Enzyme Loading Research Articles

Enzymatic production of xylose esters using degummed soybean oil fatty acids following a hydroesterification strategy

The enzymatic synthesis of xylose fatty acid esters was carried out in two steps: hydrolysis of degummed soybean oil (DSO) and esterification of the produced and purified free fatty acids (FFA) with xylose. Different lipases immobilized on a hydrophobic support were evaluated in the hydrolysis of DSO and an experimental design was used to optimize the reaction. Eversa® Transform 2.0 (EV-Purolite) yielded 93% conversion after 6 h using biocatalyst load of 6.53 wt%, water/oil mass ratio of 5.5 and temperature of 48.4 °C. High operational stability for EV-Purolite was found using a sequential batch strategy and the possibility of concentrating glycerol. In the esterification stage, lipase from Thermomyces lanuginosus (TLL-Purolite) and lipase B from Candida antarctica showed better performance than lipase from porcine pancreas and EV-Purolite. About 17% of FFAs (0.85 molecules of FAA per xylose molecule) was consumed using an enzyme load of 5 wt% of TLL-Purolite and FFA/xylose molar ratio of 5. Sequential esterification batches showed low operational stability of TTL-Purolite due to the desorption of TLL from the support. The final mixture of the reaction medium containing xylose fatty acid esters showed emulsifying properties similar to those of commercial surfactant.

Mild γ-Butyrolactone/Water Pretreatment for Highly Efficient Sugar Production from Corn Stover.

In this study, γ-butyrolactone/water (GBL/H2O) was explored as a mild, efficient, and cost-effective binary solvent pretreatment to enhance hydrolyzability of corn stover (CS). Key pretreatment parameters-reaction time, temperature, and H2SO4 concentration-were systematically investigated for their effects on the physicochemical properties of CS. Specifically, increased temperature and acid concentration significantly decreased cellulose crystallinity (from 1.39 for untreated CS to 1.04 for CS pretreated by GBL/H2O with 100 mM H2SO4 at 120°C for 1h) and promoted lignin removal (47.3% for CS pretreated by GBL/H2O with 150 mM H2SO4 at 120°C for 1h). Acknowledging the cellulase's limited hydrolysis efficiency, a dual-enzyme scheme using a low cellulase dosage (10 FPU/g) supplemented with β-glucosidase or xylanase was tested, enhancing hydrolysis of CS pretreated under low temperature-long duration and high temperature-short duration conditions, respectively. Optimum sugar release was obtained from CS pretreated with GBL/H2O and 150 mM H2SO4 at 120°C for 1h, achieving 98% glucan and 82.3% xylan conversion, compared with 53.9% and 17% of glucan and xylan conversion from untreated CS. GBL/H2O pretreatment outperformed other binary systems in literature, achieving the highest sugar conversions with lower enzyme loading. These results highlight the potential of GBL/H2O pretreatment for efficient biomass conversion, contributing to the goals of the green economy.

Lipase from Rhizomucor miehei onto home-made hydrophobic polymers: Stable and efficient biocatalysts

