High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

BACKGROUND: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. RESULTS: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20 C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. CONCLUSIONS: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

The research progress of dynamic combinatorial chemistry / 药学学报

As a novel branch of combinational chemistry, dynamic combinatorial chemistry (DCC) can be viewed as a technique which combines library synthesis and screening in one pot. By addition of molecular target, ligangds, which show binding affinity or strong interaction with the molecular target, can be amplified an young but rapidly growing branch of combinatorial chemistry, has been widely used in organic chemistry, biochemistry, material fields. Ligands in the library can be amplified, since synthesis of the library is screened by a molecular target. Therefore, these structures could be identified easily. Consequently DCC has been widely used in the lead discovery, material chemistry and other fields. On the basis of the principle and method of DCC, this review emphasizes the three factors of DCC, including molecular targets (bio-enzyme, lectin, nucleic acid, organic molecule, inorganic molecule); reaction (disulphide chemistry, ammoniation reduction reaction, hydrazone chemistry, etc.) and analytical method. Meanwhile, limitation, current situation and future development of DCC were also discussed in this paper.

Application of efficient synthetic techniques in drug research / 药学学报

Compound libraries and chemical synthesis play important roles in drug discovery and development, and efficient synthetic techniques can greatly facilitate the drug research. This review highlights the application of some efficient synthetic techniques in drug research including microwave chemistry, click chemistry, combinatorial chemistry, cascade reactions and multicomponent reactions, as well as the construction of diverse and drug-like compound libraries.

Application of NMR technique in the discovery and pharmacological studies of active substances from natural products / 药学学报

The application of HPLC-NMR-MS hyphenated technique in the structural identification of trace substances from complex mixtures and the identification of endogenous and exogenous substances in the establishment process of metabolic profiling have become effective analytical tools in pharmaceutical chemistry, pharmacological and pharmacokinetic studies of active substances from natural products. Metabolomics method based on NMR technology can accurately portray metabolic phenotypes with the characteristics of diseases and a variety of disease-related pathways, and it can greatly enrich and supplement the traditional disease evaluation methods. So it can be used for pharmacological studies of active substances from natural products, such as toxicological studies, the dose optimization, active substances screening and pharmacodynamic evaluation. Hyphenated technique associated with metabolomics method based on NMR technology will accelerate the speed of the discovery of active substances from natural products, and improve the efficiency of their pharmacological evaluation.

Optimization of Douchi fibrinolytic enzyme production by statistical experimental methods / 华中科技大学学报（医学）（英德文版）

Thrombus disease, one of the common cardiovascular diseases, has attracted worldwide attention for its rising mortality and morbidity. Due to the distinct shortages of current fibrinolytic drugs, new fibrinolytic agents warrant investigation. In this study, 8 fibrinolytic enzyme-producing strains were isolated from Douchi-a traditional Chinese food, and strain XY-1 which produced the largest amount of the enzyme was chosen for the following experiments. The enzyme produced by strain XY-1 was named Douchi fibrinolytic enzyme (DFE). We optimized the liquid culture medium of strain XY-1 for enzyme production using Plackett-Burman and Box-Behnken design. The predicted maximal DFE yield was 19.78 FU/mL with 11.4 g/L peptone, 0.5 g/L magnesium sulfate and 1 g/L sodium chloride. However, we acquired maximal production of 21.33 FU/mL in actual experiments, equal to 107.84% of the theoretical value, and the yield had been increased by 79.55% as compared to the yield of un-optimized culture. It was demonstrated that the combined use of Plackett-Burman design and response surface methodology in fermentation optimization can effectively and rapidly increase DFE production.

Biosurfactants production by yeasts using soybean oil and glycerol as low cost substrate

Biosurfactants are bioactive agents that can be produced by many different microorganisms. Among those, special attention is given to yeasts, since they can produce many types of biosurfactants in large scale, using several kinds of substrates, justifying its use for industrial production of those products. For this production to be economically viable, the use of residual carbon sources is recommended. The present study isolated yeasts from soil contaminated with petroleum oil hydrocarbons and assessed their capacity for producing biosurfactants in low cost substrates. From a microbial consortium enriched, seven yeasts were isolated, all showing potential for producing biosurfactants in soybean oil. The isolate LBPF 3, characterized as Candida antarctica, obtained the highest levels of production - with a final production of 13.86 g/L. The isolate LBPF 9, using glycerol carbon source, obtained the highest reduction in surface tension in the growth medium: approximately 43% of reduction after 24 hours of incubation. The products obtained by the isolates presented surfactant activity, which reduced water surface tension to values that varied from 34 mN/m, obtained from the product of isolates LBPF 3 and 16 LBPF 7 (respectively characterized as Candida antarctica and Candida albicans) to 43 mN/m from the isolate LPPF 9, using glycerol as substrate. The assessed isolates all showed potential for the production of biosurfactants in conventional sources of carbon as well as in agroindustrial residue, especially in glycerol.

