Mei Lehe

Tolerance of immobilized baker’s yeast in organic solvents

Abstract By determining the metabolic activity retention of baker’s yeast after 24 h exposure to various organic solvents, the tolerances of immobilized baker’s yeast and free cells in organic solvents were studied, respectively. The effects of pre-incubation time and temperature on the tolerance of immobilized baker’s yeast were examined in detail. The results showed that the tolerance of immobilized baker’s yeast was higher than that of free cells and increased with the increase of logP value of organic solvent. In addition, the effect of temperature on the tolerance of immobilized baker’s yeast was similar to its effect on the growth of baker’s yeast. The tolerance of immobilized baker’s yeast reached maximum at 30°C and decreased when it was in the climax of budding.

Recovery of antibiotics by aqueous two-phase partition —Partitioning behavior of pure acetylspiramycin solution in polyethylene glycol/potassium phosphate aqueous two-phase systems

The effects of average molecular weight of PEG, concentrations of PEG and KH2PO4 and pH on the partition equilibrium of acetylspiramycin in PEG/KH2PO4 aqueous two-phase systems were studied in detail. The partition coefficients of acetylspiramycin in PEG/ KH2PO4 systems were measured at room temperature 25 °C. It was found that acetylspiramycin partitioned unevenly in the aqueous two-phase systems composed of PEG and KH2PO4 and could be purified by this technique. A suitable phase-forming system (pH=6.7, 12w/w% PEG2000, 11w/w% KH2PO4) was found out after partition coefficient (Kp=42) , extraction ratio (η=96%) and recovery ratio(R=98.8%) were investigated comprehensively in this paper.

Kinetic and thermodynamic studies of sulforaphane adsorption on macroporous resin.

The adsorption equilibrium, kinetic and thermodynamic of sulforaphane (SF) adsorption onto macroporous resin in aqueous phase were studied. The SP850 resin was screened as the appropriate resin for SF purification. From the equilibrium studies, the Redlich-Peterson model was found to be the best for description of the adsorption behavior of SF onto SP850 resin, followed by the Freundlich model and the Langmuir model. Batch equilibrium experiments demonstrated that, in the examined temperature range, the equilibrium adsorption capacity of SP850 resin decreased with increasing adsorption temperature. Thermodynamics studies indicated that the adsorption of SF was a physical, exothermic, and spontaneous process. The adsorption kinetics revealed that the pseudo-second-order kinetic model was suitable to characterize the kinetics of adsorption of SF onto SP850. Finally, the intra-particle diffusion model demonstrated that SF diffused quickly into macropores, and that diffusion slowed down in the meso- and micropores.

Purification of Cytochrome P450 BM-3 as a Monooxygenase

After investigating two anion-exchange resins, the purification factor and activity yields of P450 BM-3 were higher with Resource Q than with DEAE-Sepharose FF. Screening of HIC media showed that Source 15ISO was the most suitable for purification of P450 BM-3. An effective isolation and purification procedure of P450 BM-3 was developed and included three steps: 35%-70% saturation (NH(4))(2)SO(4) precipitation, Source 15ISO hydrophobic interaction chromatograph and Sephacryl S-200 gel filtration chromatography. Using this protocol, the purification factor and P450 BM-3 activity recovery was 13.5 and 13.7%, respectively.

删除Loop区域表面不稳定氨基酸提高( R )-ω-转氨酶热稳定性

Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T₅₀¹⁰ (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.