Evaluation of improved biological properties of chemically modified exopolysaccharides from Lactobacillus plantarum BR2.

This work engrosses the production and further chemical modifications of EPS produced by Lactobacillus plantarum BR2 and subsequent evaluation of their biological properties showed greater antioxidant properties for the derivatives compared to its native unmodified form. Of the three derivatives, acetylated EPS (a-EPS), carboxymethylated EPS (Cm-EPS), and sulphated EPS (s-EPS), a-EPS exhibited the highest DPPH radical scavenging and total antioxidant activity in a dose-dependent manner. At all tested concentrations, a-EPS showed higher scavenging activity, and a maximum activity of 73.81% at 2 mg/mL. Meanwhile, s-EPS showed the highest reducing power potential and hydroxyl radical scavenging activities. At 2 mg/mL concentration, the order of reducing power was observed to be s-EPS (41.39%) > a-EPS (37.43%) > Cm-EPS (24.02) > BR2 control EPS (16%) and the hydroxyl radical scavenging activity for the s-EPS was 54.43%. The highest reducing power activity exhibited by s-EPS is 2.6-fold higher and a 1.5-fold increase in the scavenging activity of native BR2 EPS after the sulphonyl group addition was observed. The increase in these activities is due to the addition of various functional groups that contributes largely to the scavenging abilities of different free radicals. The s-EPS and Cm-EPS derivatives also exhibited increased cholesterol-lowering activity of 40 and 34.5%, respectively, than the native EPS. Interestingly, there were hardly any inhibitions on cell growth and viability of normal L929 fibroblast cell lines upon treatment with these EPSes. The improved antioxidant properties resulting from chemical modification opened better avenues for EPS application in the food and pharma sectors. Thus, the potentiality of chemically modified EPS may be explored further in the development of functional foods. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03718-5.

Direct utilization and conversion of raw starch to exopolysaccharides by a newly isolated amylolytic Streptococcus sp.

A newly isolated culture is identified as Streptococcus lutetiensis with significant starch saccharifying activity. Along with considerable amylolytic property (∼ 2.71 U/mL), the culture exhibited significant production of exopolysaccharide (EPS) in starch medium. Interestingly, the glycosyl transferase activity which is essential in the biosynthesis of polysaccharide is also detected in the culture and after screening and process optimization, a maximum EPS titre of 19.92 ± 0.5 g/L was obtained from cassava starch. The crude EPS, after purification and characterization (monosaccharide analysis, FT-IR, TGA, GPC NMR, and SEM) was found to be of dextran nature with a Mw of 1275.36 kDa. Dextran type exopolysaccharide are synthesized by dextransucrase enzyme by the transfer of glucosyl residues from sucrose to dextran polymer. Interestingly, the glycosyl transferase enzyme activity which is essential in the biosynthesis of EPS is also detected in the culture. The particle size (447.8 dnm) and the zeta potential (-33.4) analysis of the purified EPS showed that the EPS produced is a stable molecule and has a random coil confirmation when exposed to alkaline condition with shear thinning property. One step conversion of sustainable low-cost starchy raw materials without adding external enzymes for hydrolysis, improved the economic viability of EPS production.

Fusarium biocontrol: antagonism and mycotoxin elimination by lactic acid bacteria.

Mycotoxins produced by Fusarium species are secondary metabolites with low molecular weight formed by filamentous fungi generally resistant to different environmental factors and, therefore, undergo slow degradation. Contamination by Fusarium mycotoxins in cereals and millets is the foremost quality challenge the food and feed industry faces across the globe. Several types of chemical preservatives are employed in the mitigation process of these mycotoxins, and they help in long-term storage; however, chemical preservatives can be used only to some extent, so the complete elimination of toxins from foods is still a herculean task. The growing demand for green-labeled food drives to evade the use of chemicals in the production processes is getting much demand. Thus, the biocontrol of food toxins is important in the developing food sector. Fusarium mycotoxins are world-spread contaminants naturally occurring in commodities, food, and feed. The major mycotoxins Fusarium species produce are deoxynivalenol, fumonisins, zearalenone, and T2/HT2 toxins. Lactic acid bacteria (LAB), generally regarded as safe (GRAS), is a well-explored bacterial community in food preparations and preservation for ages. Recent research suggests that LAB are the best choice for extenuating Fusarium mycotoxins. Apart from Fusarium mycotoxins, this review focuses on the latest studies on the mechanisms of how LAB effectively detoxify and remove these mycotoxins through their various bioactive molecules and background information of these molecules.

