Enzyme Reusability Research Articles

Second-generation biorefineries: single platform for the conversion of lignocellulosic wastes to environmentally important biofuels.

The continuously increasing demands for various fossil fuels to achieve the day-to-day needs of the human population are growing and causing adverse effects on the environment and leading to the depletion of their natural resources. To overcome such drastic problems and minimize the production of greenhouse gases, lignocellulose biomass, which is an abundant and bio-renewable source present on earth with excellent properties and composition, has been used for decades to develop biofuels that can easily take over the place of conventional fuels. Lignocellulose biomass comprises polymeric sugars, i.e., cellulose and hemicellulose, and aromatic polymer, lignin, which are responsible for producing various bio-based products. However, utilizing lignocellulosic wastes for such purposes is needed but their recalcitrant structure makes it difficult to achieve their full usage. For this, several pretreatment approaches are developed to loosen the complexity between sugars and lignin. In some way, few of the conventional pretreatment methods are expensive, non-eco-friendly, and produce undesired by-products, causing a lower yield and reusability of enzymes used in the reaction. Utilizing novel pretreatment strategies that are cost-effective, help in increasing the yield of products, and are environment-friendly is required. Thus, incorporating nanoparticles and nanomaterials in the development of pretreatment and other strategies for the production of bio-based products is currently thriving. This review is designed in such a way that the readers can easily get brief knowledge about the production of important biofuels developed within second-generation biorefineries using lignocellulosic biomass. It also summarizes the importance of nanotechnology in different steps of biofuel development.

Emerging Nanomaterials as Versatile Nanozymes: A New Dimension in Biomedical Research.

The enzyme-mimicking nature of versatile nanomaterials proposes a new class of materials categorized as nano-enzymes, ornanozymes. They are artificial enzymes fabricated by functionalizing nanomaterials to generate active sites that can mimic enzyme-like functions. Materials extend from metals and oxides to inorganic nanoparticles possessing intrinsic enzyme-like properties. High cost, low stability, difficulty in separation, reusability, and storage issues of natural enzymes can be well addressed by nanozymes. Since 2007, more than 100 nanozymes have been reported that mimic enzymes like peroxidase, oxidase, catalase, protease, nuclease, hydrolase, superoxide dismutase, etc. In addition, several nanozymes can also exhibit multi-enzyme properties. Vast applications have been reported by exploiting the chemical, optical, and physiochemical properties offered by nanozymes. This review focuses on the reported nanozymes fabricated from a variety of materials along with their enzyme-mimicking activity involving tuning of materials such as metal nanoparticles (NPs), metal-oxide NPs, metal-organic framework (MOF), covalent organic framework (COF), and carbon-based NPs. Furthermore, diverse applications of nanozymes in biomedical research are discussed in detail.

Key Takeaways on the Cost-Effective Production of Cellulosic Sugars at Large Scale

The production of cellulosic sugars in lignocellulose biorefinery presents significant economic and environmental challenges due to the recalcitrant nature of biomass. The economic and facile production of renewable sugars with high yield and productivity is pivotal for the success of biorefinery. The cellulosic sugars are valorized either by biochemical routes or chemical routes or by hybrid (biological and chemical) routes into renewable chemicals, fuels, and materials. This manuscript focuses on the critical parameters affecting the economic viability of cellulosic sugar production at large scale, including biomass-specific pretreatment strategies and enzyme cost efficiency. High pretreatment costs, carbohydrate loss, and inhibitors production during pretreatment are identified as major contributors to overall production costs. To address these issues, we highlight the importance of developing cost-effective and efficient pretreatment methods tailored to specific biomass types and strategies for enzyme reuse and recycling. Future research should focus on innovations in pretreatment technologies, improved logistics for high-density feedstocks, biomass feeding systems, and advancements in enzyme technology to enhance the economic and environmental sustainability of lignocellulosic biorefineries. The findings highlight the need for continued innovation and optimization to make the commercial-scale production of cellulosic sugars more viable and sustainable.

