Table Of ContentBiocatalytic
Applications in
Biotechnology
Edited by
Emmanuel M. Papamichael and Panagiota-Yiolanda Stergiou
Printed Edition of the Special Issue Published in Catalysts
www.mdpi.com/journal/catalysts
Biocatalytic Applications in
Biotechnology
Biocatalytic Applications in
Biotechnology
Editors
Emmanuel M. Papamichael
Panagiota-Yiolanda Stergiou
MDPI•Basel•Beijing•Wuhan•Barcelona•Belgrade•Manchester•Tokyo•Cluj•Tianjin
Editors
EmmanuelM.Papamichael Panagiota-YiolandaStergiou
UniversityofIoannina UniversityofIoannina
Greece Greece
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Contents
AbouttheEditors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
ChanMiPark,HyunSeoPark,GunSuCha,KiDeokParkandChul-HoYun
RegioselectiveHydroxylationofRhododendrolbyCYP102A1andTyrosinase
Reprintedfrom: Catalysts2020,10,1114,doi:10.3390/catal10101114 . . . . . . . . . . . . . . . . . 1
Tha´ıs Carvalho Maester, Mariana Rangel Pereira, Aliandra M. Gibertoni Malaman,
Janaina Pires Borges, Paˆmela Aparecida Maldaner Pereira and Eliana G. M. Lemos
Exploring Metagenomic Enzymes: A Novel Esterase Useful for Short-Chain Ester Synthesis
Reprintedfrom: Catalysts2020,10,1100,doi:10.3390/catal10101100 . . . . . . . . . . . . . . . . . 13
Ekaterina Yu. Bezsudnova, Alena Yu. Nikolaeva, Sergey Y. Kleymenov, Tatiana E. Petrova,
SofiaA.Zavialova,KristinaV.Tugaeva,NikolaiN.Sluchanko,etal.
Counterbalance of Stability and Activity Observed for Thermostable Transaminase from
ThermobaculumterrenuminthePresenceofOrganicSolvents
Reprintedfrom: Catalysts2020,10,1024,doi:10.3390/catal10091024 . . . . . . . . . . . . . . . . . 31
Olga A. Gkini, Panagiota-Yiolanda Stergiou, Athanasios Foukis, Panayiotis V. Ioannou
and Emmanuel M. Papamichael
Kinetic Analysis of the Lipase-Catalyzed Hydrolysis of Erythritol and Pentaerythritol
Fatty Acid Esters: A Biotechnological Application for Making Low-Calorie Healthy Food
Alternatives
Reprintedfrom: Catalysts2020,10,965,doi:10.3390/catal10090965 . . . . . . . . . . . . . . . . . . 43
Kamila Wlizło, Jolanta Polak, Justyna Kapral-Piotrowska, Marcin Graz, Roman Paduch
and Anna Jarosz-Wilkołazka
Influence of Carrier Structure and Physicochemical Factors on Immobilisation of Fungal Laccase
in Terms of Bisphenol A Removal
Reprintedfrom: Catalysts2020,10,951,doi:10.3390/catal10090951 . . . . . . . . . . . . . . . . . . 53
PedroL.Valencia,Bastia´nSepu´lveda,DiegoGajardoandCarolinaAstudillo-Castro
Estimating the Product Inhibition Constant from Enzyme Kinetic Equations Using the Direct
LinearPlotMethodinOne-StageTreatment
Reprintedfrom: Catalysts2020,10,853,doi:10.3390/catal10080853 . . . . . . . . . . . . . . . . . . 75
Nadya Dencheva, Joana Braz, Dieter Scheibel, Marc Malfois, Zlatan Denchev
and Ivan Gitsov
Polymer-Assisted Biocatalysis: Polyamide 4 Microparticles as Promising Carriers of Enzymatic
Function
Reprintedfrom: Catalysts2020,10,767,doi:10.3390/catal10070767 . . . . . . . . . . . . . . . . . . 85
Thanawat Phuadraksa, Jurairat Chittrakanwong, Kittitouch Tullayaprayouch,
Naruthai Onsirisakul, Sineewanlaya Wichit and Sakda Yainoy
Engineering of Bifunctional Enzymes with Uricase and Peroxidase Activities for Simple and
Rapid Quantification of Uric Acid in Biological Samples
Reprintedfrom: Catalysts2020,10,428,doi:10.3390/catal10040428 . . . . . . . . . . . . . . . . . . 115
Victor N. Emenike, Dominik Hertweck, Rene´ Schenkendorf and Antje C. Spiess
Rigorous Model-Based Design and Experimental Verification of Enzyme-Catalyzed
Carboligation under Enzyme Inactivation
Reprintedfrom: Catalysts2020,10,96,doi:10.3390/catal10010096 . . . . . . . . . . . . . . . . . . 133
v
Chien Thang Doan, Thi Ngoc Tran, I-Hong Wen, Van Bon Nguyen, Anh Dzung Nguyen
andSan-LangWang
Conversion of Shrimp Head Waste for Production of a Thermotolerant, Detergent-Stable,
AlkalineProteasebyPaenibacillussp.
