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Cellobiose lipids (CBLs) are glycolipid biosurfactants that are widely applicable across diverse industries.CBLs have remarkable gelling, surface-active, and antifungal properties, making them ideal compounds for developing novel biotechnologies (e.g., biomaterials, detergents, and emulsifiers) with commercial potential.Various production methodologies have been developed via an interplay between producing organisms, media, and production approaches, each with its unique advantages and limitations toward scalability.Significant exploration is still required regarding downstream processing of CBLs because an optimised and scalable downstream technique is needed to complete the biotechnological cycle of CBL production.Finally, the industrialisation of CBL production requires the implementation of techno-economic and life-cycle assessments, which are currently understudied. Cellobiose lipids (CBLs) are glycolipid biosurfactants that have garnered attention due to their potential applications in diverse industries. Here, we review the current state of CBL research, from production and purification, to the potential applications of CBLs. We elucidate CBL functionality and consider some commercial applications, as well as how operating conditions (e.g., media and organism, or production approaches) impact productivity. Methodologies based on enzymatic synthesis or postproduction chemical modification of CBL variants are also presented. Given the importance of purity in current CBL applications, we discuss CBL separation and purification techniques. Finally, we highlight the importance of techno-economic and life-cycle assessments for the industrialisation of CBLs, while suggesting potential future routes for investigation. Cellobiose lipids (CBLs) are glycolipid biosurfactants that have garnered attention due to their potential applications in diverse industries. Here, we review the current state of CBL research, from production and purification, to the potential applications of CBLs. We elucidate CBL functionality and consider some commercial applications, as well as how operating conditions (e.g., media and organism, or production approaches) impact productivity. Methodologies based on enzymatic synthesis or postproduction chemical modification of CBL variants are also presented. Given the importance of purity in current CBL applications, we discuss CBL separation and purification techniques. Finally, we highlight the importance of techno-economic and life-cycle assessments for the industrialisation of CBLs, while suggesting potential future routes for investigation.

Cellobiose lipids (CBLs) are a class of glycolipid biosurfactants produced by various fungal strains. These compounds have gained significant interest due to their surface-active and antifungal properties, which are comparable to traditional synthetic surfactants and antimicrobials. Despite their potential applicability in various cosmetic, pharmaceutical, and agricultural formulations, significantly less research has been focused on their production and purification in comparison to other glycolipid biosurfactants, such as mannosylerythritol lipids (MELs) and sophorolipids. Hence, this work proposes the development of a bioprocess that involves the microbial production and high-level chromatographic purification of CBLs from a submerged culture of Ustilago maydis DSM 4500. After a highly purified CBL product was obtained, the factors affecting the production of this glycolipid were investigated. It was demonstrated that U. maydis DSM 4500 produces a specific structural variant of CBLs at a concentration of 1.36 g/L on an optimized the growth medium. Also, it was established that when the C/N ratio was decreased, the CBL titer increased by 2.3-fold. Furthermore, supplementing the culture with ZnSO 4 at a concentration of 0.04 mg/L further increased CBL concentration to 4.95 g/L, representing the highest CBL titer achieved in a single-stage bioprocess to date. This study developed a methodology for utilizing U. maydis as a high-level CBL producer, which could challenge other familiar CBL producers, such as Sporisorium scitamineum and Cryptococcus humicola .

Product inhibition by cellobiose decreases the rate of enzymatic cellulose degradation. The optimal reaction conditions for two Emericella (Aspergillus) nidulans-derived cellobiohydrolases I and II produced in Pichia pastoris were identified as CBHI: 52 °C, pH 4.5–6.5, and CBHII: 46 °C, pH 4.8. The optimum in a mixture of the two was 50 °C, pH 4.9. An almost fourfold increase in enzymatic hydrolysis yield was achieved with intermittent product removal of cellobiose with membrane filtration (2 kDa cut-off): The conversion of cotton cellulose after 72 h was ~19 % by weight, whereas the conversion in the parallel batch reaction was only ~5 % by weight. Also, a synergistic effect, achieving ~27 % substrate conversion, was obtained by addition of endo-1,4-β-D-glucanase. The synergistic effect was only obtained with product removal. By using pure, monoactive enzymes, the work illustrates the profound gains achievable by intermittent product removal during cellulose hydrolysis.

