Title: Immobilization of Aspergillus terreus lipase in self-assembled hollow nanospheres for enantioselective hydrolysis of ketoprofen vinyl ester Hu C, Wang N, Zhang W, Zhang S, Meng Y, Yu X Ref: J Biotechnol, 194:12, 2015 : PubMed
The aim of this study was to improve the ability of Aspergillus terreus lipase to separate the racemic ketoprofen vinyl ester into individual enantiomers using hollow self-assembly alginate-graft-poly(ethylene glycol)/alpha-cyclodextrins (Alg-g-PEG/alpha-CD) spheres as enzyme immobilization carriers. The morphology and size of the Alg-g-PEG/alpha-CD particles were investigated by transmission electron microscopy (TEM) and were found to be nanoscale. To facilitate recycling, calcium alginate (CA) beads were developed to encapsulate Alg-g-PEG/alpha-CD particles, thereby producing Alg-g-PEG/alpha-CD/CA composite beads. The influence of buffer pH and enzyme concentration during immobilization was studied along with the biocatalyst's kinetic parameters. When the immobilized enzyme was under optimal conditions in the resolution reaction, maximal conversion (approximately 45.9%) and enantioselectivity (approximately 128.8) were obtained. The immobilized A. terreus lipase maintained excellent performance even after 20 reuses and retained nearly 100% of its original activity after 24 weeks of storage at 4 degrees C.
Title: Insight into substituent effects in Cal-B catalyzed transesterification by combining experimental and theoretical approaches Ni Z, Lin X Ref: J Mol Model, 19:349, 2013 : PubMed
Candida antarctica lipase B (Cal-B) is one of the most recognized biocatalysts because of its high degree of selectivity in a broad range of synthetic applications of industrial importance. Herein, the substituent effects involved in transesterification catalyzed by Cal-B are explored in detail using a combination of experimental analysis and theoretical modeling. The transesterification ability of Cal-B was experimentally determined with 22 vinyl ester analogs and ribavirin as substrates and, on this basis, a series of quantitative structure-activity relationship (QSAR) models are developed using various structural parameters characterizing the variation in substituent groups of the substrate molecules. The resulting models exhibit a good stability and predictive power, from which five most important properties are highlighted and engaged to ascertain the structural basis and reaction mechanism underlying the transesterification. From the modeling analysis it is seen that the size, geometry, and charge distributions of substrate exert a significant effect on reaction yield, where, the size of the substituent group was the most significant impact factor on the reaction yield, the charge distribution was the second, and then the topological structure of the substrate.
Title: Integrating In Silico and In vitro approaches to dissect the stereoselectivity of Bacillus subtilis lipase A toward ketoprofen vinyl ester Ni Z, Zhou P, Jin X, Lin XF Ref: Chemical Biology Drug Des, 78:301, 2011 : PubMed
The asymmetric catalysis, as the character of enzyme, attracts increasing attention from the scientific and industrial communities. In this study, the Bacillus subtilis lipase A, as a model enzyme, is studied systematically to dissect its stereoselectivity toward (rac)-ketoprofen vinyl ester using a combination scheme of molecular docking and quantum mechanical/molecular mechanical (QM/MM) analysis. In this procedure, the rational orientation of the two enantiomers of ketoprofen vinyl ester is obtained with the AutoDock performing, and then, the steric contacts between the enzyme and substrate in the docking outputs are examined visually at the atomic level with a small-probe technique. Subsequently, the binding energies of the enzyme-substrate complexes are calculated using an ONIOM (Our own N-layered Integrated Molecular Orbital + Molecular mechanics)-based QM/MM protocol. The results obtained from the theoretical studies show that the B. subtilis lipase A prefer to hydrolyze the (R )-ketoprofen vinyl ester when compared to its (S )-enantiomer, with a relatively high E (stereoselectivity) value of 31.28 charactering its enantioselectivity. Furthermore, to verify the conclusions from the computational analysis, the B. subtilis lipase A gene is cloned to overexpress the recombinant B. subtilis lipase A, and its stereoselectivity was determined. Satisfactorily, the experimental results are in well agreement with the theoretical predictions because the (R )-ketoprofen vinyl ester is found as the preferring enantiomer of the B. subtilis lipase A, with experimentally measured E value of 36.7. We therefore expect that this in silico-in vitro hybrid approach can provide a new and effective avenue to predict the catalytic activity of and to investigate the molecular mechanism of enzyme-mediated asymmetric catalysis and help in understanding the enzymatic process and in rational enzyme design.