Reprintedfrom: Catalysts2019,9,798,doi:10.3390/catal9100798 . . . . . . . . . . . . . . . . . . . 151
Raphael I. Adeoye, Dunsin S. Osalaye, Theresia K. Ralebitso-Senior, Amanda Boddis,
AmandaJ.Reid,AmosA.Fatokun,AndrewK.Powell,etal.
CatalyticActivitiesofMultimericG-QuadruplexDNAzymes
Reprintedfrom: Catalysts2019,9,613,doi:10.3390/catal9070613 . . . . . . . . . . . . . . . . . . . 165
YanfeiMiao,YuewangLiu,YushuHeandPuWang
BiotransformationwithaNewAcinetobactersp.IsolateforHighlyEnantioselectiveSynthesisof
aChiralIntermediateofMiconazole
Reprintedfrom: Catalysts2019,9,462,doi:10.3390/catal9050462 . . . . . . . . . . . . . . . . . . . 179
AslıYenenler,HasanKurtandOsmanUg˘urSezerman
Enhancing Enzymatic Properties of Endoglucanase I Enzyme from Trichoderma Reesei via
SwappingfromCellobiohydrolaseIEnzyme
Reprintedfrom: Catalysts2019,9,130,doi:10.3390/catal9020130 . . . . . . . . . . . . . . . . . . . 191
YoungChanJeon,AnhDucNguyenandEunYeolLee
BioproductionofIsoprenoidsandOtherSecondaryMetabolitesUsingMethanotrophicBacteria
asanAlternativeMicrobialCellFactoryOption: CurrentStageandFutureAspects
Reprintedfrom: Catalysts2019,9,883,doi:10.3390/catal9110883 . . . . . . . . . . . . . . . . . . . 209
Wahhida Latip, Victor Feizal Knight, Norhana Abdul Halim, Keat Khim Ong,
Noor Azilah Mohd Kassim, Wan Md Zin Wan Yunus, Siti Aminah Mohd Noor, et al.
Microbial Phosphotriesterase: Structure, Function, and Biotechnological Applications
Reprintedfrom: Catalysts2019,9,671,doi:10.3390/catal9080671 . . . . . . . . . . . . . . . . . . . 223
vi
About the Editors
EmmanuelM.Papamichael
Prof.EmmanuelM.Papamichael,graduated(DiplomaofChemistry)fromAthens’Kapodistrian
University in 1972, and received his Doctoral degree (PhD) from University of Ioannina (1981).
Currently, he is Professor Emeritus in the Department of Chemistry (University of Ioannina). His
research interests comprise: the kinetics of enzymatic and heterogeneous reactions, Investigation
of their mechanisms and the development of optimal mathematical models, the isolation and
purificationofhydrolyticenzymes,enzymebiotechnology,parametricandnon-parametricstatistical
treatment of experimental data, experimental design, error analysis, computer programming.
Prof. Papamichael has published 131 original papers (after peer-review) in international journals,
internationalbooksandinternationalconferences,hasreceived<1300citations,andparticipatesas
anEditorialBoardMemberinmanyISIJournals.Heisalsoareviewerinmorethan100international
journals, and he was supervisor on < 30 PhD/Master/Diplomas. In the last 5 years he was/is
scientificdirectorinthreecompetitiveresearchprogramsfundedbytheEU.
