T. Doohun Kim

Scalable One-pot Bacteria-templating Synthesis Route toward Hierarchical, Porous-Co3O4 Superstructures for Supercapacitor Electrodes

Template-driven strategy has been widely used to synthesize inorganic nano/micro materials. Here, we used a bottom-up controlled synthesis route to develop a powerful solution-based method of fabricating three-dimensional (3D), hierarchical, porous-Co3O4 superstructures that exhibit the morphology of flower-like microspheres (hereafter, RT-Co3O4). The gram-scale RT-Co3O4 was facilely prepared using one-pot synthesis with bacterial templating at room temperature. Large-surface-area RT-Co3O4 also has a noticeable pseudocapacitive performance because of its high mass loading per area (~10 mg cm−2), indicating a high capacitance of 214 F g−1 (2.04 F cm−2) at 2 A g−1 (19.02 mA cm−2), a Coulombic efficiency averaging over 95%, and an excellent cycling stability that shows a capacitance retention of about 95% after 4,000 cycles.

Amyloid formation and disaggregation of α-synuclein and its tandem repeat (α-TR).

The aggregation of α-synuclein is clearly related to the pathogenesis of Parkinsons disease. Therefore, detailed understanding of the mechanism of fibril formation is highly valuable for the development of clinical treatment and also of the diagnostic tools. Here, we have investigated the interaction of α-synuclein with ionic liquids by using several biochemical techniques including Thioflavin T assays and transmission electron microscopy (TEM). Our data shows a rapid formation of α-synuclein amyloid fibrils was stimulated by 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [BIMbF(3)Im], and these fibrils could be disaggregated by polyphenols such as epigallocatechin gallate (EGCG) and baicalein. Furthermore, the effect of [BIMbF(3)Im] on the α-synuclein tandem repeat (α-TR) in the aggregation process was studied.

Ionic liquids promote amyloid formation from α-synuclein

The slow process required for alpha-synuclein to form amyloid fibrils is a major obstacle in the development of therapeutic compounds for alpha-synuclein-related neurodegenerative diseases such as Parkinsons disease (PD). Here we have developed an efficient method by which amyloid fibrils can be formed from alpha-synuclein using ionic liquids (ILs). This report indicates that ILs could potentially be used as a stimulator for the amyloid formation of alpha-synuclein.

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 Å resolution; it exhibits an α/β-hydrolase fold consisting of a central β-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.

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.

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.

Amyloid formation using 1-butyl-3-methyl-imidazolium-based ionic liquids.

Amyloid fibrils are highly organized protein filaments that can be used as novel biomaterials. In this study, we show that proteins could be selectively induced to form amyloid fibrils at room temperature by the introduction of imidazolium salts, which could trigger the self-assembly process with their hydrophobic and ionic properties.

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.

Purification, crystallization and preliminary crystallographic analysis of Est25: a ketoprofen-specific hormone-sensitive lipase

Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form. Est25 was crystallized from 2.4 M sodium malonate pH 7.0 and X-ray diffraction data were collected to 1.49 A using synchrotron radiation. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 197.8, b = 95.2, c = 99.4 A, beta = 97.1 degrees.

Coaggregation of amyloid fibrils for the preparation of stable and immobilized enzymes.

Highly stable enzyme coaggregates were developed using amyloid fibrils as support materials. Amyloid fibril formation was induced by ionic liquids, and immobilization was done by the coaggregation of enzymes and amyloid fibrils followed by chemical cross-linking. Transmission and scanning electron microscopy studies were carried out to characterize the coaggregates. The amyloid fibril-linked enzymes showed significantly increased stability against various deactivating conditions. In addition, a high level of reusability was clearly observed. This study clearly demonstrated that amyloid fibrils can be used as biomaterials for enzyme immobilization and that amyloid fibril-linked enzyme coaggregates have good potential for industrial applications.