Immobilized lipases are biotechnological spotlights as tools to improve and enhance enzyme applicability. Here, lipase from Rhizomucor miehei (RML) was immobilized onto core/shell supports, named poly(methyl methacrylate) (PMMA/PMMA); poly(methyl methacrylate) copolymerized with divinylbenzene (PMMA-co-DVB/PMMA-co-DVB); and polystyrene copolymerized with divinylbenzene (PS-co-DVB/PS-co-DVB), and on a commercial non-core/shell porous support (polypropylene - Accurel MP 1000). Different enzyme loadings were evaluated (400 U.g−1, 500 U.g−1, 600 U.g−1, 4000 U.g−1, and 6000 U.g−1), and the immobilized biocatalysts were used in hydrolysis (using p-nitrophenyl laurate (p-NPL) and p-nitrophenyl butyrate (p-NPB) as substrates) and in esterification reactions (using oleic acid and ethanol as substrates). The influence of pH, temperature, and buffer concentration in the hydrolysis of p-NPL was also investigated. The immobilized biocatalysts were compared to free RML and/or to commercially immobilized RML (RM-IM) and/or to Novozym 435. RML interacted differently with each support, being RML-PMMA-co-DVB/PMMA-co-DVB and RML-PMMA/PMMA the biocatalysts with the highest enzymatic activities (hydrolytic activity: >400 U.g−1, using p-NPL; esterification activity: 25,532 U.g−1). Immobilization improved the thermal and pH tolerance and buffer concentration tolerance of RML. Both free RML and the immobilized biocatalysts exhibited their maximum activity at pH 7, with the exception of RML-Accurel MP 1000, which displayed optimal performance at pH 9.0. In addition, RML-PS-co-DVB/PS-co-DVB demonstrated increased activity at 70 °C. Notably, RML-PMMA/PMMA and RML-PMMA-co-DVB/PMMA-co-DVB showcased a maximum activity of 120 U/g, and this was achieved at different temperatures, 30°C and 50°C, respectively. This diverse library of immobilized biocatalysts shows promising potential for application in various industrial processes.

Lipase-Catalyzed Preparation and Optimization of Structured Phosphatidylcholine Containing Nervonic Acid.

This study investigated the incorporation of nervonic acid into the chemical structure of phosphatidylcholine via a lipase-catalyzed acidolysis reaction to obtain a functional phospholipid. Lipase immobilization was conducted, and Amberlite XAD7-HP was selected as a carrier to immobilize phospholipase A1 (PLA1) for subsequent experiments. The main acidolysis reaction parameters, including enzyme load, substrate ratio, temperature, and water content, were studied against the reaction time. The optimum reaction conditions obtained were enzyme load, 20%; reaction temperature, 55 °C; water content, 1%; and reaction time, 9 h. The maximum incorporation of nervonic acid into phosphatidylcholine was 48 mol%, with PC recovery at 61.6 mol%. The positional distribution of structured phosphatidylcholine shows that nervonic acid was found in the sn-1 position due to enzyme specificity and in the sn-2 position, possibly due to acyl migration.

Immobilization of fungal mycelial and laccase from Trametes hirsuta EDN082 in alginate-cellulose beads and its use in Remazol Brilliant Blue R dye decolorization

In this study, laccase from Trametes hirsuta EDN 082 was immobilized onto alginate-cellulose beads (CAC-L) and employed for the decolorization of Remazol Brilliant Blue R (RBBR) dye. The characterization and decolorization efficiency of the CAC-L beads were compared to immobilized laccase without cellulose (CA-L), immobilized fungal mycelia (CAC-FM), alginate-cellulose (CAC), and alginate beads (CA), and examined using light microscopy, FTIR, and SEM. The results showed that laccase was successfully immobilized onto alginate-cellulose beads with an enzyme loading of 1.9 U/mL and an immobilization efficiency of 97.7 %. The optimal decolorization (89.2 %) was achieved with 2 g CAC-L beads and 1.9 U/mL laccase activity at 40 °C and pH 5 for 6 h of incubation. Furthermore, the decolorization extent was >70–80 % after five cycles. The phytotoxicity test using Vigna radiata and Artemia salina revealed biodetoxification, indicating that CAC-L beads could be used as a biocatalyst for dye removal.

Reaction kinetics involved in esterification between the fatty acids in castor oil and furfuryl alcohol

Kinetics of esterification reaction between furfuryl alcohol (FA) and castor oil fatty acid (COFA) using immobilized Candida antartica lipase B in a solvent free system is investigated in present study. The well-known ping-pong bi bi mechanism with two-substrate inhibition has been confirmed to correctly describe the governing reaction pathway. Subsequently, the rate equation was used to evaluate pertinent kinetic parameters. Parametric optimization study was also carried out with respect to temperature, molar ratio of reactants and enzyme loading to maximize the equilibrium yield of COFA ester. Furthermore, functional variations of the kinetic parameters, which include Michaelis-Menten constants and inhibition coefficients, were evaluated with respect to three process parameters. Corresponding inferences are of high utility, specifically, in cases where the global optimum is beyond the scope of the existing production facility in any industrial platform.

