Immobilized Biocatalysts Research Articles

Development of a Bio-Selecting Agent Based on Immobilized Bacterial Cells with Amidase Activity for Bio-Detection of Acrylamide

Actinobacteria cells Rhodococcus erythropolis 4-1 and Rhodococcus erythropolis 11-2 and Proteobacteria Alcaligenes faecalis 2, which have amidase activity, were immobilized by entrapping barium alginate and agarose into the gel structure, as well as by obtaining biofilms on thermally expanded graphite (TEG). The operational stability of such immobilized biocatalysts after storage in frozen and dehydrated form was determined, and a prototype of a conductometric acrylamide biosensor based on such a bioselective agent was developed. The most preferred method for storing immobilized cells was freezing at temperatures from –20 to –80°C; long-term storage is also possible wet at 4–25°C. It was shown that these cells were most preferable for the biodetection of acrylamide A. faecalis 2, immobilized in an agarose gel structure. An agarose gel with bacterial cells immobilized in its structure had greater mechanical strength and stability during successive cycles of conversion of acrylamide into acrylic acid compared to barium alginate gel. The mechanical strength of barium alginate gel can be enhanced by the addition of carbon nanomaterials during cell immobilization. Growing biofilms on carbon materials used for manufacturing electrodes is also promising. Biofilms of R. erythropolis 11-2 on TEG are capable of converting acrylamide into acrylic acid in more than 20 reaction cycles while maintaining at least 50% amidase activity.

Microbial Immobilized Enzyme Biocatalysts for Multipollutant Mitigation: Harnessing Nature's Toolkit for Environmental Sustainability.

The ever-increasing presence of micropollutants necessitates the development of environmentally friendly bioremediation strategies. Inspired by the remarkable versatility and potent catalytic activities of microbial enzymes, researchers are exploring their application as biocatalysts for innovative environmental cleanup solutions. Microbial enzymes offer remarkable substrate specificity, biodegradability, and the capacity to degrade a wide array of pollutants, positioning them as powerful tools for bioremediation. However, practical applications are often hindered by limitations in enzyme stability and reusability. Enzyme immobilization techniques have emerged as transformative strategies, enhancing enzyme stability and reusability by anchoring them onto inert or activated supports. These improvements lead to more efficient pollutant degradation and cost-effective bioremediation processes. This review delves into the diverse immobilization methods, showcasing their success in degrading various environmental pollutants, including pharmaceuticals, dyes, pesticides, microplastics, and industrial chemicals. By highlighting the transformative potential of microbial immobilized enzyme biocatalysts, this review underscores their significance in achieving a cleaner and more sustainable future through the mitigation of micropollutant contamination. Additionally, future research directions in areas such as enzyme engineering and machine learning hold immense promise for further broadening the capabilities and optimizing the applications of immobilized enzymes in environmental cleanup.

Rapid Degradation of Hazardous Amides by Immobilized Engineered Pseudomonas putida KT2440 Based on a Novel Gene Expression Vector.

The amides 4-trifluoromethylnicotinamide, acrylamide, and benzamide are widely used in agriculture and industry, posing hazards to the environment and animals. Immobilized bacteria are preferred in wastewater treatment, but degradation of these amides by immobilized engineered bacteria has not been explored. Here, engineered Pseudomonas putida KT2440 pLSJ15-amiA was constructed by introducing a new amidase gene expression vector into environmentally safe P. putida KT2440. P. putida KT2440 pLSJ15-amiA had high amidase activity, even at 80 °C. P. putida KT2440 pLSJ15-amiA immobilized with calcium alginate exhibited a greater environmental tolerance than free cells. The amides were rapidly degraded by the immobilized cells, but the activity was inhibited by high concentrations of substrates. The substrate inhibition model revealed that the optimum initial concentrations of 4-trifluoromethylnicotinamide, acrylamide, and benzamide for degradation by immobilized cells were 197.65, 350.76, and 249.40 μmol/L, respectively. This study develops a novel and excellent immobilized biocatalyst for remediation of wastewater containing hazardous amides.

