黑曲霉丝氨酸羧肽酶的重组表达及其水解大豆蛋白的效果分析

Recombinant Expression of Serine Carboxypeptidase in Aspergillus niger and Analysis of Hydrolysis Effect on Soybean Protein

丝氨酸羧肽酶能够剪切肽链C末端的氨基酸,在大豆蛋白脱苦中有着广泛的应用。该研究将黑曲霉中3个丝氨酸羧肽酶基因(CPG、CPF、CPA)在低背景的无孢黑曲霉HL-1中进行重组表达。利用启动子PnaⅡ、终止子Ttef和营养缺陷型筛选标记pyr G构建分别含自身信号肽和糖化酶信号肽的丝氨酸羧肽酶表达载体;利用PEG介导的转化法转化无孢黑曲霉宿主HL-1,构建了丝氨酸羧肽酶重组表达菌株。通过摇瓶发酵筛选得到高表达重组菌株HL-CPG,其丝氨酸羧肽酶酶活达到163.71 U/mL。利用6ⅹHis标签进行镍柱亲和层析得到单一的丝氨酸羧肽酶CPG并研究了其酶学性质,该酶最适反应温度为40℃,最适p H为3.5,Cu^(2+)具有明显抑制效果。另外,将重组表达的丝氨酸羧肽酶CPG与胃蛋白酶复配水解大豆蛋白,水解液中疏水性氨基酸Leu、Tyr和Phe的含量分别增加606.47μg/m L、434.06μg/m L和205.11μg/m L。综上所述,该研究在黑曲霉中成功实现丝氨酸羧肽酶的高效表达,对大豆蛋白水解液脱苦处理工艺具有一定的借鉴意义。

Serine carboxypeptidase could cleave the amino acid at the C-terminus of the peptide chain and has a wide range of applications in debittering soybean protein.In this study,three serine carboxypeptidase genes(CPG,CPF,CPA)from Aspergillus niger were recombinantly expressed in low-background Aspergillus niger HL-1;promoter PnaⅡ,terminator Ttef and auxotrophic selection marker pyr G were used to construct serine carboxypeptidase expression vector containing its own signal peptide and glucoamylase signal peptide;PEG-mediated transformation method was used to transform into host HL-1 to construct a serine carboxypeptidase recombinant expression strain;the high expressional recombinant strain HL-CPG was obtained by fermentation screening,and its serine carboxypeptidase activity reached 163.71 U/m L;the 6ⅹHis tag was used for nickel column affinity chromatography to obtain a single serine carboxypeptidase CPG and its enzymatic properties.The optimal reaction temperature of the enzyme was 40℃,the optimal pH was 3.5,and Cu^(2+)had a significant inhibitory effect.In addition,when serine carboxypeptidase CPG and pepsin were compounded to hydrolyze soybean protein,the content of hydrophobic amino acids Leu,Tyr and Phe in the hydrolysate increased by 606.47μg/m L,434.06μg/m L and 205.11μg/m L,respectively.In summary,this study successfully achieved high-efficiency expression of serine carboxypeptidase,and provided support for solving the debittering treatment process of soybean protein hydrolysate.