Rapid High-Throughput Screening of Curdlan-Producing Mutants via a Microscale Aniline Blue Colorimetric Assay

Curdlan is an industrially important β-1,3-glucan with applications in the food, pharmaceutical, and biomaterial industries. However, the identification of high-yielding curdlan-producing strains is hindered by the absence of rapid and efficient screening methods. To address this limitation, we developed a systematically optimized integrated microscale workflow combining 48-well plate fermentation with a quantitative aniline blue-based colorimetric assay in 96-well plates for high-throughput screening of curdlan-producing strains. Fermentation was miniaturized using 48-well plates, while curdlan quantification was performed in 96-well plates through formation of a curdlan–aniline blue complex. Key parameters were systematically optimized. Under optimal conditions, curdlan dissolved in 1.0 mol/L NaOH was reacted with 2.0 mg/mL aniline blue in 0.5 mol/L phosphate buffer (pH 7.0) for 90 min, and absorbance was measured at 550 nm. The assay demonstrated excellent linearity between curdlan concentration and absorbance (y = 1.0008x + 0.1536, R2 = 0.9957, p < 0.001). To validate the method, curdlan yields from nine mutants derived from ATCC31749 were determined using both gravimetric and colorimetric approaches, revealing a strong correlation (R2 = 0.8683, p < 0.001), that confirmed the assay’s reliability for rapid screening. Application of this platform to 132 UV-mutagenized strains identified nine mutants with enhanced curdlan production. The best-performing strain, UV150824-02, produced 46.8 ± 0.08 g/L curdlan, an 11.4% increase over the wild-type strain ATCC31749 (41.6 ± 1.54 g/L), and maintained stable curdlan production over nine laboratory passages (coefficient of variation = 2.72%). This method significantly improves the efficiency of mutagenesis-based strain screening and provides a practical and efficient tool for accelerating strain improvement in industrial polysaccharide fermentation.