Molecular cloning, overexpression, characterization, and In silico modelling analysis of a novel GDSL autotransporter-dependent outer membrane lipase (OML) of Pseudomonas guariconensis.

The outer membrane lipase (oml) gene, encoding a novel autotransporter-dependent lipase from Pseudomonas guariconensis, was cloned and sequenced. The oml gene has an open reading frame of 1866 bp. It encodes the 621 amino acid autotransporter-dependent GDSL lipase (OML), which has the highest sequence similarity (64.08 %) with the EstA of Pseudomonas aeruginosa (PDB:3kvn.1. A). OML was expressed and purified, which showed a purified band of approximately 70 kDa. The purified enzyme showed maximum activity at pH 9 and 40 °C. Substrate specificity studies and kinetic study by Lineweaver-Burk plot of purified OML showed Km of 1.27 mM and Vmax of 333.33 U/mL with p-nitrophenyl palmitate. The purified enzyme showed good stability in the presence of hexane, methanol, and ethanol, while the presence of the metal ion Mg2+ showed maximum lipase activity. Bioinformatics analysis supported the in vitro findings by predicting enzyme substrate specificity towards long-chain fatty acids and fatty acids with shorter chain lengths. The stability of the interaction of the protein-ligand complex (OML-ricinoleic acid) was confirmed using MDS and castor oil bioconversion using purified OML was confirmed using High-Performance Liquid Chromatography (HPLC).

Sortase E-mediated site-specific immobilization of green fluorescent protein and xylose dehydrogenase on gold nanoparticles.

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV-vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

The implication of Sortase E in the morphology and physiology of Corynebacterium glutamicum.

In this study, we analyzed the srtE gene from Corynebacterium glutamicum ATCC 13032, which codes for class E sortase, a transpeptidase involved in attaching surface proteins to the cell wall peptidoglycan. The surface proteins contain an N-terminal leader sequence and a C-terminal sorting signal which consist of a LAXTG motif, a transmembrane region, and a few positively charged amino acids. Sortase E deletion or its overexpression alters the attachment of the surface proteins to the cell wall peptidoglycan; however, the effects on morphology and bacterial physiology have not been studied. Thus, we constructed three C. glutamicum derivatives such as srtE deletion mutant, complemented and overexpressed strains to monitor the possible impact of the gene on cell growth, morphology, and physiological changes. Interestingly, deletion of the gene did not show any change in growth or morphology in C. glutamicum but showed a decrease in cell surface hydrophobicity and heat stress. However, the cells overexpressing the protein not only showed elongated cell morphology and a reduction in hydrophobicity when compared to wild-type and complemented strain, but also showed an increased sensitivity to heat. These results suggest that C. glutamicum sortase E deletion or overexpression causes sorting intermediates to accumulate, altering cellular morphology and physiology and adversely impacting the membrane integrity.

Accomplishment of probiotics in human health pertaining to immunoregulation and disease control.

It is a well-established fact that the microbiome harboring the human body plays a critical role in maintaining human health and can influence treatments against various ailments. Human microbiome-based research contemplates the possibility of selecting and administering specific commensal bacterial strains to modulate the gut microbiota to attain favorable outcomes to the therapies. Consumption of probiotics and probiotic-based dietary supplements as functional foods has been a promising treatment strategy against various diseases. Clinical studies demonstrate that probiotic administration alters gut microbiota composition and instigates immune modulation in the host. The benefits of probiotics are reported to be strain-specific and depend on the host's baseline immune competence. This review explores the role of probiotics in alleviating symptoms of allergy, cancer, cardio vascular (CV) diseases, diabetes mellitus (DM), bowel diseases (IBD and IBS), periodontal disease, diseases affecting liver and kidney, neuroinflammatory diseases, and viral infections. Also, it surveyed the broad spectrum bioactive compounds produced by probiotics and possible mechanisms that trigger the immune system.

Therapeutic and biotechnological applications of substrate specific microbial aminopeptidases.