Xylose Dehydrogenase Immobilized on Ferromagnetic Nanoparticles for Bioconversion of Xylose to Xylonic Acid.

d-Xylonic acid (XA), derived from pentose sugar xylose, is a multifunctional high-value chemical with a wide range of applications in the fields of medicines, food, agriculture and is a valuable chemical reagent for the synthesis of other useful commodity chemicals. In the bacterial system, xylose dehydrogenase (XDH) catalyzes the oxidation of d-xylose into d-xylonolactone, consuming NAD+ or NADP+ as a cofactor. The d-xylonolactone then undergoes auto-oxidation into d-xylonic acid. Herein, the XDH enzyme overexpressed in Escherichia coli is purified and immobilized on ferromagnetic nanoparticles, effectively converting xylose into xylonic acid. Parameters deciding the bioconversion were statistically optimized and obtained a maximum of 91% conversion rate. Kinetic parameters of immobilized xylose dehydrogenase showed a 2-fold increase in the maximum velocity of the reaction and catalytic efficiency compared to free enzyme. The operational stability test for the enzyme-nanoparticle conjugate retained 93% relative activity after 10 successive experiments, exhibiting the good recyclability of the biocatalyst for XA production.

In silico analysis of nitrilase-3 protein from Corynebacterium glutamicum for bioremediation of nitrile herbicides.

BACKGROUND: The nitrile compounds are produced either naturally or synthetically and are highly used in many manufacturing industries such as pharmaceuticals, pesticides, chemicals, and polymers. However, the extensive use and accumulation of these nitrile compounds have caused severe environmental pollution. Nitrilated herbicides are one such toxic substance that will persist in the soil for a long time. Therefore, effective measures must be taken to avoid its pollution to the environment. A variety of nitrile-converting bacterial species have the ability to convert these toxic substances into less toxic ones by using enzymatic processes. Among the bacterial groups, actinobacteria family members show good degradation capacity on these pollutants. The soil-dwelling Gram-positive industrial microbe Corynebacterium glutamicum is one such family member and its nitrile-degradation pathway is not well studied yet. In order to understand the effectiveness of using C. glutamicum for the degradation of such nitrile herbicides, an in silico approach has been done. In this perspective, this work focus on the structural analysis and molecular docking studies of C. glutamicum with nitrilated herbicides such as dichlobenil, bromoxynil, and chloroxynil. RESULTS: The bioinformatics analysis using different tools and software helped to confirm that the genome of C. glutamicum ATCC 13032 species have genes (cg 3093) codes for carbon-nitrogen hydrolase enzyme, which specifically act on non-peptide bond present in the nitrile compounds. The conserved domain analysis indicated that this protein sequence was nitrilase-3 and comes under the nitrilase superfamily. The multiple sequence alignment analysis confirmed that the conserved catalytic triad residues were 40E, 115K, and 151C, and the existence of nitrilase-3 protein in the genome of Corynebacterium sp. was evaluated by a phylogenetic tree. The analysis of physico-chemical properties revealed that alanine is the most abounded amino acid (10.20%) in the nitrilase-3 protein, and these properties influence the substrate specificity of aliphatic and aromatic nitrile compounds. The homology modelled protein showed better affinity towards nitrile herbicides such as 2,6-dichlorobenzamide (BAM) and 3,5-dichloro-4-hydroxy-benzamide (CIAM) with the affinity value of - 5.8 and - 5.7 kcal/mol respectively. CONCLUSIONS: The in silico studies manifested that C. glutamicum ATCC 13032 is one of the promising strains for the bioremediation of nitrilated herbicides contaminated soil.

