Yuran Zhang

Preparation and characterization of affinity sorbents based on isoalloxazine‐like ligands for separation of flavoenzymes

Affinity ligands for flavoenzymes were synthesized based on the natural structure of flavo-coenzymes. Two typical flavoenzymes, cholesterol oxidase from Brevibacterium sp. and xanthine oxidase from bovine milk, were employed as standard enzymes. Fluorescent probes were synthesized from eight isoalloxazine-like chemicals and 5-aminofluorescein. Probe-enzyme interactions were analyzed via fluorescence spectra. Chemicals with high binding abilities to flavoenzymes were coupled with Sepharose through spacers composed of epichlorohydrin, ethylenediamine, 1,3-diaminopropane, 2-hydroxy-1,3-diaminopropane, and 1,4-diaminobutane, and subjected to adsorption analysis with flavoenzymes. The results indicated that ligands synthesized from 2,4-dioxohexahydropyrimidine-5-carboxylic acid, cytosine, 7-chloroalloxazine, and 8-chloroalloxazine had high binding abilities to the flavoenzymes. The affinity sorbent based on these ligands revealed a high theoretical maximum adsorption (Q(max)). Protein and bioactivity recoveries were tested after one step of affinity binding via chromatographic analysis on small columns. Results showed that ligands linked with sorbents through long hydrophilic spacers had higher activity recoveries.

Novel affinity purification of xanthine oxidase from Arthrobacter M3

An affinity protocol for purification of xanthine oxidase (XOD) from Arthrobacter M3 was developed. The isolation procedure consisted of only three steps, ammonium sulfate precipitation, affinity extraction to exclude the major impurities, and the final refining procedure with DEAE ion-exchange chromatography for removal of minor contaminants. In this affinity preparation, guanine, an analogue of xanthine, was chosen as the affinity ligand, and was coupled with Sepharose 4B through spacers composed of epichlorohydrin and ethylenediamine. Crude protein has been run through ammonium sulfate precipitation and the affinity column, 99.1% of proteins were removed. After DEAE ion-exchange chromatography, the purity of the refined XOD was 97.5% by Native-PAGE analysis. The activity recovery of purified XOD (36.1%) was almost higher than that of other methods reported. Reducing SDS-PAGE analysis showed that the purified XOD (one band in Native-PAGE analysis) showed two polypeptides with the molecular weights ∼35kDa and ∼100kDa, respectively. The desorption constant K(d) and the theoretical maximum absorption Q(max) on the affinity medium were 3.0μg/ml and 2.2mg/g medium in absorption analysis.

Expression and comparison of recombinant cholesterol oxidases (COD) in Escherichia coli with native cholesterol oxidase expressed in Brevibacterium sp.

The structure and bio-activity of an endogenous cholesterol oxidase from Brevibacterium sp. was compared to the same enzyme exogenously expressed in Escherichia coli BL21 (DE3) with and without N- or C-terminal his-tags. The different proteins were purified with affinity and subtractive protocols. The specific activity of the natural enzyme from Brevibacterium sp. was 17.5 ± 0.2 U/mg, while the activities of the exogenously expressed forms were 16 ± 0.3 U/mg for non-tagged enzyme from E. coli , 12 ± 0.1 U/mg for the N-terminal his-tagged enzyme, and 4 ± 0.3 U/mg for C-terminal his-tagged enzyme. Circular dichroism revealed that the added histidine residues altered the natural folding of the enzyme. The natural cholesterol oxidase was composed of 39% α-helix, 40% β-sheet, and 20% random coil, while the non-tagged enzyme was composed of 40% α-helix, 35% β-sheet, and 24% random coil. In contrast, the N-terminal his-tagged enzyme was composed of 45% α-helix, 29% β-sheet, and 25% random coil, and the C-terminal his-tagged enzyme was composed of 55% α-helix, 16% β-sheet, and 28% random coil. Hydrophobic fluorescence analysis revealed that the hydrophobicity of the enzyme was reduced by his-tags. Coenzyme-like fluorescent probe binding analysis indicated that the coenzyme binding site should be blocked by his-tags. The his-tag method for protein isolation can disrupt the catalytic activity of the cholesterol oxidase. Key words : Cholesterol oxidase; Brevibacterium sp.; Escherichia coli; structural disruption, His-tags.

Thermal inactivation of xanthine oxidase from Arthrobacter M3: mechanism and the corresponding thermostabilization strategy

The mechanism of thermal inactivation about xanthine oxidase (XOD) from Arthrobacter M3 was investigated. Results of reducing SDS-PAGE indicated that the inactivation of XOD was not related to the peptide degradation. Meanwhile, fluorimetry and circular dichroism spectroscopy suggested that XOD inactivation might be associated with the exposure of hydrophobic residues to surface and partial loss of secondary structure. Specific formation of soluble aggregates of XOD was detected by size exclusion chromatography. In addition, the thermal-dynamic analysis showed that the inactivation kinetics of XOD followed the first-order model. Therefore, trehalose (cosolute) and betaine (osmolyte) were accordingly employed to attenuate the inactivation of this enzyme. The results associated with these two reagents further confirmed that the loss of XOD activity was mainly due to the exposure of hydrophobic residues and formation of aggregation. Owing to the added trehalose and betaine, half-life could be significantly increased, and the inactivation rate constant (k) was detected as decreased.