Yu Xin

Jiangnan University, Wu-hsi, Jiangsu Sheng, China

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Publications (24)56.41 Total impact

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    ABSTRACT: Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3 mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02 × 10−4 and 1.19 × 10−4 μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%.
    No preview · Article · Jan 2016
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    ABSTRACT: The codon-optimized sarcosine oxidase from Thermomicrobium roseum (TrSOX) was successfully expressed in Escherichia coli and its soluble expression was significantly enhanced via the co-expression of chaperones. With the assistance of whole-genome analysis of T. roseum DSM 5159, the sox gene was predicated and its sequence was optimized based on the codon bias of E. coli . The TrSOX gene was successfully constructed in the pET28a plasmid. After induction with IPTG for 8h, SDS-PAGE analysis of crude enzyme solutions showed a significant 43kDa protein band, indicating SOX was successfully expressed in E. coli . However, the dark band corresponding to the intracellular insoluble fraction indicated that most of TrSOX enzyme existed in the inactive form in "inclusion bodies" owing to the "hot spots" of TrSOX. Furthermore, the co-expression of five different combinations of chaperones indicated that the soluble expression of TrSOX was greatly improved by the co-expression of molecular chaperones GroES-GroEL and DnaK-DnaJ-GrpE-GroES-GroEL. Additionally, the analysis of intramolecular forces indicated that the hydrophobic amino acids, hydrogen bonds, and ionic bonds were favorable for enhancing the interaction and stability of TrSOX secondary structure. This study provides a novel strategy for enhancing the soluble expression of TrSOX in E. coli .
    Full-text · Article · Nov 2015 · Journal of Biotechnology
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    ABSTRACT: Product inhibition is the main problem of high-strength rice vinegar fermentation by Acetobacter pasteurianus. An optimized semi-continuous operation mode was proposed with a modified starting-up protocol which can improve the average acetification rate 35 % higher than original one. First, we revealed PQQ-ALDH is the rate-limiting enzyme responsible for ethanol oxidation route, whose apparent K m of PQQ-ADH and PQQ-ALDH is 1.89 and 7.80 mM, respectively. Further work elucidated that PQQ-ALDH follows ordered Bi–Bi mechanism which indicated that Co Q can improve the reaction. Among five Co Q precursors, supplementation of 20 mM isopentenyl alcohol got the best result, which improved strain acid resistance ability that accelerates biomass accumulation. A two-stage starting-up protocol was developed, which reduced the duration of the starting-up process from 45 to 34 h. An optimized semi-continuous operation mode was proposed whose whole operation time reduced from 116 to 98 h and average acetification rate has been raised to 1.77 g/Lh, while the original is 1.31 g/Lh. The average activity of ALDH is 43 % higher than original process, while ADH is 19 %, which shows a good correlation with acetification rate.
    No preview · Article · Oct 2015 · European Food Research and Technology
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    ABSTRACT: Sarcosine oxidase (SOX) was an important diagnostic enzyme in the renal function examination. An integrated strategy coupling codon and fermentation optimization was firstly proposed for improving SOX production from recombinant E. coli in 3-L fermentor. The expression suppression (gene phase) and poor balance between SOX expression and cell growth (fermentation phase) in the traditional SOX production were respectively improved by the multiple strategies. Based on the codon bias, the expression suppression was weakened via codon optimization and SOX activity reached 1,521 U/L. The induction toxicity was reduced with the optimal induction condition and SOX production increased to 4,015 U/L. Based on the kinetic analysis of μ x and μ p , a better balance between cell growth and expression was achieved by the two-stage pH-stat control strategy. The SOX activity was further improved to 8,490 U/L and fermentation cycle was also significantly shortened from 44 to 32 h. The substrate inhibition was weakened with a constant feeding fed-batch. With the assistance of integrated strategy, the activity and productivity reached 12,466 U/L and 389.6 U/(L h), respectively, or 3.1-fold and 4.3-fold of the uncontrolled fermentation. The strategy would be also useful in the industrial application of other similar enzymes.
