Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions

BACKGROUND: Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts. RESULTS: Two types of procedures were used for the preparation of biocatalysts from purified wild type LS (WT LS) B. subtilis and the R360K and Y429N LS mutants: purified enzymes aggregated with glutaraldehyde (cross-linked enzyme aggregates: CLEAs), and covalently immobilized enzymes in Eupergit C(®). The biocatalysts were characterized and used for fructoside synthesis using xylose as an acceptor model. CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes. The specific activity of CLEAs prepared from wild type LS (44.9 U/mg of CLEA), R360K (56.5 U/mg of CLEA) and Y429N (1.2 U/mg of CLEA) mutants were approximately 70, 40 and 200-fold higher, respectively, than equivalent Eupergit C(® )immobilized enzyme preparations (U/mg of Eupergit), where units refer to global LS activity. In contrast, the specific activity of the free enzymes was 160, 171.2 and 1.5 U/mg of protein, respectively. Moreover, all CLEAs had higher thermal stability than corresponding soluble enzymes. In the long term, the operational stability was affected by levan synthesis. CONCLUSION: This is the first report of cross-linked transglycosidases aggregates. CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs) reported in the literature. CLEAs from R360K and Y429N LS mutants were particularly suitable for fructosyl-xyloside synthesis as the absence of levan synthesis decreases diffusion limitation and increases operational stability

New insights in bacillus subtillis levansucrase mechanism and applications

B. subtilis levansucrase (SacB) is a widely studied glycoside hydrolase from Family 68 family. Although reports on SacB properties date back to the 70’s (Chambert & Gonzy-Tréboul, 1976), questions regarding levan synthesis mechanism are still open. These questions refer to the factors influencing reaction specificity, including the effect of sucrose and levan hydrolysis, product structure and levan molecular weight. In this conference we review recent findings regarding the modulating effect of SacB concentration on levan molecular weight distribution (Porras-Domínguez et al., 2015; Raga-Carbajal et al., 2016). In effect, we demonstrated that high enzyme concentrations (\u3e1.0 µM), direct levan synthesis exclusively to low molecular weight products (av 7.6 KDa), while low enzyme concentrations (\u3c 0.1µM) favor the synthesis of a high molecular weight levan fraction (\u3e2000 kDa). From a detailed HPAEC-PAD analysis of product evolution, a shift from a clear non-processive elongation mechanism at high protein concentrations to a -most likely- processive mechanism when low protein concentrations are used in the reaction. Trough calorimetric experiments we demonstrate that these changes in enzyme performance do not involve protein-protein interactions (Raga-Carbajal et al., 2016). We demonstrated, through an extensive characterization of the levan hydrolysis reaction by SacB, that the wide diversity of products derives also from fructosyl transfer to free sugars available from sucrose and levan hydrolysis. Actually, levan is an efficient fructosyl donor for fructosylation reactions, in which FOS such as levanbiose, inulobiose, blastose, …, are formed (Méndez-Lorenzo et al., 2015). The efficiency of SacB fructosylation with levan as donor was applied for the synthesis of blastose, a sucrose analogue with potential prebiotic properties. For this reaction, fructose was transferred to trehalose to produce a (2-6) fructosylated trehalose, which was later hydrolysed by trehalase to yield blastose (Miranda-Molina et al, 2017). Up to now there is not an efficient enzyme for the synthesis of levan-type FOS, in spite of intensive efforts to modify SacB or other levansucrases specificity by site directed mutagenesis. For this purpose, after a complete characterization of a combined bi-enzymatic reaction between SacB and an endolevanase produced by B.licheniformis. (LevB1) (Porras-Domínguez et al., 2014) we designed a fusion enzyme containing both activities. This fusion enzyme is able to produce levan-type FOS from sucrose, with molecular weights in the range of DP2 to DP10 including mainly 1-kestose, 6-kestose, neokestose, levanbiose and blastose, with 40% w/w yields. Chambert, R., & Gonzy-Tréboul, G. (1976). European Journal of Biochemistry / FEBS, 62(1), 55–64. Méndez-Lorenzo, L., Porras-Domínguez, J. R., Raga-Carbajal, E., Olvera, C., Rodríguez-Alegría, M. E., Carrillo-Nava, E.. López Munguía, A. (2015). PLoS ONE, 10(11), 1–15. Miranda-Molina, A., Castillo, E., & Lopez Munguia, A. (2017). Food Chemistry, 227, 202–210. Porras-Domínguez, J. R., Ávila-Fernández, Á., Miranda-Molina, A., Rodríguez-Alegría, M. E., & Munguía, A. L. (2015). Carbohydrate Polymers, 132(October), 338–344. Porras-Domínguez, J. R., Ávila-Fernández, Á., Rodríguez-Alegría, M. E., Miranda-Molina, A., Escalante, A., González-Cervantes, R., López Munguía, A. (2014). Process Biochemistry, 49(5), 783–790. Raga-Carbajal, E., Carrillo-Nava, E., Costas, M., Porras-Dominguez, J., López-Munguía, A., & Olvera, C. (2016). Glycobiology, 26(4), 377–385