Evaluation of biomass production, carotenoid level and antioxidant capacity produced by Thermus filiformis using fractional factorial design

A fractional factorial design 2(5-1) was used to evaluate the effect of temperature, pH, and concentrations of yeast extract, tryptone and Nitsch's trace elements on the biomass, total carotenoids and protection against singlet oxygen by carotenoid extracts of the bacterium Thermus filiformis. In addition, the carotenoid composition was determined by high-performance liquid chromatography connected to a diode array and mass spectrometer detectors (HPLC-DAD-MS/MS). The production of biomass ranged from 0.113 to 0.658 g/L, the total carotenoid from 137.6 to 1,517.4 mg/g and the protection against singlet oxygen from 4.3 to 85.1 %. Results of the fractional factorial design showed that temperature had a negative effect on biomass production and a positive effect on carotenoid content and protection against singlet oxygen, besides, high levels of pH value, concentrations of yeast extract and tryptone had a positive effect on biomass production only at lower temperatures. The main carotenoids of T. filiformis were thermozeaxanthins. In the tested conditions, changes in the levels of the variables influenced the biomass, carotenoid production, and protection against singlet oxygen, although they did not influence the carotenoid profile. The results of this study provide a better understanding on the interactions among certain nutritional and cultivation conditions of a thermophile bacterium, Thermus filiformis, on biomass and carotenoid amounts, as well as on the antioxidant capacity.

Genesis, development and application prospect of antibody library: a review / 生物工程学报

Antibodies are immunoglobulins specifically introduced by immunity response of high animals, with the responsibility for recognising and cleaning out specific antigens. Antibody is not only a powerful weapon against pathogen invasion in the organism, but also a tool for specific molecular recognition used in basic scientific research. The diversity of antibody molecules resulted in the concept of antibody library; each individual animal is a natural antibody library. In the post-genome era, in order to fit various "omics", especially for proteomics requirement of high throughput technology, some gene engineering antibody libraries and antibody alternative libraries have been constructed based on phage display technology. Yet, more and more in vitro display systems such as ribosome display, mRNA display have been used for antibody library study, and that present more advantages than phage display. This mini review outlines the genesis, development and application prospect of antibody libraries according to the published reviews and research articles, and offers up to date development and application prospect of antibody library technology.

A novel recombinant human interferon alpha2b with high antivirus activity by combinatorial mutagenesis / 生物医学工程学杂志

In order to create a novel recombinant human interferon alpha2b (rh-IFN alpha2b) with higher biological activity, we subjected the rational designed sequence of rh-IFN alpha2b to direct evolution by strategy of the combinatorial mutagenesis. The amino acid residues at multiple sites of 52-53-55, 103-107, and 121-125 were simultaneously mutated. The resulted gene of the mutated rh-IFN a2b was cloned into the pET28a and expressed in E. coli BL21 Condon plus (RIL). The anti-virus activity of the novel interferon alpha2b was 9.3 x 10(7) IU/mg, 93 times higher than the wild type (1 x 10(6) IU/mg). The results showed that the multiple point mutation used in this study could effectively combine the site effects of rh-IFN alpha2b and increase its biological activity.

Applications of chemical genetics in biomedical research / 生物医学工程学杂志

Chemical genetics is the science which takes the small molecular compounds as tools to solve the genetics problems or to disturb/adjust normal biological process so as to find out protein functions. Because the small molecules have the diverse chemical characters and the ability to identify the target proteins, they also can be filtrated on the basis of phenotype. So the methods of chemical genetics have been applied in almost all of the researches on biology and medicine. In this paper, the methods to acquire small molecular compounds are introduced. The enormous progress achieved in the field of combinatorial chemistry, which has allowed the rapid production of a large number of chemically diverse molecules, is an important prerequisite to make chemical libraries available to academic researchers. And the applications of the compounds in early embryo development, cell differentiation, on-set and course of disease are discussed, too. The application of small molecules has an enormous impact on our understanding of cell biology. There are many examples where small molecules, in combination with genetic screens, have facilitated the dissection of complex cellular processes.