Deciphering the immobilization of lipases on hydrophobic wrinkled silica nanoparticles

In this work, the adsorption of Candida antarctica B (CALB) and Rhizomucor miehei (RML) lipases into hydrophobic wrinkled silica nanoparticles (WSNs) is investigated. WSNs are hydrophobized by chemical vapor deposition. Both proteins are homogeneously distributed inside the pores of the nanoparticles, as confirmed by Transmission Electron Microscopy and Energy Dispersive X-ray measurements. The maximum enzyme load of CALB is twice that obtained for RML. Fourier Transform Infrared Spectroscopy confirms the preservation of the enzyme secondary structure after immobilization for both enzymes. Adsorption isotherms fit to a Langmuir model, resulting in a binding constant (KL) for RML 4.5-fold higher than that for CALB, indicating stronger binding for the former. Kinetic analysis reveals a positive correlation between enzyme load and RML activity unlike CALB where activity decreases along the enzyme load increases. Immobilization allows for enhancing the thermal stability of both lipases. Finally, CALB outperforms RML in the hydrolysis of ethyl-3-hydroxybutyrate. However, immobilized CALB yielded 20 % less 3-HBA than free lipase, while immobilized RML increases 3-fold the 3-HBA yield when compared with the free enzyme. The improved performance of immobilized RML can be explained due to the interfacial hyperactivation undergone by this lipase when immobilized on the superhydrophobic surface of WSNs.

Lipase in-situ immobilized in covalent organic framework: Enzymatic properties and application in the preparation of 1, 3-dioleoyl-2-palmitoylglycerol

In this study, the critical importance of designing an appropriate immobilized carrier and method for free lipase to ensure exceptional biological catalytic activity and stability was emphasized. Covalent organic frameworks (COF-1) were synthesized as a novel porous carrier with an azine structure (-CN-NC-) through the condensation of hydrazine hydrate and benzene-1,3,5-tricarbaldehyde at room temperature. Simultaneously, Rhizomucor miehei lipase (RML) was immobilized within the COF-1 carrier using an in-situ aqueous phase method. Characterization of the carrier and RML@COF-1 and evaluation of the lipase properties of RML and RML@COF-1 through p-Nitrophenyl palmitate hydrolysis were conducted. Additionally, application in the synthesis of 1, 3-dioleoyl-2-palmitoylglycerol (OPO) was explored. The results showed that RML@COF-1 exhibited a high enzymatic loading of 285.4 mg/g. Under 60℃ conditions, the activity of RML@COF-1 was 2.31 times higher than that of free RML, and RML@COF-1 retained 77.25% of its original activity after 10 cycles of repeated use, indicating its excellent thermal stability and repeatability. Under the optimal conditions (10%, 1:8 PPP/OA, 45℃, 5 h), the yield of OPO reached 47.35%, showcasing the promising application prospects of the novel immobilized enzyme synthesized via in-situ aqueous phase synthesis for OPO preparation.

Modeling and design of fed-batch strategies for achieving 255 g/L sugar concentration from high-solid enzymatic hydrolysis of pretreated corn stover

Achieving high sugar concentrations through enzymatic hydrolysis of pretreated lignocellulose with low enzyme loadings is crucial for reducing the costs of lignocellulosic biorefineries. However, the adverse effects of the “solid effect” pose essential challenges in this regard. In this study, the objective is to improve the sugar concentrations through enzymatic hydrolysis of Densifying Lignocellulose with Ca(OH)2 or H2SO4 followed by Autoclave pretreated corn stover (DLCA(ch)-CS or DLCA (sa)-CS) at 40 % w/w solid loading through kinetic analysis and a statistically designed fed-batch feeding strategy. Initially, the hydrolysis kinetics of different pretreated biomass were analyzed using fractal kinetic modeling, aiming to elucidate the reasons for the observed differences in hydrolysis. Furthermore, leveraging the kinetic differences, the optimal initial solid loading was determined, and statistical models were employed to optimize feeding modes for enzymatic hydrolysis. The result revealed that compared with H2SO4, Ca(OH)2 serves as an better catalyst for improving the hydrolysability of biomass, enhancing cellulose accessibility to cellulases and reduce the mass transfer rate during enzymatic hydrolysis. Interestingly, the optimal feeding mode for less digestible substrate (DLCA(sa)-CS) is different from the more digestible substrate (DLCA(ch)-CS). Notably, by using the optimized fed-batch feeding mode, the sugar concentration can reach a maximum of 255 g/L, with glucan conversion of 89.8 %, at low enzyme loading (10 mg protein/g glucan). This is the highest total sugar concentration achieved through 72 h enzymatic hydrolysis with low enzyme loadings using a fed-batch strategy. This approach proves highly effective in designing fed-batch strategies for pretreated biomass and has great potential to serve as a universal tool for designing suitable feeding schemes for various bionconversion process.

