catalysts Article Application of Immobilized Cholest-4-en-3-one D1-Dehydrogenase from Sterolibacterium Denitrificans for Dehydrogenation of Steroids Mateusz Tataruch 1, Patrycja Wójcik 1, Agnieszka M. Wojtkiewicz 1 , Katarzyna Zaczyk 1,2, Katarzyna Szyma ´nska 3 and Maciej Szaleniec 1,* 1 Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Kraków, Poland; [email protected] (M.T.); [email protected] (P.W.); [email protected] (A.M.W.); [email protected] (K.Z.) 2 Faculty of Energy and Fuels, AGH University of Science and Technology, 30 A. Mickiewicza Av., 30-059 Kraków, Poland 3 Department of Chemical Engineering and Process Design, Silesian University of Technology, Ks. M. Strzody 7, 44-100 Gliwice, Poland; [email protected] * Correspondence: [email protected] Received: 30 November 2020; Accepted: 12 December 2020; Published: 14 December 2020 Abstract: Cholest-4-en-3-one D1-dehydrogenase (AcmB) from Sterolibacterium denitrificans was successfully immobilized on 3-aminopropyltrimethoysilane functionalized mesoporous cellular foam (MCF) and Santa Barbara Amorphous (SBA-15) silica supports using adsorption or covalently with glutaraldehyde or divinyl sulfone linkers. The best catalyst, AcmB on MCF linked covalently with glutaraldehyde, retained the specific activity of the homogenous enzyme while exhibiting a substantial increase of the operational stability. The immobilized enzyme was used continuously in the fed-batch reactor for 27 days, catalyzing 1,2-dehydrogenation of androst-4-en-3-one to androst-1,4-dien-3-one with a final yield of 29.9 mM (8.56 g/L) and 99% conversion. The possibility of reuse of the immobilized catalyst was also demonstrated and resulted in a doubling of the product amount compared to that in the reference homogenous reactor. Finally, it was shown that molecular oxygen from the air can efficiently be used as an electron acceptor either reoxidizing directly the enzyme or the reduced 2,4-dichlorophenolindophenol (DCPIPH2). Keywords: 3-ketosteroid D1-dehydrogenase; KSTD; KSDH; AcmB; 1,2-dehydrogenation; cholest-4-en-3-one D1-dehydrogenase; enzyme immobilization; FAD-dependent enzymes; enzyme immobilization 1. Introduction Steroids are an important group of naturally occurring or synthetic compounds belonging to nonsaponifiable lipids. Their structure is based on a cyclopenta[a]phenanthrene carbon skeleton which can be partially unsaturated and is usually substituted with a methyl group at C10 and C13 as well as with an alkyl group at C17 [1]. Steroid compounds are important cell membrane components, precursors for the synthesis of vitamins and hormones. As a result, steroid derivatives form one of the largest group of drugs currently on the market and their synthesis and modification are of utmost importance for the pharmaceutical industry. Due to their structural complexity and often multiple substituents, synthesis and modification of steroids were never entirely based on solely chemical methods. For example, the first efficient synthetic methods of progesterone and cortisone started with natural sapogenin, diosgenin, derived from a Catalysts 2020, 10, 1460; doi:10.3390/catal10121460 www.mdpi.com/journal/catalysts Catalysts 2020, 10, x FOR PEER REVIEW 2 of 14 from a plant source (Cabeza de negro, Annona purpurea). Progesterone was obtained by chemical means (via Marker degradation) and finally cortisone by fermentation with Rhizopus mold (Upjohnʹs process) [2,3]. In addition, 1,2‐dehydrogenation, for example for androst‐4‐en‐3‐one (AD) to androst‐1,4‐dien‐3‐one (ADD) conducted by means of microbial biotransformation, has a very long history [4]. Currently, sex hormones and corticosteroid drugs are synthesized by a combination of chemicalCatalysts and biotechnological2020, 10, 1460 methods [5]. For several decades, biotransformation is gaining2 of 13 more importance in the synthesis of steroid APIs and these methods have been recently reviewed by Fernándezplant‐Cabezón source (Cabeza et al. [6]. de negro, Annona purpurea). Progesterone was obtained by chemical means Most(via of Markerthe already degradation) developed and finally biotechnological cortisone by fermentationapproaches withare basedRhizopus onmold biotransformation (Upjohn’s or applicationprocess) of the [2 ,whole3]. In‐ addition,cells [7–9]. 