High Enzyme Loading Research Articles

Binding Peptide-Guided Immobilization of Lipases with Significantly Improved Catalytic Performance Using Escherichia coli BL21(DE3) Biofilms as a Platform

Developing novel immobilization methods to maximize the catalytic performance of enzymes has been a permanent pursuit of scientific researchers. Engineered Escherichia coli biofilms have attracted great concern as surface display platforms for enzyme immobilization. However, current biological conjugation methods, such as the SpyTag/SpyCatcher tagging pair, that immobilize enzymes onto E. coli biofilms seriously hamper enzymatic performance. Through phage display screening of lipase-binding peptides (LBPs) and co-expression of CsgB (nucleation protein of curli nanofibers) and LBP2-modified CsgA (CsgALBP2, major structural subunit of curli nanofibers) proteins, we developed E. coli BL21::ΔCsgA-CsgB-CsgALBP2 (LBP2-functionalized) biofilms as surface display platforms to maximize the catalytic performance of lipase (Lip181). After immobilization onto LBP2-functionalized biofilm materials, Lip181 showed increased thermostability, pH, and storage stability. Surprisingly, the relative activity of immobilized Lip181 increased from 8.43 to 11.33 U/mg through this immobilization strategy. Furthermore, the highest loading of lipase on LBP2-functionalized biofilm materials reached up to 27.90 mg/g of wet biofilm materials, equivalent to 210.49 mg/g of dry biofilm materials, revealing their potential as a surface with high enzyme loading capacity. Additionally, immobilized Lip181 was used to hydrolyze phthalic acid esters, and the hydrolysis rate against dibutyl phthalate was up to 100%. Thus, LBP2-mediated immobilization of lipases was demonstrated to be far more advantageous than the traditional SpyTag/SpyCatcher strategy in maximizing enzymatic performance, thereby providing a better alternative for enzyme immobilization onto E. coli biofilms.

Modification of Electrospun Regenerate Cellulose Nanofiber Membrane via Atom Transfer Radical Polymerization (ATRP) Approach as Advanced Carrier for Laccase Immobilization.

This study aimed to modify an electrospun regenerated cellulose (RC) nanofiber membrane by surface grafting 2-(dimethylamino) ethyl methacrylate (DMAEMA) as a monomer via atom transfer radical polymerization (ATRP), as well as investigate the effects of ATRP conditions (i.e., initiation and polymerization) on enzyme immobilization. Various characterizations including XPS, FTIR spectra, and SEM images of nanofiber membranes before and after monomer grafting verified that poly (DMAEMA) chains/brushes were successfully grafted onto the RC nanofiber membrane. The effect of different ATRP conditions on laccase immobilization was investigated, and the results indicated that the optimal initiation and monomer grafting times were 1 and 2 h, respectively. The highest immobilization amount was obtained from the RC-Br-1h-poly (DMAEMA)-2h membrane (95.04 ± 4.35 mg), which increased by approximately 3.3 times compared to the initial RC membrane (28.57 ± 3.95 mg). All the results suggested that the optimization of initiation and polymerization conditions is a key factor that affects the enzyme immobilization amount, and the surface modification of the RC membrane by ATRP is a promising approach to develop an advanced enzyme carrier with a high enzyme loading capacity.

Rational design of non-toxic GOx-based biocatalytic nanoreactor for multimodal synergistic therapy and tumor metastasis suppression.

