β-Glucosidase catalyzed synthesis of octyl-β-D-glucopyranoside using whole cells of Pichia etchellsii in micro aqueous media

Octyl-β-D-glucopyranoside was synthesized by transglucosylation between p-nitrophenyl β-D-glucopyranoside (pNPG) and octanol as an acceptor using whole cells of thermo tolerant yeast Pichia etchellsii displaying cell wall bound β-glucosidase. Effect of several parameters such as glucosyl donor concentration, enzyme units and initial water activity was studied to optimize product yield. An initial water activity interval of 0.33-0.64 was favorable and increase in total enzyme units had marginal effect on conversion yield. An empirical model was developed to describe the relationship between various parameters and octyl glucoside yield. These factors were combined in a batch replacement strategy whereby octyl-β-D-glucopyranoside was synthesized in 4h to a concentration of 30 mM (9.25 mg/ml) with a conversion yield of nearly 70% with pNPG as a glucosyl donor. Quantitative analysis was done by a highly reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method and detection was achieved using refractive index detector. The structure of the product was confirmed by 13C and 1H NMR spectroscopy. Additional products like octyl diglucoside were also formed, the structure of which was confirmed by mass spectrometry.     

The reducing component BoxA of benzoyl-coenzyme A epoxidase from Azoarcus evansii is a [4Fe-4S] protein

BoxA is the reductase component of the benzoyl-coenzyme A (CoA) oxidizing epoxidase enzyme system BoxAB. The enzyme catalyzes the key step of an hitherto unknown aerobic, CoA-dependent pathway of benzoate metabolism, which is the epoxidation of benzoyl-CoA to the non-aromatic 2,3-epoxybenzoyl-CoA. The function of BoxA is the transfer of two electrons from NADPH to the epoxidase component BoxB. We could show recently that BoxB is a diiron enzyme, whereas here we demonstrate that BoxA harbors an FAD and two [4Fe-4S] clusters per protein monomer. The characterization of BoxA was hampered by severe oxygen sensitivity; the cubane [4Fe-4S] clusters degrade already with traces of oxygen. Interestingly, the adventitiously formed [3Fe-4S] centers could be reconstituted in vitro by adding Fe(II) and sulfide to retrieve the native cubane centers. BoxA is the first example of a reductase of this type that has an FAD and two bacterial ferredoxin-type [4Fe-4S] clusters. In other cases within the catalytically versatile family of diiron enzymes, the related reductases have plant-type ferredoxin or Rieske-type [2Fe-2S] centers only.     

In vitro complex formation of human PYRIN domain-only protein 3 prevented by self-oligomerization of ASC PYD domain

The formation of inflammasome complexes contributes inactivation of inflammatory caspases viz caspase 1, which is generally considered essential for the innate response. Three proteins constituted this inflammasome complex, such as Nod-like receptors (NLRP or AIM2), ASC possessing caspase-recruiting domain, and caspase-1. The ASC proteins comprise two domains, the N-terminal PYD domain responsible for the interaction of various proteins, including PYD only protein 3 (POP3), and the CARD domain for association with other proteins. The PYRIN Domain-Only Protein POP3 negatively regulates responses to DNA virus infection by preventing the ALR inflammasome formation. POP3 directly interacts with ASC, therefore inhibiting ASC recruitment to AIM2-like receptors (ALRs). In the current study, we designed various constructs of the PYRIN Domain-Only Protein 3 (POP3) and ASC PYD domain to find the best-overexpressed construct for biochemical characterization as well as our complex studies. We cloned, purified, and characterized the PYD domain of pyrin only protein 3 and ASC PYD domain under physiological conditions. Our in vitro study clearly shows that the ASC PYD domain of corresponding amino acid 1–96 aa with ease self-oligomerization in physiological buffer conditions, and complex formation of POP3 PYD (1–83 aa) was inhibited by ASC PYD domain. Besides, we purified the PYD of POP3 protein in low and high salt conditions and different pH values for their biochemical characterization. Our results showed that POP3 formed a dimer under normal physiological conditions and was stable under normal buffer conditions; however, the purification in extremely low pH (pH5.0) conditions shows unstable behavior, the high salt conditions (500 mM NaCl) influence the protein aggregation. SDS PAGE arbitrated the homogeneity of the PYD domain of pyrin only protein 3 and ASC PYD domain of corresponding amino acids 1–83 and 1–96, respectively. Furthermore, our native PAGE shows the PYD domain of pyrin; only protein 3 did not form a complex with ASC PYD domain because of oligomerization mediated by the PYD domain.     

Putrescine Importer PlaP Contributes to Swarming Motility and Urothelial Cell Invasion in Proteus mirabilis

Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437–446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.     

