Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042.
Pediococcus acidilactici ATCC 8042 is a lactic acid bacteria that inhibits pathogenic microorganisms such as Staphylococcus aureus through the production of two proteins with lytic activity, one of 110 kDa and the other of 99 kDa. The 99-kDa one has high homology to a putative peptidoglycan hydrolase (PGH) enzyme reported in the genome of P. acidilactici 7_4, where two different lytic domains have been identified but not characterized. The aim of this work was the biochemical characterization of the recombinant enzyme of 99 kDa. The enzyme was cloned and expressed successfully and retains its activity against Micrococcus lysodeikticus. It has a higher N-acetylglucosaminidase activity, but the N-acetylmuramoyl-L-alanine amidase can also be detected spectrophotometrically. The protein was then purified using gel filtration chromatography. Antibacterial activity showed an optimal pH of 6.0 and was stable between 5.0 and 7.0. The optimal temperature for activity was 60 °C, and all activity was lost after 1 h of incubation at 70 °C. The number of strains susceptible to the recombinant 99-kDa enzyme was lower than that susceptible to the mixture of the 110- and 99-kDa PGHs of P. acidilactici, a result that suggests synergy between these two enzymes. This is the first PGH from LAB that has been shown to possess two lytic sites. The results of this study will aid in the design of new antibacterial agents from natural origin that can combat foodborne disease and improve hygienic practices in the industrial sector.
Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., México.
Israel
I
García-Cano
Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., México.
Manuel
M
Campos-Gómez
Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., México.
Mariana
M
Contreras-Cruz
Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., México.
Carlos Eduardo
CE
Serrano-Maldonado
Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México y Hospital General de México, 06720, México D.F., México.
Augusto
A
González-Canto
Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., México.
Carolina
C
Peña-Montes
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., México.
Romina
R
Rodríguez-Sanoja
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., México.
Sergio
S
Sánchez
Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., México. farres@unam.mx.
Amelia
A
Farrés
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D050296:Microbial Viability / Q000187:drug effects; D008837:Micrococcus / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010376:Pediococcus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D011994:Recombinant Proteins; D009238:N-Acetylmuramoyl-L-alanine Amidase
4-Nitrophenyl N-acetyl-β-D-glucosamine; N-acetylglucosaminidase; N-acetylmuramoyl-L-alanine amidase; Pediococcus acidilactici ATCC 8042; Peptidoglycan hydrolase; Zymogram