Direct and single-step sensing of primary ovarian cancers related glycosidases

High sensitive, accurate detection for tumor-associated overexpressed enzyme activity is highly significant for further understanding enzyme function, discovering potential drugs, and early diagnosis and prevention of diseases. In this work, we developed a facile, direct and single-step detection platform for primary ovarian cancers related glycosidase activity based on the inner filter effect(IFE) between glycosidase catalytic product and black phosphorus quantum dots(BPQDs). Highly fluorescent BPQDs were successfully synthesized from bulk black phosphorus by a simple liquid exfoliation method. Under the catalysis of β-galactosidase, p-nitrophenyl-β-D-galactopyranoside(PNPG) was transformed into pnitrophenol(PNP) and β-D-galactopyranoside. Meanwhile, the absorption of catalytic product PNP greatly overlapped with the excitation and emission spectra of fluorescent BPQDs, leading to the fluorescence quenching of BPQDs with a high quenching efficiency. The proposed sensing strategy provided a low detection limit of 0.76 U/L, which was 1 — 2 orders of magnitude lower than most unmodified sensing platforms. D-Galactal was selected as the inhibitor for β-galactosidase to further assess the feasibility of screening potential inhibitors. The fluorescence recovery of BPQDs suggests that the unmodified sensing platform is feasible to discover potential drugs of β-galactosidase. Our work paves a general way in the detection of glycosidase activity with fluorescent BPQDs, which can be promising for glycosidase-related disease diagnosis and pathophysiology elucidation.     

Catalytic enantioselective synthesis of β-amino alcohols by nitrene insertion

Chiral β-amino alcohols are important building blocks for the synthesis of drugs, natural products, chiral auxiliaries, chiral ligands and chiral organocatalysts. The catalytic asymmetric β-amination of alcohols offers a direct strategy to access this class of molecules. Herein, we report a general intramolecular C(sp³)–H nitrene insertion method for the synthesis of chiral oxazolidin-2-ones as precursors of chiral β-amino alcohols. Specifically, the ring-closing C(sp³)–H amination of N-benzoyloxycarbamates with 2 mol% of a chiral ruthenium catalyst provides cyclic carbamates in up to 99% yield and with up to 99% ee.The method is applicable to benzylic, allylic, and propargylic C–H bonds and can even be applied to completely non-activated C(sp³)–H bonds, although with somewhat reduced yields and stereoselectivities. The obtained cyclic carbamates can subsequently be hydrolyzed to obtain chiral β-amino alcohols. The method is very practical as the catalyst can be easily synthesized on a gram scale and can be recycled after the reaction for further use. The synthetic value of the new method is demonstrated with the asymmetric synthesis of a chiral oxazolidin-2-one as intermediate for the synthesis of the natural product aurantioclavine and chiral β-amino alcohols that are intermediates for the synthesis of chiral amino acids, indane-derived chiral Box-ligands, and the natural products dihydrohamacanthin A and dragmacidin A.     

Metal-seed assistant photodeposition of platinum over Ta3N5 photocatalyst for promoted solar hydrogen production under visible light

Cocatalysts play a vital role in accelerating the reaction kinetics and improving the charge separation of photocatalysts for solar hydrogen production.The promotion of the photocatalytic activity largely relies on the loading approach of the cocatalysts.Herein,we introduce a metal-seed assistant photodeposition approach to load the hydrogen evolution cocatalyst of platinum onto the surface of Ta₃N₅ photocatalyst,which exhibits about 3.6 times of higher photocatalytic proton reduction activity with respect to the corresponding impregnation or photodeposition loading.Based on our characterizations,the increscent contact area of the cocatalyst/semiconductor interface with metal-seed assistant photodeposition method is proposed to be responsible for the promoted charge separation as well as enhanced photocatalytic H2 evolution activity.It is interesting to note that this innovative deposition strategy can be easily extended to loading of platinum cocatalyst with other noble or non-noble metal seeds for promoted activities,demonstrating its good generality.Our work may provide an alternative way of depositing cocatalyst for better photocatalytic performances.     

