Comparison between different hybrid organic/inorganic bioceramics containing microbial hydrolases, synthesized via the sol-gel process

Abstracts / Journal of Biotechnology 131S (2007) S98–S121

diminishes down to 40%. A similar behavior is appreciated for all E/S ratio analyzed. When esterification was carried out at 45 ◦ C, FFA phase containing 35% of GLA was obtained, yielding a recovery of 80%. A concentrate of 45% of GLA, with a 98% of GLA yield, was achieved with this technology.

S111

References Kondo, A., Ueda, M., 2004. Yeast cell-surface display-applications of molecular display. Appl. Microbiol. Biotechnol. 64, 28–40. Kim, H.C., Kim, H.J., Choi, W.B., Nam, S.W., 2006. Inulooligosaccharide production from inulin by Saccharomyces cerevisiae strain displaying cellsurface endoinulinase. J. Microbiol. Biotechnol. 16, 360–367.

doi:10.1016/j.jbiotec.2007.07.191 References Barre, D.E., 2001. Potential of evening primrose, borage, black currant, and fungal oils in human health. Annu. Nutr. Metab. 45, 47–57. Shimada, Y., Sugihara, A., Tominaga, A., 2001. Enzymatic purification of polyunsaturated fatty acids. J. Biosc. Bioeng. 1, 19–23.

doi:10.1016/j.jbiotec.2007.07.190

24. Comparison between different hybrid organic/inorganic bioceramics containing microbial hydrolases, synthesized via the sol-gel process Zoltan Dudas a,∗ , Beatrice Oros a , Gabriela Preda a , Monica Dragomirescu b , Janos Halasz c , Adrian Chiriac a a

a

West University of Timisoara, Pestalozzi 16, 300115 Timisoara, Romania b Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and Biotechnologies, Timisoara, Romania c University of Szeged, Department of Applied and Environmental Chemistry, Szeged, Hungary

Cell-surface display of heterologous proteins by using microorganisms has been widely utilized in various areas (Kondo and Ueda, 2004; Kim et al., 2006). Expression of proteins on the cell surface of Saccharomyces cerevisiae offers more advantages than other microbial systems, since it allows the folding and glycosylation of expressed heterologous eukaryotic proteins and can be subjected to many genetic manipulations. The endoxylanase gene from Bacillus sp. was expressed on the cell surface of S. cerevisiae by fusing with Aga2p linked to the membrane anchored protein, Aga1p. The endoxylanase gene was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTXYN (6.8 kb) was introduced to S. cerevisiae EBY100 cell and then yeast transformants were selected on the synthetic defined media lacking uracil. The endoxylanase gene under the control of GAL1 promoter was successfully expressed in the yeast transformant. When the yeast transformants were grown on YPD medium, the total activities of the enzymes reached about 1.92 unit/ml. To produce efficiently xylooligosaccharide from xylan, various reaction conditions such as temperature, amount of enzyme, substrate type and concentration, and reaction time were examined. When the oat spelts xylan was hydrolyzed by treatment of cell surface-displayed endoxylanase, it was found that xylose, xylobiose, and xylotriose were produced, and xylotriose was the main product. The optimized conditions for the production of xylooligosaccharide were as follows: temperature, 50 ◦ C; amount of enzymes, 10 unit; substrate concentration, 4%; reaction time, 6 h.

Catalysis by sol-gel doped materials - porous metaloxides confining active species, obtained by hydrolysis-condensation reactions of suitable precursors - has become in the last 20 years a prominent tool to synthesize a vast number of useful molecules both in the laboratory and in industrial biotechnologies (Pierre, 2004; Zhao et al., 2006). In the recent past sol-gel derived materials have been gradually gaining importance for several biological applications such as biocompatible implants, biomineralization, biocatalysts and biosensors. The sol-gel technique is particularly advantageous to these applications because of a number of desirable features it offers such as room temperature processability, control of surface architecture, ability to form films, monoliths, chemical inertness, thermal and photochemical stability, tunable porosity, and negligible swelling in aqueous and organic solvents and resistance to microbial attack (Kirk et al., 2002). The domains of organic chemistry and ceramic materials were merging and the new area of inorganic-organic hybrid materials had started. A true control and tailor of the chemical and physical properties of sol-gel molecular catalysts is the unique feature of these materials. The catalyst textural properties, shape and composition are thus to be planned by a rational choice of the preparation conditions, the precursors and the templating agents (Bommarus and Polizzi, 2006; Cao, 2005). The main objective of the present work was to apply hybrid silica composites for immobilization of practically important hydrolases, namely proteases and amylases. The selected enzymes differ not only in the specificity, but also in their stability and conditions for functioning (optimal pH and ionic strength of aqueous media). It was examined whether these factors influence the immobilization and activity of immobilized enzymes. The experimental data showed that both enzymes could be successfully entrapped in hybrid silica xerogels.

23. Production of xylan-based oligosaccharides by cell-surface engineered yeast Hyun-Jin Kim a,∗ , Jae Hyung Lee a , Soo-Wan Nam b Department of Biomaterial Control (BK21 program), 995 Eomgwangno, Busan jin-gu, Dong-Eui University, 614-714 Busan, Korea, South b Dept. Biomaterial Control and Dept. Biotechnology and Bioengineering, Dong-Eui University, Busan, 614-714, Korea, Republic of

