一株贝莱斯芽孢杆菌的分离、鉴定及发酵产蛋白酶工艺研究OA

This study aimed to screen high-yield protease-producing strains from marine symbiotic microbiota to provide microbial re-sources 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 identi-fied as Bacillus velezensis via morphological observation,metabolic fingerprint analysis,and phylogenetic tree construction using concatena-ted sequences of the 16S rRNA and gyrB gene.The protease produced by this strain exhibits excellent characteristics:it is halotolerant(re-taining 81.46％relative enzyme activity in 100 g/L NaCl),alkaliphilic(with an optimal pH around 11.0),and cold-adapted(maintai-ning 82.12％relative enzyme activity at 25 ℃).The fermentation medium for protease production was optimized through single-factor ex-periments 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 in-hibit 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 environ-ments.The optimized fermentation process prozides technical support for its industrial application.