This study aimed to screen high-yield protease-producing strains from marine symbiotic microbiota to provide microbial resources for developing novel protease preparations.Using the intestinal contents of marine tilapia as materials, a strain LfF-1 with high yield of metalloproteinase was isolated through plate screening and secondary fermentation-based activity assessment.The strain was identified as Bacillus velezensis via morphological observation, metabolic fingerprint analysis, and phylogenetic tree construction using concatenated sequences of the 16S rRNA and gyrB gene.The protease produced by this strain exhibits excellent characteristics:it is halotolerant (retaining 81.46% relative enzyme activity in 100 g/L NaCl), alkaliphilic (with an optimal pH around 11.0), and cold-adapted (maintaining 82.12% relative enzyme activity at 25 ℃).The fermentation medium for protease production was optimized through single-factor experiments and response surface methodology (RSM).The optimal medium composition was determined as follows:lactose (34.36 g/L), beef extract (18.54 g/L), KH2PO4 (5.40 g/L), wheat bran (33.20 g/L), with an initial pH of 7.0-7.5.The study also found that the suitable temperature for enzyme production of this strain was in the range of 27-33 ℃, and higher than 36 ℃ would significantly inhibit protease synthesis.Temperature-shift control (33 ℃ for 0-24 h;27 ℃ after 24 h) can effectively coordinate the growth of bacteria and enzyme production, and significantly improve the enzyme activity of fermentation broth.Notably, excess Mn2+, Zn2+, or Fe3+ in the culture medium can lead to the denaturation and inactivation of its protease, thus significantly reducing the enzyme activity of fermentation broth.In 5 L fermenter trials, using the optimized medium with temperature-shift control and fed-batch operation, the fermentation cycle was shortened to 60 h, achieving a maximum protease activity of 11 500 U/mL and increasing total protease yield by 76.9% compared to batch fermentation, showing significant advantages over the reported enzyme production levels of similar strains.The results showed that B.velezensis LfF-1 possesses high protease-producing capacity, and the produced protease has outstanding adaptability to extreme environments.The optimized fermentation process prozides technical support for its industrial application.
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