Strategy of molecular drug design: dual-target drug design / 药学学报

Physiology-based and target-based drug discovery constitutes two principal approaches in drug innovation, which are mutually complementary and collaborative. With the target-based approach, a lot of new molecular entities have been marketed as drugs. However, many complicated diseases such as cancer, metabolic disorders, and CNS diseases can not be effectively treated or cured with one medicine acting on a single target. As simultaneous intervention of two (or multiple) targets relevant to a disease has shown improved therapeutic efficacy, the innovation of dual-target drugs has become an active field. Dual-target drug can modulate two receptors, inhibit two enzymes, act on an enzyme and a receptor, or affect an ion channel and a transporter. From viewpoint of molecular design, there are three approaches to construct a dual-target drug molecule. A connective molecule can simply be realized by combining two active molecules or their pharmacophores with a linker; while an integrated molecule comes into an entity either by fusing or by merging the common structural or pharmacophoric features of two active molecules, depending on the extent of the common features. The latter approach facilitates the reduction of molecular size and molecular weight and the optimal overlap between the pharmacodynamic and pharmacokinetic spaces, which will certainly elevate the probability of being a drug.

Structure-activity relationships analysis of thienorphine and its derivatives / 药学学报

Thienorphine is a chemically-new opioid developed in Beijing Institute of Pharmacology and Toxicology. To elucidate the chemical basis for the unique pharmacological effects of thienorphine, 15 derivatives were synthesized according to combinatorial chemistry and the structure-activity relationships of these compounds were studied. It is demonstrated that thienorphine is a potent long-acting partial agonist. N-Cyclopropylmethyl is responsible for the antagonist effect of thienorphine. More importantly, thiophene at the end of side chain is most likely the pharmacophore accounts for the long-lasting effect of thienorphine. Change of the connection of thiophene and the side chain does not result in changes in the antinociceptive activity.

Advances in the study of affinity selection-ultrafiltration/HPLC-MS / 药学学报

Affinity selection-ultrafiltration/HPLC-MS is the combination of the ultrafiltration and HPLC-MS, mainly used for screening small active molecular substances from combinatorial libraries and natural product extracts, which can bind to solution-phase targets. Besides, it can be used in metabolic screening and characterization of ligand-receptor binding. It is a complement to the traditional methods of screening and identifying drugs. This review describes its principle and application in drug study.

Fmoc solid-phase synthesis of cyclopeptide FIK / 生物工程学报

We study the techniques of synthesis of disulfide bond-bearing cyclopeptides FIK. This experimentation with the material of Fmoc-aa use Solid-Phase synthesis after condensation by HBTU/HOBt/DIEA to synthesize linear peptide, then cyclopeptide was synthesized by creation of intramolecular disulfide bond by means of 12 oxidation of bis cysteine sulfhydryl of the linear peptide. The crude production was cleaved from the resin together with all protecting group and identified and separated by MALDI-MS and RP-HPLC. The peptide yield was 18%, after purification the purity was more than 97%. It was identified on MALDI-MS and Ellman reagent detection. This method is effective, simple, rapid and obtained good yield, and it's fit for the large-scale production.

Exploring conformational space using a mean field technique with MOLS sampling.

The computational identification of all the low energy structures of a peptide given only its sequence is not an easy task even for small peptides,due to the multiple-minima problem and combinatorial explosion. We have developed an algorithm, called the MOLS technique,that addresses this problem, and have applied it to a number of different aspects of the study of peptide and protein structure. Conformational studies of oligopeptides, including loop sequences in proteins have been carried out using this technique. In general the calculations identified all the folds determined by previous studies,and in addition picked up other energetically favorable structures. The method was also used to map the energy surface of the peptides. In another application, we have combined the MOLS technique, using it to generate a library of low energy structures of an oligopeptide, with a genetic algorithm to predict protein structures. The method has also been applied to explore the conformational space of loops in protein structures.Further, it has been applied to the problem of docking a ligand in its receptor site, with encouraging results.

1, 4- Addition of diazomethane to a heterodiene: a direct preparation of the oxazolic ring

The reaction of naphthoquinone-oximes (3) and (4) with diazomethane yields directly, in one step, the oxazoles (5) and (6), respectively.

A reação das naphthoquinona-oximas (3) e (4) com diazometano fornece diretamente, em uma etapa, os oxazóis (5) e (6), respectivamente.

Study on the reactions of azo compounds with acyl halides mediated by Sm/TiCl4 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Amides can be obtained in good to excellent yield by Sm/TiCl(4) mediated reductive cleavage of N=N bond in azo compounds and successive acylation in one pot. It offers an alternative method for the synthesis of amides from very simple starting materials directly.