Panagiota-YiolandaStergiou
Dr. Panagiota-Yiolanda Stergiou, graduated (Diploma of Chemistry) from the University of
Ioannina in 2008, and received her Master of Science Diploma (2011) and her Doctoral degree
(2017) from University of Ioannina. At present, she is a Postdoctoral Researcher at the Department
of Chemistry, University of Ioannina, Greece (Enzyme Biotechnology and Genetic engineering
group). Her MSc is in Chemistry and her PhD is in Enzyme Biotechnology, entitled “Analytical
and kinetic methods, and Structural studies for the investigation of specific biotechnological
applicationsoffreeandimmobilizedproteolyticenzymes”. Herresearchinterestsareintheareasof
enzymebiotechnology(foodbiotechnology,biofuel),thekineticsandmechanismsofenzymaticand
heterogeneousreactionsandtheexperimentaldesignofbioprocesses.Shehaspresentedmorethan30
papersininternationalandnationalconferences,andshehaspublished20papersinpeer-reviewed
scientificjournals.
vii
catalysts
Article
Regioselective Hydroxylation of Rhododendrol by
CYP102A1 and Tyrosinase
ChanMiPark1,HyunSeoPark1,GunSuCha2,KiDeokPark3andChul-HoYun1,*
1 SchoolofBiologicalSciencesandTechnology,ChonnamNationalUniversity,77Yongbongro,
Gwangju61186,Korea;[email protected](C.M.P.);[email protected](H.S.P.)
2 NamhaeGarlicResearchInstitute,2465-8Namhaedaero,Namhae,Gyeongsangnamdo52430,Korea;
[email protected]
3 GwangjuCenter,KoreaBasicScienceCenter,77Yongbongro,Gwangju61186,Korea;[email protected]
* Correspondence: [email protected]
Received: 12August2020;Accepted: 24September2020;Published: 25September2020
Abstract: Rhododendrol(RD)isanaturallyoccurringphenoliccompoundfoundinmanyplants.
Tyrosinase(Ty)convertsRDtoRD-catecholandsubsequentlyRD-quinoneviatwo-stepoxidation
reactions, after which RD-melanin forms spontaneously from RD-quinone. RD is cytotoxic in
melanocytesandlungcancercells,butnotinkeratinocytesandfibroblasts. However,thefunction
of RD metabolites has not been possible to investigate due to the lack of available high purity
metabolites. In this study, an enzymatic strategy for RD-catechol production was devised using
engineered cytochrome P450 102A1 (CYP102A1) and Ty, and the product was analyzed using
high-performance liquid chromatography (HPLC), LC-MS, and NMR spectroscopy. Engineered
CYP102A1 regioselectively produced RD-catechol via hydroxylation at the ortho position of RD.
AlthoughRD-quinonewassubsequentlyformedbytwostepoxidationinTycatalyzedreactions,
L-ascorbicacid(LAA)inhibitedRD-quinoneformationandcontributedtoregioselectiveproduction
ofRD-catechol. WhenLAAwaspresent,theproductivityofRD-catecholbyTywas5.3-foldhigher
thanthatbyengineeredCYP102A1. TheseresultsindicatethatengineeredCYP102A1andTycan
beusedaseffectivebiocatalyststoproducehydroxylatedproducts,andTyisamorecost-effective
biocatalystforindustrialapplicationsthanengineeredCYP102A1.
Keywords: rhododendrol;cytochromeP450;tyrosinase;biocatalyst;hydroxylation;bioconversion
1. Introduction
Biocatalystsarebiologicalsystems,suchaspurifiedenzymesandwholecells,thatcancatalyze
diversechemicalreactions. Enzymescanacceptawiderangeofmoleculesassubstrates,andthey
exhibithighcatalyticactivitiestowardssimpleandcomplexchemicals. Consequently,theyareusedas
biocatalystsinfieldsofchemicalsynthesis,biosensorproduction,andbioremediation. Thebiocatalysts
canalsobeusedtosupportseveralcascadereactionsduringorganiccompoundsynthesis. Inparticular,
theyareeffectivetoolsinthefinechemicalandpharmaceuticalindustriesbecausetheyexhibitchemo-,
stereo-,andregioselectivityforchemicalproductionineco-friendlyways[1–5].