Comparative oil extraction from mutt (Myliobatis goodei) liver by enzymatic hydrolysis: free versus immobilized biocatalyst.

The development and fine-tuning of biotechnological processes for fish oil extraction constitute a very important focus to contribute to the development of a food industry based on fish consumption. This work lies in a comparative analysis of the oil extraction yield of Myliobatis goodei livers using free and immobilized enzymes. An immobilized biocatalyst was designed from the cell-free extract of a Bacillus sp. Mcn4. A complete factorial design was used to study the components of the bacterial culture medium and select the condition with the highest titers of extracellular enzymatic activities. Wheat bran had a significant effect on the culture medium composition for enzymatic production. The immobilized biocatalyst was designed by covalent binding of the proteins present in the cocktail retaining a percentage of different types of enzymatic activities (Mult.Enz@MgFe2 O4 ). Among the biocatalyst used, Alcalase® 2.4 L and Purazyme® AS 60 L (free commercial proteases) showed extraction yields of 87.39% and 84.25%, respectively, while Mult.Enz@MgFe2 O4 achieved a better one of 89.97%. The oils obtained did not show significant differences in their physical-chemical properties while regarding the fatty acid content, the oil extracted with Purazyme® AS 60 L showed a comparatively lower proportion of polyunsaturated fatty acids. Our results suggest that the use of by-products of M. goodei is a valid alternative and encourages the use of immobilized multienzyme biocatalysts for the treatment of complex substrates in the fishing industry. © 2023 Society of Chemical Industry.

Rational design of biocatalysts based on covalent immobilization of acylase enzymes

Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports. Predictions of the RDID1.0 software and the experimental results led to the selection of glyoxyl-Sepharose CL 4B support and pH 10.0. One of the predicted clusters of reactive amino groups generates an enzyme-support configuration with highly accessible active sites, contributing with 82% of the biocatalyst’s total activity. For Escherichia coli PGA, the predictions and experimental results show similar maximal amounts of immobilized protein and activity at pH 8.0 and 10.0 on glyoxyl-Sepharose CL 10B. However, thermal stability of the immobilized derivative is higher at pH 10.0 due to an elevated probability of multipoint covalent attachment. In this case, two clusters of amino groups are predicted to be relevant for PGA immobilization in catalytically competent configurations at pH 10.0, showing accessible active sites and contributing with 36% and 44% of the total activity, respectively. Our results support the usefulness of the RDID strategy to model different protein engineering approaches (site-directed mutagenesis or obtainment of fusion proteins) and select the most promising ones, saving time and laboratory work, since the in silico-designed modified proteins could have higher probabilities of success on bioconversion processes.

Hexavalent chromium detoxification by haloalkaliphilic Nesterenkonia sp strain NRC-Y immobilized in different matrices

To develop a bioprocess for Cr(VI) detoxification in industrial effluents, a previously isolated potent Cr(VI) reducing haloalkaliphilic Nesterenkonia sp strain NRC-Y was immobilized and investigated for Cr(VI) detoxification. The immobilization matrices included; natural, modified natural, synthetic, and mixtures of natural and synthetic polymers. Among the tested matrices and immobilization approaches, strain NRC-Y cells encapsulated in 1.5% (w/v) amidated pectin beads exhibited the highest Cr(VI) reduction efficiency (47.02% of initial Cr(VI) concentration 150 mg/L after 20 h) followed by alginate (34.4%), alginate-PVA-chitosan (29.6%), alginate-PVA (25.1%), PVA-PVP (23.2%), and PVA (9.5%). Chromate reductase enzyme was poorly immobilized by covalent binding on the tested materials. Therefore, entrapment in amidated pectin was selected for further investigation and immobilization of both whole cells and partially purified chromate reductase. Operational stability study revealed that the immobilized cells were more efficient and stable than the immobilized chromate reductase, and the free cells. For instance, about 60%, 27.0% and 11.5% of these samples initial activities were retained after four successive batches, respectively. The temperature and pH optima for the immobilized cells were 35°C and 7.0, respectively. The pH and thermal stability of strain NRC-Y cells were significantly enhanced upon immobilization in amidated pectin beads by up to 2.3- and 1.4-fold, respectively. The developed immobilized biocatalyst was applied for Cr(VI) reduction in industrial effluent samples, and it completely reduced Cr(VI) within 4 and 8 h for effluents of initial Cr(VI) concentrations 10 and 30 mg/L, respectively. The developed immobilized biocatalyst is promising and has the potential for large-scale Cr(VI) detoxification application.