Biotechnology in tomorrow food

Tema del mesEste artículo forma parte de uno de los temas (conferencias) impartidos dentro del programa "La Biotecnología llega a Palacio" en referencia al Palacio de Minería, recinto en el que anualmente la feria tiene lugar. El objetivo de esta plática, como el de este artículo, es traer a la atención y análisis del lector la forma en la que la tecnología ha influenciado nuestra alimentación y lo que comemos, con particular énfasis en la influencia de la biotecnología en lo que comemos. El objetivo es demasiado ambicioso para las limitaciones de espacio, sin embargo se hace una breve reseña histórica que inicia en los mismos principios de la especie humana, para llegar en unos cuantas líneas a los paradigmas de la era actual. Es en ese contexto que se hacen algunos planteamientos del autor sobre el actual dilema: OGMs o no OGMs.This article was written in the context of the XX International Book Fair celebrated in México in February 2014. Among the activities involved in the program: Biotechnology reaches the Palace, (in reference to the Palacio de Minería, where the fair takes place each year) the idea of this conference in particular was to discuss the way technology has influenced what we eat, with emphasis in the biotechnological applications to food production. After a brief historical review, starting from the very beginning of the human era the analysis is taken to the actual environmental and food security paradigms that the world is facing. In this context some of the implications and consequences of modern biotechnology are exposed, particularly the question: to OGM or not to OGM

Evaluation of cross-linked aggregates from purified <it>Bacillus subtilis </it>levansucrase mutants for transfructosylation reactions

Abstract Background Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts. Results Two types of procedures were used for the preparation of biocatalysts from purified wild type LS (WT LS) B. subtilis and the R360K and Y429N LS mutants: purified enzymes aggregated with glutaraldehyde (cross-linked enzyme aggregates: CLEAs), and covalently immobilized enzymes in Eupergit C®. The biocatalysts were characterized and used for fructoside synthesis using xylose as an acceptor model. CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes. The specific activity of CLEAs prepared from wild type LS (44.9 U/mg of CLEA), R360K (56.5 U/mg of CLEA) and Y429N (1.2 U/mg of CLEA) mutants were approximately 70, 40 and 200-fold higher, respectively, than equivalent Eupergit C® immobilized enzyme preparations (U/mg of Eupergit), where units refer to global LS activity. In contrast, the specific activity of the free enzymes was 160, 171.2 and 1.5 U/mg of protein, respectively. Moreover, all CLEAs had higher thermal stability than corresponding soluble enzymes. In the long term, the operational stability was affected by levan synthesis. Conclusion This is the first report of cross-linked transglycosidases aggregates. CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs) reported in the literature. CLEAs from R360K and Y429N LS mutants were particularly suitable for fructosyl-xyloside synthesis as the absence of levan synthesis decreases diffusion limitation and increases operational stability.</p

Thermodynamically controlled chemoselectivity in lipase-catalyzed aza-Michael additions

Chemoselective synthesis of N-protected beta-amino esters involving lipase-catalyzed aza-Michael additions and alpha,beta-unsaturated precursors is mainly hampered by the two electrophilic sites present on these compounds. In order to control the chemoselectivity a solvent engineering strategy based on the thermodynamic behaviour of products in media of different polarity was designed. This strategy allowed to obtain aza-Michael adducts from benzylamine and different acrylates with high selectivity. In almost all reactions carried out in n-hexane, a non-polar solvent, aminolysis was avoided while the corresponding Michael adducts were exclusively synthesized in 53-78% yields. On the contrary, in reactions carried out in a polar solvent such as 2-methyl-2-butanol the aminolysis products were favoured. Thermodynamic analyses of these processes using the COSMO-RS method helped to understand some of the key factors affecting chemoselectivity and confirmed that a reliable estimation of the thermodynamic interactions of solutes and solvents allows an adequate selection of a reaction media that may lead to chemoselectivity

Binding assays of native IslA and the truncated form IslA3 with non induced CW28 cells

Molecular weight control (M). Gel deals with native IslA (line 1) while gel with IslA3 (line 1). In both gels line 2 refers to the non induced CW28 cells; line 3 refers to the washed non induced cells after contact with the protein; and line 4 refers to the protein solution after contact with non induced cells.<p><b>Copyright information:</b></p><p>Taken from "Functional role of the additional domains in inulosucrase (IslA) from CW28"</p><p>http://www.biomedcentral.com/1471-2091/9/6</p><p>BMC Biochemistry 2008;9():6-6.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2270844.</p><p></p