Immobilised Alcohol Dehydrogenase and Ene‐Reductase Work in Concert to Reduce Cyclohex‐2‐enone in Bulk Organic Solvent

Enone reduction by ene reductase (ERED) has not been reported yet in bulk organic media probably due to expected challenges with the cofactor recycling using a second enzyme and the exchange of the NAD(P)‐cofactor between the two enzymes. Herein, the combination of an ERED from Zymomonas mobilis (NCR‐ERED) and Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) has been found as an efficient biocatalytic redox system for the reduction of cyclohex‐2‐enone into cyclohexanol in bulk organic solvents. Both enzymes were successfully immobilised on three EziG supports, finding Coral as the most promising carrier for both of them. The highest enzymatic activities were found in hydrophobic solvents such as toluene and isooctane. It was observed that the control of water content and post‐immobilisation treatments enabled retention of enzyme activity. Both enzymes were co‐immobilised on the same bead together with NADP+, leading to the production of a self‐sufficient catalytic system, although the external supplementation of the nicotinamide cofactor was necessary for an efficient enzyme reuse. In this case, no ERED activity loss was detected after two additional cycles, while the ADH experienced less than 20% activity loss upon reuse.

Development of an immobilized laccases - CDI CIMmultus® monolithic reactor for ecofriendly producing gallic acid-dextran conjugate

Enzymatic modification of polysaccharides is an efficient and environmentally friendly method to enhance their bioactivities. Immobilized enzymes represent a technology that enables the reuse of enzymes and increase their stability. In this study, laccases from Trametes versicolor were covalently immobilized on CIMmultus® CDI monolithic column, which was employed to graft gallic acid (GA) onto the backbone of T40 dextran. 1.43 mg laccases were successfully immobilized, yielding a mass conversion rate of 32.17 %. The kinetic parameters of free and immobilized laccases were respectively 0.0056 and 0.1770 U for Vmax and 0.0121 and 0.7510 mmol·l−1 for Km. Remarkably, 91.29 % of the enzymatic activity of immobilized laccases was preserved after catalyzing 1200 ml of reactant. A central composite design was carried out to investigate the influence of T40 dextran concentration and flow rate on the phenolization. Response surface methodology was used for predicting the optimal conditions, showed that a 2.07 % mass ratio of phenolization could be achieved with 2 % dextran and a flow rate of 0.89 ml·min−1. The GA-dextran conjugate, produced under the best conditions, revealed a DPPH radical inhibition activity of 63.97 % and an IC50 of 73.39 µg·ml−1. This work describes a new IMER for ecofriendly producing phenolized polysaccharides.

Enhanced Enzyme Immobilization Using a Novel Agarose‐binding Tag Leads to Improved Flow Reactor Performance

Continuous enzymatic synthesis has gained increasing prominence, extending its applicability to the chemical industry. A practical approach for integrating enzymes into continuously operated reactors is their entrapment within polymer‐based hydrogels. In this study, we present a novel approach to enhance enzyme retention within agarose hydrogels, employing a carbohydrate‐binding module sourced from the Microbulbifer thermotolerans thermostable β‑agarase I (MtCBM) as a versatile agarose‐binding tag. Among the four tested carbohydrate‐binding modules, MtCBM exhibited superior binding affinity to solid agarose and fusion proteins incorporating MtCBM demonstrated significantly enhanced retention within agarose hydrogels. Employing a newly developed fluidic flat‐bed agarose reactor, we demonstrate that, due to increased retention in agarose hydrogels, a phenacrylate decarboxylase tagged with MtCBM yielded nearly six times more product compared to a similarly sized fusion enzyme lacking the MtCBM‐tag. The higher leaching rate of the unlabeled enzyme could not be compensated even by tripling the hydrogel layer thickness, clearly documenting the effectiveness of MtCBM labeling for retention in the gel matrix. In addition, we performed theoretical modelling of the reactor, which led to a deeper understanding of the retention kinetics. The results suggest that this innovative approach is promising for improving stability and reusability of enzymes in agarose‐based carriers.

