Combining analytical approaches for better lignocellulosic biomass degradation: a way of improving fungal enzymatic cocktails?

PURPOSE: In this study, a combinatory approach was undertaken to assay the efficiency of fungal enzymatic cocktails from different fermentation conditions to degrade different lignocellulosic biomasses with the aim of finely characterizing fungal enzymatic cocktails. METHODS: Enzymatic assays (AZO and pNP-linked substrates and ABTS) were used to assess the composition of the fungal enzymatic cocktails for cellulase, xylanase and laccase activities. Comparisons were made with a new range of chromogenic substrates based on complex biomass (CBS substrates). The saccharification efficiency of the cocktails was evaluated as a quantification of the sugar monomers released from the different biomasses after incubation with the enzymatic cocktails. RESULTS: The results obtained showed striking differences between the AZO and pNP-linked substrates and the CBS substrates for the same enzymatic cocktails. On AZO and pNP-linked substrates, different hydrolysis profiles were observed between the different fungi species with Aspergillus oryzae being the most efficient. However, the results on CBS substrates were more contrasted depending on the biomass tested. Altogether, the results highlighted that assessing laccase activities and taking into account the complexity of the biomass to degrade were key in order to provide the best enzymatic cocktails. CONCLUSION: The complementary experiments performed in this study showed that different approaches needed to be taken in order to accurately assess the ability of an enzymatic cocktail to be efficient when it comes to lignocellulosic biomass degradation. The saccharification assay proved to be essential to validate the data obtained from both simple and complex substrates.

Sensopeptidomic Kinetic Approach Combined with Decision Trees and Random Forests to Study the Bitterness during Enzymatic Hydrolysis Kinetics of Micellar Caseins.

Protein hydrolysates are, in general, mixtures of amino acids and small peptides able to supply the body with the constituent elements of proteins in a directly assimilable form. They are therefore characterised as products with high nutritional value. However, hydrolysed proteins display an unpleasant bitter taste and possible off-flavours which limit the field of their nutrition applications. The successful identification and characterisation of bitter protein hydrolysates and, more precisely, the peptides responsible for this unpleasant taste are essential for nutritional research. Due to the large number of peptides generated during hydrolysis, there is an urgent need to develop methods in order to rapidly characterise the bitterness of protein hydrolysates. In this article, two enzymatic hydrolysis kinetics of micellar milk caseins were performed for 9 h. For both kinetics, the optimal time to obtain a hydrolysate with appreciable organoleptic qualities is 5 h. Then, the influence of the presence or absence of peptides and their intensity over time compared to the different sensory characteristics of hydrolysates was studied using heat maps, random forests and regression trees. A total of 22 peptides formed during the enzymatic proteolysis of micellar caseins and influencing the bitterness the most were identified. These methods represent simple and efficient tools to identify the peptides susceptibly responsible for bitterness intensity and predict the main sensory feature of micellar casein enzymatic hydrolysates.

Principal Component Analysis from Mass Spectrometry Data Combined to a Sensory Evaluation as a Suitable Method for Assessing Bitterness of Enzymatic Hydrolysates Produced from Micellar Casein Proteins.

Enzymatic hydrolysis of food proteins generally changes the techno-functional, nutritional, and organoleptic properties of hydrolyzed proteins. As a result, protein hydrolysates have an important interest in the food industries. However, they tend to be characterized by a bitter taste and some off-flavors, which limit their use in the food industry. These tastes and aromas come from peptides, amino acids, and volatile compounds generated during hydrolysis. In this article, sixteen more or less bitter enzymatic hydrolysates produced from a milk protein liquid fraction enriched in micellar caseins using commercially available, food-grade proteases were subjected to a sensory analysis using a trained and validated sensory panel combined to a peptidomics approach based on the peptide characterization by reverse-phase high-performance liquid chromatography, high-resolution mass spectrometry, and bioinformatics software. The comparison between the sensory characteristics and the principal components of the principal component analysis (PCA) of mass spectrometry data reveals that peptidomics constitutes a convenient, valuable, fast, and economic intermediate method to evaluating the bitterness of enzymatic hydrolysates, as a trained sensory panel can do it.

