RESUMO
The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat(H-Ala-Pro-pNA) L(-1), which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkat(H-Ala-pNA) L(-1). After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn(2+)-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co(2+)>Zn(2+)>Mn(2+)>Ca(2+)>Mg(2+). PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.
Assuntos
Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Caseínas/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Lactobacillus helveticus/enzimologia , Proteínas Recombinantes/metabolismo , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Lactobacillus helveticus/genética , Dados de Sequência Molecular , Peso Molecular , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , TermodinâmicaRESUMO
The aminopeptidase P (PepP, EC 3.4.11.9) gene from Lactococcus lactis ssp. lactis DSM 20481 was cloned, sequenced and expressed recombinantly in E. coli BL21 (DE3) for the first time. PepP is involved in the hydrolysis of proline-rich proteins and, thus, is important for the debittering of protein hydrolysates. For accurate determination of PepP activity, a novel gas chromatographic assay was established. The release of L-leucine during the hydrolysis of L-leucine-L-proline-L-proline (LPP) was examined for determination of PepP activity. Sufficient recombinant PepP production was achieved via bioreactor cultivation at 16 °C, resulting in PepP activity of 90 µkatLPP Lculture-1. After automated chromatographic purification by His-tag affinity chromatography followed by desalting, PepP activity of 73.8 µkatLPP Lculture-1 was achieved. This was approximately 700-fold higher compared to the purified native PepP produced by Lactococcus lactis ssp. lactis NCDO 763 as described in literature. The molecular weight of PepP was estimated to be ~ 40 kDa via native-PAGE together with a newly developed activity staining method and by SDS-PAGE. Furthermore, the kinetic parameters Km and Vmax were determined for PepP using three different tripeptide substrates. The purified enzyme showed a pH optimum between 7.0 and 7.5, was most active between 50°C and 60°C and exhibited reasonable stability at 0°C, 20°C and 37°C over 15 days. PepP activity could be increased 6-fold using 8.92 mM MnCl2 and was inhibited by 1,10-phenanthroline and EDTA.