Tissue-targeting lead generation and optimization from random and directed screening of technetium-99m labeled tripeptide complex libraries in vivo / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Screening libraries against a molecular target in vitro are idealized models that cannot reflect the real state in vivo where biomolecules coexist and interact. C-terminal amide tripeptides labelled with Technetium-99m can provide a unique noninvasive approach to trace a large number of compounds in vivo.</p><p><b>METHODS</b>The C-terminal amide tripeptide libraries were synthesized on Rink Amide-MBHA resin using iterative and pooling protocol. Technetium (V) oxo core [TcO(3+)] was bound to each tripeptide via 4 deprotonated nitrogen atoms to form a library of 8000 (99m)Tc tripeptoid complexes. The radiocombinatorial screening (RCS) in vivo was carried out on SD rats and A549 tumour bearing mice.</p><p><b>RESULTS</b>Signals of tissue distribution and metabolism of libraries were recorded by counting or imaging and tissue targeting leads identified by both random and directed RCS. Among them, (99m)Tc RPA, (99m)Tc VIG and (99m)Tc RES had specific tissue targeting in kidney, liver and tumour respectively. The percent injected dose per gram tissue of (99m)Tc labelled leads in their target tissue was highly structure dependent. Because the nontarget tissue binding and the metabolism of (99m)Tc tripeptoid sublibraries were simultaneously monitored successfully by RCS, the interference of background activity was limited to the lowest level. Optimization of renal function agent from the labelled libraries was carried out by directed screening. (99m)Tc DSG was finally identified the most promising agent for renal function studies.</p><p><b>CONCLUSIONS</b>RCS in vivo is a powerful tool for the discovery of tissue targeting drugs. The potential screening bias is probably the major limitation of labelled libraries.</p>

Optimization of angiotensin I-converting enzyme (ACE) inhibition by rice dregs hydrolysates using response surface methodology / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Angiotensin I-converting enzyme (ACE) inhibitory peptides have been shown to have antihypertensive effects and have been utilized for physiologically functional foods and pharmaceuticals. The ACE inhibitory ability of a hydrolysate is determined by its peptide composition. However, the peptide composition of a hydrolysate depends on proteolytic enzyme and the hydrolysis conditions. In this study, the effect of process conditions on the ACE inhibitory activity of rice dregs hydrolyzed with a trypsin was investigated systematically using response surface methodology. It was shown that the ACE inhibitory activity of rice dregs hydrolysates could be controlled by regulation of five process conditions. Hydrolysis conditions for optimal ACE inhibition were defined using the response surface model of fractional factorial design (FFD), steepest ascent design, and central composite design (CCD).

Optimization of technological conditions for one-pot synthesis of (S)-alpha-cyano-3-phenoxybenzyl acetate in organic media / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Optically active form of alpha-cyano-3-phenoxybenzyl (CPB) alcohol, building block of pyrethroid insecticides, was synthesized as its acetate by the combination of anion-exchange resin (D301)-catalyzed transcyanation between m-phenoxybenzaldehyde (m-PBA) and acetone cyanohydrin (AC), and lipase (from Alcaligenes sp.)-catalyzed enantioselective transesterification of the resulting cyanohydrin with vinyl acetate. Through optimizing technological conditions, the catalyzing efficiency was improved considerably compared to methods previously reported. Concentrations of CPB acetate were determined by gas chromatograph. The enantio excess (e.e.) values of CPB acetate were measured by NMR (nuclear magnetic resonance) method. Effects of solvents and temperatures on this reaction were studied. Cyclohexane was shown to be the best solvent among the three tested solvents. 55 degrees C was the optimal temperature for higher degree of conversion. External diffusion limitation was excluded by raising the rotational speed to 220 r/min. However, internal diffusion could not be ignored, since the catalyst (lipase) was an immobilized enzyme and its particle dimension was not made small enough. The reaction rate was substantially accelerated when the reactant (m-PBA) concentration was as high as 249 mmol/L, but decreased when the initial concentration of m-PBA reached to 277 mmol/L. It was also found that the catalyzing capability of recovered lipase was high enough to use several batches. Study of the mole ratio of AC to m-PBA showed that 2:1 was the best choice. The strategy of adding base catalyst D301 was found to be an important factor in improving the degree of conversion of the reaction from 20% to 80%. The highest degree of conversion of the reaction has reached up to 80%.