Design and fabrication of magnetic cross-linked laccase aggregate using superparamagnetic metal-organic frameworks for phenol removal

Phenolic pollutants represent a significant environmental concern due to their widespread presence in industrial effluents, agricultural runoff, and urban waste streams. Enzymatic bioremediation emerges as a promising approach for the efficient removal of phenolic pollutants from contaminated environments. Laccases exhibit remarkable catalytic capabilities in degrading phenolic compounds into less harmful or even inert products. In this study, magnetic cross-linked laccase aggregate was prepared using superparamagnetic metal-organic frameworks and then was applied for phenol removal. The obtained results showed that the magnetic cross-linked laccase aggregate retains its activity across a broad pH range. Interestingly, (NH4)2SO4 exhibited a positive influence on enzyme activity, resulting in a relative activity of 137.7 %. The findings suggest that an optimal concentration range of 0.4–0.8 % can achieve maximum enzyme activity while avoiding potential negative impacts associated with excessive cross-linking. Optimal pH and temperature conditions were identified for enhanced phenol removal efficiency as pH 4.5 and 45℃, respectively. The investigation revealed the remarkable reusability of the cross-linked enzyme over multiple cycles. Harnessing the power of enzymes for phenolic pollutant remediation holds immense promise for mitigating environmental pollution and safeguarding public health.

Evaluation of free and immobilized cellulase on chitosan-modified magnetic nanoparticles for saccharification of sorghum residue.

Enzymatic hydrolysis plays a pivotal role in transforming lignocellulosic biomass. Addressing alternate techniques to optimize the utilization of cellulolytic enzymes is one strategy to improve its efficiency and lower process costs. Cellulases are highly specific and environmentally benign biocatalysts that break down intricate polysaccharides intosimple formsofsugars. In contrast to the most difficult and time-consuming enzyme immobilization processes, in this research, we studied simple, mild, and successful techniques for immobilization of pure cellulase on magnetic nanocomposites using glutaraldehyde as a linker and used in the application of sorghum residue biomass. Fe3O4 nanoparticles were coated with chitosan from the co-precipitation method, which served as an enzyme carrier. The nanoparticles were observed under XRD, Zeta Potential, FESEM, VSM, and FTIR. The size morphology results presented that the Cs@Fe3O4 have 42.2nm, while bare nanoparticles (Fe3O4) have 31.2nm in size. The pure cellulase reaches to 98.07% of loading efficiency and 71.67% of recovery activity at optimal conditions. Moreover, immobilized enzyme's pH stability, thermostability, and temperature tolerance were investigated at suitable conditions. The kinetic parameters of free and immobilized enzyme were estimated as Vmax; 29 ± 1.51 and 27.03 ± 2.02µmolmin-1mg-1, Km; 4.7 ± 0.49mM and 2.569 ± 0.522mM and Kcat; 0.13s-1, and 0.89s-1. Sorghum residue was subjected to 2% NaOH pre-treatment at 50℃. Pre-treated biomass contains cellulose of 64.8%, used as a raw material to evaluate the efficiency of reducing sugar during hydrolysis and saccharification of free and immobilized cellulase, which found maximum concentration of glucose 5.42g/L and 5.12g/L on 72h. Thus, our study verifies the use of immobilized pure cellulase to successfully hydrolyze raw material, which is a significant advancement in lignocellulosic biorefineries and the reusability of enzymes.

Recent Progress on Conversion of Lignocellulosic Biomass by MOF-Immobilized Enzyme.

The enzyme catalysis conversion of lignocellulosic biomass into valuable chemicals and fuels showed a bright outlook for replacing fossil resources. However, the high cost and easy deactivation of free enzymes restrict the conversion process. Immobilization of enzymes in metal-organic frameworks (MOFs) is one of the most promising strategies due to MOF materials' tunable building units, multiple pore structures, and excellent biocompatibility. Also, MOFs are ideal support materials and could enhance the stability and reusability of enzymes. In this paper, recent progress on the conversion of cellulose, hemicellulose, and lignin by MOF-immobilized enzymes is extensively reviewed. This paper focuses on the immobilized enzyme performances and enzymatic mechanism. Finally, the challenges of the conversion of lignocellulosic biomass by MOF-immobilized enzyme are discussed.

