ole Xia

High efficient treatment of citric acid effluent by Chlorella vulgaris and potential biomass utilization

The efficiency of treating citric acid effluent by green algae Chlorella was investigated. With the highest growth rate, Chlorella vulgaris C9-JN2010 that could efficiently remove nutrients in the citric acid effluent was selected for scale-up batch experiments under the optimal conditions, where its maximum biomass was 1.04 g l(-1) and removal efficiencies of nutrients (nitrogen, phosphorus, total organic carbon, chemical oxygen demand and biochemical oxygen demand) were above 90.0%. Algal lipid and protein contents were around 340.0 and 500.0 mg · g(-1) of the harvested biomass, respectively. Proportions of polyunsaturated fatty acids in the lipids and eight kinds of essential amino acids in algal protein were 74.0% and 40.0%, respectively. Three major fatty acids were hexadecanoic acid, eicosapentaenoic acid and docosadienoic acid. This specific effluent treatment process could be proposed as a dual-beneficial approach, which converts nutrients in the high strength citric acid effluent into profitable byproducts and reduces the contaminations.

Affinity purification of a cholesterol oxidase expressed in Escherichia coli

A cholesterol oxidase (COD) gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), an affinity protocol was developed for the preparation, and industrial application of this method was of great potential. Riboflavin was chosen as the affinity ligand, and it was coupled with Sepharose 4B through some spacers. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture the target protein. The purified cholesterol oxidase was 99.5% pure analyzed on HPLC Vydac C4 column, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 9.8% of crude extracted proteins; the recovery of typical cholesterol oxidase activity was 90.1%, higher than that of other reported traditional protocols. Reducing SDS-PAGE analysis showed that the enzyme was a single polypeptide with the mass of ∼50 kDa. The desorption constant K(d) and the theoretical maximum absorption Q(max) on the affinity medium were 1.0 μg/g medium and 74.5 mg/g medium in absorption analysis. K(m) and V(max) of cholesterol oxidase activity for the purified enzyme were 25.5 μM and 16.4 μmol/(min mg), respectively.

High strength vinegar fermentation by Acetobacter pasteurianus via enhancing alcohol respiratory chain

Seeking high strength vinegar fermentation by acetic acid bacteria (AAB) is still the mission of vinegar producers. AAB alcohol respiratory chain, located on intracellular membrane, is directly responsible for vinegar fermentation. In the semi-continuous vinegar fermentation by Acetobacter pasteurianus CICIM B7003, acetification rate showed positive correlation with the activity of the enzymes in alcohol respiratory chain. Aiming at achieving high strength fermentation process, a series of trials were designed to raise the activity of AAB alcohol respiratory chain. Finally, acetification was enhanced by adding some precursors (ferrous ions and β-hydroxybenzoic acid) of alcohol respiration associated factors and increasing aeration rate (0.14 vvm). As final result, average acetification rate has been raised to 2.29 ± 0.02 g/L/h, which was 28.7% higher than the original level. Simultaneously, it was found that the oxidization of alcohol into acetic acid in AAB cells was improved by well balancing of three factors: enzyme activity in alcohol respiratory chain, precursor of ubiquinone biosynthesis, and aeration rate.

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.

Improving fermented quality of cider vinegar via rational nutrient feeding strategy

This work aimed to find a rational nutrient feeding strategy for cider vinegar fermentation based on adequate information on the nutritional requirement of acetic acid bacteria. Through single nutrient lack experiment assay, necessary nutrient recipe for Acetobacter pasteurianus CICIM B7003 in acetous fermentation was confirmed. Compounds from the essential nutrient recipe were tested further to find out the key substrates significantly influencing cider vinegar fermentation. The findings showed that aspartate, glutamate, proline and tryptophan should be considered in detail for optimizing nutritional composition of cider. Finally, a nutrient feeding strategy that simultaneously adds proline, glutamate, aspartate and tryptophan to form final concentrations of 0.02g/L, 0.03g/L, 0.01g/L and 0.005g/L in cider was achieved by orthogonal experiment design. Comparing to the original fermentation, the yield of acetic acid from alcohol reached 93.3% and the concentration of most volatile flavor compounds increased with the rational nutrient feeding strategy.

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.