Aminopeptidases (EC 3.4.11.) belongs to exoprotease family, which can catalyze the cleavage of peptide bond which connects the N-terminal amino acid to the penultimate residue in a protein. Aminopeptidases catalyze the process of removal of the N-terminal amino acids of target substrates by sequential cleavage of one amino acid residue at a time. Microbial aminopeptidase are of great acceptance as industrial enzymes with varying applications in food and pharma industry since these enzymes possess unique characteristics than aminopeptidases from other sources. This review describes the various applications of microbial aminopeptidases in different industrial sectors. These enzymes are widely used in food industry as a debittering agent as well as in the preparation of protein hydrolysates. In baking, brewing, and cheese making aminopeptidases are extensively used for removing the bitterness of peptides. The inhibitors of these enzymes are found great clinical applications against various diseases such as cancer, diabetes, and viral infections. Aminopeptidases are widely used for the synthesis of biopeptides and amino acids, and found to be efficient than chemical synthesis. These enzymes are capable of hydrolyzing organophosphate compounds, thus having biological as well as environmental significance.Key Points• Cleaves the amino-terminal amino acid residues from proteins and peptides.• Microbial aminopeptidase are of great acceptance as both therapeutic and industrial enzyme.• Review describes the potential applications of microbial aminopeptidases.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum.

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A-F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

Valorization of lignocellulosic residues from the olive oil industry by production of lignin, glucose and functional sugars.

Spent olive pomace from the two-phase extraction system of virgin olive oil and olive pomace oil, is a major agro-industrial residue. Present study aimed at the valorization of residual olive pomace and stones (seeds) by hydrothermal treatment and enzymatic hydrolysis of glucans. Both residues contain lignin (31.2% and 42.1%), glucans (13.8% and 15.3%) and xylans (18.9% and 20.3%). After hydrothermal pretreatment (130 °C, 30 min; severity factor log R0 = 2.99), 65% and 75% of hemicelluloses (65% of xylan) were hydrolysed into xylo-oligosaccharides in pomace and stones, respectively. Cellulose and lignin were not substantially affected. Three commercial enzyme preparations, Saczyme Yield, Ultimase BWL 40 and Celluclast 1.5 L, were evaluated for saccharification of pomace or stones at three biomass loads (10, 20 and 30%, w/v). Saczyme and Ultimase were active with high solid loads (30%), reaching 80 and 90% of glucan conversion in pomace, and 40 and 55% in stones, respectively, after 5 h.

Molecular advances in microbial aminopeptidases.

Aminopeptidases are exopeptidases that catalyze the hydrolysis of amino acid residues from the N terminus of peptides and proteins. They are widely and diversely used for protein hydrolysis in industrial and research applications. They form a large enzyme family in microorganisms and most of the sequenced microbial genomes contain several aminopeptidase coding genes. Various approaches are being used to enhance the yield and desired properties of these enzymes to make it more suited for industrial applications. Novel aminopeptidases are being developed by site directed mutagenesis and recombinant DNA technology with improved substrate specificity and stability. This review focuses on its classification and recent advancements in the molecular studies pertaining to this enzyme.

Antifungal, Anticancer and Aminopeptidase Inhibitory Potential of a Phenazine Compound Produced by Lactococcus BSN307.

A bioactive compound was purified from the culture medium of a new strain of Lactococcus BSN307 by solvent extraction followed by chromatographic techniques. This bioactive compound was identified to belong to phenazine class of compounds by MS, NMR and FTIR. The phenazine compound showed antifungal activity against Aspergillus niger, Penicillium chrysogenum as well as Fusarium oxysporum by disc diffusion assay in addition to antioxidant potential as demonstrated by DPPH scavenging assay. The compound demonstrated selective cytotoxicity against cancer cell lines HeLa and MCF-7 where IC50 was achieved with 20 and 24 µg/mL respectively. At the same time no cytotoxicity was occurred in normal H9c2 cells. The bioactive found to be inhibitory to both leucine and proline aminopeptidases and thus revealed its potential as metalloenzyme inhibitor. This study, for the first time reports the production of phenazine class of compounds by lactic acid bacteria.

Lactococcus garvieae subsp. bovis subsp. nov., lactic acid bacteria isolated from wild gaur (Bos gaurus) dung, and description of Lactococcus garvieae subsp. garvieae subsp. nov.

