Thermostable trypsin conjugates for high-throughput proteomics: synthesis and performance evaluation

Conjugating bovine trypsin with oligosaccharides maltotriose, raffinose and stachyose increased its thermostability and suppressed autolysis, without affecting its cleavage specificity. These conjugates accelerated the digestion of protein substrates both in solution and in gel, compared to commonly used unmodified and methylated trypsins.     

When peptides fly: Advances in Drosophila proteomics

In the past decade, improvements in genome annotation, protein fractionation methods and mass spectrometry instrumentation resulted in rapid growth of Drosophila proteomics. This review presents the current status of proteomics research in the fly. Areas that have seen major advances in recent years include efforts to map and catalog the Drosophila proteome and high-throughput as well as targeted studies to analyze protein–protein interactions and post-translational modifications. Stable isotope labeling of flies and other applications of quantitative proteomics have opened up new possibilities for functional analyses. It is clear that proteomics is becoming an indispensable tool in Drosophila systems biology research that adds a unique dimension to studying gene function.     

Studies on immobilized trypsin in high concentrations of organic solvents.

Trypsin was covalently immobilized on porous glass in the presence and absence of a specific substrate and reacted in various organic solvents of different dielectric constants. Optimum solvent concentration, pH profile, Km(app), Vmax(app), productivity versus temperature, activity, and reaction rates were determined. Reaction rates of six lysyl dipeptides were compared. Crystalline trypsin was dansylated for studies by nanosecond fluorescence techniques to determine the effects of introducing high concentrations of organic solvents on the molecule. The results indicated that greater reaction rates were observed with dipeptides having more acidic carboxyl terminal groups. The data also indicated that greater reaction rates were observed in higher concentrations of solvents of lower dielectric constants. Nanosecond fluorescence spectroscopy of trypsin in high concentrations of a low dielectric constant solvent indicated major dehydration even though maximal enzyme activity was achieved under these conditions.     

Application of proteomics in biotechnology--microbial proteomics

The genomes of most economically important microbial cells are already sequenced and proteomic technologies can be applied during various process development steps, starting with the selection and optimization of the functions of the industrial strains, application of the knowledge of cell function in response to the changes of production parameters, validation of the downstream processing, and thorough characterization of the final product. Unfortunately, there are only a few direct examples in the literature that present the optimization of the production process based on proteomics. In this review, we discuss the potential of this technology for the design of future bioprocesses and for optimization of existing ones.     

Procyclins, proteases and proteomics: dissecting trypanosomes in the tsetse fly.

The forms of African trypanosomes that live in tsetse fly vectors are coated with lipid-anchored proteins and glycoproteins known collectively as procyclins. Procyclins are expressed during development in the fly in a multiplicity of isoforms yet their functions remain unknown. Recent studies involving a multidisciplinary synthesis of tsetse biology, immunochemistry, biological chemistry and mass spectrometry have yielded much new information about procyclins, which could now provide an unparalleled view of the dynamic molecular interactions between this parasite and its insect vector.     

Effects of organic solvents and substrate binding on trypsin in acetonitrile and hexane media

In this work, we used molecular dynamic (MD) simulation to study trypsin with and without a six-amino-acid peptide bound in three different solvents (water, acetonitrile and hexane) in order to provide molecular information for well understanding the structure and function of enzymes in non-aqueous media. The results show that the enzyme is more compact and less native-like in hexane than in the other two polar solvents. The substrate could stabilize the native protein structure in the two polar media, but not in the non-polar hexane. There are no significant differences in the conformation of the S1 pocket upon the substrate binding in water and acetonitrile media while a reverse behavior is observed in hexane media, implying a possible induced fit binding mechanism in the non-polar media. The substrate binding enhances the stability of catalytic H-bond network since it could expel the solvent molecules from the active site. The enzyme and the substrate appear to be more appropriate to the reactive conformation in the organic solvents compared with aqueous solution. There is much greater substrate binding strength in hexane media than the water and acetonitrile ones since the polar solvent significantly weakens electrostatic interactions, which are observed to be the main driving force to the binding. In addition, some residues of the S1 pocket could remain favorable contribution to the binding despite the solvent change, but with differences in the contribution extent, the number and the type of residues between the three media.

胰蛋白酶镜像酶LysargiNase的开发及其在蛋白质组学研究中的应用

蛋白质组学是系统鉴定、定量蛋白质及其翻译后修饰形式,并研究这些蛋白质生物学功能的学科。目前,基于质谱的鸟枪法蛋白质组学技术是蛋白质组学研究的主要手段之一,其技术流程是先将蛋白质组样品经位点特异性蛋白酶消化形成肽组,再进行高效液相色谱分离和质谱检测。而位点特异性蛋白酶对蛋白质样品的消化是质谱检测的前提和基础。随着蛋白质组学研究的深入,多种位点特异性蛋白酶被先后开发利用;而切割发生在相应氨基酸的N端,与传统的C端蛋白酶互为镜像的蛋白酶的鉴定、开发、特性研究和广泛使用更是为蛋白质组学研究提供了新的工具。文中对最近发现的胰蛋白酶的镜像酶——赖氨酸精氨酸N端蛋白酶(LysargiNase)的特点及其应用进行综述,为国内外学者更加广泛的使用创造条件。     

