Screening of a collagenase-producing strain from the sediments in fishing grounds of freshwater lakes and optimization of the fermentation conditions

Abstract:

[Background] Collagenase can efficiently degrade water-insoluble collagen and is widely used in various industries. Most of the collagen from animal remains in fishing grounds of freshwater lakes will settle into sediments, and thus the ecological environment should be rich in collagenase-producing strains. [Objective] The collagenase-producing strains were screened from the sediments of freshwater lakes around Dongting Lake. Among them, a strain with high enzyme production and biosafety was identified and the fermentation conditions for enzyme production were optimized. The result is expected to lay a foundation for the industrial production and application of collagen and collagenase. [Methods] The collagenase-producing strains were screened with the spread plate method and the spot inoculation method. The collagenase activity of fermentation broth was determined by the ninhydrin colorimetry. Enzyme-producing strains were identified by 16S rRNA gene sequence alignment and phylogenetic analysis. Antibiotic sensitivity and haemolytic activity were detected with the disk diffussion method, amino acid decarboxylase activity by the bromocresol purple chromogenic assay, and nitrate reductase activity by nitrate reductase activity assay kit. Single factor experiment and orthogonal experiment were carried out to optimize the fermentation conditions for producing collagenase. [Results] A total of 113 collagenase-producing strains were screened out. Among them, a strain with high enzyme production and biosafety was selected from the sediment of East Lake in Huarong County, which was identified an Exiguobacterium strain and named Exiguobacterium sp. DJ1. The optimal enzyme production conditions of strain DJ1 were as follows: in 100 mL medium of 30.0 g/L gelatin, 10.0 g/L peptone, 5.0 g/L fish meal, 0.5 g/L KH₂PO₄, 0.2 g/L MgSO₄·7H₂O, 0.1 g/L NaCl in a 250 mL flask, with the initial pH of 8.5, inoculum of 3%, and culture temperature of 28 ℃. Under the optimized conditions, the activity of collagenase reached (185.45±23.87) U/mL, which was about 3.7 times higher than that before optimization. [Conclusion] There are abundant collagenase-producing strains in the sediments in fishing grounds of freshwater lakes. Strain DJ1 has high enzyme-producing capacity and biosafety. After optimization of fermentation conditions, the enzyme activity of strain DJ1 is increased by about 3.7 times, which lays a theoretical foundation for the industrial application of collagen and collagenase.

Key words:collagenase;collagenase-producing bacteria;lake sediment;fermentation process