EnZolv delignification of cotton spinning mill waste and optimization of process parameters using response surface methodology (RSM)

BackgroundEnZolv is a novel enzyme-based, eco-friendly biomass pretreatment process that has shown great potential in the field of textile engineering and biotechnology. It employs laccase from Hexagonia hirta MSF2 and 2% ethanol in the process of delignification. The process is designed to evaluate optimal conditions to remove lignin and other impurities from cotton spinning mill waste (CSMW), without compromising the quality and strength of the fibers. CSMW is a low-cost and readily available source of cellulose, making it an ideal candidate for delignification using EnZolv. By optimizing the pretreatment conditions and harnessing the potential of enzymatic delignification, this research aims to contribute to more sustainable and efficient ways of utilizing lignocellulosic biomass in various industries for the production of biochemical and bioproducts.ResultsThe present study emphasizes the EnZolv pretreatment in the delignification of cotton spinning mill wastes irrespective of the cellulose content. EnZolv process parameters such as, moisture content, enzyme load, incubation time, incubation temperature, and shaking speed were optimized. Under pre-optimized conditions, the percent lignin reduction was 61.34%, 61.64%, 41.85%, 35.34%, and 35.83% in blowroom droppings (BD), flat strips (FS), lickerin fly (LF), microdust (MD) and comber noils (CN), respectively. Using response surface methodology (RSM), the statistically optimized EnZolv pretreatment conditions showed lignin reduction of 59.16%, 62.88%, 48.26%, 34.64%, and 45.99% in BD, FS, LF, MD, and CN, respectively.ConclusionTraditional chemical-based pretreatment methods often involve harsh chemicals and high energy consumption, which can have detrimental effects on the environment. In contrast, EnZolv offers a greener approach by utilizing enzymes that are biodegradable and more environmentally friendly. The resulting fibers from EnZolv treatment exhibit improved properties that make them suitable for various applications. Some of the key properties include enhanced cellulose recovery, reduced lignin content, and improved biophysical and structural characteristics. These improvements can contribute to the fiber's performance and processability in different industries and future thrust for the production of cellulose-derived and lignin-derived bioproducts.Graphical

Influence of Cellulose-Cellulase Enzyme Digestibility in the Production of Glucose from Lignocellulosic Biomass Waste

A comprehensive understanding of the factors and ability of cellulase enzyme to completely hydrolyze the structural lignocellulosic biomass has been a major research focus over the years. A comparative analysis of cellulose-cellulase digestibility of Kraft-pretreated sawdust from twenty different Nigeria wood wastes was carried out with increasing enzyme (Trichorderma viride) loading of 0.4 mg cm−3, 1.0 mg cm−3, 2.0 mg cm−3, and 4.0 mg cm−3 at constant substrate application of 10 mg cm−3 biomass concentration, temperature of 40°C and 2 h incubation period. This was carried out in order to establish the optimum cellulase-cellulose ratio for maximum biodegradation of the lignocellulosic biomass to produce glucose, a fermentable sugar. The influence of the cellulose-cellulase interaction from 0.4 mg cm−3 to 1.0 mg cm−3 enzyme treatment resulted in 144% increase in glucose yield from E. suaveolen and 121% from S. pustlatas. while 2.0 mg cm−3 cellulase concentration gave 674%, 641% and 617% increase from E.suaveolen, H. ciliate and A. germinans respectively. A general trend of increasing sugar formation was observed with an increasing enzyme loading due to enhanced cellulose accessibility by the cellulase enzyme leading to effective saccharification of the lignocellulosic materials for more sugar production. However, further increase of 4.0 mg cm−3 enzyme concentration failed to produce a commiserate amount of fermentable sugar.