Enhanced growth and ß-galactosidase production on Escherichia coli using oxygen vectors.

The addition of n-dodecane (between 1-3%) to the Escherichia coli fermentation broth in a mechanically agitated and aerated bioreactor revealed improved DO (dissolved oxygen) levels induced during fermentation which lead to an increase in biomass productivity and faster glucose consumption. The maximum values for enzyme activity (increased with 43% compared with the control) and k L a (the volumetric mass transfer coefficient) were obtained for the addition of 2% v/v n-dodecane in the bioreactor, due to the fact that oxygen limitation during the exponential growth phase of the bacterium can repress ß-galactosidase production. The oxygen vector addition increased the available dissolved oxygen and activated a redox-sensitive regulation and an elevated intracellular oxidative metabolism that lead to the enhancement in E. coli biomass accumulation and a more accurate protein folding of ß-galactosidase that would increase its activity. In addition to the experimental analysis, a complex model, developed using an improved version of Bacterial Foraging Algorithm and Artificial Neural Networks, was proposed, with a good average absolute value (6.2% in the training phase and 7.28% in the testing phase) between the process dynamic and the predictions generated by the model.

Enzymatic depolymerization of industrial lignins by laccase-mediator systems in 1,4-dioxane/water.

Lignin is the second most abundant polymer after cellulose in lignocellulosic biomass. Its aromatic composition and recalcitrant nature make its valorization a major challenge for obtaining low molecular weight aromatics compounds with high value-added from the enzymatic depolymerization of industrial lignins. The oxidation reaction of lignin polymer using laccases alone remains inefficient. Therefore, researches are focused on the use of a laccase-mediator system (LMS) to facilitate enzymatic depolymerization. Until today, the LMS system was studied using water-soluble lignin only (commercial lignins, modified lignins, or lignin model compounds). This work reports a study of three LMS systems to depolymerize the three major industrial lignins (organosolv lignin, Kraft lignin, and sodium lignosulfonate). We show that an enzymatic depolymerization of these lignins can be achieved by LMS using laccase from Trametes versicolor, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt as mediator and a cosolvent (25% of 1,4-dioxane) to enhance the solubilization of lignins.

Innovative microscale workflow from fungi cultures to Cell Wall-Degrading Enzyme screening.

This study aimed at developing a complete miniaturized high-throughput screening workflow for the evaluation of the Cell Wall-Degrading Enzyme (CWDE) activities produced by any fungal strain directly cultivated on raw feedstock in a submerged manner. In this study, wheat straw was selected as model substrate as it represents an important carbon source but yet poorly valorised to yield high added value products. Fungi were grown in a microbioreactor in a high-throughput (HT) way to replace the fastidious shaking flask cultivations. Both approaches were compared in order to validate our new methodology. The range of CWDE activities produced from the cultures was assayed using AZO-died and pNP-linked substrates in an SBS plate format using a Biomek FXp pipetting platform. As highlighted in this study, it was shown that the CWDE activities gathered from the microbioreactor cultivations were similar or higher to those obtained from shake flasks cultures, with a lower standard deviation on the measured values, making this new method much faster than the traditional one and suitable for HT CWDE production thanks to its pipetting platform compatibility. Also, the results showed that the enzymatic activities measured were the same when doing the assay manually or using the automated method.

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis.

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

Sustainable efficient way for opioid peptide LVV-h7 preparation from enzymatic proteolysis in a microfluidic-based reaction-extraction process with solvent recycling.

