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Sucrose Phosphorylase As a Cross-Linked Sucrose phosphorylase as a cross-linked enzyme aggregate: Improved thermal stability for industrial applications an G Cerdobbel, Karel de Winter, Tom Desmet, Wim Soetaert To cite this version: an G Cerdobbel, Karel de Winter, Tom Desmet, Wim Soetaert. Sucrose phosphorylase as a cross-linked enzyme aggregate: Improved thermal stability for industrial applications. Biotechnology Journal, Wiley-VCH Verlag, 2010, 5 (11), pp.1192. 10.1002/biot.201000202. hal-00579475 HAL Id: hal-00579475 https://hal.archives-ouvertes.fr/hal-00579475 Submitted on 24 Mar 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biotechnology Journal Sucrose phosphorylase as a cross-linked enzyme aggregate: Improved thermal stability for industrial applications For Peer Review Journal: Biotechnology Journal Manuscript ID: biot.201000202.R1 Wiley - Manuscript type: Research Article Date Submitted by the 19-Aug-2010 Author: Complete List of Authors: Cerdobbel, An De Winter, Karel; Ghent University Desmet, Tom; Ghent University Soetaert, Wim; Ghent University Primary Keywords: White/Industrial Biotechnology Secondary Keywords: Biocatalysis Keywords: Immobilization Wiley-VCH Page 1 of 21 Biotechnology Journal 1 2 3 4 5 6 Sucrose phosphorylase as cross-linked enzyme aggregate: Improved thermal 7 8 stability for industrial applications 9 10 11 12 * 13 An Cerdobbel , Karel De Winter, Tom Desmet, Wim Soetaert 14 15 16 17 18 Centre of Expertise for Industrial Biotechnology and Biocatalysis, Department of 19 20 Biochemical andFor Microbial Peer Technology, Review Faculty of Biosciences Engineering, 21 22 Ghent University, Coupure Links 653, B-9000 Ghent, Belgium 23 24 25 26 27 *Corresponding author: 28 29 Tel.: +3292649921; Fax: +3292646032; E-mail: [email protected] 30 31 32 33 34 Keywords 35 36 37 CLEA, sucrose phosphorylase, thermostability, enzyme immobilization, 38 39 biocatalysis 40 41 42 43 44 Abbreviations 45 46 SPase: sucrose phosphorylase; G1P: α-glucose-1-phosphate; GA: glutaraldehyde; 47 48 CLEA: cross-linked enzyme aggregate 49 50 51 52 53 54 55 56 57 58 59 60 Wiley-VCH Biotechnology Journal Page 2 of 21 1 2 3 4 5 6 Abstract 7 8 Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety 9 10 of small molecules using sucrose as a cheap but efficient donor substrate. The low 11 12 13 thermostability of the enzyme, however, limits its industrial applications, as these 14 15 are preferably performed at 60 °C to avoid microbial contamination. Cross-linked 16 17 18 enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium 19 20 adolescentis wereFor found to Peerhave a temperature Review optimum that is 17 °C higher than 21 22 that of the soluble enzyme. Furthermore, the immobilized enzyme displays an 23 24 25 exceptional thermostability, retaining all of its activity after one week incubation 26 27 at 60 °C. Recycling of the biocatalyst allows its use in at least ten consecutive 28 29 reactions, which should dramatically increase the commercial potential of its 30 31 32 glycosylating activity. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Wiley-VCH Page 3 of 21 Biotechnology Journal 1 2 3 4 5 6 1. Introduction 7 8 Enzymes are powerful biocatalysts that find increasing applications in various 9 10 industrial processes. The major advantages of the use of enzymes for chemical 11 12 13 transformations are their high chemo-, regio- and stereospecificity, as well as their 14 15 environmentally friendly properties. Unfortunately, natural enzymes are often not 16 17 18 optimally suited for industrial applications, which can be hampered by their lack 19 20 of long-term stabilityFor under Peer process conditions Review and by their difficult recovery and 21 22 recycling. These drawbacks can, however, be overcome by immobilization of the 23 24 25 enzymes [1-4]. 26 27 28 29 Cross-linked enzyme aggregates (CLEAs) have emerged as a novel class of 30 31 32 immobilized biocatalysts for use in both aqueous and non-aqueous environments 33 34 [5]. Such preparations are obtained by the physical aggregation of the enzymes 35 36 37 followed by chemical cross-linking (Fig. 1). This procedure allows enzymes to be 38 39 immobilized without the use of a carrier, which not only decreases the cost but 40 41 also avoids “dilution” of the enzymes’ activity [6]. CLEAs share their beneficial 42 43 44 properties with cross-linked enzyme crystals (CLECs) but do not require tedious 45 46 crystallization procedures [7, 8]. 