ABSTRACTS

New Biotechnology Volume 25S September 2009

2.3.094 Glycosidase enzymes stabilization through immobilization onto nanoparticulated bimodal organosilicas M. Tortajada 1, , D. Ramn 1 , D. Beltrn 2 , P. Amors 2
1

2.3.095 Engineering of a Bacillus independent of calcium -amylase whose stability is

M. Ghollasi , K. Khajeh, H. Naderi-Manesh

Tarbiat Modares University, Tehran, Islamic Republic of Iran

BIOPOLIS, S.L., Paterna, Spain Universitat de Valencia, Spain

Glycosidases are hydrolases capable of releasing monoterpenes from diglycoside conjugates. For their biotechnological interest in different agrochemical sectors, including oenological applications such as varietal avour improvement, several glycosidases ( -L-arabinofuranosidases, -D-apiosidases, -L-rhamnosidases and -D-glucosidases) from different microbial sources have been characterized. However, some of these enzymes are partially inhibited by typical winemaking conditions such as low pH and high ethanol and glucose concentration. Conversely, immobilized enzymes are commercially advantageous because of possibility of reuse, ease of separation, and potential increase in thermal and pH stability. Recently, mesoporous silicas have been used as promising host materials because of their large pore sizes and open nature of the inorganic support. Covalent immobilization of the enzymes - Larabinofuranosidase (ABF) and -glucosidase (BGL) has been accomplished inside bimodal porous organosilicas. The enzymes were produced by batch fermentation of genetically engineered Saccharomyces cerevisiae yeast strains. Different silica supports of the HPNO family showing surface nitrogen moieties were assayed. The enzymes were later covalently bound to the silica surface by carbodiimide addition. Individual and co-immobilized active preparations were produced and characterized for their kinetic constants, optimum pH and temperature, thermal stability and resistance to common inhibitors such as ethanol and glucose. Materials were further characterized by XRD techniques, TEM, and N2 isotherms. The HPNO materials show quick and high enzyme loading and retain signicant enzyme activity. Moreover, the optimum pH is shifted towards more acid conditions and increased thermal and ethanol resistance are found. The functionalized materials retain their structure and open nature and ABF/BGL enzymes covalently bound are located inside the larger pores, close to the silica surface. This work shows the ability of aminated porous silica-based networks for glycosidic enzyme immobilization. Their open morphology, combined to highly accessible amino groups, is the key to achieve the high and quick enzyme loading. The covalent immobilization promotes the optimum enzymatic working conditions shift towards lower pH values and higher temperatures than the free enzyme, together with an increased resistance to ethanol concentration. Such an improved enzymatic performance is probably because of the presence of amine groups at the surface which generate an increased local pH, together with the stability provided by the covalent linkages. These improved working conditions, close to those of winemaking industrial processes, are favorable for oenological applications. The synthesis strategy here presented is likely to be adapted to the preparation of a diversity of hybrid materials of biotechnological interest. doi:10.1016/j.nbt.2009.06.477
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Successful industrial use of amylases requires that they are sufciently stable and active at application conditions, for example, at high temperature in starch-liquefaction process. In this study, site-directed mutagenesis was used to enhance the thermostability and calcium independency of a mesophilic -amylase from Bacillus megaterium WHO. Mutations (A53S and H58I) were designed at the calcium-binding site. Kinetic and thermostability parameters were calculated in mutants and compared with the wild-type. In the presence of calcium, the afnity of starch to the enzymes (wild-type and mutants) was increased. In comparison to the wildtype calcium ion improved the catalytic efciency, kcat /Km , and the half-life (at 60 C) of A53S mutant. In A53S, the dependence of halflife on calcium concentration showed that the enhanced calcium binding is likely to be responsible for the increased stability. In contrast, the present study revealed that the H58I mutant retained the high thermostability in spite of calcium independency. In addition, thermodynamic parameters of amylolytic reaction exhibited an increase in the activation energy and the entropy of the system. Kinetics of Irreversible thermal inactivation suggests that the activation energy increased by 1.4-fold in the most stable variant. doi:10.1016/j.nbt.2009.06.478

2.3.096 Comparative analysis of bacterial whole-cell biocatalysts with para-nitrophenyl palmitate assay using methanol, ethanol and no alcohol for biodiesel production V.E. Lpez , Y. Lpez, M.D.C. Cruz Lpez, J. Dinorn Tllez Girn
Centro de Investigacin en Biotecnologa Aplicada del I.P.N., Tepetitla de Lardizabal, Mexico

Biodiesel is a mixture of mono-alkyl esters obtained from vegetable oils in alkali catalyzed transesterication process. Although alkali transesterication promotes high conversion rates of triglycerides to their corresponding alkali esters within short reaction times, the process has several drawbacks, including energy intensiveness and difculty of glycerol recovery, removal of alkaline catalyst from product and treatment of waste water. Also biodiesel production from unrened feedstocks and greases is not appropriated as a result of their properties and/or free fatty acid content. Enzymatic transesterication process under mild conditions can overcome those problems of the chemical process; however the cost of lipase enzyme remains a barrier for industrial implementation. Hence, the utilization of whole-cell biocatalyst could be both more costeffective and more advantageous for industrial application. In this sense, our research group had isolated several microorganisms that presented lipolytic activity with the potential of been used as a whole-cell biocatalyst in biodiesel production. One bacterial iso-