Molecular Basis for Stereospecific Hydrolysis of Ethyl Mandelate by Thermophilic Esterase

The stereospecific hydrolysis of mandelate can be effectively catalyzed by hyperthermophilic acylpeptide esterase APE 1547（Aeropyrum pernix esterase 1547）. APE 1547 used in this reaction showed a remarkable stereodi-scrimination in favour of R-mandelic acid（99% e.e.） with an enantiomeric ratio E〉200. The results of computer simulation are consistent with the experimental results. It can be inferred that the R-substrate adopted a binding mode productive of the reaction due to the formation of the hydrogen bond at the active site of APE 1547.

Degradation of amyloid β-peptides catalyzed by nattokinase in vivo and in vitro

Amyloid-β 1-42(Aβ42)plays a pivotal role in Alzheimer disease(AD)pathogenesis. Peripheral clearance of Aβ42 largely affects its level in the brain and affects AD progression. Although nattokinase(NK)degrades Aβ40, the details of NK's capture of various Aβ species and reduction of plasma Aβ42/Aβ40 are uncharacterized. In this study, the Aβ42/Aβ40-degrading ability of NK was investigated using five Aβs and AD model mice. The C-terminal region of Aβ42/Aβ40(Gly29 to Val40)was primarily required for NK capture, and the integrated conformation in Aβ42/Aβ40 aggregates was a more efficient target for NK catalysis. Further, suspended Aβ42/Aβ40 oligomers were more easily captured by NK than suspended Aβ42/Aβ40 fibrils, while deposited Aβ42/Aβ40 fibrils recruited more NK than deposited Aβ42/Aβ40 oligomers. Although most NK was likely lost during NK uptake and/or entry into the blood, a small fraction of NK showed good plasma Aβ42/Aβ40-degrading efficacy after entering the blood due to NK's stability in the plasma of AD mice for at least 9 days. It was concluded that oral administration of NK is a feasible approach for peripheral Aβ42/Aβ40 clearance. This implies that NK might serve as an anti-Aβ42 agent for the treatment of Aβ42/Aβ40-related diseases such as AD.

Discovery of Kaempferol,a Novel ADAM10 Inhibitor,as a Potential Treatment for Staphylococcus aureus Infection

Host-directed therapy(HDT)is an emerging novel approach for treating multidrug-resistant Staphylococcus aureus(S.aureus)infection.Functioning as the indispensable specific cellular receptor for a-toxin(Hla),a-disintegrin and metalloproteinase 10(ADAM10)is exploited to accelerate S.aureus infection through diverse mechanisms.The extraordinary contribution of ADAM10 to S.aureus pathogenesis renders it an attractive HDT target for combating S.aureus infection.Our study is the first to demonstrate the indispensable role of ADAM10 in S.aureus-induced necroptosis,and it enhances our knowledge of the role of ADAM10 in S.aureus infection.Using a fluorogenic substrate assay,we further identified kaempferol as a potent ADAM10 inhibitor that effectively protected mice from S.aureus infection by suppressing Hla-mediated barrier disruption and necroptosis.Collectively,our work presents a novel hostdirected therapeutic strategy for using the promising candidate kaempferol to treat S.aureus infection and other diseases relevant to the disordered upregulation of ADAM10.

Comparison Between the Effects of 2-Selenium Bridged β-Cyclodextrin and Ebselen on Treating SHRsp Stroke