47 48 49 50 51 Sucrose phosphorylase (SPase) catalyses the reversible phosphorolysis of sucrose 52 53 into α-D-glucose-1-phosphate (G1P) and fructose. This enzyme is mainly found in 54 55 lactic acid bacteria and bifidobacteria, where it contributes to an efficient energy 56 57 58 metabolism [9]. SPase is formally classified as a glycosyl transferase (EC 2.4.1.7), 59 60 Wiley-VCH Biotechnology Journal Page 4 of 21 1 2 3 4 5 6 although it belongs to glycoside hydrolase family 13 [10] and follows the typical 7 8 double displacement mechanism of retaining glycosidases [11]. The crystal 9 10 structure of the enzyme from Bifidobacterium adolescentis has been determined 11 12 13 and consists of a ( β/α)8 barrel containing two carboxylic amino acids as catalytic 14 15 residues [12]. 16 17 18 19 20 A number of practicalFor applications Peer have Reviewbeen developed for SPase [13, 14]. The 21 22 enzyme has long been used for the production of G1P, an efficient donor for 23 24 25 chemical and enzymatic glycosylation reactions [15]. Furthermore, SPase can also 26 27 transfer a glucosyl group to different carbohydrate and non-carbohydrate 28 29 acceptors thanks to its broad substrate specificity [16, 17]. Goedl et al. , for 30 31 32 example, have developed an exceptionally efficient and selective process for the 33 34 production of glyceryl α-D-glucoside, a moisturizing agent for cosmetics that is 35 36 37 marketed under the tradename Glycoin® [18, 19]. 38 39 40 41 Unfortunately, no SPases have so far been reported that can resist the extreme 42 43 44 process conditions that are preferred by the industry. Indeed, carbohydrate 45 46 conversions are typically performed at elevated temperatures (60 °C or higher), 47 48 mainly to avoid microbial contamination [20-22]. Here, we describe the 49 50 51 production of CLEAs of the SPase from B. adolescentis that display exceptional 52 53 thermal and operational stability. 54 55 → Fig. 1 56 57 58 59 60 Wiley-VCH Page 5 of 21 Biotechnology Journal 1 2 3 4 5 6 2. Materials and methods 7 8 2.1. Materials 9 10 Tert -butyl alcohol, glutaraldehyde, and sodium borohydride were purchased from 11 12 13 Aldrich-Chemie, Fisher and Acros, respectively. All other reagents were 14 15 purchased from Sigma-Aldrich. 16 17 18 19 20 2.2. Enzyme productionFor Peer Review 21 22 The SPase gene from B. adolescentis LMG 10502 was recombinantly expressed 23 24 25 in E. coli XL10-Gold, under control of the constitutive promotor P34 [23]. The 26 27 construction of the corresponding expression vector pCXhP34_SPBa will be 28 29 described elsewhere. Transformed cells were cultivated in 1 l shake flasks at 37 30 31 −1 32 °C using LB medium supplemented with 100 mg l ampicillin. After 8 h of 33 34 expression, the cells were harvested by centrifugation (7000 rpm, 4 °C, 20 min). 35 36 37 Crude enzyme preparations were prepared by enzymatic lysis of frozen pellets 38 39 using the EasyLyse Bacterial Protein Extraction Solution (Epicentre). Cell debris 40 41 was removed by centrifugation (12000 rpm, 4 °C, 30 min). The crude enzyme 42 43 44 preparation was heat purified by incubation at 60 °C for 60 min. Denaturated 45 46 proteins were removed by centrifugation (12000 rpm, 4 °C, 15 min). 47 48 49 50 51 2.3. CLEAs production 52 53 Aggregates of SPase were prepared by adding 6 ml of tert -butyl alcohol under 54 55 agitation to 4 ml of heat-purified SP enzyme (1.2 mg ml -1) at pH 7. After 30 min, 56 57 58 varying amounts of a 25 % (v/v) glutaraldehyde solution were added to cross-link 59 60 Wiley-VCH Biotechnology Journal Page 6 of 21 1 2 3 4 5 6 the enzyme aggregate, and the mixture was kept under stirring for 15, 30, 60 or 7 8 120 min. Reduction of the formed imine bond was achieved by adding 10 ml of a 9 10 solution containing 1 mg ml -1 sodium borohydride in 0.1 M sodium bicarbonate 11 12 13 buffer at pH 10. After 15 min, another 10 ml was added and allowed to react for 14 15 15 min. Finally, the CLEAs were separated by centrifugation (15 min at 12000 16 17 18 rpm) and washed five times with 0.1 M phosphate buffer at pH 7. All the steps 19 20 were performedFor in a thermoshaker Peer (Eppendorf) Review at 750 rpm and 4 °C. The 21 22 immobilization yield is defined as the ratio of the activity detected in the CLEA 23 24 25 preparation and that present in the original enzyme solution.
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