Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

The couplings of N-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence of Bacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

Summary The couplings ofN-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence ofBacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Peptide syntheses mediated by Bacillus subtilis protease

Bacillus subtilis protease (Amano protease N) was examined as a catalyst for peptide bond formation via both the kinetically and thermodynamically controlled approaches. In general, the latter approach proved to be superior to the former, and a series of dipeptide syntheses and several segment condensations were achieved in good to high yields using the immobilized enzyme on Celite in acetonitrile with low water content.     

Peptide syntheses mediated by Bacillus subtilis protease

Summary Bacillus subtilis protease (Amano protease N) was examined as a catalyst for peptide bond formationvia both the kinetically and thermodynamically controlled approaches. In general, the latter approach proved to be superior to the former, and a series of dipeptide syntheses and several segment condensations were achieved in good to high yields using the immobilized enzyme on Celite in acetonitrile with low water content.     

Utilization of Proteases from Aspergillus Species for Peptide Synthesis via the Kinetically Controlled Approach

We examined Aspergillus melleus protease (Amano protease P) and A. oryzae protease (Amano protease A) as catalysts for peptide bond formation via the kinetically controlled approach. As the coupling efficiency was only moderate, even with a good amino acid substrate as the carboxyl component, in acetonitrile as a solvent (with or without a small amount of added water) that we had mainly employed previously in α-chymotrypsin catalyzed couplings, other solvent systems were sought. In 1,1,1,3,3,3-hexafluoro-2-propanol-DMF (1:1) without added water, these Aspergillus proteases were found to remain active for a long period of time and to be utilizable for peptide synthesis when the carbamoylmethyl ester was employed as the acyl donor, though the coupling efficiencies were dependent rather largely on the combination of the amino acid residues at the coupling site. The superiority of the carbamoylmethyl ester to conventional esters, for example the methyl ester, was once again established. Furthermore, some segment condensations were also achieved by the same procedure.     

Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.     

Facile Synthesis of N α -Protected Amino/Peptide Hydroxamic Acids Mediated by COMU

One-pot preparation of N ^α -protected amino/peptide hydroxamic acids from corresponding carboxylic acids is described using uronium-type coupling reagent COMU. The present protocol is simple and mild conditions are used. Thus the resulting hydroxamic acids are obtained in good yields without racemization.     

Characterization of bacterial proteases with a panel of fluorescent peptide substrates

Bacteria produce a range of proteolytic enzymes. In an attempt to detect and identify bacteria on the basis of their protease activity, a panel of protease substrates was investigated. Peptides conjugated to the fluorophore 7-amino-4-methylcoumarin (AMC) are well-established substrates for measuring protease activity. Although peptide-AMC substrates are generally not specific for a single protease, a unique pattern can be achieved for both highly specific enzymes and those with a broader substrate range by comparing different peptide substrates. The panel of 7 peptide-AMC substrates chosen exhibited a unique pattern for nine microbial proteases. The selected peptides were used to determine protease activity in cultured strains of Pseudomonas aeruginosa and Staphylococcus aureus. A signal pattern obtained with peptides with arginine, lysine, and tyrosine in the P1 position characterized the bacterial protease activities in these samples. The kinetic parameters for the three best substrates for the P. aeruginosa sample were calculated. Further information about substrate specificity was gained by the selective use of protease inhibitors. The results presented show that peptide-AMC substrates provide a simple and sensitive tool to characterize protease activity in microbiological samples and that they have the potential to identify and distinguish different bacterial species.     

Kinetically controlled synthesis of dipeptides using ficin as biocatalyst.

The application of the sulfhydryl protease ficin as biocatalyst is proposed as a novel method for enzyme-catalyzed synthesis of dipeptides. The negligible peptidase but considerable esterase activity at alkaline pH facilitated the kinetically controlled formation of peptide bonds by coupling the ester substrates Z-Ala-OMe and Z-Gly-OMe with L-alanine, D-alanine, L-glutamine, D-glutamine and L-Cys(acetamidomethyl) respectively. The reaction is accomplished without the occurrence of secondary peptide hydrolysis. Under optimum reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 4.8% ethanol, 40 degrees C), the peptide yields ranged from 5 to 91%, depending on the structure of the amino and/or carboxyl component. No racemization was observed in the enzymatic reaction. Application of short-chain peptides has been advocated recently in clinical nutrition. Ficin-catalyzed peptide synthesis might be an attractive biotechnological approach for the synthesis of suitable dipeptides in this respect.     

