Identification and Characterization of a New Alkaline Thermolysin-Like Protease, BtsTLP1, from Bacillus thuringiensis Serovar Sichuansis Strain MC28.

Thermolysin and its homologs are a group of metalloproteases that have been widely used in both therapeutic and biotechnological applications. We here report the identification and characterization of a novel thermolysin-like protease, BtsTLP1, from insect pathogen Bacillus thuringiensis serovar Sichuansis strain MC28. BtsTLP1 is extracellularly produced in Bacillus subtilis, and the active protein was purified via successive chromatographic steps.

Dansylation of unactivated alcohols for improved mass spectral sensitivity and application to analysis of cytochrome P450 oxidation products in tissue extracts.

Chemical derivatization is useful for improving the ionization characteristics of poorly or nonionizable analytes in liquid chromatography-mass spectrometry (LC-MS). Dansyl chloride has been widely used as a derivatizing reagent for fluorescence detection and for facilitating the MS detection of phenols and amines, but not for general alcohols. A new dansylation method for improving the mass spectral sensitivity of unactivated alcohols was developed.

Characterizing proteins of unknown function: orphan cytochrome p450 enzymes as a paradigm.

With the rapid completion of genomic sequences of organisms today, we have far more gene products than functions we can ascribe. A number of experimental strategies have been developed and applied, both in vitro and in vivo, to put functions to these orphan proteins. The "deorphanization" of human and Streptomyces cytochrome P450 enzymes is considered quite important for pharmacology, with ramifications for the use of clinical therapeutics.

Approaches to deorphanization of human and microbial cytochrome P450 enzymes.

One of the general problems in biology today is that we are characterizing genomic sequences much faster than identifying the functions of the gene products, and the same problem exists with cytochromes P450 (P450). One fourth of the human P450s are not well-characterized and therefore considered "orphans." A number of approaches to deorphanization are discussed generally.

Human cytochrome P450 4F11: heterologous expression in bacteria, purification, and characterization of catalytic function.

Human cytochrome P450 (P450) 4F11 is still considered an "orphan" because its function is not well characterized. A bacterial expression system was developed for human P450 4F11, producing approximately 230nmol P450 from a 3-l culture of Escherichia coli. P450 4F11 was purified and utilized for untargeted substrate searches in human liver extract using a liquid chromatography/mass spectrometry-based metabolomic and isotopic labeling approach (Tang et al.

Elucidation of functions of human cytochrome P450 enzymes: identification of endogenous substrates in tissue extracts using metabolomic and isotopic labeling approaches.

One of the central problems in biochemistry in the postgenomic era is the elucidation of functions of proteins, including "orphan" human cytochromes P450 (P450s), when the substrates are unknown. A general strategy for identification of endogenous substrates of P450s in tissue extracts using metabolomic and isotopic labeling approaches is described, involving four main steps: (1) In vitro incubation of a P450 enzyme system with cofactor and tissue extract is done under a mixture of (18)O(2)/(16)O(2) (1:1). (2) Liquid chromatography/mass spectrometry (LC/MS) assay of an organic extract of the reaction mixture is performed.

Immobilized capillary enzyme reactor based on layer-by-layer assembling acetylcholinesterase for inhibitor screening by CE.

A method for creating an immobilized capillary acetylcholinesterase (AChE) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. The unique capillary AChE reactor was easily prepared by the instrument in three steps: first, a 0.5 cm long plug of a solution of the cationic polyelectrolyte polydiallyldimethylammonium (PDDA) was injected into the capillary to produce a positively charged coating on the surface of the capillary; subsequently, the enzyme solution with the same plug length was injected into the capillary and incubated for 10 min to immobilize the enzyme on the capillary wall via electrostatic interaction; third, PDDA solution with the same plug length was injected again into the capillary to cover the immobilized enzyme by forming PDDA-AChE-PDDA sandwich-like structure.

Screening of acetylcholinesterase inhibitors in natural extracts by CE with electrophoretically mediated microanalysis technique.

An electrophoretically mediated microanalysis (EMMA) method for screening acetylcholinesterase (AChE) inhibitors in natural extracts is described. In this method, solutions of AChE and the mixture of the substrate and the natural extract were successively injected into the capillary, and mixed electrophoretically by applying a voltage for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract.

Enzyme inhibitor screening by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique.

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding.

Enantioseparation of chiral allenic acids by micellar electrokinetic chromatography with cyclodextrins as chiral selector.

Enantioseparation of chiral aryl allenic acids by micellar electrokinetic chromatography (MEKC) with cyclodextrins (CDs) as chiral selectors was described. The screen of chiral selectors (beta-CD, gamma-CD, and hydroxypropyl (HP)-gamma-CD) showed that the enantioseparation was not only dependent on the type of CD but also the presence of 2-propanol in the buffer. In order to optimize the operational parameters, the effect of the concentration of CDs, sodium dodecyl sulfate (SDS), and 2-propanol, as well as the buffer ionic strength and pH on enantioseparation were studied.