A taxonomic investigation was performed on a Gram-stain-positive coccus, designated strain BSN307T, isolated from gaur (Indian bison, Bos gaurus) dung based on phenotypic and molecular approaches. Based on the biochemical tests, cellular morphology and 16S rRNA gene sequence similarity, this strain was found to be a member of the genus Lactococcus and closely related to Lactococcus garvieae ATCC 49156T (99.6 % 16S rRNA gene sequence similarity) and L.actococcus formosensis 516T (99.0 %). However, DNA-DNA hybridization studies showed that the level of relatedness between strain BSN307T and L. garvieae ATCC 49156T was 75.8 %, suggesting that it represented a novel subspecies of L. garvieae. The inability to grow in brain heart infusion (BHI) medium at pH 9.6, in tryptic soy agar (TSA) with 4 % (w/v) NaCl and at 42 °C (MRS agar) clearly differentiated BSN307T from L. garvieae ATCC 49156T. Rep-PCR fingerprint patterns, substantial differences in summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C19 : 0 cyclo ω8c and C16 : 0 also differentiated strain BSN307T from the reference strain of L. garvieae. Moreover, analysis of the housekeeping genes pheS and rpoA revealed sequence similarities that were at the limit for species differentiation (92.2 and 97.8 %, respectively). Combined genotypic and phenotypic data indicate that strain BSN307T represents a subspecies of L. garvieae for which the name Lactococcus garvieae subsp. bovis subsp. nov. is proposed. The type strain is BSN307T (=DSM 100577 T=MCC 2824T=KCTC 21083T).

Engineering of Corynebacterium glutamicum for xylitol production from lignocellulosic pentose sugars.

Xylitol is a non-fermentable sugar alcohol used as sweetener. Corynebacterium glutamicum ATCC13032 was metabolically engineered for xylitol production from the lignocellulosic pentose sugars xylose and arabinose. Direct conversion of xylose to xylitol was achieved through the heterologous expression of NAD(P)H-dependent xylose reductase (xr) gene from Rhodotorula mucilaginosa. Xylitol synthesis from arabinose was attained through polycistronic expression of l-arabinose isomerase (araA), d-psicose 3 epimerase (dpe) and l-xylulose reductase (lxr) genes from Escherichia coli, Agrobacterium tumefaciens and Mycobacterium smegmatis, respectively. Expression of xr and the synthetic araA-dpe-lxr operon under the control of IPTG-inducible Ptac promoter enabled production of xylitol from both xylose and arabinose in the mineral (CGXII) medium with glucose as carbon source. Additional expression of a pentose transporter (araTF) gene enhanced xylitol production by about four-fold compared to the parent strain. The constructed strain Cg-ax3 produced 6.7±0.4g/L of xylitol in batch fermentations and 31±0.5g/L of xylitol in fed-batch fermentations with a specific productivity of 0.28±0.05g/g cdw/h. The strain Cg-ax3 was also validated for xylitol production from pentose rich, acid pre-treated liquor of sorghum stover (SAPL) and the results were comparable in both SAPL (27±0.3g/L) and mineral medium (31±0.5g/L).

Production and characterization of poly(3-hydroxy butyrate-co-3 hydroxyvalerate) (PHBV) by a novel halotolerant mangrove isolate.

A halophilic mangrove isolate identified by 16S rRNA sequence as a Bacillus spp. was found to be capable of using a broad range of carbon sources including monosaccharides (glucose and fructose), disaccharides (sucrose), pentoses (xylose and arabinose), various organic acids (acetic acid, propionic acid and octanoic acid) and even the acid pre-treated liquor (APL) of sugarcane trash, a lignocellulosic biomass, for growth and the production of polyhydroxyalkanoates (PHAs) such as poly(3-hydroxybutyrate, P3HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate, PHBV), and 4-hydroxyhexanoate, 4HHX). The study describes the innate ability of a wild-type culture for PHBV production by both propionate dependent and propionate independent pathways. The biopolymer was extracted and characterized physico-chemically. The PHBV yield from glucose was estimated to be 73% of biomass weight with a high 3-hydroxyvalerate fraction of 48mol%. Thereafter, spherical homogenous PHBV nanoparticles of ∼164nm size were prepared for future applications.

