Recent advances in mapping environmental microbial metabolisms through 13C isotopic fingerprints

After feeding microbes with a defined ¹³C substrate, unique isotopic patterns (isotopic fingerprints) can be formed in their metabolic products. Such labelling information not only can provide novel insights into functional pathways but also can determine absolute carbon fluxes through the metabolic network via metabolic modelling approaches. This technique has been used for finding pathways that may have been mis-annotated in the past, elucidating new enzyme functions, and investigating cell metabolisms in microbial communities. In this review paper, we summarize the applications of ¹³C approaches to analyse novel cell metabolisms for the past 3 years. The isotopic fingerprints (defined as unique isotopomers useful for pathway identifications) have revealed the operations of the Entner–Doudoroff pathway, the reverse tricarboxylic acid cycle, new enzymes for biosynthesis of central metabolites, diverse respiration routes in phototrophic metabolism, co-metabolism of carbon nutrients and novel CO₂ fixation pathways. This review also discusses new isotopic methods to map carbon fluxes in global metabolisms, as well as potential factors influencing the metabolic flux quantification (e.g. metabolite channelling, the isotopic purity of ¹³C substrates and the isotopic effect). Although ¹³C labelling is not applicable to all biological systems (e.g. microbial communities), recent studies have shown that this method has a significant value in functional characterization of poorly understood micro-organisms, including species relevant for biotechnology and human health.     

Banana peel: an effective biosorbent for aflatoxins

This work reports the application of banana peel as a novel bioadsorbent for in vitro removal of five mycotoxins (aflatoxins (AFB1, AFB2, AFG1, AFG2) and ochratoxin A). The effect of operational parameters including initial pH, adsorbent dose, contact time and temperature were studied in batch adsorption experiments. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and point of zero charge (pH_pzc) analysis were used to characterise the adsorbent material. Aflatoxins’ adsorption equilibrium was achieved in 15 min, with highest adsorption at alkaline pH (6–8), while ochratoxin has not shown any significant adsorption due to surface charge repulsion. The experimental equilibrium data were tested by Langmuir, Freundlich and Hill isotherms. The Langmuir isotherm was found to be the best fitted model for aflatoxins, and the maximum monolayer coverage (Q₀) was determined to be 8.4, 9.5, 0.4 and 1.1 ng mg^?1 for AFB1, AFB2, AFG1 and AFG2 respectively. Thermodynamic parameters including changes in free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were determined for the four aflatoxins. Free energy change and enthalpy change demonstrated that the adsorption process was exothermic and spontaneous. Adsorption and desorption study at different pH further demonstrated that the sorption of toxins was strong enough to sustain pH changes that would be experienced in the gastrointestinal tract. This study suggests that biosorption of aflatoxins by dried banana peel may be an effective low-cost decontamination method for incorporation in animal feed diets.     

Technological Implications of Modifying the Extent of Cell Wall-Proanthocyanidin Interactions Using Enzymes

The transference and reactivity of proanthocyanidins is an important issue that affects the technological processing of some fruits, such as grapes and apples. These processes are affected by proanthocyanidins bound to cell wall polysaccharides, which are present in high concentrations during the processing of the fruits. Therefore, the effective extraction of proanthocyanidins from fruits to their juices or derived products will depend on the ability to manage these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the role of pure hydrolytic enzymes (polygalacturonase and cellulose) and a commercial enzyme containing these two activities on the extent of proanthocyanidin-cell wall interactions. The results showed that the modification promoted by enzymes reduced the amount of proanthocyanidins adsorbed to cell walls since they contributed to the degradation and release of the cell wall polysaccharides, which diffused into the model solution. Some of these released polysaccharides also presented some reactivity towards the proanthocyanidins present in a model solution.     

Effect of a cold‐active pectinolytic system on colour development of Malbec red wines elaborated at low temperature

The effect of a new cold‐active pectinolytic system on colour of Malbec wines was studied under the following winemaking conditions: (i) fermentation at low temperature (20 °C) and (ii) prefermentative cold maceration (PCM) (5 °C–7 days) followed by traditional fermentation (28 °C). The pectinolytic system was mainly composed of polymethylgalacturonase and pectin lyase activities, detected under similar conditions to those in winemaking (pH 3.6–20 °C). The results show that the enzyme system significantly accelerated colour extraction by reducing the maceration time necessary for vinification at low temperature and shortening the PCM stage. Enzyme‐treated wines exhibited better chromatic parameters than their controls at devatting and after 6 months of storage. The cold‐active enzyme compensated the decrease in colour extraction due to the low maceration temperature, achieving high‐quality wines with chromatic characteristics similar to those of traditional wines.     