S112

Abstracts / Journal of Biotechnology 131S (2007) S98–S121

References Bommarus, A.S., Polizzi, K.M., 2006. Novel biocatalysts: Recent developments. Chem. Eng. Sci. 61, 1004. Cao, L., 2005. Immobilised enzymes: science or art? Curr. Opin. Chem. Biol. 9, 217. Kirk, O., Borchert, T.V., Fuglsang, C.C., 2002. Industrial enzyme applications. Curr. Opin. Biotechnol. 13, 345. Pierre, A.C., 2004. The sol-gel encapsulation of enzymes. Biocatal. Biotransform. 22 (3), 145. Zhao, X.S., Bao, X.Y., Guo, W., Lee, F.Y., 2006. Immobilizing catalysts on porous materials. Mater. Today 9 (3), 32.

doi:10.1016/j.jbiotec.2007.07.192 25. Biocatalysts entrapped in silica gels Monica Dragomirescu a,∗ , Teodor Vintila a , Zoltan Dudas b , Beatrice Vlad-Oros b , Gabriela Preda b a

BANAT”S University of Agricultural Science and Biotechnology and Veterinary Medicine Timisoara, Calea Aradului, 119, 300645 Timisoara, Romania b West University of Timi¸soara, Faculty of Chemistry-BiologyGeography, Timisoara, Romania Entrapment of biocatalysts (as enzymes and whole cells) in silica gels fabricated using sol-gel methods by polymerization of hybrid organic-inorganic precursors makes the bond between living and not living world, between biological systems and modern inorganic materials (Avnir et al., 1994; Livage et al., 2001). The sol-gel methods are very attractive for applications in biotechnology because hybrid silica glasses are obtained under mild chemical conditions, at room temperature, at which biomolecules are active. Silica mesoporous matrices are biocompatible with biomolecules and living cells, allow the free flow of small substrate and product molecules through the pores, are chemically inert, resistant to microbial attack, have a stabile structure and exhibit improved mechanical strength (Livage et al., 2001; Ferreira et al., 2003). Entrapped enzymes and cells in silica gels have been shown to retain their biocatalitic activity, to remain accessible to external reagents and to often exhibit increased stability at environmental effects (Ferreira et al., 2003; Dragomirescu et al., 2006). Biotechnological processes are using in many cases immobilized biocatalysts because they are easy to separate and reuse. Silica gels provide an aqueous environment close to that observed in biological media and the entrapment in silica glasses directly of whole bacteria cells, really very small bioreactors, might be advantageous (Livage et al., 2001; Fennouh et al., 1999; Coiffier et al., 2001; Kabaivanova et al., 2005). In this way we can immobilize the enzymes in their natural environment and avoid enzyme separation and purification procedures. In this study we have investigated the viability and the functionality of some Bacillus sp. cells entrapped in silica gels, obtained by using different alkoxide precursors (TEOS, MTES, etc.) and a two-step procedure, the acid hydrolysis of alkoxisilane, followed by encapsulation at neutral pH. The enzymatic (amylase) activity of free and entrapped bacteria cells was then

analyzed. We have investigated the stability of the immobilized biocatalysts and its ability to produce enzymes under repeated batch cultivation conditions. References Avnir, D., Braun, S., Lev, O., Ottolenghi, M., 1994. Enzymes and other proteins entrapped in sol-gel materials. Chem. Mater. 6, 1605–1614. Coiffier, A., Coradin, T., Roux, C., Bouvet, O.M.M., Livage, J., 2001. Solgel encapsulation of bacteria: a comparison between alkoxide and aqueous routes. J. Mater. Chem. 11, 2039–2044. Dragomirescu, M., Preda, G., Oros, B., Peter, F., 2006. Rev. Chim., Bucuresti 57 (6), 610. Ferreira, L., Ramos, M.A., Dordick, J.D., Gil, M.H., 2003. Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis protease (Subtilisin Carlsberg). J. Molec. Catal. B: Enzymatic 21, 189–199. Fennouh, S., Guyon, S., Jourdat, C., Livage, J., Roux, C., 1999. C.R. Acad. Sci. Paris, t.2, Serie IIc, 625. Kabaivanova, L., Dobreva, E., Dimitrov, P., Emanuilova, E., 2005. Immobilization of cells with nitrilase activity from a thermophilic bacterial strain. J. Ind. Microbiol. Biotechnol. 32, 7–11. Livage, J., Coradin, T., Roux, C., 2001. J. Phys.: Condens. Matter. 13, 673.

doi:10.1016/j.jbiotec.2007.07.193 26. Production of fructo-oligosaccharides from sucrose by two levansucrases from Pseudomonas aurantiaca and Zymomonas mobilis Sun-Ho Byun a , Woo-Cheul Han a , Soon Ah Kang c , Chul Ho Kim b , Ki-Hyo Jang a,∗ a

Kangwon National University, Samchuck, Korea, Republic of Daejeon, Korea, Republic of c Seoul University of Venture & Information, Seoul, Korea, Republic of b KRIBB,

The use of levansucrase (EC 2.4.1.10) as a biocatalyst in fructan (␤-D-fructofuranosyl polymers with a terminal Dglucosyl moiety) synthesis has been the object of increasing interest in recent years. The enzyme splits sucrose, releasing fructose and glucose and concomitantly polymerizes the fructose molecules to acceptor molecules. The resulting fructose homopolymer, levan and its fructo-oligosaccharides, is attractive biopolymer due to its potential usage as prebiotics. In this study, we optimized the conditions for the formation of fructooligosaccharides from sucrose by transfructosylation reaction using levansucrase of Pseudomonas aurantiaca and Zymomonas mobilis. Fructo-oligosaccharides formation from both levansucrases was sucrose concentration dependent; high sucrose concentration was favorable. Optimum conditions for the formation of fructo-oligosaccharides (degree of polymerization (DP) of 3–6) were: 40 ◦ C(45 ◦ C for Pseudomonas aurantiaca), pH7, and 70% of sucrose. Under optimized conditions, yields of fructo-oligosaccharides were 24–26%. doi:10.1016/j.jbiotec.2007.07.194