Extraction,purification and characterization of a novel cysteine protease from the latex of plant <Emphasis Type="Italic">Vallaris solanacea</Emphasis>

Plant proteases with excellent catalytical properties perform many functions in biological systems. A novel plant protease Vallaris solanacea, was identified. Its proteolytic activity was screened using the substrate casein. This protein activity was specifically inhibited by p-chloromercuribenzoate, which showed that it is a cysteine protease. Preliminary investigations such as pH effect and temperature dependence on the caseinolytic activity of crude protease were done. Stability towards temperature and pH were also evaluated. The activity curves drawn in relation to pH, temperature and stability suggested the presence of one protease in the latex of Vallaris solanacea. In the present study, separation and purification of the latex cysteine protease solanain from Vallaris solanacea to a state of near homogeneity was also done using ion exchange and size exclusion chromatography. SDS PAGE was used to determine molecular weight of the solanain (28–29 kDa). The molecular weight was confirmed as 28.9 kDa using MALDI-TOF. Purified protease was named solanain and it was further characterized. An internal tryptic fragment was identified by MALDI-TOF, and this peptide showed a homology (66% sequence similarity) with target sequence of cysteine endopeptidase from Ricinus communis.     

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, <Emphasis Type="Italic">Antheraea assamensis</Emphasis>

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> signal peptidase recognizes and cleaves archaeal signal sequence

Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction. Tk1884 was purified from the extracellular medium, and its molecular mass determined by electrospray ionization mass spectrometry indicated the cleavage of a few amino acids. The N-terminal amino acid sequence of the purified Tk1884 was determined, which revealed that the signal peptide was cleaved between Ala26 and Ala27 by E. coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase.     

<Emphasis Type="Italic">A2144G</Emphasis> Is the Main Mutation in the <Emphasis Type="Italic">23S</Emphasis> rRNA Gene of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Associated with Clarithromycin Resistance

To detect point mutations A2115C, A2143G/C, and A2144G in the 23S rRNA gene of Helicobacter pylori associated with resistance of the microorganism to clarithromycin, a new powerful way of analysis was used. This method involved the reaction of minisequencing followed by MALDI-TOF mass spectrometry of reaction products. In ten analyzed clarithromycin-resistant clinical isolates of H. pylori obtained in Russia, the resistance was found to be mediated only by mutation A2144G in the 23S rRNA gene.     

Identification of calmodulin binding proteins in the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Calmodulin (CaM) is a primary Ca²⁺ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.     

Detection of Fluoroquinolone Resistance Single-Nucleotide Polymorphisms in <Emphasis Type="Italic">Neisseria gonorrhoeae gyr</Emphasis>A and <Emphasis Type="Italic">par</Emphasis>C Using MALDI-TOF Mass Spectrometry

Many known mechanisms of drug resistance in microorganisms have genetic markers, which are specific genomic changes, mostly single-nucleotide polymorphisms (SNPs). A search for new methods of detecting SNPs is necessary for more efficient identification of resistant strains. A new method was proposed for SNP detection on the basis of minisequencing and/or sequencing with subsequent matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. The method was tested with a set of Neisseria gonorrhoeae clinical isolates in experiments on detecting the gyrA and parC SNPs that are markers of fluoroquinolone resistance. The results fully coincided with data previously obtained by gold-standard methods (sequencing and gel electrophoresis of sequencing products). Sequencing of short DNA fragments with MALDI-TOF mass spectrometry is of special interest. The new method considerably improves the efficiency of identification and genotyping of microorganisms and determination of their drug resistance markers.     

Extracellular chitinases of mutant superproducing strain <Emphasis Type="Italic">Serratia marcescens</Emphasis> M-1

Four extracellular proteins with chitinase activity capable of binding chitin substrates have been revealed in the culture liquid of chitinase superproducing mutant strain M-1 of Serratia marcescens. Proteins were analyzed by SDS-PAGE and MALDI-TOF mass spectrometry. Based on the data obtained, the proteins were identified as typical chitinases of S. marcescens: ChiA, ChiB, ChiC, and CBP21.     

