Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications.

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.

Immobilization strategies of photolyases: Challenges and perspectives for DNA repairing application.

Photolyases are enzymes that repair DNA damage caused by solar radiation. Due to their photorepair potential, photolyases added in topical creams and used in medical treatments has allowed to reverse skin damage and prevent the development of different diseases, including actinic keratosis, premature photoaging and cancer. For this reason, research has been oriented to the study of new photolyases performing in extreme environments, where high doses of UV radiation may be a key factor for these enzymes to have perfected their photorepair potential. Generally, the extracted enzymes are first encapsulated and then added to the topical creams to increase their stability. However, other well consolidated immobilization methods are interesting strategies to be studied that may improve the biocatalyst performance. This review aims to go through the different Antarctic organisms that have exhibited photoreactivation activity, explaining the main mechanisms of photolyase DNA photorepair. The challenges of immobilizing these enzymes on porous and nanostructured supports is also discussed. The comparison of the most reported immobilization methods with respect to the structure of photolyases show that both covalent and ionic immobilization methods produced an increase in their stability. Moreover, the use of nanosized materials as photolyase support would permit the incorporation of the biocatalyst into the target cell, which is a technological requirement that photolyase based biocatalysts must fulfill.

Immobilization of Aspergillus oryzae ß-galactosidase in cation functionalized agarose matrix and its application in the synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized in in-house quaternary ammonium agarose (QAA) and used for the first time in the synthesis of lactulose. A biocatalyst was obtained with a specific activity of 24,690 IUH∙g-1; protein immobilization yield of 97% and enzyme immobilization yield of 76% were obtained at 30 °C in 10 mM phosphate buffer pH 7 for standard size agarose at 100 mgprotein∙gsupport-1 which the maximum protein load of QAA. Highest yield and specific productivity of lactulose were 0.24 g∙g-1 and 9.78 g∙g-1 h-1 respectively, obtained at pH 6, 100 IUH∙g lactose-1 enzyme/lactose ratio and 12 lactose/fructose molar ratio. In repeated-batch operation with the immobilized enzyme, the cumulative mass of lactulose per unit mass of contacted protein and cumulative specific productivity were higher than obtained with the soluble enzyme since the first batch. After enzyme activity exhaustion, the enzyme was desorbed and QAA support was reused without alteration in its maximum enzyme load capacity and without detriment in yield, productivity and selectivity in the batch synthesis of lactulose with the resulting biocatalyst. This significantly decreases the economic impact of the support, presenting itself as a distinctive advantage of immobilization by ionic interaction.