Pyranose 2-oxidases catalyze the oxidation of various pyranose sugars at the C2 position. However, their potential application for detecting sugars other than glucose in blood is hindered by relatively high activity towards glucose. In this study, in order to find a mutant enzyme with enhanced specificity for 1,5-anhydro-D-glucitol (1,5-AG), which is a biomarker for diabetes mellitus, we conducted site-directed mutagenesis of pyranose 2-oxidase from the basidiomycete Phanerochaete chrysosporium (PcPOX). Considering the three-dimensional structure of the substrate-binding site of PcPOX and the structural difference between glucose and 1,5-AG, we selected alanine 551 of PcPOX as a target residue for mutation. Kinetic studies of the 19 mutants of PcPOX expressed as recombinant proteins in E. coli revealed that the ratio of kcat/Km for 1,5-AG to kcat/Km for glucose was three times higher for the A551L mutant than for wild-type PcPOX. Although the A551L mutant has lower specific activity towards each substrate than the wild-type enzyme, its increased specificity for 1,5-AG makes it a promising lead for the development of POX-based 1,5-AG detection systems.
Phosphoryl oligosaccharides of calcium (POs-Ca) is a calcium salt of phosphoryl maltooligosaccharides made from potato starch. POs-Ca is highly water-soluble and can supply both the calcium ion and acidic oligosaccharides in an aqueous solution. In this study, we investigated the effects of POs-Ca on the mycelial growth and fruiting body yield of Pleurotus ostreatus, which is one of the most widely cultivated edible mushrooms in the world. We cultivated the mushroom using both potato dextrose agar (PDA) medium and sawdust-based medium, with added calcium salts. The addition of POs-Ca into the PDA medium with a calcium concentration of 10 mg increased mycelial growth significantly (p < 0.05, vs. control). POs-Ca addition to the sawdust-based medium at concentrations of 1.0 to 3.0 g/100 g medium increased the amount of calcium in the fruiting bodies but did not affect the length of the cultivation period or the weight of the fruiting body. The calcium content in the fruiting body increased 12-fold when compared to the control. On the other hand, neither the CaHPO4･2H2O group nor the CaHPO4･2H2O with oligosaccharides group showed changes in the calcium content of the fruiting bodies. Our results indicate that the use of POs-Ca in mushroom cultivation allows for the possibility of developing new functional foods like calcium-enriched edible mushrooms. This is the first report describing the effects of POs-Ca on mushroom cultivation.