A glutathione peroxidase（GPX） mimic, 2-selenium bridged β-cyclodextrin（2-SeCD）, was synthesized. In order to examine its role and mechanism in treating stroke we chose stroke-prone spontaneously hypertensive rats（SHRsp） as animal model. 56 SHRsps of 8-week olds were randomly divided into several groups： test groups （low, moderate, high dose of 2-SeCD） and control groups（positive and negative）. After onset of the stroke, the rats in test groups were orally administrated with different amounts of 2-SeCD, the positive control group with ebselen, and the negative control group with drinking water. The treatment lasted two weeks, followed by observation of the rats for 10 days, meanwhile blood pressure, biochemical parameters of plasma, and the contents of nitric oxide（NO） and malondialdehyde（MDA） in plasma and brain were determined. The results show that there were significant differences in contents of NO and MDA in plasma and brain between the test groups（high, moderate dose of 2-SeCD） and negative control group. The NO contents of the test groups were obviously higher than that of the negative control group （P〈0.01）. The MDA contents of the test groups（high, moderate dose of 2-SeCD） were obviously lower than that of the negative control group（P〈0.01）. The mechanism of 2-SeCD in treating stroke was discussed, which maybe related to the increase of NO and the decrease of MDA in plasma and brain tissue, but the exact mechanism should be further studied. Moreover, the tendencies of changes in systolic blood pressure, contents of NO and MDA, and other physiological parameters for the test groups were shown to be much better than the corresponding parameters for the positive group（the group with ebselen）（P〈0.05）, indicating that the treatment effect of 2-SeCD is better than that of ebselen.

Preparation and Anti-oxidative Effects of Corn Peptides

Corn peptides(CP) were prepared from zein via proteolysis by means of a type of alkaline protease, Alcalase, at 50 ℃ and pH 8.0. The anti oxidative activity of the hydrolysate was examined. It was found that the anti oxidative activity reached the maximum at the fourth hour of the hydrolysis and then decreased. The effect of the enzyme dosage on the solubility of corn protein, the dependence of hydrolysis time on the degree of the hydrolysis(DH) and molecular weight distribution were examined respectively. 30% DH was controlled on the basis of anti oxidative activity. The hydrolysate was fractionated on a Sephadex G 15 column. The anti oxidative activities were examined for all the fractions. The main fraction for anti oxidative activity was collected and characterized by the methods of amino acid analysis, the measurement of average chain length and etc .. The main M W distribution of the main fraction was 300-600 Daltons. The anti oxidative properties of the small peptides in vitro were studied. It was found that they could inhibit the auto oxidation of pyrogallol and the oxidation of hemoglobin. The hemolysis degree of erythrocyte caused by hydroxyl free radicals was decreased greatly, indicating a protective effect of the anti oxidative peptides on the cell membrane damage of erythrocyte.

Cellular Adhesion Tripeptide RGD Inhibits Growth of Human Ileocecal Adenocarcinoma Cells HCT-8 and Induces Apoptosis

The tripeptide,Arg-Gly-Asp（RGD）motif is an integrin-recognition site found in adhesive proteins present in extracellular matrices（ECM）and in the blood.HCT-8 cells were treated with cellular adhesion tripeptide RGD at various concentrations.MTT assay was performed to examine the growth and proliferation of HCT-8 cells after treatment with RGD for 48 h.Haematoxylin and Eosin（HE）staining and electromicroscope were used to observe the morphology of apoptotic cells.Survivin and flow cytometry were also used to analyze the HCT-8 apoptosis.Cellular adhesion tripeptide RGD significantly inhibits the growth and proliferation of HCT-8 cells in a dose-dependent manner and induces apoptosis of HCT-8.These results indicate that cellular adhesion tripeptide RGD inhibits the growth and proli-feration of tumor HCT-8 cell,probably by the aid of inducing apoptosis of HCT-8 cell.

Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1

The changes in the activity and the conformation of the hyperthermophilic esterase derived from aerobic thermophilic Aeropyrum pernix K1 （ APE1547 ） were studied during denaturation by guanidine hydrochlofide （ GdnHC1 ） and urea. The denaturation course of APE1547 was followed by the steady-state and time resolved fluorescence methods. An increase in the denaturant concentration in the denatured system can significantly enhance the inactivation and unfolding of APE1547. The enzyme can be completely inactivated with a urea concentration of 2.7 mol/L or a GdnHCl concentration of 7.5 mol/L. The fluorescence emission maximum of the enzyme protein red shifts in magnitude to a maximum value（355 nm） when the concentration of GdnHCl is 5.1 mol/L. The experimental results indicate that APE1547 has a high resistance to urea. Unfolding of APE1547 in GdnHCI（4. 2-6.0 mol/L） was shown to be an irreversible process. The present results indicate that the ion pairs in this protein may be a key factor for the stability of this esterase.