Generally, cytochrome P450 (P450 or CYP) enzymes catalyze the monooxygenase reaction
(i.e.,theinsertionofoneatomofmolecularoxygenintoasubstrateandthereductionoftheother
oxygenatomtowater)[6]. Requiredelectronsforthereactionareprovidedfromnicotinamideadenine
dinucleotidecofactors(NADPHorNADH)viacytochromeP450reductase(CPR).Inaddition,P450s
catalyze diverse reactions such as C-hydroxylation; N-oxidation; N-, O-, and S-dealkylation; C–C
bondcleavage; alcoholoxidationusingdiversenaturalandnon-naturalcompoundsassubstrates.
Accordingly, CYPs have considerable potential as biocatalysts for novel drug development [7–9].
CYP102A1fromBacillusmegaterium(BM3)consistsofthecatalytichemedomainandthereductase
Catalysts2020,10,1114;doi:10.3390/catal10101114 www.mdpi.com/journal/catalysts
1
Catalysts2020,10,1114
domaininasinglepolypeptide[10]. CYP102A1ismoreeffectiveasabiocatalystbecauseofitshigh
activityandsolubility. TheengineeringofCYP102A1throughsite-directedandrandommutagenesis
is reported to improve their activities toward various marketed drugs, steroids, and non-natural
substrates. However,bioconversionsviaCYP102A1havedrawbacksonanindustrialscalebecause
theystillrequiretheuseofahigh-costcofactor,NADPH[11–15].
Tyrosinase(Ty)isacopper-containingdioxygenaseresponsibleformelaninformationinmany
organisms. Twocopperions,CuAandCuB,coordinatesixconservedhistidineresiduesintheactive
sites[16,17]. Tycatalyzestheortho-hydroxylationofmonophenolstoortho-diphenols(catechols)via
monophenolaseactivity,aswellasthesubsequentoxidationofortho-diphenolstoortho-quinonesvia
diphenolaseactivity. Thehighlyreactiveortho-quinonesconverttomelaninspontaneously,andare
involved in skin pigmentation and fruit and vegetable browning [18,19]. Ty has a considerable
potentialtosynthesizebioactivecatecholderivativesbecauseofregioselectiveortho-hydroxylationvia
monophenolaseactivity. However,Ty-associatedmelaninformationshouldbeinhibitedforindustrial
applicationsofthebioactivecatecholderivatives[20,21]. Proteinengineeringusingsite-directedand
randommutagenesishasbeenattemptedtoimproveTy’sactivityandsubstratespecificity[22,23].
Furthermore,theinhibitionofTy’sdiphenolaseactivityusingcatechol,NADH,andascorbicacidwas
foundtoenhancetheproductionofcatecholderivatives[24–26]. FortheindustrialuseofTytoproduce
catecholcompounds,theissueoffurtheroxidationofcatecholneedstobesolved,butTyseemstobe
amorecost-effectivebiocatalystcomparedwithP450becauseitdoesnotrequiretheredoxpartners
andNADPH.
Rhododendrol(RD,4-(4-hydroxyphenyl)-2-butanol)isanaturally-occurringphenoliccompound
found in many plants such as Acer nikoense and Betula pubescens [27–29]. In melanocytes, RD is
metabolized to RD-quinone via RD-catechol by a Ty-catalyzed reaction. Subsequent spontaneous
reactionsconvertRD-quinonetomelanin[30]. RDmetabolitesandRD-derivedmelaningeneratedby
Tycauseoxidativestressandexhibitcytotoxicityinmelanocytes[31]. RDisalsocytotoxicinhuman
lungcancercells[32],butnotinkeratocytesandfibroblasts[33]. However,thebioactiveoradverse
effectsofvariousRDmetabolitesarenotwellestablishedbecausemetaboliteswithhighpuritycannot
beobtainedonalargescale.