LVV-h7 (LVVYPWTQFR) is a bioactive peptide that can be obtained from blood as waste of food industry, more precisely from hemoglobin hydrolysis by pepsin. This opioid peptide belongs to the hemorphins family and have strong physiological effects that bring its use in pharmaceutics and various therapeutic treatments attractive, in particular for substituting its costly chemically synthetized analogous. Hemoglobin hydrolysis by pepsin generates a huge variety of peptides among whose LVV-h7 can be purified by liquid-liquid extraction (LLE). Herein, selective preparation of this peptide is proposed by a microfluidic-based continuous reaction-separation process. Hemoglobin hydrolysis in microreactor was firstly coupled to LVV-h7 LLE in octan-1-ol and then coupled to LVV-h7 back LLE in acidic water. This continuous process allowed to prepare pure LVV-h7, as confirmed by liquid chromatography and mass spectrometry. The microfluidic circuit also allowed octan-1-ol recycling in a closed loop, making this method more sustainable than similar biphasic batch process.

Ion-pairing separation of bioactive peptides using an aqueous/octan-1-ol micro-extraction system from bovine haemoglobin complex hydrolysates.

The ion-pair concept was applied on complex haemoglobin hydrolysates to extract two opioid peptides, LVV-haemorphin-7 and VV-haemorphin-7, in an aqueous/octan-1-ol micro-extraction system in the presence of alkyl-sulfonic acid as a surfactant agent and in relation to the haemorphin physico-chemical properties (charge, hydrophobicity). The effect of combined alkyl chain length/aqueous phase pH and the haem behaviour during the extraction, on the haemorphin recovery yield and enrichment has been determined. It has proved that transport over the organic phase is mediated by the alkyl-sulfonic acids, whatever be the aqueous phase pH. However, increasing both the alkyl chain length and the pH in the aqueous phase shows an haemorphin enrichment ratio increase but a recovery decrease of the extracted opioid peptides in the organic phase. Therefore, the best conditions to extract LVVh-7 and VVh-7 are the use of the octane-sulfonic acid at aqueous phase pH of 5 or 7 and the octane or the heptane-sulfonic acid with an aqueous phase pH of 5 or 7 respectively. In these conditions, a partition coefficient of 1.64 and 1.60 respectively for LVVh-7 and VVh-7 are obtained and represent about 40 times that acquired without agent.

Continuous production of a peptidic fraction containing the intermediate opioid peptide LVV-haemorphin-7 (LVVh-7) by peptic hydrolysis of bovine haemoglobin in a continuous membrane reactor.

Peptic hydrolysis of native bovine haemoglobin at pH 3 yields the LVV-haemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe; LVVh-7) opioid peptide corresponding to the residues-31-40 fragment of the beta-chain of haemoglobin. This peptide is intermediate in the course of batch hydrolysis and is rapidly degraded. Indeed, it shows an optimum at 3% degree of hydrolysis (i.e. 2 min of reaction time). The hydrolysis was carried out in a continuous membrane reactor with a space time (ratio of the flux to the reactor volume) set to 2 min (corresponding to optimum LVVh-7 production). This process allows the continuous production of a constant fraction of intermediate peptides containing LVVh-7 for 48 min.

Kinetic study of the appearance of an anti-bacterial peptide in the course of bovine haemoglobin peptic hydrolysis.

The kinetics of the alpha (1-23) peptide, which is the first anti-bacterial peptide to be isolated from a haemoglobin hydrolysate, was studied in the course of peptic hydrolysis at pH 4.5 and 23 degrees C in an homogeneous-phase system. A one-step reversed-phase HPLC coupled with photodiode array detector method was applied to identify and isolate this anti-bacterial peptide. The kinetics of peptide appearance were investigated in acetate buffer alone and in urea as a haemoglobin-denaturing agent. Two different mechanisms, 'one-by-one' for native haemoglobin hydrolysis and 'zipper' for denatured haemoglobin hydrolysis, were observed. Whatever the haemoglobin state, native or denatured, and whatever the hydrolytic mechanism, one-by-one or zipper, the anti-bacterial alpha (1-23) peptide is a transient peptide. To prepare the alpha (1-23) peptide it is suitable to hydrolyse haemoglobin in the presence of urea at a corrected degree of hydrolysis (DH(c)) of 13.5%. The amount of peptide produced in the presence of urea was twice as high as for the hydrolysis of native haemoglobin. The yields of alpha (1-23) peptide with respect to haemoglobin at the optimal DH(c) values were 55 and 25% respectively.