Expression and Characterization of an Active Chimeric Protein of Human Extracellular Superoxide Dismutase and hCuZnSOD in Pichia pastoris

Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases（SODs）, CuZn-SOD and extracellular SOD（EC-SOD）, involved in the defense system against reactive oxygen species（ROS）. The two SODs have structurally homologous centre domain with distinct N- and C-terminuses, resulting in the different characteristics of the structure and function of the two molecules. We generated a hybrid SOD molecule（namely hy- SOD） via replacing the N- and C-terminuses of hCuZnSOD with the counterparts of hEC-SOD. The hySOD was expressed in host Pichia pastoris and the purified protein was a dimer with a molecular weight of about 34000. A series of activity analyses indicates that the hySOD is similar to hEC-SOD in heat-stability, and has the activity of protecting the host cell against heat shock and oxidative stress. Our results show evidence for the study on the compound activity of multiple SOD molecules, and may be important for understanding the relationship between structure and function of hEC-SOD and hCuZnSOD.

Isolation and Characterization of Two Novel Gene Encoded Alkaline Esterases from an Alkaline Soil Metagenomic Library

The development of industrial biotechnology has created an increasing demand for alkaline lipolytic enzymes with functional diversity. In this study, an alkaline soil metagenomic library was constructed to search for new lipolytic enzymes. Two novel gene encoded alkaline esterases（designated as estA and estB） were isolated by functional screening from the library. The estA gene consisted of 834 bp and coded for 277 amino acids with a molecular mass of 29998. Amino acid sequence homology analysis indicates that EstA belongs to α/β hydrolase fold family 4.4（abH4.4）, with EstA being the smallest member of that family yet reported. The estB gene consisted of 1185 bp and encoded 394 amino acids with a theoretical molecular mass of 40090. Its conserved domain analysis shows that EstB belongs to the GDSL hydrolase superfamily. Both EstA and EstB exhibit only moderate identity（〈38%） in amino acid sequence to the known lipolytic enzyme genes in the database. The two genes were respectively expressed in Escherichia coli and the protein products were purified for functional characterization. While the expressed EstA did not exhibit the functional properties that were superior to those of other esterases previously reported, the EstB was stable at temperature up to 45 ℃ and its maximum activity was measured to be 53.6 U/mg at pH=10. Both the en- zymes have further enriched the diversity of the lipolytic enzymes database and also appear to be promising biocatalysts for potential biotechnological application.

Immobilization of urease in metal–organic frameworks via biomimetic mineralization and its application in urea degradation

Enzyme immobilization has been accepted as an efficient technique for improving the stability and recyclability of enzymes.Herein,biomimetic mineralization strategy was employed to achieve the immobilization of urease in a type of metal–organic frameworks(zeolite imidazolate framework-8,ZIF-8),and the immobilized enzyme urease@ZIF-8 was systematically evaluated for its structure,activity,stability and recyclability,using the hydrolysis of urea as a model.The entrapment of urease was found to be realized in a synchronous manner with the formation of ZIF-8 crystal.The loading of urease in ZIF-8 was measured to be ca.10.6%through the bicinchoninic acid(BCA)protein assay.The encapsulated urease could efficiently maintain its native conformation,which endowed the immobilized urease with excellent activity and stability,even in harsh conditions(e.g.,in the presence of trypsin,acidic or alkali conditions,or at high temperature).Further,urease@ZIF-8 exhibited good recyclability during the degradation of urea,in which it could keep 58.86%of initial activity after being used for 5 cycles.Thus,biomimetic mineralization could be potentially utilized as a promising method to prepare immobilized ureases with superior activity,stability and recyclability,thereby facilitating the construction of efficient catalysts for industrial biocatalysis and biosensing.

