为提高脱乙酰基酶的可溶性表达含量,对前期挖掘的脱乙酰基酶NAP-Das2.3基因进行异源表达及发酵优化。将脱乙酰基酶NAP-Das2.3基因克隆至枯草芽孢杆菌表达载体pP43NMK的nprB信号肽下游,转入B. subtilis WB800构建了重组工程菌B. subtilis WB800/pP43NMK/nap-das2.3,并对重组菌培养及发酵产酶条件进行了优化。重组菌最适培养和产酶条件分别为甘油6 g/L、牛肉膏30 g/L、NaCl 10 g/L;pH 7.5、培养温度37 ℃、装液量100 mL (500 mL摇瓶)、培养30 h。在优化的条件下,发酵液中脱乙酰基酶酶活达到106.42 U/L,较出发条件提高了424.68倍,在5 L发酵罐培养30 h后,脱乙酰基酶酶活达到116.13 U/L。研究实现了脱乙酰基酶的异源高效可溶性表达,为脱乙酰基酶的应用提供了基础。
In order to improve the soluble expression of deacetylase, a deacetylase NAP-Das2.3 was heterologously expressed in Bacillus subtilis, and the fermentation condition was optimized. The genes of NAP-Das2.3 were cloned into the downstream of the nprB signal peptide in pP43NMK (an expression vector of B. subtilis), and the resulting expression plasmid was transformed into B. subtilis WB800 to construct the recombinant strain B. subtilis WB800/pP43NMK/nap-das2.3. The optimum conditions for the recombinant strain for cultivation and enzyme production were as follows: 6 g/L glycerol, 30 g/L beef extract, 10 g/L NaCl, 100 mL/500 mL shake flask of medium and incubated at pH 7.5 and 37 ℃ for 30 h. Under the optimal conditions, the deacetylase activity reached 106.42 U/L, which was 424.68 times higher than that of the original conditions. After 30 h incubation in a 5 L fermenter, the deacetylase activity reached 116.13 U/L. This study achieved heterologous soluble overexpression of recombinant deacetylase, which is helpful for its development and application.
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