The aim of this study was to produce a highly pure RD-catechol product using engineered
CYP102A1 and Ty as biocatalysts. We found that engineered CYP102A1 and Ty can catalyze the
regioselectivehydroxylationattheorthopositionofRDtoproduceanRD-catecholwithhighselectivity
(Figure 1). In addition, we compared the yield, final product concentration, and productivity for
RD-catecholproductionbyengineeredCYP102A1andTy. Tothebestofourknowledge,thisstudyis
thefirstreportoftheproductionofahighlypureRD-catecholusingengineeredCYP102A1andTy.
Figure1. Schemeforrhododendrol(RD)-catecholproductionusingcytochrome(CYP)102A1and
tyrosinase(Ty).
2. ResultsandDiscussion
2.1. HydroxylationofRDbyEngineeredCYP102A1
To find a CYP102A1 enzyme with a high activity towards RD, the catalytic activities of the
CYP102A1 wild type (WT) and its engineered enzymes toward RD were investigated at a fixed
substrate concentration of 200 μM for 30 min of incubation. An NADPH regenerating system
(NGS) was added to provide NADPH to the CYP102A1. The formation of RD-derived products
2
Catalysts2020,10,1114
by CYP102A1 was analyzed using high-performance liquid chromatography (HPLC). One major
productwasgeneratedbyengineeredCYP102A1. TheretentiontimesofRDanditsmajorproduct
were13.4and8.4min,respectively(Figure2). Amongthisstudy’stestedenzymes,WTCYP102A1
apparentlycouldnotoxidizeRD,whereastenengineeredCYP102A1showedhighactivitytoward
RDwithmorethan200nmolproduct/nmolP450. EngineeredCYP102A1#16(M16)wasselectedfor
furtherexperimentsbecauseitshowedthehighestactivitywith645nmolproduct/nmolP450among
thetested45engineeredCYP102A1(FigureS1).
Figure2. High-performanceliquidchromatography(HPLC)chromatogramofRDandRD-catechol
produced by engineered CYP102A1. The reaction mixtures contained 0.2 μM M16 or 200 μM
RD with or without NADPH regenerating system (NGS). RD and RD-catechol were eluted at
13.4and8.4min,respectively.
M16 might produce RD-catechol or RD-resorcinol via hydroxylation at the ortho or meta
positions of the RD’s phenol ring. The major RD-derived product was identified using liquid
chromatography-massspectrometry(LC-MS)andnuclearmagneticresonance(NMR)spectroscopy.
AlthoughthemolecularmassofRDis166.22g·mol−1,itsmasstocharge(m/z)valuewascalculatedas
149viaLC-MS[34]. Them/zvalueofthemajorproductwas165,whichwas16highercomparedto
RD(FigureS2). The1Hand13CNMRspectraofRDandtheproductofM16areshowninFigures
S3–S6. ChemicalstructureandchemicalshiftassignmentsforRDandtheproductofM16areshownin
FigureS7andTableS1. TheseresultsindicatethatengineeredCYP102A1regioselectivelyproducesan
RD-catecholviahydroxylationattheorthoposition.
2.2. RD-CatecholProductionbyEngineeredCYP102A1
To increase the product concentration, M16 catalytic reactions were performed with various
RDconcentrations. RD-catecholproductionincreasedwithRDconcentration,andtheproductivity
increasedupto200μMRD(Figure3). Inthiscondition,productyield,finalproductconcentration,
and productivity were 50.4%, 18.4 mg·L−1, and 36.7 mg·L−1·h−1, respectively. The maximum
RD-catecholwas produced when RD concentration was 200 μM. The RD-catechol yield increased
until100μMRD,andthendecreased. WhenRDconcentrationwas100μM,theyieldwashighestat
53.8%. TheseresultsindicatethatM16catalyticreactionsbelow200μMRDtoproduceRD-catecholis
efficientFurtherpurificationstepsmightbeneededtoobtainhighlypureRD-catechol,astheyield
of RD-catechol was lower than 90%. Although M16 can catalyze the regioselective hydroxylation
of RD, M16 has limitations for industrial-scale applications due to its low yield and demand for
3
