449676819bc33e11e408fabac9027bed pmc_34299429.pdf 1c0d38bf41de7d8e8061700268411b3c60510536 pmc_34299429.pdf 7d09f9da57ed17f4d533490394f9f3bca53e78a48c33bbaead2d10f45154b24e pmc_34299429.pdf Title: Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of -Galactosidase Subject: Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from -galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected -galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications. Keywords: -galactosidase; nanoimmobilization; biomineralization Author: Luigi Tavernini, Oscar Romero, Carla Aburto, Fernando López-Gallego, Andrés Illanes and Lorena Wilson Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Jul 22 10:42:46 2021 CEST ModDate: Thu Jul 22 10:58:09 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2566103 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- HJOMHV+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 HHFREY+EURM7 Type 1 Builtin yes yes no 15 0 DRZUJM+VnURWPalladioL Type 1 Custom yes yes no 19 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 23 0 PNLMVK+URWPalladioL-Ital Type 1 Custom yes yes no 27 0 BAGOID+URWPalladioL-Bold Type 1 Custom yes yes no 32 0 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subsection.2.1 Item: Title: Biomineralization of -Galactosidase with Calcium Destination: subsection.2.2 Item: Title: Effect of pH on the Biomineralization of -Galactosidase Destination: subsection.2.3 Item: Title: Thermal Stability of the -Galactosidase Nanocrystals Destination: subsection.2.4 Item: Title: In Silico Prediction of Metal-Ion Binding Regions Destination: subsection.2.5 Item: Title: Characterization of the -galactosidase Nanocrystals Destination: subsection.2.6 Item: Title: Synthesis of Galacto-Oligosaccharides Destination: subsection.2.7 Item: Title: Materials and Methods Destination: section.3 Children: Item: Title: Chemicals and Reagents Destination: subsection.3.1 Item: Title: Hydrolytic Activity of -Galactosidase Destination: subsection.3.2 Item: Title: Biomineralization of -Galactosidase Destination: subsection.3.3 Item: Title: Thermal Stability of the -Galactosidase Bioinorganic Nanocrystals Destination: subsection.3.4 Item: Title: In Silico Prediction of Calcium Binding Sites Destination: subsection.3.5 Item: Title: Characterization of the -Galactosidase Bioinorganic Nanocrystals Destination: subsection.3.6 Item: Title: Synthesis of Galacto-Oligosaccharides Destination: subsection.3.7 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: section.4 Info: Title: Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of -Galactosidase Author: Luigi Tavernini, Oscar Romero, Carla Aburto, Fernando López-Gallego, Andrés Illanes and Lorena Wilson Subject: Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from -galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected -galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications. 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