneuronapoptosisby recombinantCT105and repressionofapoptosisbycell-^free extracts: ^The ^repression êffect ôf â^cell-^free êxtract^from ^wild ^yeast ôn ^neuron âpoptosis ^was investigated. ^Three experimental groups were used: ^WT ^PC12 ^cells, recombinant PC12 cells expressingCT105andrecombinantPC12cellsexpressingCT105treatedwiththecell-^freeêxtract. Forthecell-^freeêxtract^treated^group, ^CT105âpoptosis-înduced^PC12^cells (¹⁰³/^mL) ^were^putînto 6-^well^platesând^culturedôvernight. ^Then, ⁵⁰^μg/^mLôf^the^cell-^freeêxtracts^wereâdded^to^the^wells containing the overnight cultured PC12 cells and RPMI media with 5% ^FBS ând ^penicillin/ streptomycinandthenculturedat37℃ ^for²⁴^hr. ^Finally, âll^three^groupsôf^cells^were^checked^by microscopeforapoptosis (×²⁰⁰).

RESULTS AND DISCUSSION

Screening of the AChE inhibitor-producing yeasts

ToselectpotentAChEinhibitor-^producing^yeasts, ^cell-^freeêxtractsôf^fifty-^three^non-^pathogenic^wild yeastsfrom thewatersandsoils ofthreemainriversinDaejeonmetropolitancityincludingthe DaejeoncheonweretestedfortheirAChEinhibitoryactivities. Ônly^ten^wild^yeasts^were^showed AChEinhibitoryactivityandtheotherwildyeastswerenotdetermined (^Table¹). Âmong^them, SaccharomycescerevisiaeWJSL0113exhibitedthegreatestAChEinhibitoryactivityof95.²%, ând anotherS. ^cerevisiae^WJSL0090ând^S. ^cerevisiae^WJSL0088âlsoêxhibitedâ^very^highînhibitory activity of84.²% ând ⁵³.⁵%, respectively. ^However, ^the ôther^strains êxhibited ^lowînhibitory activitiesbelow20% ôr^not^determined.

Meanwhile, ^cell-^freeêxtractsôfêleven^non-^pathogenic^wild^yeasts^from^the^watersând^soilsôf YeongsangangrivertestedfortheirAChEinhibitoryactivities. Ônly^three^wild^yeasts^were^showed AChEinhibitoryactivityandthehighestAChEinhibitoryactivitywasobservedinWickerhamomyces

Table 1. Acetylcholineseterase inhibitory activities on wild yeasts from three main rivers in Daejeon metropolitan city and Yeongsangang river in Sangju city, Korea

Strains Isolated no. AChE inhibitory activity (%) Collected sites Saccharomyces cerevisiae WJSL0088 53.5 ± 1.1 Three main rivers in

Daejeon metropolitan Saccharomyces cerevisiae WJSL0089 12.5 ± 0.1 city

Saccharomyces cerevisiae WJSL0090 84.2 ± 0.0 Saccharomyces cerevisiae WJSL0113 95.2 ± 1.6 Wickerhamomyces anomalus WJSL0120 8.9 ± 0.3 Candida pseudolambica WJSL0125 9.9 ± 0.2 Kazachstania servazzii WJSL0084 1.7 ± 0.0 Pichia membranifaciens WJSL0177 20.0 ± 0.0 Debaryomyces vindobonensis WJSL0162 4.4 ± 0.2 Rhodotorula mucilaginosa WJSL0035 3.2 ± 0.5

Lachancea thermotolerans JSF0125 13.0 ± 0.2 Yeongsangang river in Sangju city

Lachancea thermotolerans JSF0126 14.5 ± 0.4 Wickerhamomyces anomalus JSF0128 23.9 ± 0.7 AChE; acetylcholinesterase

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anomalusJSF0128 (²³.⁹%) (^Table¹). Thermophilicyeasts; ^LachanceathermotolerantsJSF0126and JSF0125exhibitedlowinhibitoryactivitiesof14.⁵ând¹³.⁰%, respectively. ^TheÂChEînhibitory activitiesofthecell-^freeêxtractsôf^the^wild^yeasts^S. ^cerevisiae^WJSL0113^from^the^three^main^rivers inDaejeonmetropolitancitywerehigherthanthoseofthecell-^freeêxtractsôf^the^wild^yeast^W. anomalus JSF0128 and thethermophilic yeasts L. thermotolerants JSF0125 and JSF0126 from Yeongsangangriverinthisstudy.

