Bum Han Ryu

Structural and biochemical characterization of a carbohydrate acetylesterase from Sinorhizobium meliloti 1021

In many microorganisms, carbohydrate acetylesterases remove the acetyl groups from various types of carbohydrates. Sm23 from Sinorhizobium meliloti is a putative member of carbohydrate esterase family 3 (CE3) in the CAZy classification system. Here, we determined the crystal structure of Sm23 at 1.75 Å resolution and investigated functional properties using biochemical methods. Furthermore, immobilized Sm23 exhibited improved stability compared with soluble Sm23, which can be used for the design of plant cell wall degrading‐systems.

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 Biochemical Characterization of an Octameric Carbohydrate Acetylesterase from Sinorhizobium meliloti.

Carbohydrate acetylesterases, which have a highly specific role among plant‐interacting bacterial species, remove the acetyl groups from plant carbohydrates. Here, we determined the crystal structure of Est24, an octameric carbohydrate acetylesterase from Sinorhizobium meliloti, at 1.45 Å resolution and investigated its biochemical properties. The structure of Est24 consisted of five parallel β strands flanked by α helices, which formed an octameric assembly with two distinct interfaces. The deacetylation activity of Est24 and its mutants around the substrate‐binding pocket was investigated using several substrates, including glucose pentaacetate and acetyl alginate. Elucidation of the structure‐function relationships of Est24 could provide valuable opportunities for biotechnological explorations.

Biochemical and Structural Analysis of a Novel Esterase from Caulobacter crescentus related to Penicillin-Binding Protein (PBP).

Considering that the prevalence of antibiotic-resistant pathogenic bacteria is largely increasing, a thorough understanding of penicillin-binding proteins (PBPs) is of great importance and crucial significance because this enzyme family is a main target of β-lactam-based antibiotics. In this work, combining biochemical and structural analysis, we present new findings that provide novel insights into PBPs. Here, a novel PBP homologue (CcEstA) from Caulobacter crescentus CB15 was characterized using native-PAGE, mass spectrometry, gel filtration, CD spectroscopy, fluorescence, reaction kinetics, and enzyme assays toward various substrates including nitrocefin. Furthermore, the crystal structure of CcEstA was determined at a 1.9 Å resolution. Structural analyses showed that CcEstA has two domains: a large α/β domain and a small α-helix domain. A nucleophilic serine (Ser68) residue is located in a hydrophobic groove between the two domains along with other catalytic residues (Lys71 and Try157). Two large flexible loops (UL and LL) of CcEstA are proposed to be involved in the binding of incoming substrates. In conclusion, CcEstA could be described as a paralog of the group that contains PBPs and β-lactamases. Therefore, this study could provide new structural and functional insights into the understanding this protein family.

Characterization of a novel SGNH-type esterase from Lactobacillus plantarum

Lactic acid bacteria (LAB) are sources of a large variety of microbial ester hydrolases because they can produce a wide range of short-chain esters, phenolic alcohols, and fatty acids. Here, a novel SGNH-type esterase (LpSGNH1) from Lactobacillus plantarum WCFS1 was identified, functionally characterized, and immobilized for biotechnological applications. Homologs of LpSGNH1 are also found in many lactic acid bacteria (LAB) species. Biochemical features of LpSGNH1 were investigated using mass spectrometry, gel filtration chromatography, enzyme kinetics, fluorescence, and circular dichroism (CD) spectroscopy. LpSGNH1 were retained its activity under conditions that would be encountered during fermentations. Interestingly, LpSGNH1 exhibited the ability to act on a broad range of substrates including ketoprofen acetate, cefotaxime (CTX), and 7-aminocephalosporanic acid (7-ACA) as well as glucose pentaacetate, acetylxylan, and acetylalginate, which make LpSGNH1 a great candidate for extensive industrial applications. Furthermore, cross-linked enzyme aggregates of LpSGNH1 (CLEA-LpSGNH1) displayed recycling ability and thermal stability compared to free LpSGNH1, which could be useful for industrial applications. This work highlights the importance of LpSGNH1 in the preparation of commercial compounds, and LpSGNH1 can be used as a model system of SGNH esterases in lactic acid bacteria.

Preparation of cobalt nanoparticles from polymorphic bacterial templates: A novel platform for biocatalysis

Nanoparticles have gathered significant research attention as materials for enzyme immobilization due to their advantageous properties such as low diffusion rates, ease of manipulation, and large surface areas. Here, polymorphic cobalt nanoparticles of varied sizes and shapes were prepared using Micrococcus lylae, Bacillus subtilis, Escherichia coli, Paracoccus sp., and Haloarcula vallismortis as bacterial templates. Furthermore, nine lipases/carboxylesterases were successfully immobilized on these cobalt nanoparticles. Especially, immobilized forms of Est-Y29, LmH, and Sm23 were characterized in more detail for potential industrial applications. Immobilization of enzymes onto cobalt oxide nanoparticles prepared from polymorphic bacterial templates may have potential for efficient hydrolysis on an industrial-scale, with several advantages such as high retention of enzymatic activity, increased stability, and strong reusability.