The yeast Trichosporon porosum suppresses growth of ascomycetes and basidiomycetes belonging to 52 genera. It is due to secretion of a thermostable fungicidal agent. The suppression was maximal at pH 3.5-4.0. Fungicidal preparation obtained from the culture broth was shown to be a mixture of cellobiosides of dihydrodecane acid with different degree of acetylation of cellobiose residue. The preparation caused the death of Candida albicans and Filobasidiella neoformans cells in the concentrations of 0.2 and 0.03 mM, respectively.

Cellobiose, a disaccharide, is a valuable product that can be obtained from cellulose hydrolysis. In this study, a simple methodology is presented to enhance the production and improve the selectivity of cellobiose during enzymatic hydrolysis of cellulose. The approach consisted of a multistage removal of filtrate via vacuum filtration and resuspension of the retentate. By this process, the remaining solid was further hydrolyzed without additional enzyme loading. Compared to the continuous hydrolysis process, the production of cellobiose increased by 45%. Increased selectivity of cellobiose is due to the loss of β-glucosidases in the filtrate, while enhanced productivity is likely due to mitigated product inhibition.

Four ion-exchange resins (Amberlite IRA 900, IRA 400, IRA 96, and IRA 67) were employed for lactic acid recovery from simultaneous saccharification and fermentation (SSF) media. The best resins (Amberlite IRA 900 and IRA 400) were assayed for capacity, regenerant consumption, percentage of lactic acid recovery, and product concentration. Almost quantitative lactic acid recoveries at constant capacities were achieved in four sequential loading/regeneration cycles. A strong-base resin (Amberlite IRA 400) was selected for intermittent lactic acid separation in a typical SSF process, in which pretreated wood was saccharified by cellulases in the presence of Lactobacillus delbrueckii. The dynamics of lactic acid generation and lactic acid recovery were established.

Thermoanaerobacterium aotearoense P8G3#4 produced β-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding β-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (β/α)₈ TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na⁺ or K⁺. The enzyme showed strong ability to hydrolyze cellobiose with a K ₘ and V ₘₐₓ of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K ᵢ of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min^sup -1^ mg^sup -1^ for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 gl^sup -1^ and fructose at 47.5 gl^sup -1^ were produced from 500 gl^sup -1^ glucose at pH 7.5 and 75°C over 3 h by the enzyme.[PUBLICATION ABSTRACT]

Cellulases play important roles in the dietary fibre digestion in pigs, and have multiple industrial applications. The porcine intestinal microbiota display a unique feature in rapid cellulose digestion. Herein, we have expressed a cellulase gene, p4818Cel5_2A , which singly encoded a catalytic domain belonging to glycoside hydrolase family 5 subfamily 2, and was previously identified from a metagenomic expression library constructed from porcine gut microbiome after feeding grower pigs with a cellulose-supplemented diet. The activity of purified p4818Cel5_2A was maximal at pH 6.0 and 50 °C and displayed resistance to trypsin digestion. This enzyme exhibited activities towards a wide variety of plant polysaccharides, including cellulosic substrates of avicel and solka-Floc ® , and the hemicelluloses of β-(1 → 4)/(1 → 3)-glucans, xyloglucan, glucomannan and galactomannan. Viscosity, reducing sugar distribution and hydrolysis product analyses further revealed that this enzyme was a processive endo-β-(1 → 4)-glucanase capable of hydrolyzing cellulose into cellobiose and cellotriose as the primary end products. These catalytic features of p4818Cel5_2A were further explored in the context of a three-dimensional homology model. Altogether, results of this study report a microbial processive endoglucanase identified from the porcine gut microbiome, and it may be tailored as an efficient biocatalyst candidate for potential industrial applications.

β-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa , and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10–15 mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100 mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.