Mycelium-bound chlorogenate hydrolase of Aspergillus niger AKU 3302 as a stable immobilized biocatalyst

CHase catalyzes chlorogenic acid (CGA) hydrolysis to yield equimolar quinic (QA) and caffeic (CA) acids, products of high value and keen industrial interest. We proposed the preparation and characterization of the nonviable mycelium of Aspergillus niger AKU 3302 containing a cell-associated CHase (CHase biocatalyst) for application in hydrolyzing the CGA from yerba mate residues to produce QA and CA. When the vegetative mycelium was heated at 55°C for 30min, no loss of CHase activity occurred, but vegetative mycelial growth and spore germination ended. The CHase biocatalyst did not limit mass transfer above 100 strokes min-1. The reaction rate increased with catalyst loading and was kinetically controlled. The CHase biocatalyst exhibited suitable biochemical properties (optimum pH 6.5at 50°C) and high thermal stability (remaining stable at up to 50°C for 8h). The cations in yerba mate extracts did not affect CHase activity. We observed no apparent loss in the activity of the CHase biocatalyst after even 11 batch cycles of continuous use. The biocatalyst retained 85% of its initial activity after 25 days of storage at pH 6.5 and 5°C. When a yerba mate extract was passed through a glass column packed with the biocatalyst, an effective bioconversion of CGA into CA and QA occurred. CHase activity produced a natural biocatalysis with considerable operational and storage stability; which capability, being a novel biotechnological process, can be used in the bioconversion of CGA from yerba mate residues into CA and QA at a substantially reduced cost.

Immobilized biocatalyst engineering: Biocatalytic tool to obtain attractive enzymes for industry

Biocatalysis can improve current bioprocesses by identifying or improving enzymes that withstand harsh and unnatural operating conditions. Immobilized Biocatalyst Engineering (IBE) is a novel strategy integrating protein engineering and enzyme immobilization as a single workflow. Using IBE, it is possible to obtain immobilized biocatalysts whose soluble performance would not be selected. In this work, Bacillus subtilis lipase A (BSLA) variants obtained through IBE were characterized as soluble and immobilized biocatalysts, and how the interactions with the support affect their structure and catalytic performance were analyzed using intrinsic protein fluorescence. Variant P5G3 (Asn89Asp, Gln121Arg) showed a 2.6-fold increased residual activity after incubation at 76 °C compared to immobilized wild-type (wt) BSLA. On the other hand, variant P6C2 (Val149Ile) showed 4.4 times higher activity after incubation in 75 % isopropyl alcohol (36 °C) compared to Wt_BSLA. Furthermore, we studied the advancement of the IBE platform by performing synthesis and immobilizing the BSLA variants using a cell-free protein synthesis (CFPS) approach. The observed differences in immobilization performance, high temperature, and solvent resistance between the in vivo-produced variants and Wt_BSLA were confirmed for the in vitro synthesized enzymes. These results open the door for designing strategies integrating IBE and CFPS to generate and screen improved immobilized enzymes from genetic diversity libraries. Furthermore, it was confirmed that IBE is a platform that can be used to obtain improved biocatalysts, especially those with an unremarkable performance as soluble biocatalysts, which wouldn't be selected for immobilization and further development for specific applications.