Furthermore, ^theÂChEînhibitoryâctivityôf^the^cell-^freeêxtract^from^S. ^cerevisiae^WJSL0113 also was higherthan thoseof Yarrowia lipolytica S-³ (³⁷.⁴%) ^from traditionalsoybean paste, Doenjang [¹⁹], ⁷⁰% êthanol êxtract^from ^the êdible ^mushroom ^Pholiota âdiposa^fruiting ^body (³⁰.⁶%) [²⁰], ^waterêxtract^from^walnuts (⁷².⁶%) [²¹], ând^jobs^tears (^CoixlachrymajobiL., ⁵⁵.¹%) [⁹]. ^Chemical^structureândîts^functionôf^theseÂChEînhibitors^were^not^yet^known^with^thoseôf ourstudy. ^Therefore, îtîs^necessary^to^study^furtherônpurificationandcharacterizationofAChE inhibitorfromtheselectedyeasts. ^Finally, ^we^selectedSaccharomycescerevisiaeWJSL0113inthe sporogenouswildyeastasanewAChEinhibitor-^producing^yeast. ^W. ânomalus^JSF0128^wasâlso selectedintheasporogenousyeastsbecauseitwasnotformedascosporeandhadsugar-^tolerant^wild yeast which was grew well in 20% ^glucose-^containing ^YPD ^medium. ^Their microbiological characteristicsand conditionsforthe productionoftheirAChEinhibitorswere investigatedand compared.

Generally, ^S. ^cerevisiae^has^long^beenûsed^for^thepreparationofalcoholicbeverages [¹⁰], ^bread, etc. Îtâlso^recently^has^received^muchâttention^becauseôfîts^variousphysiologicalfunctionalities suchasanti-ângiogenic [²²], ânti-hypertensive [²³] ândânti-^dementia [²⁴] âgents. ^Thereâre^no^studies studyonthebioactivecompoundsfromW. ânomalusêxcept^for^studiesônîtsarabinosidaseand xylosidaseactivitiesandonimprovingthearomadevelopmentofwine [²⁵]. ^Therefore, ^S. ^cerevisiae WJSL0113 and W. ânomalus^JSF0128 ⁱⁿ ^this^study ^would ^be^veryûseful ⁱⁿ^the ânti-^dementia medicinalorfunctionalfoodindustry.

Microbiological characteristics of the selected strains, Saccharomyces cerevisiae WJSL0113 and Wickerhamomyces anomalus JSF0128

The morphological, ^cultural characteristics and phylogenetic tree of Saccharomyces cerevisiae WJSL0113andWickerhamomycesanomalusJSF0128aresummarizedinTable2. ^The^S. ^cerevisiae WJSL0113 strainformedanovalshapedyeastcolony and ascospores. ^The ^strain^did^not ^form pseudomyceliumandgrewwellinYPD, ^YMând^PD^mediaâs^wellâsⁱⁿ^vitamin–^free^YPD^medium. Thestrainwashalophilicandthermophilicyeastgrowingin10% ^NaCl-^containing^YPD^mediumând at35℃.

W. ânomalus^JSF0128^formedânôval^shaped^yeast^colonyând^did ^not^formâscospores ând pseudomycelium. ^Like^the^S. ^cerevisiae^WJSL0113, ^the^strainâlso^grew^wellⁱⁿ^YPD, ^YMând^PD mediaand10% ^NaCl-^containing^YPD. Êspecially, ^the^yeast^strain^was^sugar–^tolerant^growingⁱⁿ 20% ^glucose-^containing^YPD.

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ThephylogenetictreefortheselectedyeaststrainsisshowninFig. ¹. ^S. ^cerevisiae^WJSL0113^was closely relatedtoS. cerevisiaeMG859670.¹, ^KY109393.¹ ând ^S. ellipsoideusAF005708.¹. ^W. anomalusJSF0128wasalsocloselyrelatedtoW. ânomalus^KT895982.¹ând^KX253664.¹. ^The^tree wasgeneratedbytheneighbor-^joining^method, ûsing^MEGA⁷.⁰.