An enzymatically activated AND-gate DNA logic circuit for tumor cells recognition via multi-microRNAs detection

The DNA-based logic circuit, constructed to mimic biochemical reaction networks, is highly significant in detecting biomarkers at the molecular level. The differences in the expression levels of microRNAs (miRNAs) within different types of cells provide hope for distinguishing cell subtypes. However, reliance on a single miRNA often leads to unreliable results. Herein, we constructed an enzyme-triggered cascade logic circuit based on the AND gate, which is capable of generating corresponding fluorescence signals in the presence of target miRNAs. The introduction of apurinic/apyrimidinic (AP) sites effectively reduces the likelihood of false signal generation. Amplification of the fluorescence signal relies on the catalytic hairpin assembly and the repetitive reuse of the multicomponent nucleic acid enzyme (MNAzyme). We demonstrated that the logic circuit can not only distinguish cancer cells from normal cells but also identify different types of cancer cells. The programmability of the logic circuits and the simplicity of the assay system allow us to modify the functional sequences to recognize different types of biomarkers, thus providing a reference for the identification of various cell subtypes.

Lipolytic production from solid-state fermentation of the filamentous fungus Penicillium polonicum and its applicability as biocatalyst in the synthesis of ethyl oleate.

Lipases represent versatile biocatalysts extensively employed in transesterification reactions for ester production. Ethyl oleate holds significance in biodiesel production, serving as a sustainable alternative to petroleum-derived diesel. In this study, our goal was to prospect lipase and assess its efficacy as a biocatalyst for ethyl oleate synthesis. For quantitative analysis, a base medium supplemented with Rhodamine B, olive oil, and Tween 80 was used. Solid-state fermentation utilized crambe seeds of varying particle sizes and humidity levels as substrates. In the synthesis of ethyl oleate, molar ratios of 1:3, 1:6, and 1:9, along with a total enzymatic activity of 60 U in n-heptane, were utilized at temperatures of 30°C, 37°C, and 44°C. Reactions were conducted in a shaker at 200rpm for 60min. As a result, we first identified Penicillium polonicum and employed the method of solid-state fermentation using crambe seeds as a substrate to produce lipase. Our findings revealed heightened lipolytic activity (22.5 Ug-1) after 96h of fermentation using crambe cake as the substrate. Optimal results were achieved with crambe seeds at a granulometry of 0.6mm and a fermentation medium humidity of 60%. Additionally, electron microscopy suggested the immobilization of lipase in the substrate, enabling enzyme reuse for up to 4 cycles with 100% enzymatic activity. Subsequently, we conducted applicability tests of biocatalysts for ethyl oleate synthesis, optimizing parameters such as the acid/alcohol molar ratio, temperature, and reaction time. We attained 100% conversion within 30min at 37°C, and our results indicated that the molar ratio proportion did not significantly influence the outcome. These findings provide a methodological alternative for the utilization of biocatalysts in ethyl oleate synthesis.

A hydroesterification reaction system to produce octyl esters using degummed soybean oil as substrate: Combining reusable free and immobilized lipases

This study shows the enzymatic synthesis of octyl alcohol-based biolubricants carried out in two catalytic steps: degummed soybean oil (DSO) hydrolysis and esterification of the produced and purified free fatty acids (FFA). Several lipases were evaluated as catalysts in the hydrolysis of DSO. Under optimal conditions, lipase from Pseudomonas fluorescens (PFL) yielded 97 % conversion after 23 h using 50 U/g oil and DSO:water mass ratio of 1:0.5. Sequential batch strategies have been developed for the recovery and reuse of free enzyme in further hydrolysis cycles. When hexane was used in the separation between the FFA-rich phase and the phase composed of water, enzyme and glycerol, there was better PFL recovery and FFA-conversion of 65 % was still achieved after five 24-hour hydrolytic cycles. In the esterification step, the potential of four lipases (commercial Lipozym-435, and lipase B from Candida antarctica (CALB), lipase from porcine pancreas (PPL) and Eversa® Transform 2.0 (EV) immobilized on Purolite Lifetech EC8806F) were evaluated in the production of octyl esters in a solvent-free medium (FFA/octanol molar ratio of 1:2.5) using screw-capped glass bottles as reactor. The immobilized EV gave the best ester yield (∼87 wt%). When EV-Purolite was used in a vortex flow reactor, a yield of around 94 wt% was reached after 3 h of reaction using a biocatalyst load of 1 % (w/v) and FFA/octanol molar ratio of 1:2.5. The immobilized lipase could be reused for five consecutive 3 h-batches of esterification, maintaining the octyl esters yield of the first batch.