Metabolic Engineering for Valorization of Agri- and Aqua-Culture Sidestreams for Production of Nitrogenous Compounds by Corynebacterium glutamicum.

Corynebacterium glutamicum is used for the million-ton-scale production of amino acids. Valorization of sidestreams from agri- and aqua-culture has focused on the production of biofuels and carboxylic acids. Nitrogen present in various amounts in sidestreams may be valuable for the production of amines, amino acids and other nitrogenous compounds. Metabolic engineering of C. glutamicum for valorization of agri- and aqua-culture sidestreams addresses to bridge this gap. The product portfolio accessible via C. glutamicum fermentation primarily features amino acids and diamines for large-volume markets in addition to various specialty amines. On the one hand, this review covers metabolic engineering of C. glutamicum to efficiently utilize components of various sidestreams. On the other hand, examples of the design and implementation of synthetic pathways not present in native metabolism to produce sought after nitrogenous compounds will be provided. Perspectives and challenges of this concept will be discussed.

Curved membrane structures induced by native lipids in giant vesicles.

Native lipids in cell-membrane support crucial functions like intercell communication via their ability to deform into curved membrane structures. Cell membrane mimicking Giant unilamellar vesicles (GUV) is imperative in understanding native lipid's role in membrane transformation however remains challenging to assemble. We construct two giant vesicle models mimicking bacterial inner-membrane (IM) and outer-membrane (OM) under physiological conditions using single-step gel-assisted lipid swelling. IM vesicles composed of native bacterial lipids undergo small-scale membrane remodeling into bud and short-nanotube structures. In contrast, OM vesicles asymmetrically assembled from Lipopolysaccharide (LPS) and bacterial lipids underwent global membrane deformation under controlled osmotic stress. Remarkably, highly-curved structures mimicking cell-membrane architectures, including daughter vesicle networks interconnected by necks and nano-tubes ranging from micro to nanoscale, are generated in OM vesicles at osmotic stress comparable to that applied in IM vesicles. Further, we provide a quantitative description of the membrane structures by experimentally determining membrane elastic parameters, i.e., neck curvature and bending rigidity. We can conclude that a larger spontaneous curvature estimated from the neck curvature and softer membranes in OM vesicles is responsible for large-scale deformation compared to IM vesicles. Our findings will help comprehend the shape dynamics of complex native bacterial lipid membranes.

An overview of functional genomics and relevance of glycosyltransferases in exopolysaccharide production by lactic acid bacteria.

There are many reports on exopolysaccharides of lactic acid bacteria (LAB EPS) such as isolation, production and applications. The LAB EPS have been proved to exhibit significantly improved texture and rheological properties in order to prevent syneresis of fermented foods. Furthermore, they are known to have many biological properties such as mouthwatering flavors, antioxidant activity, cholesterol lowering and antimicrobial activities. Considering their GRAS status, LAB EPS need to be explored for better titre and improved biological properties, where strain improvement by genetic engineering has a major role for making tailor-made EPS. The genetic overview of the EPS production by LAB is an auxiliary area of interest as the process and the biosynthetic pathway involves numerous genes and their proteins. Among them Glycosyltransferases (gtfs) are the key enzymes involved in EPS biosynthesis. Current knowledge of gtfs of LAB and its manipulation is limited. The present review spotlights the importance of glycosyltransferases and their specific role on the biosynthesis of LAB EPS and addresses the functionality and applicability of these enzymes and their products. It enfold the available literature including some patents in recent past to underline the fact that glycosyltransferases are un-reluctantly the key proteins involved in the EPS biosynthesis.