Immobilization of Aspergillus sp. laccase on hierarchical silica MFI zeolite with embedded macropores

Laccase from Aspergillus sp. (LC) was immobilized on functionalized silica hierarchical (microporous-macroporous) MFI zeolite (ZMFI). The obtained immobilized biocatalyst (LC#ZMFI) was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (ATR-FTIR), N2 adsorption/desorption isotherms, solid-state NMR spectroscopy and thermogravimetric analysis (TGA) confirming the chemical anchoring of the enzyme to the zeolitic support. The optimal pH, kinetic parameters (KM and Vmax), specific activity, as well as both storage and operational stability of LC#ZMFI were determined. The LC#ZMFI KM and Vmax values amount to 10.3 µM and 0.74 µmol·mg−1 min−1, respectively. The dependence of specific activity on the pH for free and immobilized LC was investigated in the pH range of 2–7, The highest specific activity was obtained at pH = 3 for both free LC and LC#ZMFI. LC#ZMFI retained up to 50 % and 30 % of its original activity after storage of 21 and 30 days, respectively. Immobilization of laccase on hierarchical silica MFI zeolite allows to carry out the reaction under acidic pH values without affecting the support structure.

Improvement of stress multi-tolerance and bioethanol production by Saccharomyces cerevisiae immobilised on biochar: Monitoring transcription from defence-related genes

The study determined the protective role of using biochar as immobilisation carrier against multiple stresses encountered by Saccharomyces cerevisiae assessing transcription from important metabolic routes involved in the molecular mechanisms triggered during inhibitory bioprocess conditions. Immobilised cells exhibited higher bioethanol titre (39 g L−1) and productivity (7.72 g L−1 h−1) at elevated temperatures compared with the suspended culture that yielded 34 g L−1 and 1.99 g L−1 h−1 respectively. Fermentation at 39 °C resulted in 2.15-fold increase of HSP104 relative mRNA expression in suspended cells, while the gene was induced by 0.5-fold using the immobilised biocatalyst. A similar response occurred for HSF1 and TPS exhibiting 3.0- and 3.8-fold increase using suspended cells as opposed to the application of immobilised cells where transcription of the aforementioned genes was raised by 0.0- and 2.6-fold upon temperature increase respectively. Transcription from MSN2/MSN4 under the aforementioned conditions indicated the protective role of cell attachment on the biomaterial against stimulation of the heat shock response route and oxidative stress. Although fermentations conducted under ethanol stress resulted in failure of the conventional process, immobilised cells produced 21 g L−1 bioethanol exhibiting 7 g L−1 h−1 productivity, while monitoring transcription of HSP12 and HSP104 demonstrated the beneficial use of the proposed technology. Proline accumulation during osmotic stress further supported the elevated bioethanol productivity achieved by the immobilised system, which was 74% higher as opposed to the conventional process. The study confirmed that S. cerevisiae immobilisation on biochar conferred cells with heat tolerance, ethanol tolerance, osmotolerance and improved fermentation capacity. The technology proposed constitutes a sustainable technological alternative to strain modification improving multiple stress-tolerance in bioethanol fermentations.

Oriented immobilization of Thermomyces lanuginosus lipase by SBPs on silica-containing carriers for efficient bioproduction of biodiesel from Jatropha curcas oil

Biodiesel is recognized as green and renewable energy with low carbon intensity and less environmental impacts. The preparation of an economical immobilized lipase becomes much urgent challenge in the enzymatic production of biodiesel. In this study, recombinant Thermomyces lanuginosus lipase (TLL) was immobilized on the surface of silica-containing materials mediated by solid-binding peptides (SBPs). The obtained immobilized biocatalysts were effective in converting the oil from Jatropha curcas into biodiesel. The appropriate attachment mode of SBPs and TLL was investigated firstly, and the results showed that the activity of TLL was not negatively affected only when SBPs were fused at the C-terminus of TLL. Secondly, the immobilization efficiency of different SBPs (CecA, EctP1, VKT) on different silica-containing materials (SiO2, zeolite ZSM-5, SAR-100, MCM-41 and Na-Y) was systematically studied and it was concluded that the best immobilization efficiency was achieved for TLL-EctP1 immobilized on zeolite ZSM-5 (TLL- EctP1 @ZSM-5). TLL-EctP1 @ZSM-5 was found great performance in stability of pH, temperature, elution and storage. Eventually, biodiesel yield of 93 wt% was achieved using TLL-EctP1 @ZSM-5 as a biocatalyst to catalyze J. curcas oil for 48 h, and 81% of its initial yield was retained after 7 cycles of reuse. These results highlight the application of solid-binding peptides in enzyme immobilization for biocatalysts used in preparation of biodiesel or other chemicals.