干细胞的蛋白质组研究

干细胞研究和蛋白质组研究同属于21世纪生命科学的热点领域。将蛋白质组学技术应用于干细胞的研究,能够为了解干细胞提供蛋白质水平的信息,揭示干细胞的增殖、定向分化和迁移的机制,为人们更好地将干细胞技术应用于组织工程、基因治疗及药物开发等领域奠定基础。     

Isolation and in vitro synthesis of trypsin from the biting fly,Stomoxys calcitrans

The synthesis of [3H]trypsinlike enzyme by the fat body was followed in Stomoxys calcitransin vitro using a radioimmunoassay (RIA) developed against mammalian trypsin. Using high specific activity [3H]valine, trypsinlike activity was followed in midgut epithelial cells, thoracic muscle, and fat body removed from sugar-fed flies. Excreta protease of S. calcitrans was partially purified using charge and hydroxylapatite gel chromatography. Seventy-five percent of the enzyme eluted from these gels was inhibited by tosyl-L-lysine chloromethyl ketone HCI (TLCK) and was classified as trypsinlike. Electrophoresis of the trypsinlike enzyme indicated that it was only 50% pure. Trypsinlike activity from S. calcitrans bound to α₁-globulin IV-I and formed a complex that was dissociated on a P-100 Bio-Gel column. Binding between the protease and the α₁-gobulin IV-I caused a 1.4-fold increase in the apparent molecular weight of the protease on the P-100 Bio-Gel column. Trypsinlike activity was characterized in the midgut and excreta by affinity binding to covalently linked TLCK and tosyl-L-lysine chloromethyl ketone HCI (TAME)Sepharose 4B gels. Between 50% and 55% of the excreta protease and 5669% of the midgut protease bound to the affinity gels and was trypsinlike. Protease activity that did not bind to the gels was not inhibited by TLCK and did not have the esterolytic activity of trypsin.     

Effect of trypsin microenvironment on the rate constants of elementary stages of Nalpha-benzoyl-L-arginine ethyl ester hydrolysis

The hydrolysis reaction of Nalpha-benzoyl-L-arginine ethyl ester catalyzed by trypsin from pig pancreas was comparatively studied in an aqueous buffer solution and in the system of reversed micelles of Aerosol OT in octane (pH 8.5) to determine the mechanisms of influence of the enzyme microenvironment on the rate constants of the elementary stages of the enzymatic reaction. The temperature dependences of the catalytic constant kcat and the rate constant of the second order kcat/Km (s, catalysis efficiency) allowed the determination of the rate constants and the activation energy of elementary stages of the enzymatic reaction. It was revealed that a decrease in the efficiency of catalytic action of trypsin in inverted mycelles in comparison with an aqueous solution is first of all determined by a decrease in the rate constant of formation of the enzyme-substrate complex k1. Possible mechanisms of the effect of the microenvironment on the elementary stages of catalytic action of the enzyme are discussed.     

Improving the industrial production of 6-APA: enzymatic hydrolysis of penicillin G in the presence of organic solvents

The hydrolysis of penicillin G in the presence of an organic solvent, used with the purpose of extracting it from the culture medium, may greatly simplify the industrial preparation of 6-APA. However, under these conditions, PGA immobilized onto Eupergit displays very low stability (half-life of 5 h in butanone-saturated water) and a significant degree of inhibition by the organic solvent (30%). The negative effect of the organic solvent strongly depended on the type of solvent utilized: water saturated with butanone (around 28% v/v) had a much more pronounced negative effect than that of methylisobutyl ketone (MIBK) (solubility in water was only 2%). These problems were sorted out by using a new penicillin G acylase derivative designed to work in the presence of organic solvents (with each enzyme molecule surrounded by an hydrophilic artificial environment) and a suitable organic solvent (MIBK). Using such solvent, this derivative kept its activity unaltered for 1 week at 32 degrees C. Moreover, the enzyme activity was hardly inhibited by the presence of the organic solvent. In this way, the new enzyme derivative thus prepared enables simplification of the industrial hydrolysis of penicillin G.     

Surface effects of solvents in hydrolysis of water-soluble lipids by candidal lipase

In the study of hydrolysis of tributyrin by the lipase of Candida cylindracea, it is shown that initial rates of hydrolysis are directly proportional to the amount of enzyme adsorbed at the substrate-water interface. As a consequence of understanding the role of the physical state of the substrate in aqueous reaction media, it was hypothesized that the inclusion of synthetic (nonsubstrate) surfaces into the reaction media may enhance the hydrolysis rate of simple liquid lipids which are partly soluble in water, like triacetin. Nonpolar n-hydrocarbons having 5-11 carbon atoms were used to create interfaces in the hydrolysis of triacetin in the soluble range. All of the C(5)-C(11) hydrocarbons showed an activating effect. For quantitative evaluation of the effects of n-hydrocarbons, n-heptane was chosen as the model n-hydrocarbon. Interrelations between the reaction kinetics and adsorption of the enzyme at the n-heptane-water interface were experimentally determined by the use of the same in-line filtration device used for the tributyrin-water system. At 35 degrees C and pH 6 the relative values of the rate constants for the decomposition of enzyme-interface-substrate complexes were calculated as 12 and 1 for the tributyrin and n-heptane-triacetin systems, respectively. The nature of activation at the solvent surfaces were accounted for by a kinetic model which assumes simultaneous adsorption of enzyme and triacetin molecules at the n-heptane-water interface. Making use of the proposed model, the value of a the apparent Michaelis constant for the soluble triacetin-n-heptane system at constant n-heptane concentration, 2 vol %, was calculated as 0.044 mol/L.