蛋白质谷氨酰胺酶的重组表达与发酵条件优化

蛋白质谷氨酰胺酶(protein-glutaminase,PG)对植物蛋白具有高效的脱酰胺作用,可提高蛋白的溶解度,进而改善其起泡性、凝胶性等功能性质,在植物性食品中有巨大的应用前景.但由于原始菌株解脘金黄杆菌PG产量低,需异源重组表达以提高产量.将PG基因重组到质粒pHT43,采用强组成型启动子P43替换pHT43的...

Recombinant expression of protein-glutaminase and optimization of fermentation conditions

Protein-glutaminase(PG)has an efficient deamidation effect on plant proteins,which can increase the solubility of the protein and improve its functional properties,such as foaming and gelation.Therefore,it has huge application prospects in the industrial production of plant foods.However,due to the low fermentation yield of the original strain of PG-producing Chryseobacterium proteolyticum,heterologous recombinant expression is required to increase its yield.In this study,the PG gene was recombined into the plasmid pHT43,and the inducible promoter P grac of pHT43 was replaced by a strong constitutive promoter P 43.Then,the recombinant plasmid was transformed into E.coli BL21(DE3)strain to obtain an expression strain-pHT43:pro-mPG(P 43)/BL21(DE3).After cultured for 16 hours,the cells were collected and ultrasonically disrupted to release PG with a leader peptide.After centrifugation,the supernatant was purified by a nickel column,after desalination and concentration,the suspension was digested with 0.3 mg/mL trypsin to be catalyzed into a mature PG.The results of SDS-PAGE showed that the molecular weights of PG before and after trypsin hydrolysis were 38 kDa and 20 kDa,respectively.Western-blot results showed that both of them could bind to his-tag antibody.The enzyme activity of mature PG was 0.250 U/mL,and the optimal conditions for enzyme activity determination were 45℃and pH 6.5.The final optimized fermentation conditions were as follows:4 g/L glutamine,4 g/L sucrose,1 g/L yeast extract,8 g/L yeast extract,2 g/L tryptone,10 g/L sodium chloride,3 g/L calcium chloride.Under above fermentation conditions for 16 h at 37℃and pH 6.5,a final enzyme activity of 0.761 U/mL was achieved.