铜绿假单胞菌产蛋白酶的发酵条件优化

Optimization of protease production by Pseudomonas aeruginosa

摘要:

【目的】鉴定一株来源于酱油曲能够分泌蛋白酶的铜绿假单胞菌CAU342A，优化其产蛋白酶的发酵条件。【方法】采用形态学观察、16S rRNA基因序列比对和生理生化方法鉴定菌株CAU342A；通过碳源、氮源、初始pH、温度、表面活性剂及发酵时间的单因素优化和正交试验获得最适发酵条件。【结果】菌株CAU342A被鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)，其最适发酵产酶条件为(质量体积比)：3%酒糟，1.5%酵母浸提物，0.05%吐温-80，0.5% NaCl，0.7% K2HPO4，0.3% KH2PO4，0.04% MnSO4，培养基初始pH 7.5，30 °C培养72 h。在最适发酵条件下，该菌株最大产酶水平达到2 653.5 U/mL。蛋白酶酶谱分析表明该菌株能够产生至少4种具有蛋白酶活性的同工酶，其中两个主要酶谱带对应分子量分别为32 kD和50 kD。【结论】铜绿假单胞菌CAU342A高产蛋白酶，具有很大的工业应用潜力。

Abstract:

[Objective] To identify a protease producing bacterium CAU342A isolated from soy sauce koji and optimize the fermentation conditions for protease production. [Methods] Strain CAU342A was identified based on its morphological characters, 16S rRNA gene sequence, physiological and biochemical characteristics. The fermentation conditions for protease production by strain CAU342A were optimized using the one-factor-at-a-time method and orthogonal test. [Results] strain CAU342A was identified as Pseudomonas aeruginosa, and the optimal fermentation conditions were obtained as follows: 3% vinasse, 1.5% yeast extract, 0.05% Tween-80, 0.5% NaCl, 0.7% K2HPO4, 0.3% KH2PO4, 0.04% MnSO4, the initial pH was 7.5 and incubation temperature was 30 °C for 72 h. Under the optimized fermentation conditions, the highest protease activity of 2 653.5 U/mL was achieved. The protease zymogram analysis showed that at least 4 proteases were secreted into the fermentation broth, and two major bands had molecular masses of 32 kD and 50 kD, respectively. [Conclusion] The high-level production of protease by Pseudomonas aeruginosa CAU342A enables its potential industrial applications.