Lipase-catalyzed solution polycondensation of 1,4-butanediol and diethyl succinate: Effect of diphenyl ether concentration on enzyme stability, reuse and PBS molar mass distribution

Polybutylene Succinate (PBS) is a biodegradable polyester which can be produced from renewable monomers (provided by fermentation routes) and has been widely spread in value-added applications, such as in the biomedical sector. Regarding the synthesis routes of PBS for biomedical applications, enzymes have been gaining prominence, due to their high selectivity, milder operating temperature, absence of toxic metals and the possibility of reuse (when immobilized). Therefore, this work seeks to investigate the enzymatic polycondensation in solution, using diethyl succinate and 1,4-butanediol as monomers, Novozym 435 as biocatalyst, and diphenyl ether as solvent. The possibility of reusing the immobilized biocatalyst was also addressed. Solvent-free reactions, or containing 5 and 50 wt% of diphenyl ether were tested, at 70, 80 and 90 °C. Average molar masses were measured by Gel Permeation Chromatography to evaluate the enzyme performance in PBS synthesis. Furthermore, the kinetic behaviour of the synthesis was monitored by the formation of the by-product. The reactions containing 5 wt% of solvent at 80 °C and 90 °C proved to be more advantageous, resulting in Mw between 2.500 and 3.350 g.mol−1, in reaction times between 60 and 90 min. In this case, small additions of diphenyl ether presented diffusional advantages when compared to solvent-free processes. In addition, reuse tests of the biocatalyst were performed at 70 °C with 5 wt% of solvent. A pronounced decrease in the enzymatic activity was observed in the N435 reuse cycles, indicating that diphenyl ether affected the stability of the immobilized biocatalyst. Therefore, additional investigation is still needed in using green solvents that keep the immobilised biocatalyst's stability, providing subsidies for future works aimed at scale-up and commercial/industry production of PBS.

Immobilization of lipase on spent coffee grounds by physical and covalent methods: A comparison study

Spent coffee grounds were treated to obtain suitable carriers for both physical and covalent immobilization of Candida rugosa lipase. In the first case, physical adsorption was directly performed after water/organic solvent extraction of the interferents from the support, while in the second case the delignification and the activation of the support with glutaraldehyde or KIO4 were necessary. The support materials obtained were characterized by Fourier transform attenuated total reflection IR, elemental analysis, and scanning electron microscope. Each immobilization method was investigated to obtain the best insoluble biocatalyst immobilized activity, efficiency, and recovery. Emphasis was given to the study of the water/organic solvent ratio in the immobilization mixture as it has proven to be a decisive parameter. The optimized immobilized biocatalyst obtained by adsorption mechanism showed up to 33 U g−1 of immobilized enzyme activity, and a higher pH, thermal, and storage stability than the free lipase. This solid biocatalyst also preserved 100% of its activity after four recycles in p-nitrophenyl palmitate hydrolysis in hexane medium and permitted an almost 60% conversion of milk fats in fatty acids after 18 h of reaction, maintaining this value for three reuses.

α‐Ketobutyrate Production under Continuous‐Flow Conditions Catalyzed by Immobilized L‐Methionine γ‐Lyase

Abstractl‐Methionine γ‐lyase (MGL) is a PLP‐dependent enzyme which catalyzes the α, γ‐elimination reaction in sulfur containing amino acids, such as L‐methionine and L‐cysteine. Its major applications are on metabolic restriction of methionine to cancer cells, food industry (cheese flavor) and for the synthesis of d and l‐homoalanine. Herein, we present the immobilization and use of this new immobilized biocatalyst under batch and continuous‐flow conditions. The best optimized continuous‐flow condition allowed 89 % conversion in 30 min of residence time with good recyclability of the biocatalyst.

