Suhyun Jung

Rational design for enhancing promiscuous activity of Candida antarctica lipase B: a clue for the molecular basis of dissimilar activities between lipase and serine-protease

Lipase and serine-protease display distinct activities toward esters and amides, although they share a common catalytic triad (Asp/Glu-His-Ser). Lipases can hydrolyze esters but scarcely amides, whereas serine-proteases catalyze the hydrolysis of esters as well as amides. This issue has remained unresolved for a long period of time. Recently, we proposed a molecular basis for the enhanced lipase-catalyzed N-acylation of 1-phenylethanamine using methoxyacetate as an acyl donor (ChemBioChem, 2006, 7, 1745). We hypothesized that the hydrogen atom connected to the nitrogen atom of the amine substrate can flip-flop and thus hamper the formation of the essential hydrogen bond between the catalytic histidine and the nitrogen atom of the substrate. The disruption could be avoided by fixing the position of the amide proton using methoxyacetate as a hydrogen-bond acceptor. Based on the hypothesis, we assumed that the introduction of a residue acting as a hydrogen-bond acceptor would improve the amidase activity (the reverse reaction of aminolysis) of lipase. Herein, we demonstrate that the introduction of a polar amino acid near the active site of Candida antarctica lipase B (CAL-B) enhances the initial rate of the hydrolysis toward 4-nitroacetanilide by a factor of up to 24. This result possibly provides a clue for understanding the molecular basis of the dissimilar activities of the two hydrolases.

Hydrogen‐Bonding‐Driven Enantioselective Resolution against the Kazlauskas Rule To Afford γ‐Amino Alcohols by Candida rugosa Lipase

Most lipases resolve secondary alcohols in accordance with the “Kazlauskas rule” to give the R enantiomers. In a similar manner to other lipases, Candida rugosa lipase (CRL) exhibits R enantioselectivity towards heptan‐2‐ol, although the enantiomeric ratio (E) is low (E=1.6). However, unexpected enantioselectivity (i.e., S enantioselectivity, E=58) of CRL towards 4‐(tert‐butoxycarbonylamino)butan‐2‐ol, which has a similar chain length to heptan‐2‐ol, has been observed. To develop a deeper understanding of the molecular basis for this unusual enantioselectivity, we have conducted a series of molecular modeling and substrate engineering experiments. The results of these computational and experimental analyses indicated that a hydrogen bond between the Ser450 residue and the nitrogen atom of the carbamate group is critical to stabilize the transition state of the S enantiomer.

Facile covalent bio-conjugation of hydroxyapatite

Hydroxyapatite, which is a major component of the bone system in the human body, is an attractive material for bio-applications because it is nontoxic as well as biocompatible. In addition, many proteins are readily adsorbed on the surface of hydroxyapatite. The protein adsorption properties of hydroxyapatite have been employed for many bio-applications such as protein purification and nanomedical application. Nevertheless, the applications are not appropriate for proteins possessing hydrophobic surfaces because the surface of hydroxyapatite is charged. For applications with hydrophobic proteins, a covalent conjugation would be an alternative approach instead of physical adsorption. However, only a few examples of the covalent conjugation of proteins onto the surface of hydroxyapatite have been reported due to the lack of a convenient method for covalent conjugation. Herein, we report a facile process for the covalent conjugation of proteins on hydroxyapatite. We have successfully activated the surface of hydroxyapatite by a simple treatment with a peptide coupling reagent (for instance, N,N′-dicyclohexyl carbodiimide, N,N′-diisopropylcarbodiimide, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). Then, we directly conjugated enhanced green fluorescent protein (EGFP) as a model protein, and the conjugation was confirmed by a fluorescent microscope. We also employed the method for a hydrophobic surface protein, lipase. In this case, we found that a linker compound is required for the conjugation of lipase because of the distinct polarities of the surfaces of hydroxyapatite and lipase. The conjugated lipase on hydroxyapatite exhibited higher activity in organic solvents than the free form of lipase by a factor of up to 30 and can be recycled without a significant loss of the activity.