Functionalized magnetic nanoparticles for Cellic Ctec2 cellulase immobilization: Allowing reusability of enzyme in the conversion of cellulosic biomass

The method of enzyme immobilization can ameliorate the overall stability and restoration of enzymes, hence facilitating their broader application in several sectors. This investigation utilized cellulase as a hydrolytic enzyme. In order to enhance the stability and performance of the cellulase enzyme, the research employed immobilization technology to secure the Cellic Ctec2 cellulase to the synthesized Cs@Fe3O4 nanocomposites. Fe3O4 nanoparticles (NPs) were coated with chitosan obtained from co-precipitation method that served as enzyme carrier. The NPs (Cs@ Fe3O4) were observed under XRD; VSM (vibrating-sample magnetometer) shows saturation magnetizations (Ms), UV–vis, field emission scanning electron microscopy (FESEM), and Fourier transform infrared spectroscopy (FT-IR). Response surface approach was applied to optimize the conditions for immobilization of cellulase. The optimum immobilization of cellulase reaches to 99.1% of loading efficiency and 69.7% of recovery activity with 2.5% of glutaraldehyde concentration. Furthermore, under ideal circumstances the immobilized enzyme’s thermostability, pH stability, temperature tolerance and reusability, were studied with respect to free cellulase. Higher relative activity of cellulase enzyme was observed at pH 5 with 50 °C temperature than free enzyme. One percent CMC hydrolysis is considered for reusability of free and immobilized enzyme and releases 222 mg glucose/g substrate at 24 h, showing great quiescence in cellulosic biomass conversion. Immobilized cellulase demonstrated high reusability by retaining almost 61.2% up to the 5th cycles and 51.2% of activity-maintained 10th cycle of hydrolysis. Reusability of cellulase enzyme can attain a gradual decrease in relative activity as number of repeats of the cycle increases to 10 during hydrolysis and increases in glucose yield after hydrolysis.

Assessment of the efficiency and stability of enzymatic membrane reaction utilizing lipase covalently immobilized on a functionalized hybrid membrane

Enzyme immobilization in membrane bioreactors has been considered as a practical approach to enhance the stability, reusability, and efficiency of enzymes. In this particular study, a new type of hybrid membrane reactor was created through the phase inversion method, utilizing hybrid of graphene oxide nanosheets (GON) and polyether sulfone (PES) in order to covalently immobilize the Candida rugosa lipase (CRL). The surface of hybrid membrane was initially modified by (3-Aminopropyl) triethoxysilane (APTES), before the use of glutaraldehyde (GLU), as a linker, through the imine bonds. The resulted enzymatic hybrid membrane reactors (EHMRs) were then thoroughly analyzed by using field-emission scanning electron microscopy (FE-SEM), contact angle goniometry, surface free energy analysis, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, attenuated total reflection (ATR), and energy-dispersive X-ray (EDX) spectroscopy. The study also looked into the impact of factors such as initial CRL concentration, storage conditions, and immobilization time on the EHMR's performance and activity, which were subsequently optimized. The results demonstrated that the CRLs covalently immobilized on the EHMRs displayed enhanced pH and thermal stability compared to those physically immobilized or free. These covalently immobilized CRLs could maintain over 60% of their activity even after 6 reaction cycles spanning 50 days. EHMRs are valuable biocatalysts in developing various industrial, environmental, and analytical processes.