Rationale: Glucose oxidase (GOx)-based biocatalytic nanoreactors can cut off the energy supply of tumors for starvation therapy and deoxygenation-activated chemotherapy. However, these nanoreactors, including mesoporous silica, calcium phosphate, metal-organic framework, or polymer nanocarriers, cannot completely block the reaction of GOx with glucose in the blood, inducing systemic toxicity from hydrogen peroxide (H2O2) and anoxia. The low enzyme loading capacity can reduce systemic toxicity but limits its therapeutic effect. Here, we describe a real 'ON/OFF' intelligent nanoreactor with a core-shell structure (GOx + tirazapamine (TPZ))/ZIF-8@ZIF-8 modified with the red cell membrane (GTZ@Z-RBM) for cargo delivery.Methods: GTZ@Z-RBM nanoparticles (NPs) were prepared by the co-precipitation and epitaxial growth process under mild conditions. The core-shell structure loaded with GOx and TPZ was characterized for hydrate particle size and surface charge. The GTZ@Z-RBM NPs morphology, drug, and GOx loading/releasing abilities, system toxicity, multimodal synergistic therapy, and tumor metastasis suppression were investigated. The in vitro and in vivo outcomes of GTZ@Z-RBM NPs were assessed in 4T1 breast cancer cells.Results: GTZ@Z-RBM NPs could spatially isolate the enzyme from glucose in a physiological environment, reducing systemic toxicity. The fabricated nanoreactor with high enzyme loading capacity and good biocompatibility could deliver GOx and TPZ to the tumors, thereby exhausting glucose, generating H2O2, and aggravating hypoxic microenvironment for starvation therapy, DNA damage, and deoxygenation-activated chemotherapy. Significantly, the synergistic therapy effectively suppressed the breast cancer metastasis in mice and prolonged life without systemic toxicity. The in vitro and in vivo results provided evidence that our biomimetic nanoreactor had a powerful synergistic cascade effect in treating breast cancer.Conclusion: GTZ@Z-RBM NPs can be used as an 'ON/OFF' intelligent nanoreactor to deliver GOx and TPZ for multimodal synergistic therapy and tumor metastasis suppression.

Selective ene-reductase immobilization to magnetic nanoparticles through a novel affinity tag.

Magnetic nanoparticles (MNPs) are becoming more important as carriers, because of their large specific surface area and easy separability. They are increasingly used in enzyme technology, diagnostics, and drug delivery. For the directed and almost irreversible immobilization of proteins on MNPs, we have developed a new selective (His-Arg)4 peptide-tag, that binds fusion proteins directly from an E. coli cell lysate to non-functionalized, low-cost MNPs. Using the immobilization of an ene-reductase as an example, we could demonstrate that the fusion with this tag increases thermostability without reducing overall activity (ER w/o tag: t1/2 =3.7 h, (HR)4 -ER: t1/2 =9.9 h). Immobilization by adsorption in Tris buffer resulted in very high enzyme loads with approx. 380 mg g-1 and 67% residual activity. The immobilization on the MNPs allowed a fast concentration, buffer exchange, and reuse. While about 50% of the activity was lost after the first reuse, we were able to show that the activity did not decrease further and was stable for another ninecycles. According to our studies, our tag highly works for any kind of immobilization on MNPs and holds the potential for enzyme immobilizations as well as for drug delivery and sensors.

One-step separation and immobilization of his-tagged enzyme directly from cell lysis solution by biomimetic mineralization approach

We report a facile approach to fabricate biocatalyst, which is the BglucLH-Cu3(PO4)2 nanoflowers (BglucH-NF) possessed microscale flower-like structure assembled with nanoscale petals, by integrating the separation and immobilization of his-tagged enzyme directly from cell lysis solution in a single biomineralization step without the preparation of support. The recombinant β-glucosidase-linker-His (BglucLH) being rich in imidazole group was constructed to enhance the specific binding of enzyme with copper ions. We have demonstrated that the activity recovery and separation efficiency of BglucLH-NF were improved by 253.2 % and 290.1 % in comparison with Bgluc-Cu3(PO4)2 nanoflowers (Bgluc-NF), indicating the positive reinforcement of his-tag for improving separation and immobilization efficiency. The BglucLH-NF could maintain 73.2 ± 2.77 % of the initial activity after repeating 15 cycles, exhibiting excellent reusability. The integrated separation and immobilization of his-tagged enzyme directly from cell lysis solution by biomimetic mineralization approach displays great potential in the convenient fabrication of biocatalyst with high enzyme loading, stability, and recyclability.

Immobilization of peroxidase on textile carrier materials and their application in the bleaching of colored whey

Textiles represent promising support materials for enzymes. The goal of the present work was to investigate the immobilization of commercial peroxidase on a polyester needle felt and the repeated use in the gentle degradation of norbixin in whey from dairy cheese as a practical application. High enzyme loads were obtained by a 2-step immobilization procedure. First, the number of functional groups on the textile surface was increased by a modification with amino-functional polyvinylamine. Second, the enzyme was immobilized by using 2 types of crosslinking agents. Due to the iron content of peroxidase, inductively coupled plasma-optical emission spectrometry was used for the quantitative determination of the enzyme load on the textile. The enzyme activity was evaluated using common 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) assay for peroxidases. By the variation of enzyme input and crosslinker concentration, a maximal enzyme load of 80 mg/g of textile was achieved, and a maximum specific activity of 57 U/g of textile. For the visualization of the enzyme on the fiber surface, fluorescence microscopy as well as scanning probe microscopy were used. The immobilized peroxidase showed significant activity, even after 50 reuse cycles. In addition, the potential of the new support and enzyme combination in commercial whey bleaching was demonstrated successfully on a 10-L scale.

