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The interaction between Gd@C82(OH)22 and serum albumin (HSA and BSA) were investigated by spectroscopic analysis. From the characteristic feature of fluorescence quenching spectra at different temperatures, the inherent binding information including quenching mechanism, association constants, number of binding site, fraction of initial fluorescence and basic thermodynamic parameters were calculated. The binding of Gd@C82(OH)22 to serum albumin caused strong quenching of protein intrinsic fluorescence and the structural changes of serum albumin. At lower concentrations, Gd@C82(OH)22 was likely to rise fluorescence quenching of serum albumin through individual static quenching process by forming a ground-state complex, while dynamic and static coexisting quenching mechanism occurred in high concentration. Bimolecular quenching (Kq) value is twice the diffusion-controlled quenching constant (2.0 × 1010 L mol−1 s−1); binding sites of BSA were slightly more than those of HAS, and all of them reached to 1; the distance r between donor and acceptor was found to be 3.1494 nm and 3.6479 nm for HSA and BSA, respectively, both of which were fewer than 7 nm. It is confirmed that binding interaction for proteins in the presence of drugs was strong, the binding ratio was 1:1, and non-radiative energy transfer from protein to drug was extremely high probability in lower density. Binding process of Gd@C82(OH)22-HSA was driven mainly through van der Waals forces and hydrogen bonding formation, however more likely to be electrostatic interaction involved in the Gd@C82(OH)22-BSA binding process; Binding sites of Gd@C82(OH)22 to serum albumin were near tryprophan (HSA) and tyrosine residues (BSA), respectively. Moreover, a theoretical model of predicting the binding rate of drug to serum albumin was estimated, further analyzed that the binding rate was dynamically altered in various dose of protein and drug. Overall, these results provide potentially significant information for elucidating the distribution, transportation, the apparent relationship between pharmacologic activity and total plasma drug concentration as well as anti-carcinogenic activity and mechanisms in vivo.

Esterases (EC 3.1.1.X) have been used as biocatalysts due to their good stability, high chemo-, regio- and stereoselectivity. In our previous studies, Bacillus megaterium WZ009 harboring esterase displayed the unique capability to convert (S)-4-Chloro-3-hydroxyethylbutyrate (CHBE) in the racemate to (S)-3-hydroxy-γ-butyrolactone (HL) through stereoselective hydrolysis, dechlorination, and lactonization. The remaining (R)-CHBE and formed (S)-HL could be obtained in a one-pot enzymatic reaction. An esterase from B. megaterium WZ009 was purified and was found to have 466 encoded amino acids and an apparent molecular mass of 55 kDa. The purified esterase exhibited maximal activity at a temperature of 25 °C and at a pH of 11.5 towards 100 mM CHBE. When the stereoselective biocatalysis of rac-CHBE was performed using the recombinant Escherichia coli BL21 (DH3) cells harboring the esterase, the catalytic activity increased by 20-fold compared with the original strain B. megaterium WZ009. With the addition of activated carbon (62 g/L) in the reaction system, the conversion was increased from 39% to 45% at a substrate concentration of 750 mM. Another remarkable advantage is that both of the obtained residual (R)-CHBE and the formed (S)-HL had high optical purities (e.e.s > 99.9%, e.e.p > 99.9%), thereby making this esterase a usable biocatalyst for industrial application.

A new esterase-producing strain ( Bacillus amyloliquefaciens WZZ002) that exhibits high hydrolytic activity, excellent enantioselectivity, and high substrate tolerance on Boc- dl -Alanine methyl ester was isolated from soil samples. The reaction temperature, pH, and neutralizer optima of the cell-mediated biocatalysis were 35 °C, pH 8.0, and NH 3 ·H 2 O, respectively. The optimal substrate concentration was 2 M, with a biocatalyst loading of 50 g/L. Results showed that the enantiomeric excess values of substrate and product were both greater than 99%. Thus, bioprocessing with the use of the isolated strain is a promising route for the commercial production of Boc- d -Ala-OMe.

A chemoenzymatic methodology for the asymmetric synthesis of d-biotin intermediate lactone ((3aS, 6aR)-tetrahydro-1,3-dibenzylhexahydro-1H-Furo[3,4-d] imidazole-2,4-dione) 1 has been demonstrated. The key step of the synthetic routes is Lipozyme RM IM catalyzed desymmetrization of meso-diols 3. The highest enantiomeric excess (e.e. > 98%) and yield (>90%) of the product was achieved with Lipozyme RM IM in Dioxane/Toluene (1:3, v/v) at 35 °C. Furthermore, Lipozyme RM IM showed an excellent operational stability, retaining above 80% of the initial activity after 10 cycles of reaction. d-Biotin intermediate lactone 1 was obtained subsequently by Jones oxidation, basic hydrolysis and lactonization.

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.