2,4-Di-tert-butyl phenol as the antifungal, antioxidant bioactive purified from a newly isolated Lactococcus sp.

The volatile organic compound 2,4-di-tert-butyl phenol (2,4 DTBP) was purified from the cell free supernatant of a newly isolated Lactococcus sp. by solvent extraction and chromatographic techniques. Molecular characterization of the compound by ESI-MS, (1)H NMR and FTIR analysis revealed the structure, C14H22O. Fungicidal activity was demonstrated against Aspergillus niger, Fusarium oxysporum and Penicillium chrysogenum by disc diffusion assay. Among the cell lines tested for cytotoxicity of this compound (normal cell line H9c2 and cancer cell lines HeLa and MCF-7), a remarkable cytotoxicity against HeLa cells with an IC50 value of 10 µg/mL was shown. A biocontrol experiment with 2,4 DTBP supplemented fraction prevented growth of the abovementioned fungi on wheat grains. The study further strengthens the case for development of biopreservatives and dietary antioxidants from lactic acid bacteria for food applications.

Folate fortification of skim milk by a probiotic Lactococcus lactis CM28 and evaluation of its stability in fermented milk on cold storage.

In order to enhance folate levels in fermented foods, a folate producing probiotic lactic acid bacterium isolated from cow's milk and identified as Lactococcus lactis CM28 by 16S rRNA sequencing was used to fortify skim milk. Optimization of medium additives such as folate precursors, prebiotics and reducing agents along with suitable culture conditions enhanced folate levels in skim milk. Optimization resulted in a four fold increase in the extracellular folate (61.02 ± 1.3 µg/L) and after deconjugation the total folate detected was 129.53 ± 1.2 µg/L. The effect of refrigerated storage on the viability of L. lactis, pH, titratable acidity (TA) in terms of percentage lactic acid and finally on the stability of folate was determined. Only a slight variation in pH (4.74 ± 0.02 to 4.415 ± 0.007) and acidity (0.28 ± 0.028 to 0.48 ± 0.014 %) was noted during folate fermentation. During storage, only less than a log unit reduction was noted in the viable count of the probiotic after 15 days and about 90 % of the produced folate was retained in an active state.

Physicochemical Characterization of an Exopolysaccharide Produced by a Newly Isolated Weissella cibaria.

A high molecular weight (3037 kDa) dextran-like polysaccharide was purified from the culture broth of a probiotic Weissella cibaria. Dry weight of the exopolysaccharides (EPS) obtained after ethanol precipitation was 6.35 g/L by using sucrose as carbon source and was further purified by phenol:chloroform:isoamyl alcohol (25:24:1) extraction and acetone precipitation to white powder with 52% recovery. Rheological characterization revealed that it has a pseudoplastic behaviour and showed a temperature tolerance of up to 280 °C in thermogravimetric analysis. Fourier transform infrared (FTIR) spectra showed the characteristic peaks of polysaccharide such as hydroxyl group, α-pyranose, asymmetrical C-H stretching vibration of methyl group and carbonyl hydrogens of D-glucopyranose. The (1)H nuclear magnetic resonance (NMR) spectrum showed resonances of hydrogen corresponding to the glucosyl residue, a repeat unit of the biopolymer. The cell viability analysis of normal L6 cells treated with the Weissella EPS showed that it is noncytotoxic even up to the 500-µg/mL level. A 0.2% (w/v) supplementation of EPS in 2% (w/v) wheat starch preparation could reduce the degree of syneresis up to 45% and up to 75% with 1% EPS.

Encapsulated Lactococcus lactis with enhanced gastrointestinal survival for the development of folate enriched functional foods.

