Regioselective Hydroxylation of Naringin Dihydrochalcone to Produce Neoeriocitrin Dihydrochalcone by CYP102A1 (BM3) Mutants
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catalysts Article Regioselective Hydroxylation of Naringin Dihydrochalcone to Produce Neoeriocitrin Dihydrochalcone by CYP102A1 (BM3) Mutants 1, 1, 1 2 1,3 Thi Huong Ha Nguyen y, Su-Min Woo y, Ngoc Anh Nguyen , Gun-Su Cha , Soo-Jin Yeom , Hyung-Sik Kang 1,3,* and Chul-Ho Yun 1,3,* 1 School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University, Yongbong-ro 77, Gwangju 61186, Korea; [email protected] (T.H.H.N.); [email protected] (S.-M.W.); [email protected] (N.A.N.); [email protected] (S.-J.Y.) 2 Namhae Garlic Research Institute, 2465-8 Namhaedaero, Gyeongsangnamdo 52430, Korea; [email protected] 3 School of Biological Sciences and Technology, Chonnam National University, Yongbong-ro 77, Gwangju 61186, Korea * Correspondence: [email protected] (H.-S.K.); [email protected] (C.-H.Y.); Tel.: +82-62-530-2195 (H.-S.K.); +82-62-530-2194 (C.-H.Y.) These authors equally contributed. y Received: 23 June 2020; Accepted: 21 July 2020; Published: 23 July 2020 Abstract: Naringin dihydrochalcone (DC) is originally derived from the flavonoid naringin, which occurs naturally in citrus fruits, especially in grapefruit. It is used as an artificial sweetener with a strong antioxidant activity with potential applications in food and pharmaceutical fields. At present, enzymatic and chemical methods to make products of naringin DC by hydroxylation reactions have not been developed. Here, an enzymatic strategy for the efficient synthesis of potentially valuable products from naringin DC, a glycoside of phloretin, was developed using Bacillus megaterium CYP102A1 monooxygenase. The major product was identified to be neoeriocitrin DC by NMR and LC-MS analyses. Sixty-seven mutants of CYP102A1 were tested for hydroxylation of naringin DC to produce neoeriocitrin DC. Six mutants with high activity were selected to determine the kinetic 1 parameters and total turnover numbers (TTNs). The kcat value of the most active mutant was 11 min− 1 and its TTN was 315. The productivity of neoeriocitrin DC production increased up to 1.1 mM h− , 1 1 which corresponds to 0.65 g L− h− . In this study, we achieved a regioselective hydroxylation of naringin DC to produce neoeriocitrin DC. Keywords: CYP102A1; naringin dihydrochalcone; neoeriocitrin dihydrochalcone; regioselective hydroxylation 1. Introduction Dihydrochalcone (DC) is a bicyclic flavonoid family with two aromatic rings and a saturated C3 bridge [1]. DC compounds are mainly found in citrus fruits, grapefruits, and apples, and they play an important role in resisting biotic or abiotic stresses in plant [2,3]. To date, more than 200 DC compounds have been identified from over 30 plant families [4]. As DC compounds show strong antioxidant activities, a large number of studies have researched the potential benefits of DC compounds to human health. They were demonstrated to be effective in preventing different physiopathological processes [3], notably diabetes [5] and bone resorption [6]. In recent years, scientists have more often been attracted by in vitro and in vivo biological activities of DC compounds. Catalysts 2020, 10, 823; doi:10.3390/catal10080823 www.mdpi.com/journal/catalysts Catalysts 2020, 10, x FOR PEER REVIEW 2 of 14 scientists have more often been attracted by in vitro and in vivo biological activities of DC Catalystscompounds.2020, 10, 823 2 of 14 Naringin DC (3,5-dihydroxy-4[3-(4-hydroxyphenyl)propanoyl]phenyl 2-O-(6-deoxy-α-L- mannopyranosyl)-Naringin DCβ-L (3,5-dihydroxy-4[3-(4-hydroxyphenyl)propanoyl]phenyl-glucopyranoside) (Figure 1) is known as a widely used artificial 2-O-(6-deoxy- sweetenerα-l -mannopyranosyl)-[7,8]. Naringin DCβ-l -glucopyranoside)is produced when (Figure naringin1) is is known treated as with a widely a strong used artificialbase, such sweetener as potassium [ 7,8]. Naringinhydroxide, DC and is produced then catalytically when naringin hydrogenated. is treated Naringin with a strong is a flavanone-7- base, such asO potassium-glycoside hydroxide,between the andflavanone then catalytically naringenin hydrogenated. and the disaccharide Naringin isneoh a flavanone-7-esperidose.O -glycosideNaringin DC between has thea sweet flavanone value naringeninapproximately and the300 disaccharidetimes higher neohesperidose.than that of sucrose Naringin [9]. Naringin DC has DC a sweethas high value antioxidant approximately activity, 300which times performs higher than better that free-radical of sucrose [9 ].scavenging Naringin DCthan has its high corresponding antioxidant activity,flavanone which naringin performs [10]. betterBesides, free-radical naringin scavenging DC is a glycoside than its correspondingof phloretin that flavanone shows an naringin inhibitory [10]. effect