Conditions for the production of AChE inhibitor

Effectofmedia; ^Theêffectsôf^the^mediumândîtsînitial^pHôn^the^productionôfÂChEînhibitor wereinvestigated. Âmong^the^YPD, ^YMând^PD^media, ^cell^freeêxtracts^from^the^YPD^medium exhibitedhigh AChEinhibitoryactivitiesof92.¹% (Saccharomyces cerevisiaeWJSL0113) ând 40.³% (WickerhamomycesanomalusJSF0128) âlthough^both^strains^grew^wellⁱⁿâll^media. ^We investigatedtheeffectofculturetemperatureandpH. ^To^determine^theôptimumtemperatureforthe productionofAChEinhibitor, ^S. ^cerevisiae^WJSL0113ând^W. ânomalus^JSF0128^were^grownⁱⁿ^the YPD medium for 3 days at several temperatures, ând ^then, ^the ÂChE înhibitoryâctivity ^was measured. ^TheôptimumtemperaturefortheproductionofAChEinhibitorinthetwostrainswas 30℃. ^Theînitial^pHôf^the^medium^for^the^productionôfÂChEînhibitor^wasâlsoêxaminedⁱⁿ^the rangeofpH4.⁰~⁹.⁰ (^data^not^shown). În^S. ^cerevisiae^WJSL0113, ^theÂChEînhibitoryâctivity^was veryhighat98% ⁱⁿ^media^from^pH⁶.⁰^to^pH⁸.⁰ândâlso^showedânînhibitoryâctivityôfâbout⁶⁰% inthepH5.⁰^medium.

However, ^the^highestÂChEînhibitoryâctivityôf⁴⁴% ^wasôbservedⁱⁿ^the^pH⁵.⁰^YPD^medium forW. ânomalus^JSF0128, ând^this^result^suggests^that^W. ânomalus^JSF0128^produces^moreâcid– tolerantAChEinhibitorthanthatofS. ^cerevisiae^WJSL0113.

Next, ^weinvestigatedtheeffectofthecultivationtime. Â^time^courseôf^the^productionôf^the Table 2. Characteristics of the selected two wild yeasts, Saccharomyces cerevisiae WJSL0113 and Wickerhamomyces anomalus JSF0128

Saccharomyces cerevisiae

WJSL0113 Wickerhamomyces anomalus JSF0128

Morphological characteristics

Shape O O

Vegetative reproduction B B

Size (μm) 2.0 ⅹ 1.4 1.2 ⅹ 0.8

Ascospore + -

Pseudomycelium − −

Cultural characteristics

Growth on YPD /YM/PD media +++/+++/+++ +++/++/+

Color on YPD medium C C

Growth on Vitamin-free medium + -

Growth on 20%/50%glucose-YPD /25%

medium ++/+ +/-

Growth on 5%/15%/25% NaCl-YPD /25%

medium ++/-/- -/-/−

O, oval; B, budding; +++, very good growth; ++ or +, good growth; -, no growth; C, cream color; YM, yeast extract-malt extract medium; YPD, yeast extract-peptone-dextrose medium; PD, potato-dextrose medium.

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Fig. 1. Phylogenetic tree of anti-dementia acetylcholinesterase inhibitor-producing yeasts, Saccharomyces cerevisiae WJSL0113 and Wickerhamomyces JSF0128, isolated from freshwater based on the nucleotide sequences of large subunit 28S ribosomal DNA. The tree was generated by the neighbor-joining method, using MEGA7

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AChEinhibitorwasdeterminedinaflaskcultureunderthecultureconditionsforAChEinhibitor production. Âs^shownⁱⁿ^Fig. ², ^the^productionôfÂChEînhibitorⁱⁿ^the^two^strainsîncreasedâs^the cellgrowthincreased, ând^the^maximum^productionôfÂChEînhibitor^wasôbtainedât¹⁸^hrôf cultivation. ^ThiscultivationtimewasshorterthanthatofYarrowialipolyticaS-³^fromtraditional Doenjang (³⁶^hr) [¹⁹].

From theseresults, ^the ^culture ^conditions ^for ^the ^production ôf ^the ÂChEînhibitor ^from ^S. cerevisiae WJSL0113isYPDmedium (înitial^pH:⁶.⁰), âcultivationtimeof18hr, ândâ^culture temperatureof30℃. ^The^culture^conditions^for^the^productionôf^theÂChEînhibitor^between^the^two wildyeaststrainswerenotdifferentexceptfortheinitialpHofthemedium (^pH⁵.⁰).

Neuron apoptosis repression (anti-dementia) of the selected wild yeast

Therepressioneffectsofthecell-^freeêxtracts^from^the^selected^wild^yeastôn^CT105-înduced^neuron apoptosiswereinvestigated (^Fig. ³).

Theneuronapoptosisratewas5.⁰% (± ¹.⁵%) ⁱⁿ^the^WT^PC12^cellsând⁹¹.⁰% (± ¹.⁸%) ⁱⁿ^the recombinantPC12cellsexpressingCT105. ^However, îtsâpoptosis^rate^wassignificantlydecreased to13% (± ¹.¹%) ^when^treated^with^the^cell-^freeêxtract (⁵⁰⁰^μg/^mL). ^These^results^suggest^that^the Fig. 2. Effect of cultural time on the acetylcholinesterase inhibitory activity of Saccharomyces cerevisiae WJSL0113 ( ) and Wickerhamomyces anomalus JSF0128 ( )