Native chemical ligation in dimethylformamide can be performed chemoselectively without racemization

Native chemical ligation of unprotected peptides in organic solvents has been previously reported as a fast, efficient, and suitable method for coupling of hydrophobic peptides. However, it has not been determined whether the reaction can be carried out without possible side reactions or racemization. Here, we present a study on the chemoselectivity of this method by model reactions designed to test the reactivity of Arg and Lys side chains as well as that of α‐amino groups. A possible racemization of the C‐terminal amino acid of the N‐terminal peptide was also investigated. The results show that ligation in organic solvents can be conducted chemoselectively without side reactions with other nucleophilic groups. Furthermore, no racemization of the C‐terminal amino acid was observed if both educts were added simultaneously. Thus, native chemical ligation can be performed either in aqueous buffer systems or in organic solvents paving the way for the synthesis of larger hydrophobic peptides and/or membrane proteins. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanism of Cis-Inhibition of PolyQ Fibrillation by PolyP: PPII Oligomers and the Hydrophobic Effect

PolyQ peptides teeter between polyproline II (PPII) and β-sheet conformations. In tandem polyQ-polyP peptides, the polyP segment tips the balance toward PPII, increasing the threshold number of Gln residues needed for fibrillation. To investigate the mechanism of cis-inhibition by flanking polyP segments on polyQ fibrillation, we examined short polyQ, polyP, and tandem polyQ-polyP peptides. These polyQ peptides have only three glutamines and cannot form β-sheet fibrils. We demonstrate that polyQ-polyP peptides form small, soluble oligomers at high concentrations (as shown by size exclusion chromatography and diffusion coefficient measurements) with PPII structure (as shown by circular dichroism spectroscopy and ³J_HN-Cα constants of Gln residues from constant time correlation spectroscopy NMR). Nuclear Overhauser effect spectroscopy and molecular modeling suggest that self-association of these peptides occurs as a result of both hydrophobic and steric effects. Pro side chains present three methylenes to solvent, favoring self-association of polyP through the hydrophobic effect. Gln side chains, with two methylene groups, can adopt a conformation similar to that of Pro side chains, also permitting self-association through the hydrophobic effect. Furthermore, steric clashes between Gln and Pro side chains to the C-terminal side of the polyQ segment favor adoption of the PPII-like structure in the polyQ segment. The conformational adaptability of the polyQ segment permits the cis-inhibitory effect of polyP segments on fibrillation by the polyQ segments in proteins such as huntingtin.     

Protease-catalyzed dipeptide synthesis from N-protected amino acid carbamoylmethyl esters and free amino acids in frozen aqueous solutions

The kinetically controlled synthesis of N-benzyloxycarbonyl (Z)-dipeptides was investigated by the use of free amino acids as nucleophiles and a cysteine protease papain as catalyst. The coupling efficiency was significantly improved by the combined use of the carbamoylmethyl (Cam) ester of a Z-amino acid as acyl donor and frozen aqueous solution (ice, −16 or −24 °C) as reaction medium. The yield of peptide synthesis became high when both P₁- and P^′₁-positions were occupied by small non-polar amino acids (Z-Gly-Gly-OH, 76%; Z-Gly-Ala-OH, 75%; Z-Ala-Ala-OH, 72%). Similar results were observed by the use of ficin as catalyst instead of papain. Furthermore, this strategy was applied to the papain-catalyzed incorporation of a d-configured amino acid such as d-alanine into the resulting peptides. Although the coupling in aqueous solution (30 °C) afforded the desired Z-dipeptides in low yields, the freezing of reaction medium reduced significantly unfavorable hydrolysis of the acyl donors, resulting in improvement of the coupling efficiency (Z-Gly-d-Ala-OH, 80%; Z-Ala-d-Ala-OH, 45%; Z-d-Ala-Ala-OH, 22%).     

Nanomechanical In Situ Monitoring of Proteolysis of Peptide by Cathepsin B

Characterization and control of proteolysis of peptides by specific cellular protease is a priori requisite for effective drug discovery. Here, we report the nanomechanical, in situ monitoring of proteolysis of peptide chain attributed to protease (Cathepsin B) by using a resonant nanomechanical microcantilever immersed in a liquid. Specifically, the detection is based on measurement of resonant frequency shift arising from proteolysis of peptides (leading to decrease of cantilever''s overall mass, and consequently, increases in the resonance). It is shown that resonant microcantilever enables the quantification of proteolysis efficacy with respect to protease concentration. Remarkably, the nanomechanical, in situ monitoring of proteolysis allows us to gain insight into the kinetics of proteolysis of peptides, which is well depicted by Langmuir kinetic model. This implies that nanomechanical biosensor enables the characterization of specific cellular protease such as its kinetics.     

