Identification, characterization, immobilization of a novel type hydrolase (LmH) from Listeria monocytogenes.

A novel type of hydrolase (LmH) from Listeria monocytogenes was identified, characterized, and immobilized for biotechnological applications. Primary sequence analysis indicated that LmH had a catalytic triad (Ser(91)-Asp(192)-His(222)) with a molecular weight of 27.8 kDa. Homologs of this enzyme are produced by many Gram-positive bacteria including Bacillus, Staphylococcus, and Enterococcus. Biochemical properties of LmH were investigated by performing mass spectrometry, dynamic light scattering (DLS), enzyme assays, enantioselective analysis, circular dichroism (CD) spectroscopy, fluorescence analysis, and macroscopic hydrogel formations. Interestingly, cross-linked enzyme aggregates (CLEAs) of LmH exhibited enhanced stability and good recycling abilities compared to free LmH. These molecular characteristics of LmH highlight its great potential for the pharmaceutical, biotechnological, and chemical industries.

Crystallographic analysis and biochemical applications of a novel penicillin-binding protein/ß-lactamase homologue from a metagenomic library.

Interest in penicillin-binding proteins and ß-lactamases (the PBP-ßL family) is increasing owing to their biological and clinical significance. In this study, the crystal structure of Est-Y29, a metagenomic homologue of the PBP-ßL family, was determined at 1.7 Šresolution. In addition, complex structures of Est-Y29 with 4-nitrophenyl phosphate (4NP) and with diethyl phosphonate (DEP) at 2.0 Šresolution were also elucidated. Structural analyses showed that Est-Y29 is composed of two domains: a ß-lactamase fold and an insertion domain. A deep hydrophobic patch between these domains defines a wide active site, and a nucleophilic serine (Ser58) residue is located in a groove defined primarily by hydrophobic residues between the two domains. In addition, three hydrophobic motifs, which make up the substrate-binding site, allow this enzyme to hydrolyze a wide variety of hydrophobic compounds, including fish and olive oils. Furthermore, cross-linked Est-Y29 aggregates (CLEA-Est-Y29) significantly increase the stability of the enzyme as well as its potential for extensive reuse in various deactivating conditions. The structural features of Est-Y29, together with biochemical and biophysical studies, could provide a molecular basis for understanding the properties and regulatory mechanisms of the PBP-ßL family and their potential for use in industrial biocatalysts.

Crystallization and preliminary X-ray analysis of a novel type of lipolytic hydrolase from Bacillus licheniformis.

With increasing demand in biotechnological applications, the identification and characterization of novel lipolytic enzymes are of great importance. The crystallization and preliminary X-ray crystallographic study of a novel type of hydrolase from Bacillus licheniformis (BL28) are described here. Recombinant BL28 protein containing a C-terminal His tag was overproduced in Escherichia coli and purified to homogeneity. BL28 was crystallized using 0.2 M ammonium acetate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, 30%(w/v) PEG 4000 as a crystallizing solution. X-ray diffraction data were collected to a resolution of 1.67 Šwith an Rmerge of 5.8%. The BL28 crystals belonged to the tetragonal space group P41212, with unit-cell parameters a = b = 57.89, c = 167.25 Å. A molecular-replacement solution was obtained and structure refinement of BL28 is in progress.

Adsorption of microbial esterases on Bacillus subtilis-templated cobalt oxide nanoparticles.

Due to low diffusion rates and large surface areas, nanomaterials have received great interest as supporting materials for enzyme immobilization. Here, the preparation of a cobalt oxide nanoparticle using Bacillus subtilis as a biological template and use of the nanostructure for microbial esterase immobilization is described. Morphological features and size distributions were investigated using electron microscopy (EM) and dynamic light scattering (DLS). Catalytic properties of enzyme-coated nanostructures were investigated using 4-methylumbelliferyl acetate and p-nitrophenyl (PNP) acetate as model substrates. Enzyme-coated nanostructures were observed to retain ∼85% of the initial activity after 15 successive reaction cycles, and enzyme immobilization processes could be repeated four times without a loss of immobilization potential. The present work demonstrates that B. subtilis-templated cobalt oxide nanoparticles have the potential to be used as biocompatible immobilization materials, and are promising candidates for the preparation of effective nanobiocatalysts.