Hetero-modification of halloysite nanoclay to immobilize endoinulinase for the preparation of fructooligosaccharides

Present investigation describes immobilization efficiency of endoinulinase onto hetero-functionalized halloysite nanoclay using 3-aminopropyltriethoxysilane and glutaraldehyde as crosslinkers. Under optimal conditions (APTES 0.75%, sonication time 2.25 h, glutaraldehyde 0.75%, activation-time 65 min, immobilized endoinulinase load 60 IU and coupling-time 1 h), maximum yield in enzyme activity (70.65%) and immobilization (89.61%) was obtained. Developed immobilized biocatalyst shown maximum activity at 65 °C and pH 5.0 with wide range thermal (50–80 °C) and pH (4.0–9.0) stability. Increase in half-life (28.70-fold) of immobilized endoinulinase was observed as compared to free enzyme. An enhanced Km and reduced Vmax of endoinulinase for inulin was recorded after immobilization. Maximum FOSs production 98.42% was obtained, under optimized conditions (inulin 10%; immobilized endoinulinase load 85 IU; hydrolysis-time 10 h and agitation rate 130 rpm) containing kestose (36.26%), nystose (27.02%), fructofuranosylnystose (9.98%) and FOSs DP 5–9 (25.15%). Developed immobilized biocatalyst exhibited a splendid operational stability for 18 batch cycles.

Highly effective Candida rugosa lipase immobilization on renewable carriers: Integrated drying and immobilization process to improve enzyme performance

The overall stability of Candida rugosa lipase (CRL) was improved via immobilization onto low-cost eco-friendly carriers (agroindustrial wastes) followed by using spouted bed dryer or spray dryer. Lipase covalently immobilized on glutaraldehyde-activated rice husk proved the greatest performance, retaining 94.1% of the initial activity, followed by sugarcane bagasse (90.3%) and green coconut fiber (89.3%). On the other hand, the enzyme activity retention of CRL-lipase bioencapsulated in magnetic chitosan treated with glutaraldehyde or sodium tripolyphosphate ranged from 73.6% to 84.6%. The dried product moisture content, and water activities ranged from 4.1% and 6.5% and from 0.2 to 0.35, respectively. After storage for six months at 5 °C, the immobilized enzyme systems retained at least 70.0% of its initial activity (vs 18.3% retained activity for the free lipase). After ten reuse cycles, enzyme immobilized onto lignocellulosic carriers retained on average 72.7% of its initial activity. The thermostability of all immobilized derivatives was significantly improved and lipase immobilized onto rice husk showed a stabilization factor of 20.44 at 80 °C. The kinetic data showed that the CRL-lipase suffered alterations in catalytic behavior after the immobilization/drying, mainly a slight decrease in affinity to the substrate (↑KM). Among all the assessed immobilized lipase for aroma synthesis the rice husk immobilized lipase displayed the best result towards the production of isoamyl caprylate (62.40 g.L−1). In conclusion, this report reinforces the urgent need for the economic development of eco-sustainable immobilized biocatalysts to boost the use of enzymatic technology on industrial scale, and the feasibility of the association of immobilization and drying processes to improve the enzyme-quality features.

Effect of Binding Modules Fused to Cutinase on the Enzymatic Synthesis of Polyesters

In relation to the development of environmentally-friendly processing technologies for the continuously growing market of plastics, enzymes play an important role as green and sustainable biocatalysts. The present study reports the use of heterogeneous immobilized biocatalysts in solvent-free systems for the synthesis of aliphatic oligoesters with Mws and monomer conversions up to 1500 Da and 74%, respectively. To improve the accessibility of hydrophilic and hydrophobic substrates to the surface of the biocatalyst and improve the reaction kinetic and the chain elongation, two different binding modules were fused on the surface of cutinase 1 from Thermobifida cellulosilytica. The fusion enzymes were successfully immobilized (>99% of bound protein) via covalent bonding onto epoxy-activated beads. To the best of our knowledge, this is the first example where fused enzymes are used to catalyze transesterification reactions for polymer synthesis purposes.