Production of Biopolyamide Precursors 5-Amino Valeric Acid and Putrescine From Rice Straw Hydrolysate by Engineered Corynebacterium glutamicum.

The non-proteinogenic amino acid 5-amino valeric acid (5-AVA) and the diamine putrescine are potential building blocks in the bio-polyamide industry. The production of 5-AVA and putrescine using engineered Corynebacterium glutamicum by the co-consumption of biomass-derived sugars is an attractive strategy and an alternative to their petrochemical synthesis. In our previous work, 5-AVA production from pure xylose by C. glutamicum was shown by heterologously expressing xylA from Xanthomonas campestris and xylB from C. glutamicum. Apart from this AVA Xyl culture, the heterologous expression of xylA Xc and xylB Cg was also carried out in a putrescine producing C. glutamicum to engineer a PUT Xyl strain. Even though, the pure glucose (40 g L-1) gave the maximum product yield by both the strains, the utilization of varying combinations of pure xylose and glucose by AVA Xyl and PUT Xyl in CGXII synthetic medium was initially validated. A blend of 25 g L-1 of glucose and 15 g L-1 of xylose in CGXII medium yielded 109 ± 2 mg L-1 putrescine and 874 ± 1 mg L-1 5-AVA after 72 h of fermentation. Subsequently, to demonstrate the utilization of biomass-derived sugars, the alkali (NaOH) pretreated-enzyme hydrolyzed rice straw containing a mixture of glucose (23.7 g L-1) and xylose (13.6 g L-1) was fermented by PUT Xyl and AVA Xyl to yield 91 ± 3 mg L-1 putrescine and 260 ± 2 mg L-1 5-AVA, respectively, after 72 h of fermentation. To the best of our knowledge, this is the first proof of concept report on the production of 5-AVA and putrescine using rice straw hydrolysate (RSH) as the raw material.

Heterologous expression of genes for bioconversion of xylose to xylonic acid in Corynebacterium glutamicum and optimization of the bioprocess.

In bacterial system, direct conversion of xylose to xylonic acid is mediated through NAD-dependent xylose dehydrogenase (xylB) and xylonolactonase (xylC) genes. Heterologous expression of these genes from Caulobacter crescentus into recombinant Corynebacterium glutamicum ATCC 13032 and C. glutamicum ATCC 31831 (with an innate pentose transporter, araE) resulted in an efficient bioconversion process to produce xylonic acid from xylose. Process parameters including the design of production medium was optimized using a statistical tool, Response Surface Methodology (RSM). Maximum xylonic acid of 56.32 g/L from 60 g/L xylose, i.e. about 76.67% of the maximum theoretical yield was obtained after 120 h fermentation from pure xylose with recombinant C. glutamicum ATCC 31831 containing the plasmid pVWEx1 xylB. Under the same condition, the production with recombinant C. glutamicum ATCC 13032 (with pVWEx1 xylB) was 50.66 g/L, i.e. 69% of the theoretical yield. There was no significant improvement in production with the simultaneous expression of xylB and xylC genes together indicating xylose dehydrogenase activity as one of the rate limiting factor in the bioconversion. Finally, proof of concept experiment in utilizing biomass derived pentose sugar, xylose, for xylonic acid production was also carried out and obtained 42.94 g/L xylonic acid from 60 g/L xylose. These results promise a significant value addition for the future bio refinery programs.

Production of low-calorie structured lipids from spent coffee grounds or olive pomace crude oils catalyzed by immobilized lipase in magnetic nanoparticles.