A new strategy to iteratively update scalable universal quantitative models for the testing of azithromycin by near infrared spectroscopy

The training set of a universal near infrared (NIR) model for quantitative analysis of a drug should cover as many samples of this drug in the market as possible. Inevitably the model may fail for new products that have different excipients and production processes. In such circumstances the model should be updated. We here propose a new strategy to iteratively update a universal NIR quantitative model for azithromycin. We prove that universal quantitative models generated from this new strategy are comparably effective for azithromycin injection powders and azithromycin tablets, compared to the strategy using hierarchical clustering method which we reported previously. Furthermore, we establish the correlation coefficient r between a new sample and the training set samples can be used to decide whether or not the model should be updated.     

Directed Evolution of L-Aspartase by Mobility of Domains

To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild-type tetrameric L-asparease, these mutants remained 19. 7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their kcat, and KM changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg^2 concentration and thermostability increased. Therefore this strategy provides a novel approach to directed evolution of enzymes.     

Sensitive detection of DNA methyltransferase activity based on supercharged fluorescent protein and template-free DNA polymerization

DNA methylation, catalyzed by DNA methyltransferases(MTases), is a key component of genetic regulation, and DNA MTases have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein(Sc GFP)/graphene oxide(GO) interaction, coupled with methylation-initiated template-free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3′-terminal(DNA-1) was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleotidyl transferase(Td T), DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA fragments quickly bind with Sc GFP and form the Sc GFP/DNA nanocomplex. Such nanocomplex can effectively protect Sc GFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of Sc GFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of Sc GFP/GO. The method was successfully used to detect the activity of DNA adenine methylation(Dam) MTase with a wide linear range(0.1–100 U/m L) and a low detection limit of 0.1 U/m L. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was successfully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect Td T activity.     

Automated screening of primary cell-based aptamers for targeting and therapy of pancreatic cancer

Although it has been developed for many years, nucleic acid aptamer screening technology still fails to be widely used, a considerable part of it is due to the variability of tumor cell morphology, which leads to the use of immortalized cell lines in the laboratory to screen nucleic acid aptamers for recognition ability of tumor cells in the diseased body. To address this, primary cells that can be stably passaged were isolated and extracted from spontaneous tumors of genetically engineered panc...     

STUDIES ON RAPID METHOD OF CULTURING POTENT MICROBIAL STRAINS

In this paper we use a new inducing method by applying the powerful and penetra-tire fast neutron and Co~(60)-γ rays on single colonies of just developed aerial hyphae from whichwe have obtained a high percentage of mutant strains. At the same time, the stimulativezone is used as the hormone screening index so that the breeding rate has been greatly acceler-ated. It has been proved in our research that this new method has many merits. It is easy toapply and to obtain a high ratio of mutants. Hence it is really a fast and effective in-ducing technique. The successful breeding of Streptomyces jingyangensis sp. nov., a highlyeffective stra.in "5406", provides us with its studies and utilization.     

Pressure controllable aptamers picking strategy by targets competition

Selection of aptamers with high affinity and good specificity requires multiple rounds of alternating steps of separation and PCR amplification.Herein,we proposed a novel high-efficiency aptamers picking strategy:One-round pressure controllable selection(OPCS).OPCS integrates four types of screening superiority,high-efficiency separation,one-round selection and PCR amplification,synchronous negative selection and targets competition.The controllable screening pressure can be achieved through two approaches,balanced competition by the regulation of protein concentration,and dominant competition by introducing a predatory protein with high concentration.In OPCS process,two proteins were co-incubated with one ssDNA library,and each protein bound its favorable sequences specifically and formed protein-ss D NA complex re spectively.Meanwhile,one protein could supply/sufferthe picking pressure of affinity and specificity to/from another,which eliminated weakly bound or unbound sequences for each other.Two complexes could be separated and collected conveniently,and aptamers for two proteins obtained synchronously with high affinity and good specificity.This strategy not only provides a more effective way for aptamers selection,but shows great potential in other ligands or drugs selection.     

