Breeding of high-yield cold-adapted lipase fungi, optimization of fermentation conditions and isolation and purification of lipase

Abstract Cold-adapted lipases have attracted extensive research attention in enzymology owing to their unique low-temperature adaptability and catalytic properties, and investigations in this area continue to deepen. This study aimed to efficiently produce a cold-adapted lipase to overcome the limitations of conventional lipases under low-temperature conditions, such as low activity, high cost, and restricted applicability. Fungal strains were isolated from naturally cold environments, and subjected to multiple rounds of screening for lipase activity. A selected strain was further improved by UV-nitrosoguanidine (NTG) combined mutagenesis, followed by optimization of fermentation conditions. The lipasewas purified by ammonium sulfate precipitation, Q-HP anion exchange chromatography, and Superdex 75 gel filtration chromatography, and its enzymatic properties were characterized. A lipase-producing strain identified as Aspergillus niger was obtained through screening. Following combined mutagenesis, lipase activity by the mutant strain increased by 127.35%. Under the optimized fermentation conditions (temperature 21℃, pH 6.0, inoculation size 6.5 × 10 6 spores/mL), the lipase activity further increased to 57.26 U/mL. After purification, the cold-adapted lipase exhibited a specific activity of 113.52 U/mg. Its optimal reaction temperature was 30℃ and pH 7.0 and retained more than 60% of its maximum activity at 15℃. Its high relative activity at low temperatures, together with its low thermal stability, supported its classification as a cold-adapted lipase. This purification strategy can produce high-purity lipase at laboratory scale, which provides a pure enzyme preparation for biochemical studies. The cold-adapted lipase obtained in this study may have promising application prospects in fields such as food processing, low-temperature laundry, and environmental management.