In this study, crude oils extracted from spent coffee grounds (SCG) and olive pomace (OP) were used as raw-material to synthesize low-calorie triacylglycerols, either by acidolysis with capric acid, or by interesterification with ethyl caprate, in solvent-free media, catalyzed by sn-1,3 regioselective lipases. The Rhizopus oryzae lipase (ROL) was immobilized in magnetite nanoparticles (MNP-ROL) and tested as novel biocatalyst. MNP-ROL performance was compared with that of the commercial immobilized Thermomyces lanuginosus lipase (Lipozyme TL IM). For both oils, Lipozyme TL IM preferred interesterification over acidolysis. MNP-ROL catalyzed reactions were faster and acidolysis was preferred with yields of c.a. 50% new triacylglycerols after 3 h acidolysis of OP or SCG oils. MNP-ROL was very stable following the Sadana deactivation model with half-lives of 163 h and 220 h when reused in batch acidolysis and interesterification of OP oil, respectively.

Engineering bio-mimicking functional vesicles with multiple compartments for quantifying molecular transport.

Controlled design of giant unilamellar vesicles under defined conditions has vast applications in the field of membrane and synthetic biology. Here, we bio-engineer bacterial-membrane mimicking models of controlled size under defined salt conditions over a range of pH. A complex bacterial lipid extract is used for construction of physiologically relevant Gram-negative membrane mimicking vesicles whereas a ternary mixture of charged lipids (DOPG, cardiolipin and lysyl-PG) is used for building Gram-positive bacterial-membrane vesicles. Furthermore, we construct stable multi-compartment biomimicking vesicles using the gel-assisted swelling method. Importantly, we validate the bio-application of the bacterial vesicle models by quantifying diffusion of chemically synthetic amphoteric antibiotics. The transport rate is pH-responsive and depends on the lipid composition, based on which a permeation model is proposed. The permeability properties of antimicrobial peptides reveal pH dependent pore-forming activity in the model vesicles. Finally, we demonstrate the functionality of the vesicles by quantifying the uptake of membrane-impermeable molecules facilitated by embedded pore-forming proteins. We suggest that the bacterial vesicle models developed here can be used to understand fundamental biological processes like the peptide assembly mechanism or bacterial cell division and will have a multitude of applications in the bottom-up assembly of a protocell.

Fermentative Production of N-Alkylated Glycine Derivatives by Recombinant Corynebacterium glutamicum Using a Mutant of Imine Reductase DpkA From Pseudomonas putida.

Sarcosine, an N-methylated amino acid, shows potential as antipsychotic, and serves as building block for peptide-based drugs, and acts as detergent when acetylated. N-methylated amino acids are mainly produced chemically or by biocatalysis, with either low yields or high costs for co-factor regeneration. Corynebacterium glutamicum, which is used for the industrial production of amino acids for decades, has recently been engineered for production of N-methyl-L-alanine and sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Here, mutation of an amino acyl residue in the substrate binding site of DpkA (DpkAF117L) led to an increased specific activity for reductive alkylamination of glyoxylate using monomethylamine and monoethylamine as substrates. Introduction of DpkAF117L into the production strain accelerated the production of sarcosine and a volumetric productivity of 0.16 g L-1 h-1 could be attained. Using monoethylamine as substrate, we demonstrated N-ethylglycine production with a volumetric productivity of 0.11 g L-1 h-1, which to the best of our knowledge is the first report of its fermentative production. Subsequently, the feasibility of using rice straw hydrolysate as alternative carbon source was tested and production of N-ethylglycine to a titer of 1.6 g L-1 after 60 h of fed-batch bioreactor cultivation could be attained.

Detecting the structural assembly pathway of human antimicrobial peptide pores at single-channel level.

