Aspergillus oryzae RIB40氨肽酶(AoAPase)异源表达及在鳕鱼肽脱苦中的应用

Heterologous expression of aminopeptidase (AoAPase) from Aspergillus oryzae RIB40 and the application in debittering cod peptides

摘要:

【目的】针对鳕鱼肽苦味这一不足之处，本研究旨在获得一种高效特异性脱苦氨肽酶，实现对鳕鱼肽苦味的去除进而提升其附加值。【方法】本研究选取Aspergillus oryzae RIB40来源的氨肽酶AoAPase在毕赤酵母KM71中进行异源表达并探究其酶学性质；荧光探针法与电子舌技术联用评估AoAPase对鳕鱼肽的脱苦效果；高效液相色谱测定鳕鱼肽酶解液中游离氨基酸含量及分子量的分布变化；利用扫描电镜观察鳕鱼肽表面微观结构的差异性；通过评价DPPH、羟基及ABTS自由基清除能力，探究鳕鱼肽经AoAPase处理后抗氧化活性的变化。【结果】AoAPase在毕赤酵母KM71中成功异源表达，其分子量约41kDa。最适温度和pH分别为70℃和8.0，在最适条件下AoAPase酶活可达到2238U/mL，Ca²⁺能够提高其酶活；AoAPase具有底物特异性，以Leu-pNA为底物时，K_m为5.95mmol/L，V_max为43.58μmol/(mL·min)；AoAPase通过切除鳕鱼肽段N端疏水性氨基酸残基和降低苦味显著的肽段(500‒1000 Da)含量，进而完全消除其苦味；经AoAPase处理后的鳕鱼肽粉质地更加细腻、松散，且抗氧化活性无明显差异。【结论】重组氨肽酶AoAPase能够特异性去除鳕鱼肽苦味且不影响其生物活性，本研究为AoAPase在蛋白脱苦中的应用奠定理论基础。

Abstract:

[Objective] To explore a specific aminopeptidase that can debitter cod peptides and further increase the value of this fish. [Methods] Aspergillus oryzae RIB40-derived aminopeptidase (AoAPase) was expressed in Pichia pastoris KM71, and its characteristics were investigated. The cod peptides-debittering ability of AoAPase was evaluated by ANS probe and electronic tongue. The content and molecular weight of free amino acids in the cod peptides hydrolysate were detected by high performance liquid chromatography (HPLC), and the surface microstructure of cod peptides was observed under scanning electron microscope (SEM). Moreover, the DPPH-, hydroxyl- and ABTS-scavenging activities were determined to evaluate the antioxidant properties of cod peptides before and after AoAPase treatment. [Results] AoAPase was successfully expressed in KM71, with the molecular weight of about 41 kDa. AoAPase activity reached 2 238 U/mL under optimal conditions of 70℃ and pH 8.0. Its activity was enhanced by Ca²⁺. In addition, AoAPase exhibited substrate specificity, and its K_m and V_max values towards Leu-pNA were 5.95 mmol/L and 43.58 μmol/(mL·min), respectively. AoAPase could completely eliminate the bitterness of cod peptides by removing N-terminal hydrophobic amino acids and reducing the content of bitter peptides (500-1 000 Da). The cod peptide powder after AoAPase treatment became more delicate and loose with no significant change in antioxidant activity. [Conclusion] The recombinant AoAPase can remove the bitter taste of cod peptides without influencing their bioactivity. This study lays a theoretical foundation for the application of AoAPase in protein debittering.