Octadecyl and sulfonyl modification of diatomite synergistically improved the immobilization efficiency of lipase and its application in the synthesis of pine sterol esters.

Phytosterols usually have to be esterified to various phytosterol esters to avoid their disadvantages of unsatisfactory solubility and low bioavailability. The enzymatic synthesis of phytosterol esters in a solvent-free system has advantages in terms of environmental friendliness, sustainability, and selectivity. However, the limitation of the low stability and recyclability of the lipase in the solvent-free system, which often requires a relatively high temperature to induce the viscosity, also increased the industrial production cost. In this context, a low-cost material, namely diatomite, was employed as the support in the immobilization of Candida rugosa lipase (CRL) due to its multiple modification sites. The Fe3 O4 was also then introduced to this system for quick and simple separation via the magnetic field. Moreover, to further enhance the immobilization efficiency of diatomite, a modification strategy which involved the octadecyl and sulfonyl group for regulating the hydrophobicity and interaction between the support and lipase was successfully developed. The optimization of the ratio of the modifiers suggested that the -SO3 H/C18 (1:1.5) performed best with an enzyme loading and enzyme activity of 84.8mg·g-1 and 54 U·g-1 , respectively. Compared with free CRL, the thermal and storage stability of CRL@OSMD was significantly improved, which lays the foundation for the catalytic synthesis of phytosterol esters in solvent-free systems. Fortunately, a yield of 95.0% was achieved after optimizing the reaction conditions, and a yield of 70.0% can still be maintained after six cycles.

A novel photo-enzyme platform based on non-metallic modified carbon nitride for removal of bisphenol A in water

A nonmetallic composite photocatalyst with 2D/2D structure was prepared by hydrothermal in-situ polymerization and used for the immobilization of cytochrome C (Cyt c). The photo-enzyme coupling system has a very high enzyme load, which can reach 528.29 mg g−1 after optimization. Compared with free Cyt c, Cytc/PEDOT/CN showed better enzymatic activity, stability and catalytic efficiency. Even after being stored at 100 °C for 60 min, the enzyme activity remained at 49.42 % and remained at 57.89 % after 8 cycles. Moreover, Cytc0.5/PEDOT3/CN showed excellent photocatalytic degradation performance in the degradation experiment of bisphenol A (BPA), reaching 68.22 % degradation rate within 60 min, which was 3.9 times higher than that of pure g-C3N4 and 1.61 times higher than that of pure PEDOT3/CN. This study shows that the introduction of conductive polymers is of great significance to the photo-enzyme coupling system and provides a new strategy for the treatment of phenol-containing wastewater.

Enzymatic hydrolysates of Agave tequilana bagasse pretreated with protic ionic liquids: Saccharification efficiency and hydrogen production

This work demonstrated for the first time that hydrogen (H2) can be generated from Agave tequilana bagasse (ATB) after ionic liquid (IL) pretreatment up to 111.1 L H2/kg hydrolyzed biomass in only 20 h. It is shown that the low-cost protic IL [EOA][OAc] (ethanolamine acetate) can effectively pretreat ATB at two different scales (bench and lab) using 2 and 250 g (dry basis) at 20% solids loading and 160 °C for 90 min resulting in significant structural changes followed by FTIR spectroscopy. The lignin content of the pretreated ATB was reduced by 28.6% after lab-scale pretreatment which ultimately improved the sugar yield with the different saccharification strategies (batch vs. fed-batch). Up to 0.643 g TS (total sugars)/g ATB were obtained in a fed-batch strategy with fixed enzymatic loading, representing an enhancing 2.8-fold when compared to the untreated biomass. Models of enzymatic saccharifications were included for both, untreated and pretreated ATB, in terms of TS concentration (g/L) and fiber length (mm) to confirm differences in accessibility and availability of both substrates. The results successfully revealed that 1.1 kg or 14 m3 of H2 can be produced from 100 kg of dry ATB after pretreatment with [EOA][OAc] indicating the feasibility of improved sugar and H2 yields where either a low-cost pretreatment agent or an adequate saccharification strategy contributes to the biorefinery scheme.