From Active Sites to Machines: A Challenge for Enzyme Chemists

As researchers who study enzyme chemistry embrace increasingly complex systems, especially biological machines, our attention is also shifting from steps involving covalent bond formation or cleavage to those that exclusively involve changes in non‐covalent bonding. Assembly line polyketide synthases are an example of this growing challenge. By now, the chemical reactions underpinning polyketide biosynthesis can be unequivocally mapped to well‐defined active sites and are, for the most part, readily explicable in the language of physical organic chemistry. Yet, all of these insights merely serve as a backdrop to the real problem of explaining how the catalytic functions of dozens of active sites are synchronized in order to allow these remarkable machines to turn over with remarkable specificity. Notwithstanding the fact that the time‐honored language of physical organic chemistry can teach us a lot, it is often insufficient to describe many of these events, and must therefore evolve.     

Resonance Assignments of Lowly Populated and Unstable Enzyme Intermediate Complex under Real-Time Conditions

Unstable and low-abundance protein complexes represent a large family of transient protein complexes that are difficult to characterize, even by means of high-resolution NMR spectroscopy. A method to assign the NMR signals of these unstable complexes through a combination of selective isotope labeling of amino acids in a protein and site-specific labeling the protein with a paramagnetic tag is presented herein. By using this method, the resonances of unstable thioester intermediate complex (lifetime <5 h and highest concentration ≈20 μm ) generated by Staphylococcus aureus sortase A and its peptide substrate under a real-time reaction have been assigned.     

Improved microstructure and properties of the gelatin film produced through prior cross‐linking induced by horseradish peroxidase,glucose oxidase and glucose

The effects of prior enzymatic cross‐linking of bovine gelatin via horseradish peroxidase, glucose oxidase and glucose on microstructure and properties of the target film (cross‐linked gelatin film) were assessed. The cross‐linked gelatin film exhibited similar film thickness and moisture content, lower water solubility and higher opacity than the gelatin film directly prepared with bovine gelatin. The cross‐linked gelatin film also demonstrated improved thermal stability and mechanical properties, characterised by higher melting point and glass transition temperature, enhanced tensile strength and elongation at break and greater storage modulus. Prior gelatin cross‐linking resulted in 30.2% and 68.6% reductions in water vapour and CO₂ permeability of the cross‐linked gelatin film, respectively, but did not affect oil permeability. Furthermore, the cross‐linked gelatin film possessed smaller cross‐sectional voids (diameter 100?360 nm vs. 200?595 nm) than the bovine gelatin film. This study shows that cross‐linking can efficiently improve film microstructure and properties of the gelatin‐like products.     

Cell-envelope proteinases from lactic acid bacteria: Biochemical features and biotechnological applications

Proteins displayed on the cell surface of lactic acid bacteria (LAB) perform diverse and important biochemical roles. Among these, the cell-envelope proteinases (CEPs) are one of the most widely studied and most exploited for biotechnological applications. CEPs are important players in the proteolytic system of LAB, because they are required by LAB to degrade proteins in the growth media into peptides and/or amino acids required for the nitrogen nutrition of LAB. The most important area of application of CEPs is therefore in protein hydrolysis, especially in dairy products. Also, the physical location of CEPs (i.e., being cell-envelope anchored) allows for relatively easy downstream processing (e.g., extraction) of CEPs. This review describes the biochemical features and organization of CEPs and how this fits them for the purpose of protein hydrolysis. It begins with a focus on the genetic organization and expression of CEPs. The catalytic behavior and cleavage specificities of CEPs from various LAB are also discussed. Following this, the extraction and purification of most CEPs reported to date is described. The industrial applications of CEPs in food technology, health promotion, as well as in the growing area of water purification are discussed. Techniques for improving the production and catalytic efficiency of CEPs are also given an important place in this review.     

Influence of bunch maturation and chemical precursors on acrylamide formation in starchy banana chips

The present study investigated the effect of ripening stages and chemical precursors on acrylamide formation in deep-fried chips of five plantains and one cooking banana. The highest level of acrylamide was found in the cooking banana, followed by False Horn plantain and French plantain, respectively. French plantain hybrids exhibited a significantly lower (P < 0.05) level of acrylamide when compared to French plantain. The ripening stage demonstrated a positive Pearson correlation (P < 0.05, r = 0.57) with acrylamide formation. As ripening progressed, the levels of glucose and fructose significantly increased (P < 0.05) and showed a positive correlation with acrylamide formation (r = 0.85 and 0.96, respectively). The level of the amino acid asparagine during ripening was not correlated with acrylamide formation. In contrast, the level of histidine, arginine, iso-leucine and cystine during ripening was positively correlated (P < 0.05, r > 0.60) with acrylamide formation in fried chips. The higher level of TP was significantly related (P < 0.05) to the lower level of acrylamide (r = −0.62). The reduced levels of carotenoid isomers, except lutein, during fruit ripening were positively correlated (P < 0.05) with acrylamide formation, especially trans-BC (r = 0.72) and 9-cis-BC(r = 0.64).