Confining the motion of enzymes in nanofiltration membrane for efficient and stable removal of micropollutants

Enzymes in living cells are highly dynamic but at the same time regularly confined for achieving efficient metabolism. Inspired by this phenomenon, we have prepared a novel biocatalytic membrane with high enzyme activity and stability by tuning the confinement strength of the membrane to enzyme, which was achieved via modifying the support layer of a polymeric nanofiltration (NF) membrane and reversely filtrating enzyme. A mussel-inspired coating was used to modify the support interior of the NF membrane to enhance charge and steric effects on enzyme, thus stabilizing enzyme in the membrane with little increment in mass transfer resistance for substrate and products (only 20% permeability loss with a high enzyme loading of 1.34 mg/cm2). A suitable confinement strength of the membrane to enzyme could delay the enzyme leakage and endow enzyme with certain mobility for efficient reaction. Thus, the obtained biocatalytic membrane exhibited a negligible decline in BPA removal efficiency for 7 reuse cycles (<3.5%) or 36 h continuous operation (<1%) in flow through mode, resulting in a long-term stability adequate for micropollutant removal. For the first time, enzyme mobility was defined and calculated to quantify the confinement strength of the membrane, which could be used to optimize the microenvironment for enzyme immobilization and predict the performance of the biocatalytic membrane. This work concluded that rationally regulating the enzyme mobility in the membrane and a periodic back-flushing operation for redistribution of enzymes could achieve a long-term stable removal of micropollutant in water by a biocatalytic membrane.

Immobilizing redox enzymes at mesoporous and nanostructured electrodes

Three key challenges are stimulating intensive research in the development of productive direct electron transfer mode enzyme electrodes: proper enzyme orientation, high enzyme loading, and full retention of enzyme activity. In this review, we summarize some significant advances that have been reported in the last years on the design of mesoporous and nanostructured electrodes as enzyme scaffolds and of innovative methodologies for wiring enzymes to electrodes. Particular attention is given to investigations on physical factors that determine a favorable enzyme immobilization, to provide rational guidelines for the design of productive enzymatic electrodes. Finally, some emerging trends focused on the spatial organization of either single enzymes or enzyme cascades are also briefly addressed.

MicroGelzymes: pH-Independent Immobilization of Cytochrome P450 BM3 in Microgels.

Microgels are an emerging class of "ideal" enzyme carriers because of their chemical and process stability, biocompatibility, and high enzyme loading capability. In this work, we synthesized a new type of permanently positively charged poly(N-vinylcaprolactam) (PVCL) microgel with 1-vinyl-3-methylimidazolium (quaternization of nitrogen by methylation of N-vinylimidazole moieties) as a comonomer (PVCL/VimQ) through precipitation polymerization. The PVCL/VimQ microgels were characterized with respect to their size, charge, swelling degree, and temperature responsiveness in aqueous solutions. P450 monooxygenases are usually challenging to immobilize, and often, high activity losses occur after the immobilization (in the case of P450 BM3 from Bacillus megaterium up to 100% loss of activity). The electrostatic immobilization of P450 BM3 in permanently positively charged PVCL/VimQ microgels was achieved without the loss of catalytic activity at the pH optimum of P450 BM3 (pH 8; ∼9.4 nmol 7-hydroxy-3-carboxy coumarin ethyl ester/min for free and immobilized P450 BM3); the resulting P450-microgel systems were termed P450 MicroGelzymes (P450 μ-Gelzymes). In addition, P450 μ-Gelzymes offer the possibility of reversible ionic strength-triggered release and re-entrapment of the biocatalyst in processes (e.g., for catalyst reuse). Finally, a characterization of the potential of P450 μ-Gelzymes to provide resistance against cosolvents (acetonitrile, dimethyl sulfoxide, and 2-propanol) was performed to evaluate the biocatalytic application potential.