Self-sufficient Cytochrome P450s and their potential applications in biotechnology

Cytochrome P450s(CYPs)are ubiquitously found in all kingdoms of life,playing important role in various biosynthetic pathways as well as degradative pathways;accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation.Significantly,CYPs catalyze chemically challenging CAH and CAC activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center,while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein.Self-sufficient CYPs,encoding their heme domain and reductase protein in a single polypeptide,facilitate increased catalytic efficiency and render a less complicated system to work with.The self-sufficient CYP enzyme from CYP102A family(CYP102A1,BM3)is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications.An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade.In this review,we introduce chemistry and biology of CYPs,followed by an overview of the characteristics of self-sufficient CYPs and representative reactions.Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural(nature-mimicking)reactions are highlighted.Lastly,the strategy and efforts that aim to circumvent the challenges for improved thermostability,regio-and enantioselectivity,and total turnover number;associated with practical use of self-sufficient CYPs are reviewed.

Effects of Vitamin E on the Activities of Protective Enzymes and Membrane Lipid Peroxidation in Leymus Chinensis under Drought Stress

Leymus chinensis seedlings were treated with 0.05--10 mmol/L vitamin E under osmotic stress in the presence of polyethylene glycol（PEG） as the stress reagent. The effects of the different concentrations of exogenous vitamin E on the activities of SOD, POD and free proline, and the MDA contents under drought stress were examined so as to ascertain the mechanism of Leymus chinensis resistance to drought stress and explore the possible preventive measures. The results indicate that the activities of SOD and POD decreased but the free proline and MDA contents increased as drought stress was accentuated, showing an enhancement of oxidative stress that may cause a decline in membrane stabilization. However, the activities of SOD and POD and the free proline content increased, whereas the MDA content reduced in Leymus chinensis pretreated with vitamin E in comparison with that of the control. This indicates that exogenous vitamin E enhanced the antioxidation of Leymus chinensis seedlings. It suggests that cytomembrane can be protected from damage by increasing the free proline content and the activities of SOD and POD that result in enhancing the drought resistance of Leymus chinensis seedlings.

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of (R,S)-2-Octanol Acetate

To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96. 2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.

Raman and UV-Vis Spectroscopy Applied to the Analysis of Liver Tissues from Rats with Myocardial Ischemia Induced by Isoproterenol

The application of the laser Raman spectroscopic（LRS） technique for the analysis of liver tissues from rats with myocardial ischemia induced by isoproterenol（ISO） was described.Animal model of myocardial ischemia was established for rats induced by ISO.Rats were randomly divided into four groups as normal group and myocardial ischemia groups.We observed the successful myocardial ischemia model via serum enzymes levels and hematoxylin-eosin（HE） staining,and detected the liver tissue of the rats from normal group and liver tissue of the rats from myocardial ischemia groups via UV-Vis spectroscopy（UV-Vis） and LRS,and the changes of the absorbance spectra were compared in the above four different groups.The results show that ISO can induce rat myocardial ischemia successfully.The spectrum of normal liver tissue supernatant exhibits a strong absorption band at 968 nm,but no absorption band appears in the spectra of liver tissue supernatant solutions from the rats with myocardial ischemia induction after 2,12 and 72 h presented at 968 nm.LRS results show that Raman intensities of the precipitates suffered from ISO-treatment after 2,12 and 72 h were obviously increased compared with that of the precipitate of the liver tissue of the normal rats suffered from 0.9 g/L normal saline（NS） treatment.These results indicate that LRS and UV-Vis can be harmless,nondestructive,rapid and effective methods for analyzing different pathological specimens of liver tissue from myocardial ischemia rats.

