High-level expression and characterization of the thermostable leucine aminopeptidase Thelap from the thermophilic fungus Thermomyces lanuginosus in Aspergillus niger and its application in soy protein hydrolysis.

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 stud y , 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. • The thermostable leucine aminopeptidase from Thermomyces lanuginosus was expressed at a high level in A. niger strain HL-1. • The recombinant Thelap activity of the shake fermentation supernatant achieved 2771.5 U/mL, mediated by the CRISPR/Cas9 tool. • Characterization of the purified enzyme was performed by measuring the optimal temperature, pH and the effects of metal ions. • Thelap could further improve the degree of hydrolysis and decrease bitterness during application in soy protein. [ABSTRACT FROM AUTHOR]