Hollow Hierarchical Porous and Antihydrolytic Spherical Zeolitic Imidazolate Frameworks for Enzyme Encapsulation and Biocatalysis.

The creation of a new metal-organic framework (MOF) with a hollow hierarchical porous structure has gained significant attention in the realm of enzyme immobilization. The present work employed a novel, facile, and effective combinatorial technique to synthesize modified MOF (N-PVP/HZIF-8) with a hierarchically porous core-shell structure, allowing for the preservation of the structural integrity of the encapsulated enzyme molecules. Scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, confocal laser scanning microscopy, and other characterization tools were used to fully explore the changes of morphological structure and surface properties in different stages of the preparation of immobilization enzyme CRL-N-PVP/HZIF-8, thus showing the superiority of N-PVP/HZIF-8 as an enzyme immobilization platform and the logic of the immobilization process on the carrier. Additionally, the maximum enzyme loading was 216.3 mg mL-1, the relative activity of CRL-N-PVP/HZIF-8 increased by 15 times compared with the CRL@ZIF-8 immobilized in situ, and exhibited quite good thermal, chemical, and operational stability. With a maximal conversion of 88.8%, CRL-N-PVP/HZIF-8 demonstrated good catalytic performance in the biosynthesis of phytosterol esters as a proof of concept. It is anticipated that this work will offer fresh concepts from several perspectives for the creation of MOF-based immobilized enzymes for biotechnological uses.

Exploring the use of supercritical carbon dioxide in enzymatic hydrolysis of cellulosic substrate

Enzymes are critical components for production of fermentable sugars in lignocellulosic biorefineries. Their activity could be influenced by various reaction parameters. Herein, a formulated cellulolytic enzyme cocktail was immobilized into mesoporous aerogel material and used under high pressure environment of supercritical CO2 (scCO2). The cocktail was immobilized at different enzyme loads (36.0 to 144.0 mg enzyme/g aerogel) using sol-gel entrapment method and scCO2 drying. The immobilized cocktail at 144.0 mg/g aerogel enzyme load retained a residual yield of >50.0 % after fourth cycle of reuse compared to first cycle under atmospheric pressure. Sugar yield by the cocktail aerogel was below 10 wt% in second cycle under scCO2 due to enzyme leaching. Additionally, surface area and porous volume of reused aerogels was decreased over the reuse cycles. The study suggests that scCO2 could be employed for aerogel preparation after optimizing the immobilization process for pure enzymes rather than crude enzymes.

Isolation, screening and optimization of alkaliphilic cellulolytic fungi for production of cellulase

Abstract This study concerns with the production and partial characterization of alkaline cellulase from alkaliphilic cellulolytic (AC) fungi isolated fromsoil in Perlis, Malaysia. The best fungi strain was selected on the basis of producing the highest cellulase at high pH conditions. Cellulase from the selected fungi strain was further characterized under saccharification but varies in operating parameters. Finally, the kinetic model describing the growth of the AC fungi strain was studied by employing the logistic model. Among the tested fungi strains, Basidiomycetes strain (BK1) showed high potentiality for the production of maximum alkaline cellulase production at pH 9 after 72 h of incubation at 30°C containing 6 g·L−1 carboxyl methyl cellulose. The saccharification process showed that the enzyme favour high alkaline condition and proves thermotolerant properties, while 15% (v/v) enzyme loading and 1% substrate concentration recorded the highest glucose production at about 1.2–1.3 mg·mL−1. The novelty of the study is to identify and optimize a unique indigenous fungi that emit alkaliphilic cellulase as alternative usage in biotechnology industries due to its capacity to adapt to the extreme conditions of specific industrial processes. There are revolutionary options for use in biotechnological businesses that involve high pH and therefore have substantial biotechnological promise.