Glutaraldehyde functionalization of halloysite nanoclay enhances immobilization efficacy of endoinulinase for fructooligosaccharides production from inulin

Current work describes the enhancement of immobilization efficacy of Aspergillus tritici endoinulinase onto halloysite nanoclay using crosslinker glutaraldehyde. Under statistical optimized immobilization conditions, viz. glutaraldehyde 1.50% (v/v), enzyme coupling-time 2.20 h, glutaraldehyde activation-time 1.00 h and endoinulinase load 50 IU, maximum activity yield (65.77%) and immobilization yield (82.45%) was obtained. An enhancement of 1.15- and 1.23-fold in both enzyme activity yield and immobilization yield of endoinulinase was observed, when compared with APTES-functionalized halloysite nanoclay immobilized endoinulinase. Immobilized biocatalyst showed maximum activity at pH 5.0 and temperature 60 °C with broad pH (4.0–8.5) and temperature (50–75 °C) stability. Further, optimal hydrolytic conditions (inulin concentration 8.0%; endoinulinase load 80 IU; agitation 125 rpm and hydrolysis-time 13 h) supported fructooligosaccharides yield (95.44%) in a batch system. HPTLC studies blueprint confirmed 95.44% fructooligosaccharides containing 35.41% kestose, 26.19% nystose and 9.69% fructofuranosylnystose. The developed immobilized biocatalyst shown good stability of 8 cycles for inulin hydrolysis.

Cold-Active Lipase-Based Biocatalysts for Silymarin Valorization through Biocatalytic Acylation of Silybin

Extremophilic biocatalysts represent an enhanced solution in various industrial applications. Integrating enzymes with high catalytic potential at low temperatures into production schemes such as cold-pressed silymarin processing not only brings value to the silymarin recovery from biomass residues, but also improves its solubility properties for biocatalytic modification. Therefore, a cold-active lipase-mediated biocatalytic system has been developed for silybin acylation with methyl fatty acid esters based on the extracellular protein fractions produced by the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania). The extracellular production of the lipase fraction was enhanced by 1% olive-oil-enriched culture media. Through multiple immobilization approaches of the cold-active putative lipases (using carbodiimide, aldehyde-hydrazine, or glutaraldehyde coupling), bio-composites (S1–5) with similar or even higher catalytic activity under cold-active conditions (25 °C) have been synthesized by covalent attachment to nano-/micro-sized magnetic or polymeric resin beads. Characterization methods (e.g., FTIR DRIFT, SEM, enzyme activity) strengthen the biocatalysts’ settlement and potential. Thus, the developed immobilized biocatalysts exhibited between 80 and 128% recovery of the catalytic activity for protein loading in the range 90–99% and this led to an immobilization yield up to 89%. The biocatalytic acylation performance reached a maximum of 67% silybin conversion with methyl decanoate acylating agent and nano-support immobilized lipase biocatalyst.

Polymerization strategies to produce new polymer biocatalysts for the biodiesel industry

AbstractOne of the major challenges regarding the enzymatic production of biodiesel is the development of more robust, active, and stable immobilized biocatalysts. Thus, the present work aims to develop new enzymatic biocatalysts for use in esterification and transesterification reactions. New polymer supports based on poly(styrene‐co‐divinylbenzene) and poly(methyl methacrylate‐co‐divinylbenzene) platforms were synthesized, incorporating distinct functional compounds into the polymer chains (1‐octene, cardanol, and vinyl benzoate). Two distinct polymerization strategies were adopted for support syntheses: (i) Combined suspension and emulsion polymerization process (core‐shell particles); and (ii) Two‐step polymerization reaction comprising a suspension polymerization in presence of porogenic agents, followed by vacuum application (porous and nonporous particles). The obtained particles were employed for immobilization of lipase B from Candida antarctica. The addition of functional compounds resulted in particles with distinct textural properties. Moreover, particles with 91 m2.g−1 (P(MMA‐co‐DVB)/P(MMA‐co‐DVB)) were produced through combined suspension and emulsion polymerization, whilst particles with 154 m2.g−1 (P[MMA‐co‐DVB‐co‐VB]) were produced through suspension polymerization performed in presence of n‐heptane. Moreover, highly active biocatalysts were produced, leading to esterification conversions above 80%. Thus, based on the performance in esterification and transesterification reactions, the new functional matrices resulted in highly active biocatalysts with good potential for use in biodiesel industry.