title: Biochemical and conformational characterization of a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2.
abstract: In order to search for valuable and extremely thermo-stable enzymes that could be used in the protein hydrolysis industry, the gene corresponding to a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2 (GtLAP) was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined. Meanwhile, potential applications of GtLAP in the hydrolysis of anchovy proteins were also investigated. GtLAP was overexpressed in IPTG-induced E. coli BL21 (pET28a-LAP) as a soluble protein, and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 125 ± 8.75 U/mg proteins. The molecular mass of GtLAP was estimated to be 55 kDa by SDS-PAGE analysis. The optimal reaction temperature and pH of GtLAP were 70 °C and 8.0, respectively. Under optimal conditions, GtLAP showed a marked preference for Leu-p-nitroanilide, followed by Met- and Phe-derivatives. Activity of GtLAP was strongly stimulated by Ni²⁺ ions, but was strongly inhibited by Hg²⁺. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of GtLAP to various extents and further induce changes in enzymatic activity. Results of hydrolytic experiment showed that combining GtLAP with endogenous enzymes could significantly increase the degree of hydrolysis to anchovy proteins and concentrations of free amino acids in hydrolysates. In this regard, GtLAP could potentially be used in the protein hydrolysis industry.
authors.affiliation: College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, Guangdong, People's Republic of China.
authors.forename: Fanghua
authors.initials: F
authors.lastname: Wang
authors.forename: Shaohua
authors.initials: S
authors.lastname: Guo
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Dongming
authors.initials: D
authors.lastname: Lan
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Wang
mesh_terms: D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D056506:Geobacillus / Q000737:chemistry / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D007931:Leucyl Aminopeptidase