Synthesis and characterization of magnetic cross-linked enzyme aggregate and its evaluation of the alternating magnetic field (AMF) effects in the catalytic activity

A magnetic cross-linked enzyme aggregate (MCLEA) of cellulases and a non-magnetic analogue (CLEA) were synthesized and further investigated. The obtained MCLEA and CLEA showed a high enzyme loading efficiency of 90% and 88%, respectively. However, the MCLEA presented an activity 33% higher than the CLEA one, suggesting a catalytic enhancement effect as a result of magnetic nanoparticle presence in cross-linked cellulase aggregates structure. In addition, MCLEA shows an unusual behavior with highest enzymatic activity at low temperatures near to 37 °C. Lastly, a full factorial design (2x2) with two variables at two levels, frequency (203–420 kHz) and amplitude (3–6 kA m−1), was used in order to better investigate the effects of an alternating magnetic field (AMF) on MCLEA activity. AMF application decreased MCLEA activity, particularly in the highest values of frequency and amplitude, when compared to condition field free. In addition, frequency implied in more significant effects on MCLEA activity when compared to AMF amplitude.

A graphene-laminated electrode with high glucose oxidase loading for highly-sensitive glucose detection

Graphene oxide (GO) has received considerable attention for glucose detection because of high surface area, abundant functional groups, and good biocompatibility. Defects and functional groups of the GO are beneficial to immobilization of glucose oxidase (GOD), but sacrificing electron-transfer capability for highly-sensitive detection. In order to obtain high GOD loading and highly-sensitive detection of biosensors, we first designed and fabricated a graphene-laminated electrode by combining GO and edge-functionalized graphene (FG) layers together onto glassy-carbon electrode. The graphene-laminated electrodes exhibited faster electron transfer rate, higher GOD loading of 3.80 × 10−9 mol∙cm-2, and higher detection sensitivity of 46.71 μA∙mM-1∙cm-2 than other graphene-based biosensors reported in literature. Such high performance is mainly attributed to the abundant functional groups of GO, high electrical conductivity of FG, and strong interactions between components in the graphene-laminated electrodes. By virtue of their high enzyme loading and highly-sensitive detection, the graphene-laminated electrodes show great potential to be widely used as high-performance biosensors in the field of medical diagnosis.

Metal-Organic Frameworks: A Potential Platform for Enzyme Immobilization and Related Applications.

Enzymes, as natural catalysts with remarkable catalytic activity and high region-selectivities, hold great promise in industrial catalysis. However, applications of enzymatic transformation are hampered by the fragility of enzymes in harsh conditions. Recently, metal–organic frameworks (MOFs), due to their high stability and available structural properties, have emerged as a promising platform for enzyme immobilization. Synthetic strategies of enzyme-MOF composites mainly including surface immobilization, covalent linkage, pore entrapment and in situ synthesis. Compared with free enzymes, most immobilized enzymes exhibit enhanced resistance against solvents and high temperatures. Besides, MOFs serving as matrixes for enzyme immobilization show extraordinary superiority in many aspects compared with other supporting materials. The advantages of using MOFs to support enzymes are discussed. To obtain a high enzyme loading capacity and to reduce the diffusion resistance of reactants and products during the reaction, the mesoporous MOFs have been designed and constructed. This review also covers the applications of enzyme-MOF composites in bio-sensing and detection, bio-catalysis, and cancer therapy, which is concerned with interdisciplinary nano-chemistry, material science and medical chemistry. Finally, some perspectives on reservation or enhancement of bio-catalytic activity of enzyme-MOF composites and the future of enzyme immobilization strategies are discussed.

Clay/au nanoparticle composites as acetylcholinesterase carriers and modified-electrode materials: A comparative study

