Screening And Expression Analysis Of Leucine Aminopeptidases Genes In Aspergillus Niger

During the enzymatic treatment of soybean protein,the peptide chains are hydrolyzed to form a large number of hydrophobic polypeptides with a bitter taste in the hydrolysate.Leucine aminopeptidase is commonly used in the debittering of protein hydrolysates by removing the hydrophobic amino acids at the N-termini of the polypeptides.The study screened out two high-level expression genes lap A and The in the host Aspergillus niger HL-1 from eight leucine aminopeptidase genes of aspergillus and thermophilic fungi.The homologous recombination method combined with CRISPR/Cas9 tool can increase their gene copy number and expression level of lap A and The.And the purification process and enzymatic properties of recombinant leucine aminopeptidase Lap A and Thelap were studied,as well as the application of recombinant Thelap and alkaline protease to hydrolyze soy protein isolate in debittering.The main research contents are as follows:?1?Eight different leucine aminopeptidase expression vectors were constructed by linking the hybrid promoter Pna II with different leucine aminopeptidase genes,respectively.These different leucine aminopeptidase expression vectors were transformed into the host A.niger HL-1 for recombinant expression.Two high-level expression genes lap A and The were screened out,and the enzyme activities of the transformants Lap A-T and The-T were 2212.3 U/m L,854.6U/m L,respectively.To increase the expression of leucine aminopeptidase,the homologous recombination method combined with CRISPR/Cas9 tool was used to improve the gene copy number in the host's genome,and transformants Lap A-C and The-C showed enzyme activities of 11701.2 U/m L,2771.5 U/m L,respectively.?2?After purification of leucine aminopeptidases Lap A and Thelap by nickel column affinity chromatography,a single band of leucine aminopeptidase Lap A?35 k Da?and Thelap?35.0-40.0 k Da?were obtained.The recombinant Lap A displayed optimal activity at p H 8.5,65°C,and obtained ordinary stability,while the recombinant Thelap showed optimum activity at p H 8.5 and 75°C,and exhibited excellent thermal stability between 65°C and 75°C and great p H stability in alkaline conditions.At a concentration of 5 mmol/L,K⁺had an active effect on the recombinant Lap A and Thelap.Co²⁺increased the activity of recombinant Thelap,but had an inhibitory effect on recombinant Lap A.Ba²⁺,Mn²⁺,Mg²⁺,Ni²⁺,Ca²⁺,and surfactant SDS inhibited the enzyme activities of recombinant Lap A and Thelap.?3?To investigate the hydrolysis effect and debittering of aminopeptidase,the co-hydrolysis of soybean protein isolate was performed by alkaline protease and Thelap at 65°C and p H 8.0.After 6 hours of hydrolysis,the degree of hydrolysis in the experimental group?co-hydrolysis of alkaline protease and Thelap?was significantly higher than that in the control group?only alkaline protease?,which increased 6%.The concentration of free hydrophobic amino acids in the reaction solution was increased because of the addition of the aminopeptidase Thelap,and the most significant increase in hydrophobic amino acids was Leu,followed by Val and Ile.The increase in the concentration of hydrophobic free amino acids indicated that the aminopeptidase Thelap could reduce the concentration of hydrophobic polypeptides in the hydrolysate of soybean protein isolate,and had a better potential of removing bitterness.