Characterization, amyloid formation, and immobilization of a novel SGNH hydrolase from Listeria innocua 11262.
A novel oligomeric hydrolase (LI22) from Listeria innocua CLIP 11262 was identified, characterized, and immobilized for industrial application. Sequence analysis of LI22 revealed a putative catalytic triad (Ser(10)-Asp(176)-His(179)), and a conserved sequence motif Ser(S)(10)-Gly(G)(77)-Asn(N)(79)-His(H)(179) with moderate identities (<30%) with other members of the SGNH-hydrolase superfamily. LI22 was able to hydrolyze p-nitrophenyl acetate, α- and β-naphthyl acetate, while the S10A mutant completely lost its activity. Structural properties of LI22 were investigated using gel filtration, circular dichroism (CD), fluorescence, molecular modeling, and gel filtration. We have shown that upon incubation in 30% TFE or 50% ethanol solution, LI22 was transformed into curly amyloid fibrils. Cross-linked enzyme aggregates of LI22 were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Higher thermal and chemical stability, as well as good durability after repeated use of the LI22-CLEA, highlight its potential applicability as a biocatalyst in the pharmaceutical and chemical industries.
Department of Molecular Science and Technology, Graduate School of Interdisciplinary Programs, Ajou University, Suwon, South Korea.
Seulgi
S
Kim
Song Yi
SY
Bae
Seung Joon
SJ
Kim
Tri Duc
TD
Ngo
Kyeong Kyu
KK
Kim
T Doohun
TD
Kim
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D052160:Benzothiazoles; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D008087:Listeria / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009596:Nitrophenols / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence / Q000379:methods; D013844:Thiazoles / Q000737:chemistry
D000682:Amyloid; D052160:Benzothiazoles; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D009596:Nitrophenols; D013844:Thiazoles; C009462:thioflavin T; C008642:4-nitrophenyl acetate; D002265:Carboxylic Ester Hydrolases