1,2-dehydrogenation, However, in this for paper, example we decided for androst-4-en-3-one to focus on the (AD) application to of the immobilizedandrost-1,4-dien-3-one cholest‐4 (ADD)‐en‐3‐one conducted Δ1‐dehydrogenase by means of microbial of the biotransformation, 3‐ketosteroid dehydrogenase has a very long family history [4]. Currently, sex hormones and corticosteroid drugs are synthesized by a combination to regioselectiveof chemical dehydrogenation and biotechnological of methods 3‐ketosteroids. [5]. For several The application decades, biotransformation of natural bacterial is gaining cells for biotransformationmore importance or a inrecombinant the synthesis ofsystem steroid for APIs biocatalysis and these methods is often have cheaper been recently and simpler. reviewed byHowever, the immobilizedFernández-Cabez enzymesón et have al. [6 ].several important advantages over bacterial catalysts such as lack of side reactionMost and of toxins, the already simplified developed product biotechnological purification, approaches enhanced are based stability on biotransformation of the enzyme, or the possibilityapplication of reuse of of the the whole-cells catalyst [7 –and9]. However, easy conversion in this paper, from we decided the batch to focus to the on the continuous application process of the immobilized cholest-4-en-3-one D1-dehydrogenase of the 3-ketosteroid dehydrogenase family mode. to regioselective dehydrogenation of 3-ketosteroids. The application of natural bacterial cells for 1 Cholestbiotransformation‐4‐en‐3‐one Δ or a recombinant‐dehydrogenase system for(AcmB) biocatalysis from is often Sterolibacterium cheaper and simpler. denitrificans However, is a surprisinglythe immobilized versatile enzymes FAD‐containing have several important oxidoreductase advantages overcapable bacterial of catalysts 1,2‐dehydrogenation such as lack of side of an unusuallyreaction wide and group toxins, of simplified 3‐ketosteroids product i.e., purification, not only enhanced the standard stability ofC20–C22 the enzyme, 3‐ketosteroids the possibility but also significantlyof reuse bulkier of the catalyst compounds and easy conversionwith the from undegraded the batch to the C17 continuous aliphatic process side mode. chain such as Cholest-4-en-3-one D1-dehydrogenase (AcmB) from Sterolibacterium denitrificans is a surprisingly cholest‐4‐en‐3‐one derivatives or even 3‐ketosaponins such as (25R)‐spirosta‐4‐en‐3‐one versatile FAD-containing oxidoreductase capable of 1,2-dehydrogenation of an unusually wide group (diosgenone)of 3-ketosteroids [10–12]. In i.e., this not paper, only the we standard demonstrate C20–C22 how 3-ketosteroids AcmB covalently but also significantly immobilized bulkier on silica support compoundscan be with theapplied undegraded to C17 aliphaticdehydrogenation side chain such asof cholest-4-en-3-one4‐androstene derivatives‐3,17‐dione to 1,4‐androstadieneor even 3-ketosaponins‐3,17‐dione such(Figure as (25R)-spirosta-4-en-3-one1), an industrially important (diosgenone) process. [10–12 ].We In demonstrate this paper, how immobilizationwe demonstrate influences how AcmB the covalently overall immobilizedproductivity on silica of supportthe system can be appliedcompared to dehydrogenation to a homogenous of 4-androstene-3,17-dione to 1,4-androstadiene-3,17-dione (Figure1), an industrially important enzyme. To the best of our knowledge, this is the first report on the immobilization of a KSTD family process. We demonstrate how immobilization influences the overall productivity of the system member comparedon a solid to carrier. a homogenous Furthermore, enzyme. we To focus the best on of the our application knowledge, thisof atmospheric is the first report O2 as on an the efficient reoxidantimmobilization of the enzyme of a KSTDthat can family support member or on potentially a solid carrier. even Furthermore, replace we2,4 focus‐dichlorophenolindophenol on the application (also calledof atmospheric 2,6‐dichloroindophenol O2 as an efficient reoxidant or DCPIP), of the which enzyme is that customarily can support or used potentially to reoxidize even replace FADH2 in KSTD enzymes2,4-dichlorophenolindophenol (Figure 1). (also called 2,6-dichloroindophenol or DCPIP), which is customarily used to reoxidize FADH2 in KSTD enzymes (Figure1). Figure 1. The schematic representation of reaction catalyzed by AcmB: Dehydrogenation of Figure 1.4-androstene-3,17-dione The schematic representation to 1,4-androstadiene-3,17-dione of reaction andcatalyzed reoxidation by of AcmB: the enzyme Dehydrogenation by DCPIP or of O as well as the reoxidation of DCPIPH by H O formed in situ or O . 4‐androstene2 ‐3,17‐dione to 1,4‐androstadiene2 ‐3,172 ‐2dione and reoxidation2 of the enzyme by DCPIP or O2 as well as the reoxidation of DCPIPH2 by H2O2 formed in situ or O2. Catalysts 2020, 10, 1460 3 of 13 2. Results 2.1. Selection of Carrier with the Highest Yield and Immobilization Efficiency 1 1 Purified AcmB with the specific activity of 12 mM min− mg− and protein concentration of 1 0.91 mg mL− was immobilized on mesoporous cellular foam (MCF) and Santa Barbara Amorphous (SBA-15) silica carriers functionalized with 3-aminopropyltrimethoysilane