    No preview · Article · Mar 2015 · World Journal of Microbiology and Biotechnology (Formerly MIRCEN Journal of Applied Microbiology and Biotechnology)
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    ABSTRACT: An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25476.0±184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6±0.1 kallikrein inactivator unit /mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15907.1 ±10.2 kallikrein inactivator unit /mg. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Feb 2015 · Journal of Separation Science
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    ABSTRACT: A novel modification method was proposed for improving the stability of sarcosine oxidase. In this process, sarcosine oxidase surface was efficiently linked with poly-lysine (poly-lys) covalently after activation with N-ethyl-N'-3-dimethylaminopropyl carbodiimide (EDC); the optimal conditions for this reaction were also investigated. The molar ratios of enzyme-COOH to EDC and enzyme-COOH to poly-lys-NH2 were 1:2 and 1:50, respectively, while the optimal reaction pH was 7.0. The covalently binding of poly-lys onto enzyme surface was confirmed by mass spectrum (MS) and fourier transform infrared spectroscopy (FTIR). The catalytic kinetic parameters (Km and Vmax) of modified enzyme were determined as 47.94mM and 0.157μmol/min, respectively. Moreover, compared to the native enzyme, the pH, thermal and storage stabilities of modified sarcosine oxidase were significantly improved. More than 90% of initial activity of modified enzyme was maintained at a broad pH range from 5.0 to 10.0. Most activity of modified enzyme could be detected after being incubated at 60°C for 10min. The storage stability was enhanced ∼12 fold after being stored at 37°C for 7 days. The novel modification was highly efficient for improving the stability of sarcosine oxidase and might be a good reference for other similar enzymes.
    No preview · Article · Mar 2014 · International journal of biological macromolecules
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    ABSTRACT: The effect of initial culture pH and inducer concentration on xanthine oxidase (XOD) fermentation in shake flasks was first carried out. The results showed that the optimum initial culture pH and inducer concentration were 8.6 and 3.6 g/l, respectively. Batch fermentation of XOD by Arthrobacter M3 in a 7.5-l fermentor was then tested under various pH conditions ranging from 7.6 to 8.6. Based on the analysis of the obtained kinetic parameters, a pH-shift strategy in batch fermentation was implemented to enhance the XOD fermentation. In this strategy, the initial culture pH was set at 8.6 without control and was maintained at 7.6 after the biomass reached 2.0 g/l DCW. XOD production (P) and final average yield coefficient for production on biomass (FAYp/x) in this strategy reached 7,415.3 U/l and 1,229.7 U/g, respectively, which were significantly higher than the results from the other four protocols. In pH-shift batch fermentation, the Luedeking–Piret equation for product accumulation and the Luedeking–Piret-like equation for substrate consumption fit well with the experimental values. The correlation coefficients (R 2) of these two fitting curves were 0.977 and 0.992, respectively.
    No preview · Article · Mar 2014 · Bioprocess and Biosystems Engineering
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    ABSTRACT: The community dynamics of attached and free cells of Acidithiobacillus sp. were investigated and compared during chalcopyrite bioleaching process. In the mixed strains system, Acidithiobacillus ferrooxidans was the dominant species at the early stage while Acidithiobacillus thiooxidans owned competitive advantage from the middle stage to the end of bioprocess. Meanwhile, compared to A. ferrooxidans, more significant effects of attached cells on free biomass with A. thiooxidans were shown in either the pure or mixed strains systems. Moreover, the effects of attached cells on key chemical parameters were also studied in different adsorption-deficient systems. Consistently, the greatest reduction of key chemical ion was shown with A. thiooxidans and the loss of bioleaching efficiency was high to 50.5%. These results all demonstrated the bioleaching function of attached cells was more efficient than the free cells, especially with A. thiooxidans. These notable results would help us to further understand the chalcopyrite bioleaching.
    Full-text · Article · Dec 2013 · Bioresource Technology
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    ABSTRACT: 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.
    No preview · Article · Sep 2013 · Bioprocess and Biosystems Engineering
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    ABSTRACT: An efficient affinity purification protocol for Bacillus monomeric sarcosine oxidase expressed in Escherichia coli BL21 (DE3) was developed. 4-Aminopyrrole-2-carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric sarcosine oxidase. The purified sarcosine oxidase was 94 and 96% pure analyzed on an HPLC Vydac C8 column and reducing SDS-PAGE. Meanwhile, the recoveries of typical sarcosine oxidase activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS-PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ~46 kDa. The desorption constant Kd and theoretical maximum absorption Qmax were 35 μg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric sarcosine oxidase on an industrial scale. This article is protected by copyright. All rights reserved.
    No preview · Article · Aug 2013 · Journal of Separation Science
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    ABSTRACT: For the cholesterol oxidase from Brevibacterium sp. M201008 was not stable as free enzyme form, it had been covalently immobilized onto chemically modified sepharose particles via N-ethyl-N'-3-dimethylaminopropyl carbodiimide(EDC). The optimum immobilization conditions were determined, and the immobilized enzyme activity obtained was 12.01U/g sepharose-ethylenediamine. The immobilization of the enzyme was characterized by Fourier transform infrared spectroscopy (FTIR). The immobilized enzyme exhibited the maximal activity at 35 °C and pH 7.5, respectively, which was found unchanged compared with the free one. After being repeatedly used 20 times, the immobilized enzyme retained more than 40.43% of its original activity. The immobilized enzyme showed better operational stability, including wider thermal and pH ranges, and remained 62.87% after 20 days of storage at 4 °C, which was longer than the free enzyme.
