Screening of α-Amylase Suitable for Evaluating the Degree of Starch Retrogradation

To estimate the degrees of starch retrogradation in the complex foods, an enzymatic method using α-amylase from Bacillus subtilis was proposed in the previous report (Tsuge, H. et al.: Starch/Stärke 42 (1990), 213–216). However, actual digestibility of the enzyme for the native starch granules was not checked at that time. A comparative study to see the digestibility of native starch granules was carried out using four different α-amylase preparations and digestion of retrograded wheat starch was tested by two α-amylase preparations. Pancreas α-amylase preparation digested some native starch granules to a great extent, while Aspergillus oryzae enzyme did not digest native starch granules virtually. In conclusion, α-amylase preparation from A. oryzae was an ideal enzyme as the tool to distinguish between raw and gelatinized starches. It was justified for the use of A. oryzae enzyme as well as B. subtilis α-amylase to evaluate the retrograded starch contents in the complex foods.     

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed. Different genotype variants induced by mutagens are encapsulated in microemulsions and cultured to secrete proteins inside the emulsions. The secreted protein of interest is detected as a fluorescence signal by the fluorescent immunosensor quenchbody (Q-body), and a cell sorter is used to select emulsions containing improved protein-secreting strains based on the fluorescence intensity. The concept of the screening method is demonstrated by culturing Corynebacterium glutamicum in emulsions and detecting the secreted proteins. Finally, productive strains of fibroblast growth factor 9 (FGF9) are screened, and the FGF9 secretion increased threefold compared to that of parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag. This is a highly simple process that requires only the addition of a Q-body to the medium and does not require the addition of any substrates or chemical treatments. Furthermore, this method shortens the development period of industrial strains for biopharmaceutical protein production.