重组基因工程菌几丁质酶C的可溶性表达研究

Soluble Expression of Chitinase C in Recombinant Genetic Engineering Strains

在不同培养温度条件下对产几丁质酶C的基因工程菌BL21(DE3)进行诱导,使其表达可溶性蛋白。随后在较佳培养温度诱导条件的基础上向培养基中添加不同浓度的甘油,甘氨酸,山梨醇/甜菜碱等成分来更进一步的提高几丁质酶C的可溶性表达。其结果是在25℃诱导条件下,以添加2 g/L葡萄糖的LB培养基作为基础培养基进行培养,在基础培养基中添加3 g/L甘油的总酶活最高,达到了18.17 U/mL,较之仅含2 g/L葡萄糖的LB培养基在37℃及25℃诱导培养条件下酶活力分别提高了41.6%和20.3%;添加0.3%甘氨酸约90%的可溶性几丁质酶表达到了胞外,胞外酶活达到14.68 U/mL;添加0.5 M山梨醇/2.5 mM甜菜碱工程菌胞内酶活达到最高,为8.43 U/mL。结果表明适当降低工程菌诱导表达时的培养温度提高了几丁质酶C的可溶性表达,在较佳温诱导表达的基础上向培养基中添加不同浓度的甘油,甘氨酸,山梨醇/甜菜碱更进一步的提高了几丁质酶C的可溶性表达。

The soluble expressing of chitinase C in different ferment temperature to obtain better culture temperature, then boosting more by adding different concentrations of glycerol, glycine and sorbitol/betaine to the culture medium in the lower ferment temperature. LB culture medium which contain 2 g/L glucose as the basic medium and induced temperature set up 25 ℃. Firstly, the activity of chitinase can reach 18.17 U/mL by adding 3 g/L glycerol, increased 41.6 % and 20.3 % at 37 ℃ and 25 ℃ respectively. Secondly, about 90 % soluble chitinase could be expressed Extracellularly by adding 0.3 % glycine, and the activity of chitinase can also reach 14.68 U/mL. Thirdly, the activity of intracellular chitinase could reach 8.43 U/mL by adding 0.5 M sorbitol/2.5 mM betaine, which is highest in this research. The results show that reducing the culture temperature could be inhance expressing the soluble chitinase C, under the culture temperature of 25 ℃, adding different concentrations of glycerol glycine sorbitol betaine in the medium could be improve the soluble expression of chitinase C.