Efficient lipase-catalyzed synthesis of ceramide III series compounds in an eco-friendly solvent

Ceramide III (CER III) series compounds are essential constituents in cosmetic formulations and dermatological applications. As the cosmetics industry trends toward more sustainable practices, the development of an eco-friendly synthesis method for these vital compounds is increasingly critical. This research introduces a sustainable technique for producing CER III series compounds, using lipase-catalyzed amidation of phytosphingosine with various fatty acid esters or natural oils in an eco-compatible solvent. Through an extensive examination of various lipases and eco-friendly solvents, we first identified Novozym 435 as the most efficient biocatalyst and cyclopentyl methyl ether (CPME) as the preferred solvent. This combination established a high-efficiency reaction system for CER III series compounds synthesis. We further optimized the enzymatic conditions - a substrate molar ratio of 1:1, lipase loading at 30 wt%, at 65 °C, which presented an impressive yield of 94 % for ceramide IIIB (CER IIIB). We also investigated the reusability of enzymes in this synthetic system and further extended the scope of acyl donors using natural oils, results indicating the method excellent potentiality in industrial settings. In summary, this study provides a green and efficient pathway for synthesizing CER III series compounds, aligning with the growing eco-conscious trends in cosmetic science and technology.

Magnetic Multienzyme@Metal-Organic Material for Sustainable Biodegradation of Insoluble Biomass.

Biodegradation of insoluble biomass such as cellulose via carbohydrase enzymes is an effective approach to break down plant cell walls and extract valuable materials therein. Yet, the high cost and poor reusability of enzymes are practical concerns. We recently proved that immobilizing multiple digestive enzymes on metal-organic materials (MOMs) allows enzymes to be reused via gravimetric separation, improving the cost efficiency of cereal biomass degradation [ACS Appl. Mater. Interfaces 2021, 13, 36, 43085-43093]. However, this strategy cannot be adapted for enzymes whose substrates or products are insoluble (e.g., cellulose crystals). Recently, we described an alternative approach based on magnetic metal-organic frameworks (MOFs) using model enzymes/substrates [ACS Appl. Mater. Interfaces 2020, 12, 37, 41794-41801]. Here, we aim to prove the effectiveness of combining these two strategies in cellulose degradation. We immobilized multiple carbohydrase enzymes that cooperate in cellulose degradation via cocrystallization with Ca2+, a carboxylate ligand (BDC) in the absence and presence of magnetic nanoparticles (MNPs). We then compared the separation efficiency and enzyme reusability of the resultant multienzyme@Ca-BDC and multienzyme@MNP-Ca-BDC composites via gravimetric and magnetic separation, respectively, and found that, although both composites were effective in cellulose degradation in the first round, the multienzyme@MNP-Ca-BDC composites displayed significantly enhanced reusability. This work provides the first experimental demonstration of using magnetic solid supports to immobilize multiple carbohydrase enzymes simultaneously and degrade cellulose and promotes green/sustainable chemistry in three ways: (1) reusing the enzymes saves energy/sources to prepare them, (2) the synthetic conditions are "green" without generating unwanted wastes, and (3) using our composites to degrade cellulose is the first step of extracting valuable materials from sustainable biomasses such as plants whose growth does not rely on nonregeneratable resources.

Resilient Enzymes through Immobilisation: Stable NDP Polyphosphate Phosphotransferase from Ruegeria pomeroyi for Nucleotide Regeneration

Immobilisation plays an important role in the industrial application of enzymes. The stabilisation and reusability of immobilised enzymes reduce the cost of the catalyst and facilitate their use in continuously operated reactors. For this purpose, an applicable type of immobilisation needs to be identified. In this study, we investigate the conversion of CDP and PolyP to CTP by NDP polyphosphate phosphotransferase 3 from Ruegeria pomeroyi (RpPPK2-3) and describe the covalent immobilisation of RpPPK2-3. In order to select a suitable carrier for the immobilisation of RpPPK2-3, a screening with different amino methacrylate (glutaraldehyde-pre-activated) and epoxy methacrylate carriers was carried out. The epoxy methacrylate carrier ECR8209M (Purolite®) was found to be the most suitable. With a half-life of 462 d when stored at 6 °C and a 50-fold reusability with a 93% residual activity, the immobilised enzyme showed a higher stability compared to the soluble enzyme with a half-life of 0.04 d. Although the half-life of the soluble enzyme could be increased to 32 d by adding PPi, it could not reach the stability of the immobilisate. Due to the resilience of the immobilisate, it is suitable for application in continuous reactor set-ups, e.g., packed-bed reactors.