Enhanced production of the nonribosomal peptide antibiotic valinomycin in Escherichia coli through small-scale high cell density fed-batch cultivation

Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L^?1 by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L^?1. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD₆₀₀ >50 and a valinomycin titer of appr. 10 mg L^?1. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli.     

Simultaneous use of 1-hydroxybenzotriazole and copper(II) chloride as additives for racemization-free and efficient peptide synthesis by the carbodiimide method.

In the carbodiimide mediated coupling of Z-Gly-L-Val-OH with H-L-Val-OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leu-enkephalin. In the 4 + 1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)-oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.     

In vitro selection and characterization of DNA aptamers recognizing chloramphenicol

Chloramphenicol (Cam), although an effective antibiotic, has lost favour due to some fatal side effects. Thus there is an urgent need for rapid and sensitive methods to detect residues in food, feed and environment. We engineered DNA aptamers that recognize Cam as their target, by conducting in vitro selections. Aptamers are nucleic acid recognition elements that are highly specific and sensitive towards their targets and can be synthetically produced in an animal-friendly manner, making them ethical innovative alternatives to antibodies. None of the isolated aptamers in this study shared sequence homology or structural similarities with each other, indicating that specific Cam recognition could be achieved by various DNA sequences under the selection conditions used. Analyzing the binding affinities of the sequences, demonstrated that dissociation constants (K_d) in the extremely low micromolar range, which were lower than those previously reported for Cam-specific RNA aptamers, were achieved. The two best aptamers had G rich (>35%) nucleotide regions, an attribute distinguishing them from the rest and apparently responsible for their high selectivity and affinity (K_d ∼ 0.8 and 1 μM respectively). These aptamers open up possibilities to allow easy detection of Cam via aptamer-based biosensors.     

Biocatalytic Knoevenagel reaction using alkaline protease from Bacillus licheniformis

Abstract

BLAP (alkaline protease from Bacillus licheniformis) was used as a biocatalyst in the Knoevenagel condensations of aromatic, hetero-aromatic and α;β-unsaturated aldehydes with less reactive acetylacetone or ethyl acetoacetate. The reactions were performed in organic solvent in the presence of water. The functionalized trisubstituted alkenes and α,β,γ,δ-unsaturated carbonyl compounds could be obtained in moderate to good yields with E/Z selectivities up to >99:1. This biocatalytic reaction provided an alternative pathway for Knoevenagel condensation, which also demonstrates a novel case of unnatural activity of existing enzymes.     

Effect of ferrous chloride on biogas production and enzymatic activities during anaerobic fermentation of cow dung and <Emphasis Type="Italic">Phragmites</Emphasis> straw

The effect of ferrous (added as FeCl₂) on the anaerobic co-digestion of Phragmites straw and cow dung was studied by investigating the biogas properties, pH values, organic matter degradation (COD) and enzyme activities (cellulase, protease and dehydrogenase) at different stages of mesophilic fermentation. The results showed that Fe²⁺ addition increased the cumulative biogas yields by 18.1 % by extending the peak period with high daily biogas yields. Meanwhile, the methane (CH₄) contents in the Fe²⁺ added groups were generally higher than the control group before the 15th day. The pH values were not significantly impacted by Fe²⁺ concentrations during the fermentation process. The COD concentrations, cellulase, protease and dehydrogenase activities varied with the added Fe²⁺ concentrations and the stages of the fermentation process. At the beginning stage of fermentation (4th day), Fe²⁺ addition increased the biogas production by improving the cellulase and dehydrogenase activities which caused a decline in COD. At the peak stage of fermentation (8th day), Fe²⁺ addition enhanced the cellulase and protease activities, and resulted in lower COD contents than the control group. When the biogas yields decreased again (13th day), the COD contents varied similar with the protease and dehydrogenase activities, whilst cellulase activities were not sensitive to Fe²⁺ concentrations. At the end of fermentation (26th day), Fe²⁺ addition decreased the cellulase activities, led to lower COD contents and finally resulted the lower biogas yields than the control group. Taking the whole fermentation process into account, the promoting effect of Fe²⁺ addition on biogas yields was mainly attributed to the extension of the gas production peak stage and the improvement of cellulase activities.