蛋白质谷氨酰胺酶的重组表达与发酵条件优化

Recombinant expression of protein-glutaminase and optimization of fermentation conditions

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P 43替换pHT43的诱导型启动子P grac,并将重组质粒转化至大肠杆菌BL21(DE3)菌株,获得表达菌株pHT43:pro-mPG(P 43)/BL21(DE3)。将发酵16 h的重组菌超声破碎,菌体上清液中含有带前导肽的PG,经镍柱纯化、脱盐及浓缩后,用0.3 mg/mL的胰蛋白酶酶切,得到成熟的PG。SDS-PAGE显示PG消化前后分子量分别约为38 kDa和20 kDa,Western-blot结果表明两者均能与his-tag抗体结合显色。成熟PG的酶活力为0.250 U/mL,酶反应的最适条件为45℃、pH 6.5。发酵条件优化后的最佳产酶培养基配方为:4 g/L谷氨酰胺,4 g/L蔗糖,1 g/L酵母提取物,8 g/L酵母浸粉,2 g/L胰蛋白胨,10 g/L NaCl,3 g/L CaCl 2。37℃、pH 6.5条件下发酵16 h,最终酶活力提高至0.761 U/mL。

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.