Molecular Characterisation of a Novel Isoform of Hepatic Antimicrobial Peptide,Hepcidin (<Emphasis Type="Italic">Le</Emphasis>-Hepc), from <Emphasis Type="Italic">Leiognathus equulus</Emphasis> and Analysis of Its Functional Properties In Silico

Hepcidin represents a family of cysteine-rich antimicrobial peptides that are mainly expressed in the liver of living organisms. In this study, we have identified and characterised a novel isoform of hepcidin from the common pony fish, Leiognathus equulus (Le-Hepc). A 261-bp fragment cDNA coding for 86 amino acids was obtained. Homologous analysis showed that Le-Hepc belongs to the hepcidin super family and shares sequence identity with other known fish pre-propeptide hepcidin sequences. The ORF encodes for a 24-amino acid (aa) signal peptide coupled to a 36-aa prodomain followed by a 26-aa mature peptide. The mature peptide region has a calculated molecular weight of 2.73 kDa, a net positive charge of +2 and a theoretical pI of 8.23. Phylogenetic analysis of Le-Hepc showed a strong relationship with other fish hepcidin sequences and clustered into HAMP2 group hepcidins. Secondary structural analysis indicated that Le-Hepc mature peptide contains two antiparallel β-sheets strengthened by four disulphide bonds formed by eight conserved cysteine residues. The physicochemical properties of the peptide and its structural parameters are in agreement with characteristic features of an antimicrobial peptide. This is the first report of an antimicrobial peptide from the common pony fish, L. equulus.     

Characterization of the complex involved in regulating V-ATPase activity of the vacuolar and endosomal membrane

Regulator of the H⁺-ATPase of the vacuolar and endosomal membranes (RAVE) is essential for the reversible assembly of H⁺-ATPase. RAVE primarily consists of three subunits: Rav1p, Rav2p and Skp1p. To characterize these subunits, in this study, four strains derived from Saccharomyces cerevisiae BY4742 were constructed with a FLAG tag on the Rav1p and Rav2p subunits. Then, the corresponding RAVE containing complex was isolated by affinity purification. Western blot and MALDI-TOF mass spectrometry analyses showed that the RAVE complex contains not only the known V₁-ATPase subunits (Vma1p and Vma2p) but also a newly found Leu1p that interacts with the RAVE subunit. Furthermore, we constructed rav1?/rav2?/vma2?/leu1-deficient recombinants by fusion PCR and homologous recombination and demonstrated that leu1 is indispensable in adjusting the microbial cell to adverse environments and that the function is similar to that of rav1/rav2 but significantly differs from that of vma2. Leu1p probably plays an important role in RAVE regulation of V-ATPase activity in conjunction with RAVE.     

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (<Emphasis Type="Italic">R</Emphasis>)-phenyl-1,2-ethanediol

Objectives

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee _p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis.

Results

The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2^A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2^A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h^?1 g^?1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee _p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2^A250I-expressing E. coli/Aueh2 ^A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee _p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee _p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h.

Conclusions

Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2^A250I is an effective method for preparing (R)-PED with high ee _p and yield.

     

Cloning and functional characterization of the <Emphasis Type="Italic">SmNCED3</Emphasis> in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

As one of the most important phytohormones, the abscisic acid (ABA) is often used to breed stress-tolerant crop lines with both higher yields and active ingredient contents. In higher plants, the 9-cis-epoxycarotenoid dioxygenase (NCED) has been found to be a regulatory enzyme involved in ABA biosynthesis. In research, the novel gene SmNCED3 was isolated from S. miltiorrhiza. The open reading frame of SmNCED3 was 1725-bp, and it was encoding 574 amino acids with a calculated molecular mass of 63,822 kDa, which was verified by the expression of SmNCED3 in E. coli. The deduced SmNCED3 amino acid sequence had high sequence homology with NCED sequences from other plants and contained a putative chloroplast transit targeting signal peptide at its N terminus. Phylogenetic analysis demonstrated that SmNCED3 had a closer affinity to NCED3 in Arabidopsis thaliana (AtNCED3). The 1732-bp 5′ flanking sequence of SmNCED3 was also cloned. It contained several phytohormone response elements, biotic or abiotic stress-related elements, and plant development-related elements. Real-time PCR revealed that SmNCED3 was highly expressed in leaves, and was strongly induced by exogenous ABA. A subcellular localization experiment indicated that SmNCED3 was located in chloroplast stroma, chloroplast membranes, and thylakoid membranes. The overexpression of SmNCED3 promoted ABA accumulation. These results indicated that SmNCED3 might be a rate-limiting gene regulating ABA biosynthesis, and improving abiotic stresses tolerance and active ingredient contents in plants.     