Synthesis of Symmetric Diketones from Imidazolinium Salt and Bis-Grignard Reagents

In our earlier paper, we reported a convenient method for the synthesis of symmetricdiketones from benzimidazolium salts and his-Grignard reagents'. The yields of productobtained by the reported method are better then that of the former methods2' 3. Howeverthe method suffers from dramatic expensive material, such as o-phenyldiamine, whichmakes it not to be useful for large-scale preparation.In order to overcome this disadvantage and find a new method which can be utilizedin industry, we have e…     

Enhanced Activity and Enantioselectivity of a Hyperthermophilic Esterase from Archaeon Aeropyrum pernix K1 by Acetone Treatment

To improve the activity and enantioselectivity of hyperthermophilic archaeon Aeropyrum pernix K1 esterase (APE1547) and its mutants, they were purified by acetone-treated method. It was found that the acetone treatment not only caused APE1547 and its mutants to display higher activity and enantioselectivity but also saved more than 90% of time spent in purifying them by Ni-chelating column. In hydrolysis of p-nitrophenyl caprylate, the acetone-treated APE1547 and mutant A containing the following substitutions R11G, L36P, V225A, I551L, and A564T showed 5.7- and 6.9-fold active increase, respectively. In the resolution of 2-octanol acetate, the acetone-treated mutant A had a 9-fold enantioselective increase relative to that purified by Ni-chelating column. In addition, the impact of pH, temperature, and chemical reagents on activity of APE1547 and mutant A was discussed in this paper.     

Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling

The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling. An agar plate assay was used to screen the mutant libraries for activity. Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE). Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic. Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate. Several mutations in the shuffled mutants had additive effects.     

The “bringer” strategy

Random mutagenesis constitutes a keystone in many strategies of directed evolution of biocatalysts and is often done by error-prone polymerase chain reaction (epPCR). Traditionally, the epPCR-generated DNA fragments are then subcloned into an expression vector to obtain a mutant library, which in turn is transformed into a suited host and screened for mutants that display the desired property. However, the vast majority of epPCR-generated fragments generally are lost during the subcloning step, making it the bottleneck in the mutant library construction procedure. Here we report a rapid and convenient strategy based on the epPCR amplification of a ring-closed expression plasmid that contains the gene of interest; after a DpnI digest the product of the epPCR reaction constitutes the mutant library and can be used directly for screening procedures. Primers binding to the beta-lactamase gene were chosen to allow application of the strategy to as broad a range of target plasmids as possible. The functionality of this approach was demonstrated by mutating the alpha-peptide coding region of the lacZ gene.     

Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Ophiostoma piliferum,esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes

Recent advances in biochemistry and molecular biology have simplified the discovery and preparation of new hydrolases. Although these hydrolases might solve problems in organic synthesis, measuring their selectivity, especially enantioselectivity, remains tedious and time consuming. Recently, we developed a colorimetric screening method to measure the enantioselectivity of hydrolases. Here we apply this rapid screening method to map the substrate selectivity of four new hydrolases: lipases from the thermophilic Bacillus thermocatenulatus (DSM 730, BTL2) and a filamentous fungus Ophiostoma piliferum (NRRL 18917, OPL) and esterases from two bacteria, Pseudomonas fluorescens (SIK-W1, esterase I, PFE) and Streptomyces diastatochromogenes (Tü 20, SDE). We screened a general library of 29 substrates and a chiral library of 23 pairs of enantiomers. All four hydrolases catalysed the hydrolysis of unnatural substrates, but the two lipases accepted a broader range of substrates than the two esterases. As expected, the two lipases favoured more hydrophobic substrates, while the two esterases showed a preference for smaller substrates. Several moderately enantioselective reactions were identified for the solketal esters: BTL2, butyrate, E=7.9 (R); octanoate, E=4.9 (R) and 3-bromo-2-methyl propionate methyl esters, PFE, E=12 (S); SDE, E=5.6 (S). OPL showed low enantioselectivity toward all substrates tested. The current colorimetric screen could not measure the selectivity for several slow-reacting substrates. Traditional screening identified high enantioselectivity of BTL2 and PFE toward one of these slow substrates, 1-phenylethyl acetate (E>50).     

The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases

In this work, we introduce the application of proton transfer reaction mass spectrometry (PTR-MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC-MS)-based methods, PTR-MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR-MS-based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR-Qi-ToF-MS were compared with GC-MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR-Qi-ToF-MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC-MS analysis. This study provides a first basis for the application of rapid PTR-Qi-ToF-MS detection, in combination with multi-well formats, in HTS screening methods for the selection of highly productive terpene synthases.     

Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations

Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach--random mutagenesis within the substrate binding site--is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.     

Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases

Environmental DNA from soil and water samples was extracted to construct a plasmid library and a fosmid library containing 19,500 and 20,400 clones, respectively. Two esterases (EstP2K and EstF4K) were finally isolated from each library based on activity screening, and both of them were characterized in this study. The esterase EstF4K consists of 396 amino acids with an SMTK motif which belongs to family VIII esterase/lipase. The amino acid sequence of EstF4K showed 83 % identity with that of EstA3, a reported esterase isolated from uncultured organisms of soil. EstP2K is composed of 224 amino acids in size and shows only 37 % identity with a putative lipase of Neisseria elongata subsp. The purified EstF4K was optimally active at pH?8.0 and 50 °C. It was remarkably active and very stable in the presence of 30 % dimethyl sulfoxide. Activity fingerprint of EstF4K displayed a higher level of activity toward short-chain fatty acid p-nitrophenyl (pNP) esters, while EstP2K preferred bias for pNP caprylate ester. The optimum reaction temperature and pH for EstP2K are 45 °C and 7.5, respectively, and the enzyme exhibited strong tolerance in the presence of 30 % methanol. EstF4K and EstP2K showed opposite enantioselectivity for methyl 3-phenylglycidate, a chiral synthon for the synthesis of Taxol® side chain.     

Expanding the substrate scope of enzymes: combining mutations obtained by CASTing

In a previous paper, the combinatorial active-site saturation test (CAST) was introduced as an effective strategy for the directed evolution of enzymes toward broader substrate acceptance. CASTing comprises the systematic design and screening of focused libraries around the complete binding pocket, but it is only the first step of an evolutionary process because only the initial libraries of mutants are considered. In the present study, a simple method is presented for further optimization of initial hits by combining the mutational changes obtained from two different libraries. Combined lipase mutants were screened for hydrolytic activity against six notoriously difficult substrates (bulky carboxylic acid esters) and improved mutants showing significantly higher activity were identified. The enantioselectivity of the mutants in the hydrolytic kinetic resolution of two substrates was also studied, with the best mutant-substrate combination resulting in a selectivity factor of E=49. Finally, the catalytic profile of the evolved mutants in the hydrolysis of simple nonbranched carboxylic acid esters, ranging from acetate to palmitate, was studied for theoretical reasons.     

Array-based split-pool combinatorial screening of potential catalysts

A new method for screening split-pool combinatorial libraries for catalytic activity is described. Site-selective detection of catalytic activity for solution-based reactions was made possible without cofunctionalizing beads or adding diffusion-limiting matrixes. This was done by spatially separating resin-bound catalysts on an adhesive array on a microscope slide and introducing the reacting liquid to the top of the slide. Convective mixing and evaporation was controlled using a cover slide and imaging both the formation of products within active beads and the diffusion of products out of the beads. Colored reaction products and pH-sensitive indicators were used to visually detect catalytically active beads in the presence of inactive ones. Quantitative analyses of the images support the assumption that color intensities can be used to assess the quality of hits from a combinatorial screen. The Knoevenagel condensation reaction catalysis as well as esterase screening using methyl red were used to validate the approach. Using the esterase data, it was shown that some information on activity could also be extracted from the colored plume surrounding individual beads although the precision is not as good as that from direct measurement of absorbance through the bead. It was also found that the distribution of products within a single bead can also be gleaned from the absorbance data for different-sized beads.     

Rapid screening of the aglycone specificity of glycosidases: applications to enzymatic synthesis of oligosaccharides.

BACKGROUND: Retaining glycosidases can catalyse glycosidic bond formation through transglycosylation from a donor sugar to an acceptor bound in the aglycone site. The aglycone specificity of a glycosidase is not easily determined, thereby complicating the choice of the most appropriate glycosidase for use as a catalyst for transglycosylation. We have developed a strategy to rapidly screen the aglycone specificity of a glycosidase and thereby determine which enzymes are best suited to catalyse specific transglycosylation reactions. RESULTS: The reactivation, or turnover, of a glycosidase trapped as a fluoroglycosyl-enzyme species is accelerated in the presence of a compound that productively binds to the aglycone site. This methodology was used to rapidly screen six glycosidases with 44 potential acceptor sugars. Validation of the screening strategy was demonstrated by the identification of products formed from a transglycosylation reaction with positively screened acceptors for four of the enzymes studied. CONCLUSIONS: The aglycone specificity of a glycosidase can be rapidly evaluated and requires only an appropriate fluorosugar inactivator, a substrate for assay of activity and a library of compounds for screening.