Leucine aminopeptidase (LAP), an exopeptidase that releases amino acid residues, especially leucine, from the N-terminus of polypeptides, is often applied to debitter protein hydrolysate in the food industry. However, there are no thermostable and high activity enzymes that can be used in the food industry. In this study, we obtained the highly active and thermostable leucine aminopeptidases screened from the thermophilic fungi Thermomyces lanuginosus, Talaromyces thermophilus, and Malbranchea cinnamomea. The activity of the recombinant leucine aminopeptidase Thelap was significantly increased to 2771.5 U/mL, as mediated by the CRISPR/Cas9 tool. The recombinant Thelap was easily purified from fermentation broth by Ni-affinity chromatography, and the specific activity of the purified Thelap was increased to 7449.6 U/mg. The recombinant Thelap showed optimal activity at pH 8.5 and 75 °C and remained above 70% of the maximum activity over a wide temperature range (30-80 °C). With regard to temperature stability, Thelap retained more than 90% activity when it was incubated at 65-75 °C for 2 h. K+ and Co2+ increased the enzyme activity of the recombinant Thelap, while Ba2+, Mn2+, Ni2+, Ca2+, Mg2+ and SDS inhibited its enzyme activity, and the inhibition capacity of Mg2+ was the weakest. Upon application in soy protein hydrolysis, Thelap could significantly increase the degree of hydrolysis and remove more hydrophobic amino acids from the N-terminal region of the polypeptide to decrease the bitterness.

中文翻译：

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黑曲霉中嗜热真菌嗜热丝霉菌热稳定亮氨酸氨基肽酶Thelap的高水平表达和鉴定及其在大豆蛋白水解中的应用。

亮氨酸氨基肽酶（LAP）是一种从多肽的N末端释放氨基酸残基（尤其是亮氨酸）的肽外酶，通常用于食品工业中的脱苦蛋白水解产物。但是，没有可用于食品工业的热稳定和高活性酶。在这项研究中，我们获得了从嗜热真菌嗜热单胞菌，嗜热嗜热菌和肉桂麦芽菌中筛选出的高活性和热稳定的亮氨酸氨基肽酶。重组亮氨酸氨基肽酶Thelap的活性显着增加至2771.5 U / mL，这是由CRISPR / Cas9工具介导的。重组的Thelap易于通过Ni-亲和色谱法从发酵液中纯化，纯化的Thelap的比活性提高到7449.6 U / mg。重组Thelap在pH 8.5和75°C时显示最佳活性，并在较宽的温度范围（30-80°C）内保持最大活性的70％以上。关于温度稳定性，当Thelap在65-75°C下孵育2小时时，保留了90％以上的活性。K +和Co2 +增加了重组酶Thelap的酶活性，而Ba2 +，Mn2 +，Ni2 +，Ca2 +，Mg2 +和SDS抑制了其酶活性，而Mg2 +的抑制能力最弱。在大豆蛋白水解中应用后，Thelap可以显着提高水解程度，并从多肽的N端区域去除更多的疏水氨基酸，从而降低苦味。当在65-75°C下孵育2小时时，lap保留了超过90％的活性。K +和Co2 +增加了重组酶Thelap的酶活性，而Ba2 +，Mn2 +，Ni2 +，Ca2 +，Mg2 +和SDS抑制了其酶活性，而Mg2 +的抑制能力最弱。在大豆蛋白水解中应用后，Thelap可以显着提高水解程度，并从多肽的N端区域去除更多的疏水氨基酸，从而降低苦味。当在65-75°C下孵育2小时时，lap保留了超过90％的活性。K +和Co2 +增加了重组酶Thelap的酶活性，而Ba2 +，Mn2 +，Ni2 +，Ca2 +，Mg2 +和SDS抑制了其酶活性，而Mg2 +的抑制能力最弱。在大豆蛋白水解中应用后，Thelap可以显着提高水解程度，并从多肽的N端区域去除更多的疏水氨基酸，从而降低苦味。