    No preview · Article · May 2013 · Journal of Microbiology and Biotechnology
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    ABSTRACT: Fermentation rate and fermentation efficiency are two key aspects for industrial vinegar production. A new target, ratio of oxygen consumption versus acid yield, is introduced to evaluate the fermentation efficiency. Semi-continuous fermentation is optimized by means of harmony between acetification rate and ratio of oxygen consumption versus acid yield. This protocol operates on initial acidity of discontinuous procedure to adjust bacterial metabolism which directly affects the fermentation process. As a result, average acetification rate is increased to 1.81 g acid/l h, which is 20% higher than the original level (1.48 ± 0.32 g acid/l h), and the air flow rate is also reduced by 41% of initial setting. In such a condition, the stoichiometry yield of vinegar is also improved to 94.3 ± 0.67%. So this method could be considered to optimize the industrial scale process in future.
    No preview · Article · May 2013 · Journal of Food Engineering
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    ABSTRACT: For the cholesterol oxidase from Brevibacterium sp. M201008 was not stable as free enzyme form, it had been covalently immobilized onto functionalized sepharose particles that were activated with N-ethyl-N'-3-dimethylaminopropyl carbodiimide (EDC).The optimal conditions of enzyme immobilization were determined, and the immobilized enzyme activity was 18.03U/g of support. The surface morphology and thermal behavior of the immobilized enzyme were observed by scanning electron microscopy and differential scanning calorimetry, respectively. The binding of the enzyme to support was confirmed using Fourier transform infrared spectroscopy. The results demonstrated that the thermal, pH, and storage stabilities of cholesterol oxidase were increased by immobilization. More than 90% of the initial activity of the immobilized enzyme was remained at the points of pH 4.0 and 9.0, and that was much more stable than it was free enzyme form. Under same storage conditions, the free enzyme lost 97.8% of its initial activity after 45h, whereas the immobilized enzyme only lost its 35.8% activity.
    No preview · Article · Feb 2013 · International journal of biological macromolecules
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    ABSTRACT: An integrated bioleaching system (stable pH-silver ion-chloride ion) was firstly proposed to for improving the efficiency of chalcopyrite bioleaching by mixed strains of Acidithiobacillus. The individual effects of stable pH, silver ion, and chloride ion on bioleaching were respectively studied. The highest copper ion concentrations in each system were 45.8, 50.2, and 45.2mg/l, respectively, when it was only 28.0mg/l the blank system. Compared with the individual stable pH, silver or chloride ion systems, the relevance of biological and chemical reactions achieved a better balance in the integrated system (stable pH 1.3, 2.0mg/l silver ion, and 2.5g/l chloride ion). Moreover, the highest ferrous and sulfate ion concentrations implied less production of S(0) membrane and jarosite precipitation. It was also demonstrated by the highest copper ion concentration 55.5mg/l. These results all indicated that this system was a novel and believable strategy for effectively operating chalcopyrite bioleaching.
    Full-text · Article · Dec 2012 · Bioresource Technology
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    ABSTRACT: A xanthine oxidase (XOD) was expressed, purified and partially characterized from Arthrobacter sp. with a negative immune protocol. To determine the optimal inducer for XOD, xanthine, hypoxanthine and uric acid were added into the medium of cultivation. The results revealed that with the inducement of about 14 mM xanthine, the highest XOD activity could be detected. To separate XOD from Arthrobacter sp., the cells were first cultured without any inducement; then the total proteins of the collected cells were extracted and immunized to rabbits for the polyclonal antibodies. These antibodies were then coupled with sepharose CL 6 B, and the medium was further employed to deplete most of the cells’ back ground proteins. Began with ∼20 mg crude protein from disrupted cells was subjected to the antibody medium, and ∼1.45 mg protein was detected in unbinding fractions with ∼92.0% of activity. The extracted xanthine oxidase was ∼85% pure with native-PAGE analysis, and ∼90% pure with SDS-PAGE analysis, the yield of protein was ∼7.4%. The specific activity of the enzyme was 36.0 U/mg. The native enzyme should be a dimer (∼280 kDa) of a protein composed with two different peptides with the mass of approximately 55.5 and 85.5 kDa, respectively. The optimal pH and temperature of this enzyme were determined at about pH 7 and 50 °C. Furthermore, EDTA revealed almost no influences on the activity.
    No preview · Article · Nov 2012 · PROCESS BIOCHEMISTRY
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    ABSTRACT: 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.