Two lactic acid bacteria (LAB) isolated from cow's milk were identified as Lactococcus lactis strains and designated as L. lactis CM22 and L. lactis CM28. They were immobilised by co-encapsulation using alginate and mannitol and by hybrid entrapment with skim milk, glycerol, CaCO3 and alginate. The encapsulated cells survived better in simulated gastrointestinal conditions compared to the free cells. The percentage survival of probiotics encapsulated by hybrid entrapment method was 62.74% for L. lactis CM22 and 68% for L. lactis CM28. Studies to check their efficacy in fermentative fortification of skim milk and ice cream revealed an enhancement in folate level.

Molecular characterization of an exopolysaccharide from a probiotic Lactobacillus plantarum MTCC 9510 and its efficacy to improve the texture of starchy food.

The weight average molecular weight (Mw), number average molecular weight (Mn) and size average molecular weight (Mz) of an exopolysaccharide from probiotic Lactobacillus plantarum MTCC 9510 was found to be 2.68 × 10(5) Da, 2.55 × 10(5) Da and 2.83 × 10(5) Da, respectively by Gel permeation Chromatography employing the third order polynomial model. The polydispersity index (Mw/Mn) of the polysaccharide was obtained as 1.05. The exopolysaccharide and the starch-exopolysaccharide hydrocolloid exhibited a non-Newtonian and pseudo-plastic behaviour with improvement in the texture of starch containing food by preventing syneresis.

Synthesis, colloidal properties and cytotoxicity of biopolymer nanoparticles.

To characterize the physicochemical and biological stability of nanodevices suitable for biomedical applications, polylactic acid (PLA) nanoparticles (NPs) of 112 ± 6 nm and polyhydroxy butyrate (PHB) of 15 ± 5 nm size were prepared by standardizing the suitable method for each. Morphology of NPs was studied by scanning and transmission electron microscopy and temperature stability by thermogravimetric analysis. Their stability in biological fluids (simulated gastrointestinal and saliva) and tolerance against 0.5 mM NaCl were analyzed. PHB NPs remained stable in all fluids, while after 24 h treatment, the PLA NPs showed the beginning of disintegration with intestinal fluid mimic. In addition to the preparation of polyethylene glycol (PEG) surface-coated NPs, PLA-PEG-PLA triblock copolymer (MW ∼ 7,366 Da) was also chemically synthesized and characterized. Cytotoxicity of all forms of nanoparticles was tested by MTT assay and by annexin pi staining.

Extracellular methionine amino peptidase (MAP) production by Streptomyces gedanensis in solid-state fermentation

A bioprocess was developed for extracellular MAP production from Streptomyces gedanensis by solid-state fermentation. Response surface methodology of Box Behken Design was performed to evaluate the interaction effects of most significant variables {inoculum size, (NH4)2SO4 concentration, MgSO4.7H2O and tryptone) on MAP production after the single parameter optimization and it resulted a maximum MAP production of 55.26 IU/g PUF after 120 h of fermentation. The concentrated crude MAP displayed a pH and temperature optimum of 8.5 and 50°C. By analyzing the thermal stability, the MAP was found to be stable in a temperature range of 50 to 55°C but lost about 50% of its activity at 65°C after 30 min. This is a first report of this kind of study for MAP.

Control of spoilage fungi by protective lactic acid bacteria displaying probiotic properties.

Thirty-six lactic acid bacteria belong to Lactococcus, Lactobacillus, Enterococcus, and Pediococcus were isolated, and the spectrum of antifungal activity was verified against Fusarium oxysporum (KACC 42109), Aspergillus niger (KACC 42589), Fusarium moniliforme (KACC 08141), Penicillium chrysogenum (NII 08137), and the yeast Candida albicans (MTCC 3017). Three isolates, identified as Pediococcus pentosaceus (TG2), Lactobacillus casei (DY2), and Lactococcus (BSN) were selected further, and their antifungal compounds were identified by ESI-MS and HPLC analysis as a range of carboxylic acids along with some unidentified, higher molecular weight compounds. An attempt to check out the shelf life extension of wheat bread without fungal spoilage was performed by fermenting the dough with the Lactococcus isolate. Apart from growth in low pH and tolerance to bile salts, probiotic potential of these three isolates was further substantiated by in vitro screening methods that include transit tolerance to the conditions in the upper human gastrointestinal tract and bacterial adhesion capacity to human intestinal cell lines.