Crystallization and preliminary X-ray analysis of a highly stable novel SGNH hydrolase (Est24) from Sinorhizobium meliloti.

The SGNH hydrolase family includes enzymes that catalyze the hydrolysis of a broad range of substrates. Here, the crystallization and preliminary X-ray crystallographic studies of a novel SGNH hydrolase (Est24) from Sinorhizobium meliloti were performed. Recombinant Est24 protein containing an N-terminal His tag was expressed in Escherichia coli and purified to homogeneity. Est24 was then crystallized using a solution consisting of 0.2 M ammonium phosphate pH 4.6, 20% polyethylene glycol 3350. X-ray diffraction data were collected to a resolution of 1.45 Å with an R(merge) of 9.4%. The Est24 crystals belonged to space group C2, with unit-cell parameters a = 129.09, b = 88.63, c = 86.15 Å, α = 90.00, β = 114.30, γ = 90.00°. A molecular-replacement solution was obtained using the crystal structure of Mycobacterium smegmatis arylesterase as a template and structure refinement of Est24 is in progress.

Identification, characterization, immobilization, and mutational analysis of a novel acetylesterase with industrial potential (LaAcE) from Lactobacillus acidophilus

Lactic acid bacteria, which are involved in the fermentation of vegetables, meats, and dairy products, are widely used for the productions of small organic molecules and bioactive peptides. Here, a novel acetylesterase (LaAcE) from Lactobacillus acidophilus NCFM was identified, functionally characterized, immobilized, and subjected to site-directed mutagenesis for biotechnological applications. The enzymatic properties of LaAcE were investigated using biochemical and biophysical methods including native polyacrylamide gel electrophoresis, acetic acid release, biochemical assays, enzyme kinetics, and spectroscopic methods. Interestingly, LaAcE exhibited the ability to act on a broad range of substrates including glucose pentaacetate, glyceryl tributyrate, fish oil, and fermentation-related compounds. Furthermore, immobilization of LaAcE showed good recycling ability and high thermal stability compared with free LaAcE. A structural model of LaAcE was used to guide mutational analysis of hydrophobic substrate-binding region, which was composed of Leu156, Phe164, and Val204. Five mutants (L156A, F164A, V204A, L156A/F164A, and L156A/V204A) were generated and investigated to elucidate the roles of these hydrophobic residues in substrate specificity. This work provided valuable insights into the properties of LaAcE, and demonstrated that LaAcE could be used as a model enzyme of acetylesterase in lactic acid bacteria, making LaAcE a great candidate for industrial applications.

Identification, Characterization, and Immobilization of an Organic Solvent-Stable Alkaline Hydrolase (PA27) from Pseudomonas aeruginosa MH38

An organic solvent-stable alkaline hydrolase (PA27) from Pseudomonas aeruginosa MH38 was expressed, characterized, and immobilized for biotechnological applications. Recombinant PA27 was expressed in Escherichia coli as a 27 kDa soluble protein and was purified by standard procedures. PA27 was found to be stable at pH 8–11 and below 50 °C. It maintained more than 80% of its activity under alkaline conditions (pH 8.0–11.0). Furthermore, PA27 exhibited remarkable stability in benzene and n-hexane at concentrations of 30% and 50%. Based on these properties, immobilization of PA27 for biotechnological applications was explored. Scanning electron microscopy revealed a very smooth spherical structure with numerous large pores. Interestingly, immobilized PA27 displayed improved thermal/chemical stabilities and high reusability. Specifically, immobilized PA27 has improved thermal stability, maintaining over 90% of initial activity after 1 h of incubation at 80 °C, whereas free PA27 had only 35% residual activity. Furthermore, immobilized PA27 showed higher residual activity than the free enzyme biocatalysts against detergents, urea, and phenol. Immobilized PA27 could be recycled 20 times with retention of ~60% of its initial activity. Furthermore, macroscopic hydrogel formation of PA27 was also investigated. These characteristics make PA27 a great candidate for an industrial biocatalyst with potential applications.

Modulating α-synuclein fibril formation using DNA tetrahedron nanostructures

The small presynaptic protein α-synuclein (α-syn) is involved in the etiology of Parkinsons disease owing to its abnormal misfolding. To date, little information is known on the role of DNA nanostructures in the formation of α-syn amyloid fibrils. Here, the effects of DNA tetrahedrons on the formation of α-syn amyloid fibrils were investigated using various biochemical and biophysical methods such as thioflavin T fluorescence assay, atomic force microscopy, light scattering, transmission electron microscopy, and cell-based cytotoxicity assay. It has been shown that DNA tetrahedrons decreased the level of oligomers and increased the level of amyloid fibrils, which corresponded to decreased cellular toxicity. The ability of DNA tetrahedron to facilitate the formation of α-syn amyloid fibrils demonstrated that structured nucleic acids such as DNA tetrahedrons could modulate the process of amyloid fibril formation. Our study suggests that DNA tetrahedrons could be used as an important facilitator toward amyloid fibril formation of α-synuclein, which may be of significance in finding therapeutic approaches to Parkinsons disease and related synucleinopathies.