Production of bacteriocin by <Emphasis Type="Italic">Virgibacillus salexigens</Emphasis> isolated from “terasi”: a traditionally fermented shrimp paste in Indonesia

A natural antibacterial-substance-producing gram-positive bacterium was isolated from terasi shrimp paste, a popular fermented product in Indonesia. This strain, a spore-forming and strictly aerobic bacterium, was identified as Virgibacillus salexigens by 16S rRNA gene sequence analysis. The antibacterial substance purified from the precipitated product in the culture supernatant of the strain using ammonium sulfate showed a broad inhibition spectrum against gram-positive bacteria, including a typical foodborne bacterium, namely, Listeria monocytogenes. The antibacterial activity of the substance was inactivated by treatments with various proteolytic enzymes. It was stable after heating or pH treatment, and approximately 60 % of the initial activity remained even after heating at 121 °C for 15 min. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis indicated that its monoisotopic mass weight was 5318.4 Da (M+H)⁺. On the basis of the results obtained by the automated Edman degradation technique and MALDI-TOF MS analysis, the substance can be classified as a member of Class IId bacteriocins, but it could not be identified as any of the previously purified substances except for the putative bacteriocin predicted from the draft genome sequence data of gram-positive bacteria such as Virgibacillus and Bacillus strains.     

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing <Emphasis Type="Italic">Leuconostoc pseudomesenteroides</Emphasis> 607 Isolated from Persimmon

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C₁₈ cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05 Da and a partial N-terminal amino acid sequence of NH₂-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97 Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.     

Isolation Purification and Characterization of Antimicrobial Peptides from <Emphasis Type="Italic">Cuminum cyminum</Emphasis> L. Seeds

In this work, antimicrobial peptides from Cuminum cyminum L. seeds were isolated and purified for the first time by 50% ethanol extraction, C18 reverse phase column chromatography and ion exchange chromatography for separation different peptides fraction. Then isolated fractions were characterized by Gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography and the peptides components and molecular weights were determined by liquid chromatography and mass spectrometry. The extracts were tested against some strains of bacteria (E. coli and Staphylococcus aureus) and one strain of fungi (Candida albicans) using well diffusion and broth dilution assays. The extracts from C. cyminum L. seeds demonstrated a high degree of activity (some antibacterial effect) against the bacteria strains and аntifungal activity against the Candida albicans. However, the study indicates that the crude peptide extracts from C. cyminum L. seeds have promising antimicrobial and antioxidant activities that can be harnessed as leads for potential bioactive compounds.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Feruloyl esterase from <Emphasis Type="Italic">Alternaria tenuissima</Emphasis> that hydrolyses lignocellulosic material to release hydroxycinnamic acids

An extracellular feruloyl esterase from the culture filtrates of the isolated fungus Alternaria tenuissima was successfully purified to apparent homogeneity by anion-exchange and size-exclusion chromatography. Peptide fragments of purified enzyme (designated as AltFAE; molecular weight of 30.3 kDa determined by SDS-PAGE) were identified by mass spectrometry using a NanoLC-ESI-MS/MS system. Michaelis-Menten constants (K_M) and catalytic efficiencies (k_cat/K_M) were determined for typical substrates of feruloyl esterase, and the lowest K_M of 50.6 μM (i.e., the highest affinity) and the highest k_cat/K_M (3.1 × 10⁵ s^—1 M^–1) were observed for methyl ^p-coumarate and methyl ferulate, respectively. Not least, AltFAE catalyzed conversion of lignocellulosic material (e.g. wood meal) to release hydroxycinnamic products, i.e. ferulic- and ^p-coumaric acids.     

Recombinant expression of a GH12 β-glucanase carrying its own signal peptide from <Emphasis Type="Italic">Stachybotrys atra</Emphasis> in yeast and filamentous fungi

The β-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 β-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete.     

Detailed characterization of the <Emphasis Type="Italic">O</Emphasis>-linked glycosylation of the neuropilin-1 c/MAM-domain

Neuropilins are involved in angiogenesis and neuronal development. The membrane proximal domain of neuropilin-1, called c or MAM domain based on its sequence conservation, has been implicated in neuropilin oligomerization required for its function. The c/MAM domain of human neuropilin-1 has been recombinantly expressed to allow for investigation of its propensity to engage in molecular interactions with other protein or carbohydrate components on a cell surface. We found that the c/MAM domain was heavily O-glycosylated with up to 24 monosaccharide units in the form of disialylated core 1 and core 2 O-glycans. Attachment sites were identified on the chymotryptic c/MAM peptide ETGATEKPTVIDSTIQSEFPTY by electron-transfer dissociation mass spectrometry (ETD-MS/MS). For highly glycosylated species consisting of carbohydrate to about 50 %, useful results could only be obtained upon partial desialylation. ETD-MS/MS revealed a hierarchical order of the initial O-GalNAc addition to the four different glycosylation sites. These findings enable future functional studies about the contribution of the described glycosylations in neuropilin-1 oligomerization and the binding to partner proteins as VEGF or galectin-1.As a spin-off result the sialidase from Clostridium perfringens turned out to discriminate between galactose- and N-acetylgalactosamine-linked sialic acid.