Green synthesis and performance evaluation of mixed fatty acids-based esters as nonionic antifoaming biosurfactants

Three different polyethylene glycol (PEG) esters of 1:1 fatty acid mixture of ricinoleic, oleic, and linseed oil fatty acids, as eco-friendly nonionic antifoaming biosurfactants were synthesized using a solventless biocatalytic route, to overcome challenges associated with chemical processes. Immobilized lipase Candida antarctica (NS 435) was found to be more effective as a biocatalyst (10 % w/w) than Rhizomucor meihei (RMIM), and Thermomyces lanuginous (TLIM) and led to the highest percentage of fatty acid conversion ∼97.5–98.5 %. The reusability of lipase enzymes was studied, and it has been found that the catalytic activity of NS 435 lipase remained almost intact up to the ninth successive cycle and gradually reduced by 4–5 % thereafter. Whereas RMIM and TLIM were effective till 5 and 4 cycles respectively. The produced esters were characterized, evaluated, and compared for surface activity, emulsification, and antifoaming properties against commercially available nonionic surfactants. The results revealed that the synthesized PEG esters have higher thermal stability, better emulsifying, and excellent antifoaming properties than conventional nonionic surfactants. Thus, the synthesized PEG esters have shown great potential to be used as environment-friendly (>90 % biodegradability) nonionic antifoaming biosurfactants to prepare emulsifiable industrial fluids.

Comparative study for enzymatic hydrolysis of sugarcane bagasse using free and nanoparticle immobilized holocellulolytic enzyme cocktail

This study aimed at a comparative analysis of enzymatic hydrolysis of sugarcane bagasse biomass by using a free holocellulase cocktail of an enzyme (Cellic CTec2) and immobilized enzyme system on IOMNP@SiO2-NH2 support to make the process cost-effective. The immobilization was substantiated by examining the nano biocatalysts using FTIR, TEM, and DLS analysis. IOMNP@SiO2 –NH2 and immobilized enzyme system have average particle sizes of 81.1, and 455.9 nm confirmed by DLS. Response surface methodology (RSM) based on the Box Behnken design was employed to optimize the saccharification process conditions. The free cellulase cocktail showed a maximum hydrolysis yield of 70.71 % at 12.5FPU/gm enzyme dose, while the immobilized enzyme at a dose of 20 FPU/gm achieved a conversion rate of 74.19 % under optimized parameters likes hydrolysis time, surfactant and substrate concentration. Pretreated sugarcane bagasse substrate was used to evaluate the reusability potential of magnetically immobilized cellulase enzyme compared to recovered free enzyme up to 5 cycles. In the second and third cycles, the conversion rates of cellulose to glucose were determined to be 68.21 %, and 52.6 %, respectively. The findings of this study demonstrate that the immobilized Cellic CTec2 enzyme cocktail on functionalized IOMNP is effective for hydrolyzing sugarcane bagasse which also provides the advantages of easy recovery and reusability of enzyme for multiple hydrolysis cycles.

Improvement of catalysis performance of pepsin and lipase enzymes by double enzyme immobilization method.

Ferrocene-coupled chitosan (CS-Fc) support polymer was synthesized for the immobilization of double enzymes and single enzymes. The immobilization study was carried out after a detailed characterization study. The binding of pepsin and lipase enzymes to the polymer was supported by the shift of peaks in the FTIR spectra of free enzymes. The particle sizes of chitosan (CS) and CS-Fc were evaluated using zeta potential measurement. The optimum conditions of the enzymes in the double enzyme system did not affect each other and each showed their activity. In the presence of double enzyme immobilization, the optimum pH remained the same with free enzyme, while the optimum temperature increased differently with an increase of 10 °C. Vmax values were 0.0020 and 0.0038 for free pepsin and lipase enzyme, respectively. However, due to double enzyme immobilization, the Vmax value increased 1.75 fold for pepsin and 3.94 fold for lipase enzyme. In the double enzyme immobilization, the substrate affinity of pepsin increased by 0.4-fold, while the substrate affinity of lipase increased by about 0.3-fold. In the 5th reuse of pepsin and lipase enzymes in the presence of double enzymes (Cs-Fc-Pep) + (Cs-Fc-Lip), it was determined as 18.21% for pepsin enzyme and 90.81% for lipase enzyme.