Optimization of fermentation conditions, purification, and biochemical characterization of a β-mannanase from Purpureocillium lilacinum

This study aimed to explore and characterize novel β-mannanase resources, so as to lay a solid enzymatic foundation for the efficient degradation of mannan-rich substrates. A β-mannanase-producing fungal strain was isolated from soil samples and identified based on morphological observation and internal transcribed spacer (ITS) sequence analysis. Liquid fermentation conditions were optimized via single-factor experiments, complex carbon source screening, and response surface methodology. The target β-mannanas was purified to electrophoretic homogeneity through ammonium sulfate precipitation followed by strong anion-exchange chromatography. Subsequently, its enzymatic properties, substrate specificity, and hydrolysis characteristics were systematically investigated. A high-yield strain, Purpureocillium lilacinum CF309, was successfully identified. The optimal fermentation medium contained 6.5% complex carbon source (konjac flour to palm kernel cake=1∶5, m/m), 2.5% tryptone, and 1.3% Triton X-100. Under optimized fermentation conditions (30 ℃, initial pH 6.0, 4 days of cultivation), the maximum β-mannanase activity reached 640.4 U/mL. The purified enzyme, designated PlManA, had an apparent molecular weight of 35.0 kDa and a specific activity of 365.7 U/mg, with a 4.1-fold purification rate and a recovery yield of 8.4%. PlManA exhibited an optimal reaction pH of 6.0 and maintained high stability over a broad pH range (5.0-9.0). Its optimal reaction temperature was 65 ℃, and the enzyme retained more than 70% of its residual activity after incubation at 50 ℃ for 30 min. PlManA showed high catalytic efficiency toward locust bean gum and konjac flour, mainly producing mannooligosaccharides with a degree of polymerization of 2-6 and above. In conclusion, the β-mannanase PlManA from P. lilacinum CF309 possesses excellent pH tolerance and favorable thermostability. Its unique hydrolysis profile endows it with great potential as a biocatalyst for food industry applications.