碱性蛋白酶工程菌发酵条件及重组酶的纯化和性质的研究

在5L发酵罐中对重组碱性蛋白酶工程菌株BP071高产碱性蛋白酶的条件进行了研究，通过提高通气量和改变搅拌转速，BP071可在发酵40 h内达到产酶高峰，酶活力最高可达24480 u/mL。利用快速蛋白液相层析（FPLC）技术，建立了快速高效纯化碱性蛋白酶的方案。发酵液通过硫酸铵沉淀、DEAE-A-50脱色及聚乙二醇浓缩得粗酶，再经过CM-Sephadex-C-50、Sephadex-G-75柱层析后得到了单一组份的重组碱性蛋白酶，酶纯度提高了76.2倍。SDS-PAGE显示重组碱性蛋白酶分子量为28 kD。酶学性质研究表明，酶的最适作用pH为11，最适作用温度为60℃，具有良好的pH稳定性和热稳定性。Ca²⁺、Mg²⁺对酶的稳定性有促进作用，Hg²⁺、Ag⁺、PMFS和DFP能强烈抑制酶的活力。SDS和Urea对酶的活力无影响。

Study on Fermentation Condition of Alkaline Protease Gene Engineering Strain and the Purification and Characterization of Recombinant Enzyme

In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.