    No preview · Article · Aug 2012 · Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
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    ABSTRACT: An extremely acidophilic sulfur-oxidizing bacterium was isolated from an industrial-scale bioheap of the Zijinshan copper mine and was named ZJJN. A tuft of flagella and a layer of thick capsule outside the cell envelope were clearly observed under transmission electron microscopy (TEM), which might be closely related to the extremely acid-proof capacity of ZJJN cells in the bioleaching system; 16S ribosomal RNA (rRNA) phylogeny showed that the isolated strain was highly homologous to the genera of Acidithiobacillus. The optimum temperature of ZJJN was determined at 30 °C and pH at 1.0. It was capable of growth at even pH 0. Strain ZJJN can utilize reduced sulfur as an energy source but not with organics or ferrous ion. Strain ZJJN was sensitive to all antibiotics with different concentrations; when it showed a certain resistance to different concentrations of Cu(2+). In the mixed strains of ZJJN and A. ferrooxidans system (initial pH 1.0), the copper-leaching efficiency was up to 60.1 %, which was far higher than other systems. Scanning electron microscopy (SEM) analysis showed that less jarosite precipitation was produced in the most efficient system. The extremely acidophilic strain ZJJN would be of great potential in the application of chalcopyrite bioleaching.
    Full-text · Article · Aug 2012 · Journal of Industrial Microbiology
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    ABSTRACT: Using glutathione (GSH) as template molecule, acrylamide (AM) and methacrylic acid (MAA) as functional monomer, different glutathione-molecularly imprinted polymers (GSH-MIPs) were developed and synthesized with molecularly imprinted technique. The effects for adsorption capacity, including molar ratio of template molecule-functional monomer, pH of loading buffer, adsorption equilibrium time and loading concentration were discussed in detail. The result demonstrated that the polymers with raw material mole ratios at 1:5 for GSH:AM, and 1:4 for GSH:MAA showed higher adsorption capacity, the optimal pH values for the adsorption of GSH-MIPAMs and GSH-MIPMAAs to GSH were 3.0 and 5.0, respectively; furthermore, the optimal GSH loading concentrations were 1.50~2.00 g/L for GSH-MIPAMs, and 1.00~ 1.50 g/L for GSH-MIPMAAs; additionally, the adsorption equilibrium time was 20 h for GSH-MIPAMs and 18 h for GSH-MIPMAAs. In addition, the binding of GSH analogues were also applied to the synthesized polymers, and the results reflected that GSH-MIPs showed low recognition to these analogues. This protocol was further employed for the extraction and separation of GSH from yeast cells samples. The extraction recoveries of GSH-MIPAMs and GSH-MIPMAAs were 41% and 77%, respectively, it was of great potential in the purification of GSH.
    Preview · Article · Mar 2012 · Acta Chimica Sinica -Chinese Edition-
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    ABSTRACT: Acidithiobacillus ferrooxidans is a Gram-negative, acidophilic, and chemolithotrophic bacterium that is active in bioleaching. The leaching efficacy is directly influenced by the biomass changes of this specie in bioleaching microbial community. In order to perform a simple and sensitive assay on A. ferrooxidans from mixed strains in this process, a novel assay was developed based on sandwich hybridization assay with the aid of S1 nuclease treatment and fluorescent labeling. In the work, a designed DNA probe complementary to the conservative region of its 16S rRNA was synthesized, which showed high accuracy for distinguishing homologous species with the exclusion of even-only two base pairs difference. The specificity of this assay was verified in different systems with mixed strains, and the quantitative result was proved by comparison of microscopic cell counting. The detection sensitivity was about 8 × 10(2) cells/ml and the inter-assay coefficient of variation of three independent assays was from 3.8 to 7.7 %, respectively. In addition, the cycle of assay was about 3-4 h when the cost estimated was less than $0.5 per sample. This assay method might be applied for identifying and monitoring any kind of bacterial strain from a mixed microbial flora in bioleaching or other areas.
    Full-text · Article · Mar 2012 · Journal of Industrial Microbiology
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    ABSTRACT: Affinity protocols for the purification of urinary trypsin inhibitor (UTI) were developed. To imitate the substrate/inhibitor-binding domain (S1 domain) of trypsin and chymotrypsin, the key amino acid residues were composed to sorbents. The sorbents were then subjected to adsorption analysis with UTI. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. The purified enzyme was subjected to SDS-PAGE, trypsin inhibitor activity and peptide map fingerprinting analysis. As calculated, the theoretical maximum adsorption (Q(max)) of two affinity sorbents entitled as S-D-G and S-S-G were 31.7 and 30.1 mg/g, respectively; the desorption constants K(d) of the two sorbents were 8.9 and 18.6 μg/mL, respectively. After the separation of UTI with S-D-G and S-S-G, reducing SDS-PAGE analysis revealed that the protein was a single polypeptide with the mass of ~66 kDa, and the purified proteins were ~95 and 97% pure, respectively; the band on gel was further confirmed with peptide map fingerprinting analysis. Protein and bioactivity recoveries were 1.3 and 75.9% with S-D-G, 1.0 and 70.2% with S-S-G, respectively.
    No preview · Article · Jan 2012 · Journal of Separation Science