Structural and functional analyses of a bacterial homologue of hormone-sensitive lipase from a metagenomic library.

Intracellular mobilization of fatty acids from triacylglycerols in mammalian adipose tissues proceeds through a series of lipolytic reactions. Among the enzymes involved, hormone-sensitive lipase (HSL) is noteworthy for its central role in energy homeostasis and the pathogenic role played by its dysregulation. By virtue of its broad substrate specificity, HSL may also serve as an industrial biocatalyst. In a previous report, Est25, a bacterial homologue of HSL, was identified from a metagenomic library by functional screening. Here, the crystal structure of Est25 is reported at 1.49 Šresolution; it exhibits an α/ß-hydrolase fold consisting of a central ß-sheet enclosed by α-helices on both sides. The structural features of the cap domain, the substrate-binding pocket and the dimeric interface of Est25, together with biochemical and biophysical studies including native PAGE, mass spectrometry, dynamic light scattering, gel filtration and enzyme assays, could provide a basis for understanding the properties and regulation of hormone-sensitive lipase (HSL). The increased stability of cross-linked Est25 aggregates (CLEA-Est25) and their potential for extensive reuse support the application of this preparation as a biocatalyst in biotransformation processes.

Identification, characterization, and application of a virulence factor (EfEstA) from Enterococcus faecalis.

Although virulence factors from pathogenic bacteria have been proposed as promising industrial enzymes, characterization and application of these enzymes have been still unexplored. The objective of this study was to identify and characterize virulence factors from pathogenic bacteria for industrial applications. Here, a virulence factor (EfEstA) from Enterococcus faecalis was identified and characterized using SDS-PAGE, enzyme assays, and molecular modeling. In addition, it has been shown that cross-linked enzyme aggregates (CLEAs) of EfEstA exhibited improved stabilities and high recycling activities compared to free EfEstA. These characteristics of EfEstA shed light on the design of new lipase-based systems for industrial applications.

A novel method for the preparation of a neurotoxic complex.

Neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) can be attributed to the specific degeneration of neuronal cells in the brain. However, the natures and action modes of toxic species remain largely unknown. Here, we present a simple and fast method for the preparation of neurotoxic complex with α-synuclein, which is implicated in PD.

Characterization and preparation of highly stable aggregates of a novel type of hydrolase (BL28) from Bacillus licheniformis.

A novel type of hydrolase (BL28) from Bacillus licheniformis was identified, expressed in Escherichia coli, characterized, and immobilized for industrial applications. Biochemical characteristics of BL28 were investigated by performing SDS-PAGE, mass spectrometry, enzyme assays, CD spectroscopy, intrinsic fluorescence, and in silico analysis. Furthermore, cross-linked enzyme aggregates (CLEAs) of BL28 were prepared. These CLEA-BL28 aggregates exhibited improved catalytic efficiencies and stabilities compared to free BL28 against harsh conditions of thermal or chemical stress as well as high reusability. The characteristics of the CLEA-BL28 aggregates highlight their great potentials in pharmaceutical and chemical industries.

Identification, crystallization and preliminary X-ray diffraction analysis of esterase A from Caulobacter crescentus CB15, a family VIII lipolytic enzyme.

The structures and functions of family VIII lipolytic enzymes, which have moderate sequence identity to class C ß-lactamases and penicillin-binding proteins, are largely unknown. Here, the X-ray crystallographic study of a family VIII esterase from Caulobacter crescentus CB15 (CcEstA) is described. Sequence analysis revealed that CcEstA has a conserved serine residue within the S-X-X-K motif which acts as a catalytic nucleophile. Recombinant protein containing an N-terminal His tag was expressed in Escherichia coli and purified to homogeneity. Functional studies showed that CcEstA acts on α- and ß-naphthyl acetate as substrates. In addition, it can catalyze the hydrolysis of ketoprofen ethyl ester, a highly useful product in industrial applications. CcEstA was crystallized using a solution consisting of 1.0 M potassium/sodium tartrate, 0.1 M imidazole pH 8.0, 0.2 M NaCl, and X-ray diffraction data were collected to a resolution of 1.62 Šwith an R(merge) of 9.4%. The crystals of CcEstA belonged to space group C222(1), with unit-cell parameters a = 172.23, b = 176.68, c = 47.93 Å. Structure determination is in progress.