Cloning, Expression, Purification And Biochemical Properties Of Leucine Aminopeptidase From Bacillus Kaustophilus

As a high efficient and environmental biological catalyst,protease has been widely usedin food processing industries. After enzymolysis, the original characteristics of protein wouldchange,and the protein resolve into peptide and free amino acid. This can further enhance itsbiological function and nutrition value. However, many food protein hydrolysate will show abitter taste, which directly affect its application value. Research found that the bitter tastemainly comes from the hydrolysis produce by hydrophobic amino acid composition of lowmolecular weight peptides. How to take off the bitter protein hydrolysate become animperative problem in food processing industry. Aminopeptidase is a exonuclease, canselectively remove the amino acid residuesthe from the resection N-end of the peptide chainsor protein. They play an important role in taking off the bitter effect. Therefore, start with theresearch of aminopeptidase have some important implications on potential application valueand scientific significance.This study synthesized the total gene of leucine aminopeptidase of Bacillus kaustophilus,using an improved overlap extension PCR method. Then successfully constructedrecombination strain pET28a-LAP-BL21and get plenty of soluble expression. Through theNi2+-NTA column chromatography we get the purity BkLAP. Experiments were carried out toinvestigate the effects of various factors on the activity and conformation of BkLAP andpotential utilization of BkLAP in the hydrolysis of anchovy protein. Optimal temperature andpH of BkLAP were70°C and8.0in potassium-phosphate buffer, respectively, and theactivity was strongly stimulated by Ni²⁺, followed by Mn²⁺and Co²⁺. Conformational studiesvia circular dichroism spectroscopy indicated that various factors could influence thesecondary structure of BkLAP to different extents and further induce the changes inenzymatic activity. The potential application of BkLAP was evaluated through combinationwith the commercial or endogenous enzyme to hydrolysis the anchovy protein. Resultsshowed that combining the BkLAP with other enzymes could significantly increase thedegree of hydrolysis and amino acid component of hydrolysate. In this regard, BkLAP is apotential enzyme that can be used in the protein hydrolysate industry. The data presented herenot only considerably expand our basic knowledge about this enzyme, but also provide the useful information for its application in food processing industry.