Caspase-9 Activation—Critical for Betulin-induced Apoptosis of Human Hepatoma Cells

Betulinic acid and its derivatives have been extensively studied in the past for their anti-tumor effects, but relatively little is known about its precursor betulin. In this study we showed that betulin, an abundant natural product, significantly inhibits the cell growth of human hepatoma HepG2 cells in a dose-dependent manner. In the presence of 10 μg/mL betulin, HepG2 cells undergo an apoptosis, as evidenced by apoptotic morphology such as cell shrinkage, membrane blebbing, nuclear condensation and fragmentation, apoptotic body formation, and caspase activation. Kinetics analysis shows that the depolarization of the mitochondrial membrane potential and the release of the mito- chondrial apoptotic protein cytochrome c occurred as early as 2 h post treatment of HepG2 cells with 10 μg/mL betulin. Proteolytic activation of caspase-9, but not caspase-8, was observed in this apoptosis process. Moreover, the inactivation of caspase-9 by its specific siRNA dramatically reduced betulin-induced caspase-3 activation and apoptosis. Taken together, our observations indicate that the activation of caspase-9 is critical for betulin-induced apoptosis of human hepatoma HepG2 cells.

Inhibition of Growth of Human Ileocecal Adenocarcinoma Cells HCT-8 and Inducing Apoptosis by Different RGD-containing Peptides

Human ileocecal adenocarcinoma cells HCT-8 were treated with RGD-containing cellular adhesion peptides including RGD, RGD（NH2）2（i.e., RGE-NH2）, RGDS, and RGDS-NH2. MTT assay was prepared to examine their inhibiting effects on HCT-8 cells after treatment. The methods including Haematoxylin and Eosin（HE） staining, transmission electron microscopy（TEM）, immunohistochemistry, flow cytometry, and Reverse TranscriptionPolymerase Chain Reaction（RT-PCR） were used to observe the morphology of the apoptotic cells and analyze the mechanism of apoptosis. The experimental results indicate that RGD-containing cellular adhesion peptides can inhibit the growth and proliferation of tumor HCT-8 cells in a dose-dependent manner and induce the apoptosis of HCT-8 cells. At the same time, the high conservative property of RGD was confirmed again.

Synthesis of Codon-optimized Human Interleukin-18 Gene by Combination of Chemical and Enzymatic Method

According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.

Permeability and selectivity synergistically enhanced nanofluidic membrane for osmotic energy harvesting

For the porous‐membrane‐based osmotic energy generator,the potential synergistic enhancement mechanism of various key parameters is still controversial,especially because optimizing the trade‐off between permeability and selectivity is still a challenge.Here,to construct a permeability and selectivity synergistically enhanced osmotic energy generator,the twodimensional porous membranes with tunable charge density are prepared by inserting sulfonated polyether sulfone into graphene oxide.Influences of charge density and pore size on the ion transport are explored,and the ionic behaviors in the channel are calculated by numerical simulations.The mechanism of ion transport in the process is studied in depth,and the fundamental principles of energy conversion are revealed.The results demonstrate that charge density and pore size should be matched to construct the optimal ion channel.This collaborative enhancement strategy of permeability and selectivity has significantly improved the output power in osmotic energy generation;compared to the pure graphene oxide membrane,the composite membrane presents almost 20 times improvement.

Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E. coil

The entire gene of carboxyltransferase（CT） domain of acetyl-CoA carboxylase（ACCase） from Chinese Spring wheat（CSW） plastid was cloned firstly, and the 2.3 kb gene was inserted into PET28a^＋ vector and expressed in E. coil in a soluble state. The （His）6 fusion protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography, and the calculated molecular mass（Mr） was 88000. The results of the sequence analysis indicate that the cloned gene（GeneBank accession No. EU124675） was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid. The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides, and further to screen new herbicides.