The pore-forming structures of an anionic human antimicrobial peptide dermcidin (DCD) in a membrane environment has not been demonstrated previously. Using single-channel electrical recordings, we characterized the structural and functional properties of the DCD peptide channel in lipid membranes. We show that a 48-residue, 8 nm long anionic DCD-1L peptide is folded in the right conformation in sodium dodecyl sulfate (SDS) that spontaneously inserts into lipid bilayers to form well-defined channels. However, the DCD-1L peptides are not properly folded in n-dodecyl-ß-d-maltoside (DDM), resulting in unstable channels suggesting the significance of specific detergent in stable channel formation. Furthermore, a 25-residue cationic DCD SSL-25 peptide formed channels both in SDS and DDM micelles as the length of the peptide matches with the thickness of the membrane. Finally, we quantified the permeation of small molecules through the DCD channels in liposome assays. Accordingly, we propose a molecular model demonstrating the structural self-assembly of the DCD channels in the membrane. We suggest that an understanding of the mechanism of action of DCD peptides at single-channel resolution will lead to developing peptide-based therapeutics.

An exopolysaccharide (EPS) from a Lactobacillus plantarum BR2 with potential benefits for making functional foods.

A high molecular weight EPS of glucomannan nature was recovered and purified to get an yield of 2.8±0.5g/L from Lb. plantarum BR2 and it displayed potent antioxidant activity with 29.8% radical scavenging activity and 19% total antioxidant capacity. At 100µg/ml concentration, it is capable of inhibiting the alpha amylase activity by 10% and at 300µg/ml, it drastically inhibited the alpha-glucosidase activity by 67% which indicates its antidiabetic potential. More interestingly, at a concentration level of 0.1%, it reduced the cholesterol level by a margin of 45% in an in vitro assay. The sample didn't reveal any cytotoxicity against H9C2 normal cells indicating its potential for safe use as a food additive.

Microbial degradation of high impact polystyrene (HIPS), an e-plastic with decabromodiphenyl oxide and antimony trioxide.

Accumulation of electronic waste has increased catastrophically and out of that various plastic resins constitute one of the leading thrown out materials in the electronic machinery. Enrichment medium, containing high impact polystyrene (HIPS) with decabromodiphenyl oxide and antimony trioxide as sole carbon source, was used to isolate microbial cultures. The viability of these cultures in the e-plastic containing mineral medium was further confirmed by triphenyl tetrazolium chloride (TTC) reduction test. Four cultures were identified by 16S rRNA sequencing as Enterobacter sp., Citrobacter sedlakii, Alcaligenes sp. and Brevundimonas diminuta. Biodegradation experiments were carried out in flask level and gelatin supplementation (0.1% w/v) along with HIPS had increased the degradation rate to a maximum of 12.4% (w/w) within 30days. This is the first report for this kind of material. The comparison of FTIR, NMR, and TGA analysis of original and degraded e-plastic films revealed structural changes under microbial treatment. Polystyrene degradation intermediates in the culture supernatant were also detected using HPLC analysis. The gravity of biodegradation was validated by morphological changes under scanning electron microscope. All isolates displayed depolymerase activity to substantiate enzymatic degradation of e-plastic.

Co-expression of endoglucanase and ß-glucosidase in Corynebacterium glutamicum DM1729 towards direct lysine fermentation from cellulose.

The aim of the present study is the development of a consolidated bioprocess for the production of lysine with recombinant Corynebacterium glutamicum DM1729 strains expressing endoglucanase and ß-glucosidase genes. Here, the endoglucanase genes from Xanthomonas campestris XCC3521 and XCC2387 and betaglucosidase gene from Saccharophagus degradans Sde1394 were cloned in C. glutamicum DM1729 and expressed either extracellularly or on cell surface. The highest ß-glucosidase activity of 9±0.5U/OD600 of 1 and endoglucanase activity of 5.5±0.8U was obtained in C. glutamicum DM 1729 (pVWEx1-TATXCC2387) (pEKEx3-PorC-Sde1394) when cellobiose (20g/L) alone or in combination with carboxymethyl cellulose (20g/L) was used as the carbon sources respectively. The overall efforts resulted in a lysine titre of 5.9±0.5mM. The ability of the constructs to utilize carboxymethyl cellulose and cellobiose for growth and amino acid production proves the concept of utilization of C. glutamicum as a biocatalyst in the lignocellulosic biorefinery.