Shuhong Mao

Rational design of cholesterol oxidase for efficient bioresolution of cholestane skeleton substrates

Cholesterol oxidase catalyzes the oxidation and isomerization of the cholestane substrates leading to the addition of a hydroxyl group at the C3 position. Rational engineering of the cholesterol oxidase from Pimelobacter simplex (PsChO) was performed. Mutagenesis of V64 and F70 improved the catalytic activities toward cholestane substrates. Molecular dynamics simulations, together with structure-activity relationship analysis, revealed that both V64C and F70V increased the binding free energy between PsChO mutants and cholesterol. F70V and V64C mutations might cause the movement of loops L56-P77, K45-P49 and L350-E354 at active site. They enlarged the substrate-binding cavity and relieved the steric interference with substrates facilitating recognition of C17 hydrophobic substrates with long side chain substrates.

Electro-ultrafiltration to remove sodium dodecyl sulfate in proteins extracted for proteomics

Sodium dodecyl sulfate (SDS) is commonly used to extract membrane proteins in proteomics studies; however, it can reduce the efficiency of tryptic digestion and interfere with the results of liquid chromatography-mass spectrometry (LC-MS) analysis. Available methods for removing surfactants, such as ultrafiltration, acetone precipitation, and gel electrophoresis, are not completely satisfactory. Therefore, in this study, a new method for the depletion of SDS was established, named electro-ultrafiltration, and its performance was compared with other conventional pretreatment methods. Electro-ultrafiltration combines electrophoresis and ultrafiltration to remove SDS from protein samples. This method uses an electric field as the driving force and an ultrafiltration membrane as the separation medium. The performance of the electro-ultrafiltration method in terms of both the signals of LC-MS and the number of proteins identified was superior to that of simple ultrafiltration, but was slightly worse than that of acetone precipitation. These results demonstrate that the electro-ultrafiltration method could help to reduce the influence of SDS on protein digestion and identification, demonstrating its feasibility for application in proteomics.

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates

BackgroundBiosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor.ResultsRecombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively).ConclusionsThe successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.

Screening for Strains Capable of 13β-ethyl-4-gonene-3, 17-dione Biotransformation and Identification of Product

Screening of strains for improvement in the yields of the desired steroid as well as production of novel steroid is highly demanded. In this study, a filamentous fungus Aspergillus oryzae capable of transforming steroid efficiently was screened from the collection of fresh bark of south China. A novel transformation product was purified, crystallized, and determined as [13, 17β-]furan-17-hydroxyl-4-gonene-3one by single-crystal X-ray diffraction. At the end of the transformation, the yields of the product reached to 95 %, which indicated A. oryzae can be efficiently applied in the field of steroid biotransformation with the novel biocatalytic ability.

Reduction Reaction of Methyl Condensation Compound by Saccharomyces cerevisiae

Microbial transformation of 13β-alkyl-3-methoxy-8, 14, secogona-1, 3, 5(10), 9-tetraen-14, 17-dion with Saccharomyces cerevisiae resulted in two metabolites: product 1 (P1) and product 2 (P2). Among these, P1 was identified to be 13β alkyl-3-methoxy-8, 14-seco-1, 3, 5(10), 9(11)-estratetraene-17β-ol, 14-one, which has been studied extensively. P2 was purified, crystallized, and identified as 13β-alkyl-3-methoxy-8, 14, secogona-1, 3, 5(10), 9-tetraen-14, 17-diol by X-ray single crystal diffraction method. It was first reported in fermentation medium and the dihydroxyl reduction is a new reaction in biotransformation of steroid.

Using Water Miscible Ionic Liquid to Improve the Efficiency of 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by Penicillium raistrickii

Improved efficiency of steroid microbial transformation by co-solvents is generally attributed to their positive effects on the solubility of substrate in aqueous media. Ionic liquids have been widely researched as possible ‘green’ replacements for organic solvents. In this study, the effect of water miscible ionic liquids on the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by Penicillium raistrickii ATCC 10490 was investigated. The results indicated that the striking improvement of biotransformation of substrates up to 94.46 % were obtained after addition of [BMIM]BF4 under the optimized conditions. This also resulted in morphological changes of P. raistrickii mycelia in the fermentation broth with or without [BMIM]BF4. Based on the above observations, we therefore conclude that [BMIM]BF4-induced changes of mycelial morphology of P. raistrickii have positive effects on the production of the desired product, thus most likely contributing to the observed higher yield of steroid biotransformation in addition to [BMIM]BF4 known effects on substrate solubility.