Isolation and characterization of a proteinaceous antifungal compound from Lactobacillus plantarum YML007 and its application as a food preservative

Korean kimchi is known for its myriad of lactic acid bacteria (LAB) with diverse bioactive compounds. This study was undertaken to isolate an efficient antifungal LAB strain among the isolated kimchi LABs. One thousand and four hundred LABs isolated from different kimchi samples were initially screened against Aspergillus niger. The strain exhibiting the highest antifungal activity was identified as Lactobacillus plantarum YML007 by 16S rRNA sequencing and biochemical assays using API 50 CHL kit. Lact. plantarum YML007 was further screened against Aspergillus oryzae, Aspergillus flavus, Fusarium oxysporum and other pathogenic bacteria. The morphological changes during the inhibition were assessed by scanning electron microscopy. Preliminary studies on the antifungal compound demonstrated its proteinaceous nature with a molecular weight of 1256·617 Da, analysed by matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF). The biopreservative activity of Lact. plantarum YML007 was evaluated using dried soybeans. Spores of A. niger were observed in the negative control after 15 days of incubation. However, fungal growth was not observed in the soybeans treated with fivefold concentrated cell‐free supernatant of Lact. plantarum YML007. The broad activity of Lact. plantarum YML007 against various food spoilage moulds and bacteria suggests its scope as a food preservative.

Significance and Impact of the Study

After screening 1400 kimchi bacterial isolates, strain Lactobacillus plantarum YML007 was selected with strong antifungal activity against various foodborne pathogens. From the preliminary studies, it was found that the bioactive compound is a low molecular weight novel protein of 1256·617 Da. Biopreservative potential of Lact. plantarum YML007 was demonstrated on soybean grains, and the results point out YML007 as a potent biopreservative having broad antimicrobial activity against various foodborne pathogens.     

Structure and mechanism of the diiron benzoyl-coenzyme A epoxidase BoxB

The coenzyme A (CoA)-dependent aerobic benzoate metabolic pathway uses an unprecedented chemical strategy to overcome the high aromatic resonance energy by forming the non-aromatic 2,3-epoxybenzoyl-CoA. The crucial dearomatizing reaction is catalyzed by three enzymes, BoxABC, where BoxA is an NADPH-dependent reductase, BoxB is a benzoyl-CoA 2,3-epoxidase, and BoxC is an epoxide ring hydrolase. We characterized the key enzyme BoxB from Azoarcus evansii by structural and Mössbauer spectroscopic methods as a new member of class I diiron enzymes. Several family members were structurally studied with respect to the diiron center architecture, but no structure of an intact diiron enzyme with its natural substrate has been reported. X-ray structures between 1.9 and 2.5 Å resolution were determined for BoxB in the diferric state and with bound substrate benzoyl-CoA in the reduced state. The substrate-bound reduced state is distinguished from the diferric state by increased iron-ligand distances and the absence of directly bridging groups between them. The position of benzoyl-CoA inside a 20 Å long channel and the position of the phenyl ring relative to the diiron center are accurately defined. The C2 and C3 atoms of the phenyl ring are closer to one of the irons. Therefore, one oxygen of activated O₂ must be ligated predominantly to this proximate iron to be in a geometrically suitable position to attack the phenyl ring. Consistent with the observed iron/phenyl geometry, BoxB stereoselectively should form the 2S,3R-epoxide. We postulate a reaction cycle that allows a charge delocalization because of the phenyl ring and the electron-withdrawing CoA thioester.     

Non-chromatographic strategies for protein refolding

Overexpression of recombinant proteins in bacterial systems (such as E. coli) often leads to formation of inactive and insoluble ' inclusion bodies' . Protein refolding refers to folding back the proteins after solubilizing/unfolding the misfolded proteins of the inclusion bodies. Protein aggregation, a concentration dependent phenomenon, competes with refolding pathway. The refolding strategies largely aim at reducing aggregation and/or promoting correct folding. This review focuses on non-chromatographic strategies for refolding like dilution, precipitation, three phase partitioning and macro-(affinity ligand) facilitated three phase partitioning. The nanomaterials which disperse well in aqueous buffers are also discussed in the context of facilitating protein refolding. Apart from general results with these methods, the review also covers the use of non-chromatographic methods in protein refolding in the patented literature beyond 2000. The patented literature generally describes use of cocktail of additives which results in increase in refolding yield. Such additives include low concentration of chaotropic agents, redox systems, ions like SO4(2-) and Cl-, amines, carboxylic acids and surfactants. Some novel approaches like use of a "pressure window" or ionic liquids for refolding and immobilized diselenide compounds for ensuring correct -S-S- bonds pairing have also been discussed in various patents. In most of the patented literature, focus naturally has been on refolding in case of pharmaceutical proteins.     

Bacterial mixture from greenhouse soil as a biocontrol agent against root-knot nematode, Meloidogyne incognita, on oriental melon

The biological control efficacy of a greenhouse soil bacterial mixture of Lactobacillus farraginis, Bacillus cereus, and Bacillus thuringiensis strains with antinematode activity was evaluated against the root-knot nematode Meloidogyne incognita. Two control groups planted in soil drenched with sterile distilled water or treated with the broadspectrum carbamate pesticide carbofuran were used for comparison. The results suggest that the bacterial mixture is effective as a biocontrol agent against the root-knot nematode.     

Quantitative proteomic analysis by iTRAQ<Superscript>®</Superscript> for the identification of candidate biomarkers in ovarian cancer serum

Background

Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.

Results

Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.

Conclusions

This study provides the first analysis of immunodepleted serum in combination with iTRAQ^® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.