Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease

The presence, microenvironment, and proximity of an essential Trp with the essential His and Cys residues in the active site of an alkaline protease have been demonstrated for the first time using chemical modification, chemo-affinity labeling, and fluorescence spectroscopy. Kinetic analysis of the N-bromosuccinimide- (NBS) or p-hydroxymercuribenzoate- (PHMB) modified enzyme from Conidiobolus sp. revealed that a single Trp and Cys are essential for activity in addition to the Asp, His, and Ser residues of the catalytic triad. Full protection by casein against inactivation of the enzyme by NBS and quenching of Trp fluorescence upon binding of the enzyme with NBS, substrate (sAAPF-pNA), or inhibitor (SSI) confirmed participation of the Trp residue at the substrate/inhibitor binding site of the alkaline protease. Comparison of the K(sv) values for the charged quenchers CsCI (1.66) and KI (7.0) suggested that the overall Trp microenvironment in the protease is electropositive. The proximity of Trp with His was demonstrated by the sigmoidal shape of the pH-dependent fluorometric titration curve with a pK(F) of 6.1. The vicinity of Trp with Cys was indicated by resonance energy transfer between the intrinsic fluorophore (Trp) and 5-iodoacetamide-fluorescein labeled Cys (extrinsic fluorophore). Our results on the proximity of Trp with essential His and Cys thus confirm the presence of Trp in the active site of the alkaline protease.     

Raman exfoliative cytology for prognosis prediction in oral cancers: A proof of concept study

Oral cancer is associated with high rates of recurrence, attributable to field cancerization. Early detection of advanced field changes that can potentially progress to carcinoma can facilitate timely intervention and can lead to improved prognosis. Previous in vivo studies have successfully detected advanced field effects in oral cancers. Raman exfoliative cytology has previously shown to differentiate normal, oral pre‐cancer and cancers. The present study explores Raman‐exfoliative‐cytology‐based detection of field effects. Exfoliated cells were collected from tumor (n = 16) and contralateral‐normal appearing mucosa (n = 16) of oral cancer patients, and healthy tobacco habitués (n = 20). After spectral acquisition, specimens were Pap‐stained for cytological evaluation. Data analysis, by Principal Component Analysis and Principal Component‐Linear Discriminant Analysis, indicate several spectral‐misclassifications between contralateral normal and tumor, which were investigated and correlated with spectral, cytological and clinical outcomes. A qualitative analysis by grouping patients with number of misclassifications with tumor (Group 1: 0, Group 2: 1 and Group 3: >1) was explored. Group 3 with highest misclassifications showed spectral and cytological similarity to tumor group — one patient was a case of early inoperable residual disease, despite clear margins on histopathology. Thus, these misclassifications could be indicative of cancer field changes, and can prospectively help to identify patients susceptible to recurrences .     

In ovo silver nanoparticle supplementation for improving the post-hatch immunity status of broiler chickens

Silver nanoparticles (AgNano) are known for their unique physical, chemical and biological properties, enabling cell penetration and anti-inflammatory response. In Experiment 1, the effect of an in ovo administration of AgNano (15 µg/egg; n = 360) at different incubation times (d 7 and d 18) on hatchability parameters was explored. In Experiment 2, post-hatch performance of broilers (42 d, n = 250) was studied after in ovo AgNano administration: Group T1 remained un-injected, Group T2 was the sham control and Groups T3, T4 and T5 were injected with 12.5, 25 and 50 µg AgNano, respectively, at 18 d of incubation. Chick weight, chick to egg weight ratio and hatchability as well average daily gain, average daily feed intake and feed conversion ratio were similar in all treatment groups. No variation was seen in the weight of thymus; however, the bursa and spleen weight was increased (p < 0.05) in Groups T4 and T5 in comparison to Group T1. The in vivo immune response to phytohaemagglutinin-P was increased in Group T3 in comparison to Groups T1 and T2 (p < 0.05), while the response to sheep red blood cells was increased in all AgNano-treated groups in comparison with Group T1 (p < 0.01). The expression of toll-like receptors 2 and 4 genes was up-regulated in AgNano groups in comparison with Groups T1 and T2 (p < 0.01). In summary, an in ovo supplementation of AgNano carried out at d 18 of incubation is effective and modulates the post-hatch immune response without affecting the hatchability, growth and other performance parameters in broilers.     

Single-step purification of a small non-mAb biologic by peptide-ELP-based affinity precipitation

Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a K_D of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.     

An improved method for the determination of NADH oxidase in the presence of NADH peroxidase in lactic acid bacteria

The complexity of the coupled NADH oxidase-NADH peroxidase enzyme system in lactic acid bacteria makes it difficult to simultaneously determine the individual levels of both these enzymes spectrophotometrically. This study describes an improved assay to accurately determine low concentrations of NADH oxidase from enzyme suspensions containing NADH oxidase and NADH peroxidase. For the standardisation of the assay, pure NADH oxidase and NADH peroxidase were mixed in various proportions and the percentage recovery was estimated by both the currently available assay as well as by the improved assay reported in this study. The recovery of NADH oxidase using the currently available assay ranged from as low as -200% to as high as +102% as against 90-102% in the improved assay. The recovery percentage of NADH peroxidase ranged from 91% to 112% in both assays. The slopes of NADH oxidation by cell-free extracts of six lactic acid bacteria were also measured by both assays for the estimation of NADH oxidase and NADH peroxidase levels. The improved assay can further distinguish between NADH-H(2)O oxidase and NADH-H(2)O(2) oxidase and was successfully applied to identify the type of NADH oxidase in the lactic acid bacteria tested.     

Immobilization of alkaline protease from Conidiobolus macrosporus for reuse and improved thermal stability

Alkaline protease from Conidiobolus macrosporus was immobilized on polyamide using glutaraldehyde as a bifunctional agent. The immobilized enzyme was optimally active at a higher temperature of 50°C than the free enzyme (40°C ) and showed a ten-fold increased thermostability at 60°C compared to that of the free enzyme. The efficiency of immobilization was 58% under the optimal conditions of pH and temperature. There was a 14-fold decrease in the K _m of immobilized enzyme compared to the free enzyme. The immobilized enzyme was fully active even after twenty-two cycles of repeated use. It retained 80% activity at 50°C in presence of 8 M urea exhibiting its stability to the denaturant and was compatible with several commercial detergents.     

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.     

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

In this work, two phage biopanning strategies were developed to identify affinity peptides for a single Fab and multiple kappa Fabs. For the biopanning rounds, protein L beads were employed to bind Fab targets in a fixed orientation, and NHS functionalized magnetic beads were used to facilitate evaluation of low pH elution conditions. The resulting peptide sequences were synthesized and the binding to different Fabs was evaluated using fluorescence polarization. The first biopanning approach yielded a peptide with similar affinities for two forms of the Fab (recombinantly expressed and post papain-digestion) as well as the intact antibody. While moderate affinity was observed toward a murine variant of the Fab with the same complementarity determining regions (CDR) region but different framework, minimal binding occurred to a Fab with high sequence homology but containing different CDR loops. The second biopanning strategy yielded a peptide with affinity for all three kappa Fabs indicating that it may be a good lead for the development of more general affinity reagents for recombinant kappa Fabs. Finally, an affinity peptide column was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The results presented in this article demonstrate that different peptide-based phage biopanning strategies can be effectively employed to identify affinity peptide leads for specific Fab and more general kappa Fab purifications.