淡水湖渔场沉积物中产胶原蛋白酶菌株的筛选及发酵优化

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

摘要:

【背景】胶原蛋白酶能够高效降解水不溶性的胶原蛋白，在各行业中都有着广泛的应用。淡水湖渔场中大部分动物残骸中的胶原蛋白都会沉降到沉积物中，因而其生态环境中应含有丰富的产胶原蛋白酶菌株。【目的】以环洞庭湖水系淡水湖渔场沉积物为样品，筛选产胶原蛋白酶菌株，并对其中一株产酶量和生物安全性均高的菌株进行鉴定及发酵产酶条件的初步研究，以期为胶原蛋白和胶原蛋白酶的工业生产及应用奠定基础。【方法】采用稀释涂布平板法和点种法筛选产胶原蛋白酶菌株，通过茚三酮显色法测定发酵液胶原蛋白酶酶活，并结合16S rRNA基因序列比对和系统发育分析确定产酶菌株的种类。采用滤纸片扩散法检测菌株抗生素敏感性和溶血性，用溴甲酚紫显色法检测菌株氨基酸脱羧酶活性，硝酸还原酶活性测定用试剂盒，通过单因素试验法和正交试验法进行菌株产胶原蛋白酶发酵条件的优化。【结果】从环洞庭湖水系中的4个淡水湖渔场的表层沉积物中共筛选分离得到113株产胶原蛋白酶菌株，并从中挑选一株来自东湖沉积物的产酶量和生物安全性均高的菌株，该菌株为Exiguobacterium属细菌，命名为Exiguobacterium sp.DJ1。菌株DJ1的最佳产酶条件为： 30.0 g/L明胶，10.0 g/L蛋白胨，5.0 g/L鱼粉，0.5 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L NaCl，pH 8.5，3%的接种量，28℃的培养温度，100 mL/250 mL的装液量。优化后的胶原蛋白酶酶活达(185.45±23.87) U/mL，约为优化前的3.7倍。【结论】淡水湖渔场沉积物中含有丰富的产胶原蛋白酶菌株。菌株DJ1具有较高的产酶能力和生物安全性，经发酵条件优化使该菌株的酶活提高了约3.7倍，为胶原蛋白和胶原蛋白酶的工业化应用奠定了理论基础，具有广阔的应用前景。

关键词:胶原蛋白酶;产胶原蛋白酶细菌;湖泊沉积物;发酵工艺

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