地衣芽孢杆菌E7氨肽酶的异源表达及其在高效蛋白水解中的应用

Heterologous expression of Bacillus licheniformis E7 aminopeptidase and its application for efficient proteolysis

摘要:

【目的】将地衣芽孢杆菌(Bacillus licheniformis) E7氨肽酶基因pepN克隆到大肠杆菌(Escherichia coli) BL21中，实现氨肽酶EcPepN的异源表达，研究重组酶的酶学性质及其与碱性蛋白酶协同作用，高效水解大豆蛋白和酪蛋白，产生小分子活性肽和游离氨基酸。【方法】以地衣芽孢杆菌E7基因组DNA为模板，将氨肽酶基因pepN克隆到载体pET28a中，构建重组表达载体pET28-pepN，转化到大肠杆菌BL21感受态细胞中，经DNA测序验证，获得重组菌E.coli BL21/pET28-pepN。利用镍离子亲和层析柱对重组酶进行分离纯化，研究纯酶的pH和温度稳定性、半衰期和NaCl的耐受性等酶学性质。以商品化氨肽酶与碱性蛋白酶协同作用为对照，重组酶EcPepN与碱性蛋白酶协同水解大豆蛋白和酪蛋白，测定水解产物中小分子活性肽和游离氨基酸的组成。【结果】EcPepN在大肠杆菌BL21中可溶性表达，SDS-PAGE分析表明纯化的重组酶在52 kDa左右显示单一条带。在7种测定底物中，EcPepN的最适底物为Ala-pNA。在最适条件(pH 9.0和50下，最高比酶活为365.04 U/mg，K_M值为1.2 mmol/L，k_cat为598.3 s^–1，k_cat/K_M为499.0 L/(mmol·s)。当重组酶EcPepN在pH 6.0−9.0孵育2 d后，相对酶活力保持85%以上，在45下半衰期为36 h。酶在3.0 mol/L NaCl中22 d后，残留酶活力为55%以上。重组EcPepN与碱性蛋白酶协同作用水解大豆蛋白和酪蛋白，和商品化氨肽酶与碱性蛋白酶协同作用水解蛋白效率相当，水解产物中超过70%为分子量小于500 Da的活性肽，同时含有超过2 000 mg/L种类丰富的游离氨基酸。【结论】构建了氨肽酶的重组菌株，重组酶EcPepN具有较好pH和温度稳定性及较强的高浓度NaCl耐受性，能够高效催化大豆蛋白和酪蛋白水解，释放小分子活性肽和游离氨基酸，该研究为提高蛋白质的深度水解和富含蛋白质生物资源的增值提供了重要研究途径。

Abstract:

[Objective] Aminopeptidase gene pepN from Bacillus licheniformis E7 was cloned into Escherichia coli BL21 for heterologous expression of recombinant aminopeptidase EcPepN. The enzymatic properties of the recombinant EcPepN were studied. The combination of EcPepN with alkaline protease efficiently improved the hydrolysis of soybean protein and casein and released multitudinous peptides and amino acids. [Methods] With the genomic DNA of E7 as template, the aminopeptidase gene pepN was cloned into the expression vector pET28a to construct recombinant expression plasmid pET28-pepN, which was transformed into the competent cells of E. coli BL21. Through DNA sequencing verification, the recombinant E. coli BL21/pET28-pepN was obtained. The recombinant enzyme was purified by nickel-affinity chromatography. The pH and temperature stability, half-life, and NaCl tolerance of the pure enzyme were tested. With the commercial aminopeptidase combined with alkaline protease as a control, the combination of EcPepN and alkaline protease was used to hydrolyze soybean protein and casein, and the constituents of active small peptides and free amino acids in the hydrolysate were determined. [Results] The recombinant EcPepN was expressed in E. coli BL21. SDS-PAGE analysis showed that the purified target protein presented a single band at about 52 kDa. Among the seven substrates tested, the preferred substrate of EcPepN was Ala-pNA. Under optimal conditions (pH 9.0 and 50. After 22 days of incubation in 3.0 mol/L NaCl, the residual activity was more than 55%. When the recombinant aminopeptidase EcPepN worked synergistically with alkaline protease to hydrolyze soybean protein and casein, its hydrolysis efficiency was similar to that of the combination of commercial aminopeptidase and alkaline protease. More than 70% of the constituents of hydrolysate were peptides with molecular weight of less than 500 Da and it also contained more than 2 000 mg/L of free amino acids. [Conclusion] The recombinant strain containing aminopeptidase EcPepN was developed. The recombinant EcPepN shows pH and temperature stability and strong tolerance to NaCl of high concentration. It can efficiently catalyze the hydrolysis of soybean protein and casein and release small molecular active peptides and abundant free amino acids. This study provides important methods for enhancing deep protein hydrolysis and improving the values of protein-rich bioresources.