The Effects of Buffer Nature on Immobilized Lipase Stability Depend on Enzyme Support Loading

The lipases from Thermomyces lanuginosus (TLL) and Candida antarctica (B) (CALB) were immobilized on octyl-agarose beads at 1 mg/g (a loading under the capacity of the support) and by overloading the support with the enzymes. These biocatalysts were compared in their stabilities in 10 mM of sodium phosphate, HEPES, and Tris-HCl at pH 7. Lowly loaded CALB was more stable than highly loaded CALB preparation, while with TLL this effect was smaller. Phosphate was very negative for the stability of the CALB biocatalyst and moderately negative using TLL at both loadings. The stability of the enzymes in HEPES and Tris-HCl presented a different response as a function of the enzyme loading (e.g., using lowly loaded CALB, the stabilities were similar in both buffers, but it was clearly smaller in HEPES using the highly loaded biocatalysts). Moreover, the specific activity of the immobilized enzymes versus p-nitrophenol butyrate, triacetin and R- or S-methyl mandelate depended on the buffer, enzyme loading, and interaction between them. In some cases, almost twice the expected activity could be obtained using highly loaded octyl-CALB, depending on the buffer. A co-interaction between the effects on enzyme activity and the specificity of support enzyme loading and buffer nature was detected.

Dual assistance of surfactants in glycerol organosolv pretreatment and enzymatic hydrolysis of lignocellulosic biomass for bioethanol production

This study investigated an innovative strategy of incorporating surfactants into alkaline-catalyzed glycerol pretreatment and enzymatic hydrolysis to improve lignocellulosic biomass (LCB) conversion efficiency. Results revealed that adding 40 mg/g PEG 4000 to the pretreatment at 195 °C obtained the highest glucose yield (84.6%). This yield was comparable to that achieved without surfactants at a higher temperature (240 °C), indicating a reduction of 18.8% in the required heat input. Subsequently, Triton X-100 addition during enzymatic hydrolysis of PEG 4000-assisted pretreated substrate increased glucose yields to 92.1% at 6 FPU/g enzyme loading. High-solid fed-batch semi-simultaneous saccharification and co-fermentation using this dual surfactant strategy gave 56.4 g/L ethanol and a positive net energy gain of 1.4 MJ/kg. Significantly, dual assistance with surfactants rendered 56.3% enzyme cost savings compared to controls without surfactants. Therefore, the proposed surfactant dual-assisted promising approach opens the gateway to economically viable enzyme-mediated LCB biorefinery.

Cheese and Yogurt By-Products as Valuable Ingredients for the Production of Prebiotic Oligosaccharides

The growing global market of dairy products has led to the need for alternative approaches regarding whey valorization, which is the primary by-product of cheese and strained yogurt production. In this context, prebiotic galactooligosaccharides can be produced enzymatically from whey using commercially available β-galactosidases. A comparative study was conducted to assess the production of galactooligosaccharides from sweet and acid whey, thereby employing two commercial β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis. The study considered the initial lactose content and enzyme load as variables. The maximum yields of galactooligosaccharides in concentrated sweet whey (15% w/v initial lactose) and raw acid whey (3.1% w/v initial lactose) reached 34.4 and 14.7% with lactase from Kluyveromyces lactis (0.13 U/mL), respectively. The corresponding galactooligosaccharide yields for lactase from Aspergillus oryzae were equal to 27.4 and 24.8% in the most concentrated sweet and acid whey, respectively, using enzyme loads of 2 U/mL in sweet whey and 1 U/mL in acid whey. Concerning the profile of the produced galactooligosaccharides, the Kluyveromyces lactis lactase hydrolyzed lactose more rapidly and resulted in higher levels of allolactose and lower levels of 6-galactosyl-lactose, compared to the lactase from Aspergillus oryzae, and achieved in both cases a polymerization degree of up to six.