Rongxiu Li

In Situ Biodiesel Production from Fast-Growing and High Oil Content Chlorella pyrenoidosa in Rice Straw Hydrolysate

Rice straw hydrolysate was used as lignocellulose-based carbon source for Chlorella pyrenoidosa cultivation and the feasibility of in situ biodiesel production was investigated. 13.7 g/L sugar was obtained by enzymatic hydrolyzation of rice straw. Chlorella pyrenoidosa showed a rapid growth in the rice straw hydrolysate medium, the maximum biomass concentration of 2.83 g/L was obtained in only 48 hours. The lipid content of the cells reached as high as 56.3%. In situ transesterification was performed for biodiesel production. The optimized condition was 1 g algal powder, 6 mL n-hexane, and 4 mL methanol with 0.5 M sulfuric acid at the temperature of 90°C in 2-hour reaction time, under which over 99% methyl ester content and about 95% biodiesel yield were obtained. The results suggested that the method has great potential in the production of biofuels with lignocellulose as an alternative carbon source for microalgae cultivation.

Affinity purification of egg yolk immunoglobulins (IgY) with a stable synthetic ligand

Chicken IgY (egg yolk immunoglobulin) is a functional equivalent of mammalian IgG. Traditional methods for IgY purification involve multi-step procedures that result in low recovery of IgY. After a large scale screening of our 700-member synthetic ligand library synthesized by epichlorohydrin and cyanuric chloride methods, a high efficiency ligand of IgY was found. By one-step purification with this ligand, the purity of IgY could reach 92.1%, and the recovery of IgY could reach 78.2%. This synthetic ligand had a higher binding capacity of 74.8 mg IgY/ml and had no negative effects on immunoreactivity. Remarkably, this ligand was also highly stable and could resist 1M NaOH, thus having great potential for the industrial-scale production of IgY.

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase. Methodology/Principal Findings To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage. Conclusions/Significance This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.

Newly Combined Method of Molecularly Imprinted Solid-Phase Extraction with ELISA for Rapid Detection of Clenbuterol in Animal-Tissue Samples

Abstract A new method using molecularly imprinted polymers (MIPs) as specific adsorbent materials coupled with ELISA analysis is being reported for the first time for the detection of clenbuterol (CLB) residue in the pig muscles. After optimization of the posttreatments, the total amount of template bleeding in the CLB MIPs was decreased to only 3.0 ng CLB/60 mg MIPs, which is 10 times lower than that of the previous report. Moreover, compared to the methods of C18-ELISA and single ELISA, the combined molecularly imprinted solid-phase extraction (MISPE)–ELISA exhibited high precision and robust accuracy for CLB at all three spiked levels of 0.5, 5.0, and 10.0 ng g−1.

Design and synthesis of affinity ligands and relation of their structure with adsorption of proteins

Affinity chromatography has played an increasingly important role both in the pharmaceutical industry and academic research. In the present study, we report our preliminary investigation on the relationship between the affinity ligand structure and its adsorption to multi-protein samples. The structure of the ligands, including the size of the ring (cyclic group) and the length of the chain (linear group), has a great impact on the adsorption of ligands to proteins. Meanwhile, the functional groups that the ligands carry are also closely related to the adsorption of ligands to proteins. This research provides good guidance for the design and synthesis of affinity materials in affinity chromatography. It is also useful to other protein-ligand interaction-related research.

Phenazine-1-carboxylic acid derivatives: design, synthesis and biological evaluation against Rhizoctonia solani Kuhn.

Rhizoctonia solani Kuhn is the pathogen that causes sheath blight and results in significant yield reduction in rice and in nearly 50 other crops. In order to develop a new fungicide effective against this pathogen, a series of structurally diverse phenazine-1-carboxylic acid derivatives, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, and 2k, were designed, synthesized and evaluated for their antifungal activity. The two most active compounds 2i and 2j were selected as lead compounds for further antifungal research.

Biomimetic affinity purification of cardiotoxin and its pharmacological effects on the nervous system

Cobra venom is a very precious natural resource. The traditional method for purification of cardiotoxin from cobra venom is a multi‐step, high cost, and low recovery procedure. By molecular modeling and docking with SYBYL software, we designed and synthesized an affinity ligand, m‐aminobenzoic acid, for high efficiency purification of this therapeutically useful Chinese cobra venom cardiotoxin. The one‐step recovery of cardiotoxin reached 64% and the purity reached 92% upon purification. The binding capacity of this synthetic ligand was 9.1 mg cardiotoxin/g moist weight gel and the affinity constant for cardiotoxin was 5.5 × 103 M−1. Unlike a natural affinity ligand, this synthetic ligand is highly stable, and has great potential for industrial scale production of cardiotoxin. In addition, we examined the effects of cardiotoxin on the nervous system in a mouse model. Results showed that cardiotoxin could maintain analgesic effects for 120 min with a dose of less than 0.06 mg/kg (2.8% of the LD50). Administration of 0.12 mg/kg cardiotoxin could improve scopolamine impairments of memory in mice. These results suggest that cardiotoxin may be a potential drug for nervous system diseases. Copyright

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700‐member library of synthetic ligands in detail. We selected 297 ligand columns from a 700‐member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one‐step purification effect, and 58 columns had a one‐step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one‐step purification of special proteins. Copyright

Biomimetic affinity purification of Candida antarctica lipase B

Candida antarctica lipase B (CalB) is one of the most widely used biocatalysts in organic synthesis. The traditional method for purification of CalB is a multi-step, high cost and low recovery procedure. Biomimetic affinity purification had high efficiency purification. We selected 298 ligand columns from a 700-member library of synthetic ligands to screen Pichia pastoris protein extract. Of the 298, three columns (named as A9-14, A9-10, and A11-33) had one-step purification effect, and A9-14 of these affinity ligands, had both high purification and recovery. The one-step recovery of CalB reached 73% and the purification reached 91% upon purification. The active groups of A9-14 were cyclohexylamine and propenylamine. Furthermore, both A9-14 and A9-10 had the same R1 active group of cyclohexylamine which might act the main binding role for CalB. The synthetic ligand A9-14 had a binding capacity of 0.4 mg/mL and had no negative effects on its hydrolytic activity. Unlike a natural affinity ligand, this synthetic ligand is highly stable to resist 1M NaOH, and thus has great potential for industrial scale production of CalB.

Vitreous and serum levels of transthyretin (TTR) in high myopia patients are correlated with ocular pathologies.

PURPOSE To detect serum and vitreous transthyretin (TTR) in high myopia patients and to evaluate potential associations between TTR and clinical parameters and ocular pathologies, including different ocular pathologies. DESIGN AND METHODS Serum samples from 16 high myopia patients and 4 controls were analyzed by LTQ-MASS. Serum samples from 116 high myopia patients and 86 healthy controls were tested by Western blots and ELISA. Eight healthy and 40 pathologic vitreous samples were analyzed by ELISA. And corresponding serum samples were also analyzed by ELISA. RESULTS Significant increased TTR serum levels were detected in high myopia patients compared to healthy controls. The high levels of serum TTR were associated with ocular pathologies, long axial length, and low visual acuity. TTR in high myopia patients with macular hole and macular detachment was upregulated in both vitreous and the corresponding serum samples. TTR levels in serum samples of high myopia patients with long axial lengths were higher than in the vitreous. CONCLUSIONS Serum TTR may be a biomarker for high myopia patients with ocular pathologies.