Besides, on active naringin transport DC is a of glycosideglucose ofinto phloretin cells by thatSGLT1 shows and an SGLT2 inhibitory [8]. Nari effectngin on activeDC was transport suggested of glucose as a promising into cells therapeutic by SGLT1 andagent SGLT2 for [Alzheimer’s8]. Naringin DCdisease was suggestedtreatment asagainst a promising multiple therapeutic effects that agent reduce for Alzheimer’s Aβ levels, suppress disease treatmentneuroinflammation, against multiple and eenhanceffects that neurogenesis reduce Aβ levels, [8]. The suppress antioxidant neuroinflammation, and noncalorie and sweetener enhance neurogenesisabilities of naringin [8]. The DC antioxidant can make and it be noncalorie a potential sweetener compound abilities for applications of naringin in DC food, can beverages, make it be and a potentialpharmaceuticals compound [11]. for applications in food, beverages, and pharmaceuticals [11]. CytochromeCytochrome P450 P450 (CYP (CYP or or P450) P450) is is known known as as one one of of the the largest largest enzyme enzyme families families found found in in all all organisms.organisms. P450s P450s catalyze catalyze the the oxidation oxidation of variousof variou endogenouss endogenous and and xenobiotic xenobiotic compounds compounds [12]. [12]. Due Due to theirto their diversity diversity of substrates, of substrates, P450s P450s are are attractive attractive as biocatalystsas biocatalysts for for producing producing chemicals, chemicals, including including bioactivebioactive compounds compounds and and pharmaceuticals pharmaceuticals [13 ,[13,14].14]. CYP102A1 CYP102A1 (P450 (P450 BM3) BM3) from fromBacillus Bacillus megaterium megateriumis a self-suis a ffiself-sufficientcient monooxygenase monooxygenase enzyme, whichenzyme, is naturally which fusedis naturally to its redox fused partner, to its a mammalian-likeredox partner, a diflavinmammalian-like reductase. diflavin Engineered reductase. CYP102A1 Engineered mutants CYP102A1 have been mutants extensively have obtained been extensively through rationalobtained designthrough and rational directed evolutiondesign and to catalyzedirected the evolution oxidation to of catalyze several non-natural the oxidation substrates, of several environmental non-natural chemicals,substrates, and environmental pharmaceuticals chemical [15–19s,]. and It was pharmaceuticals also suggested [15–19]. that the engineeredIt was also CYP102A1suggested canthat bethe developedengineered as aCYP102A1 potential biocatalystcan be developed for biotechnology as a potent applicationsial biocatalyst [20 ,21for]. biotechnology applications [20,21].In this study, we have tried to find an enzymatic strategy for the production of products from naringinIn this DC. study, A large we set have of CYP102A1 tried to find mutants an enzymatic were used strategy for the for e ffithecient production synthesis ofof products potentially from valuablenaringin products DC. A large from set naringin of CYP102A1 DC. To themutants best of were our knowledge,used for the the efficient enzymatic synthesis hydroxylation of potentially of naringinvaluable DC products has not from previously naringin been DC. reported. To the best This of work our isknowledge, the first report the enzymatic on enzymatic hydroxylation synthesis of of neoeriocitrinnaringin DC DC, has a not major previously product ofbeen naringin reported. DC This (Figure work1). is the first report on enzymatic synthesis of neoeriocitrin DC, a major product of naringin DC (Figure 1). Figure 1. Chemical structures of naringin DC and neoeriocitrin DC. The conversion of the substrate, Figure 1. Chemical structures of naringin DC and neoeriocitrin DC. The conversion of the substrate, naringin DC, to its corresponding product, neoeriocitrin DC, is catalyzed by CYP102A1 in the presence naringin DC, to its corresponding product, neoeriocitrin DC, is catalyzed by CYP102A1 in the of NADPH. An enzymatic reaction site on naringin DC is marked by a star. presence of NADPH. An enzymatic reaction site on naringin DC is marked by a star. 2. Results and Discussion 2. Results and Discussion 2.1. Hydroxylation of Naringin DC by CYP102A1 Mutants 2.1. Hydroxylation of Naringin DC by CYP102A1 Mutants First, to determine the ability of CYP102A1 to hydroxylate naringin DC, the catalytic activity of the wild typeFirst, (WT) to determine and its 60 mutantsthe ability [12 of,14 CYP102A1,19,22–25] towardto hydroxylate naringin naringin DC were DC, tested the at catalytic 200 µM substrateactivity of forthe 30 wild min at type 37 ◦ C(WT) (Figure and2 ).its The 60 60mutants mutants [12,14,19,22–25] used for first screening toward werenaringin selected DC basedwere ontested our previousat 200 µM workssubstrate showing for 30 their min improved at 37 °C catalytic(Figure activities2). The 60 on mu a numbertants used of non-naturalfor