This work presents the first comparative study on syntheses, characterization, and applications of different clay/gold nanoparticle composites (clay/AuNPs) as acetylcholinesterase (AChE) carriers and modified-electrode materials to detect chlorpyrifos, an organophosphate pesticide. The selected based-clays were plate-like kaolinite (Kaol; 1:1 aluminum phyllosilicate), globular montmorillonite (Mt; 2:1 aluminum phyllosilicate), globular bentonite (Bent; 2:1 aluminum phyllosilicate), and fibrous sepiolite (Sep; 2:1 inverted ribbons of magnesium phyllosilicate). The clay/AuNPs were synthesized using in situ chemical reduction of the gold precursor with anchoring 3-aminopropyl triethoxysilane (APTES) on the clay surfaces. The studied materials were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), nitrogen adsorption/desorption, Fourier transformed infrared (FTIR) spectroscopy, thermogravimetric analyzer (TGA), zeta-potential analysis, and electrochemical impedance spectroscopy (EIS). Surface area, surface charges, and hydrophilic/hydrophobic nature of based-clays and the clay/AuNPs played major roles on AChE loading, residual activity, and sensor storage stability. The Mt/AuNPs was determined, among the studied clays, as the best supports for AChE loading and residual activities due to its high specific surface area and weak electrostatic interaction with the immobilized enzyme. Since apparent catalytic efficiencies kcatapp/KMappof the immobilized AChE in the four clay/AuNPs were found statistically similar, Mt/AuNPs which contained the highest enzyme loading was the favorite modified-electrode material. For chlorpyrifos detection, Kaol/AuNPs-based sensors resulted in the lowest detection limit, presumably due to the strong hydrophobic surface that helps to concentrate the nonpolar pesticide to the vicinity of the immobilized enzyme. Unfortunately, the highest electrostatic repulsion between the Kaol/AuNPs and AChE, and the dehydrated Kaol surface resulted in the lowest storage stability of the AChE pesticide sensors. Overall, Mt/AuNPs showed the highest potential as the AChE carrier and the modified electrode material for chlorpyrifos determination.

Preparation and characterization of amino and carboxyl functionalized core-shell Fe3O4/SiO2 for L-asparaginase immobilization: A comparison study

Magnetic nanoparticles are well known as facile and effective support for enzyme immobilization since they have a high surface area, large surface-to-volume ratio, easy separation, a fast and high enzyme loading. This study aims to provide insights on whether acidic or basic modified particles are more effective for L-asparaginase (ASNase) immobilization. Therefore, amino (Fe3O4/SiO2/NH2) and carboxyl-functionalized (Fe3O4/SiO2/COOH) particles were prepared. The functional groups, crystalline structure, magnetic properties, morphology, chemical composition and thermal behaviour of the prepared modified nanoparticles were examined via Fourier-transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD), vibrating-sample magnetometer (VSM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDAX). Under the optimum conditions, the immobilized enzymes were more stable within a certain range of temperatures and pH values in comparison to free enzyme. On the other hand, the immobilized enzymes showed greater stability after incubation for 3 h at 50 °C. The free enzyme maintained only 30% of its initial activity for 4 weeks at 4 °C, while Fe3O4/SiO2/NH2/ASNase and Fe3O4/SiO2/COOH/ASNase retained more than 78.9% and 56.5% of initial activities under the same conditions, respectively. Moreover, Fe3O4/SiO2/NH2/ASNase (77.2%) and Fe3O4/SiO2/COOH/ASNase (57.4%) displayed excellent operational stability after 17 repeated cycles. These findings suggested that the Fe3O4/SiO2/NH2 and Fe3O4/SiO2/COOH may be utilized as efficient and sustainable supports to developed immobilized ASNase in several biotechnological applications.

Production of cellulose nanocrystals integrated into a biochemical sugar platform process via enzymatic hydrolysis at high solid loading

This work evaluated the viability of integrating the isolation of cellulose nanocrystals (CNCs) via enzymatic hydrolysis at a high solid loading into the biochemical platform process for the production of sugars from sugarcane bagasse (SCB). SCB was first processed at a biochemical conversion pilot plant and bleached to yield a cellulose-rich pulp, which was enzymatically hydrolyzed at high solid and low enzyme loadings. The resulting hydrolysate had high sugar concentration (>120 g/L glucose) and the CNCs (20 nm in diameter) isolated directly from the hydrolysis residue showed superior properties (higher thermal stability, higher crystallinity index, and higher particle diameter uniformity) than the CNCs prepared from commercial bleached eucalyptus Kraft pulp. These findings demonstrate the technical viability of the proposed integrated process that combined with the high CNCs yield (approximately50%) and the no need for the costly ultrasonic dispersion treatment step to obtain nanoparticles can further contribute for improving the economic and environmental viability of the proposed enzyme-mediated isolation process.

In Vivo Enzyme Entrapment in a Protein Crystal.

Cry3Aa is a protein that forms crystals naturally in the bacterium Bacillus thuringiensis. Here we report that coexpression of Cry3Aa and a Proteus mirabilis lipase without recombinant fusion results in the efficient passive entrapment of the lipase within the nanoporous channels of the resulting crystals. This Cry3Aa crystal-mediated entrapment provides multiple benefits to the lipase including a high enzyme loading, significantly improved thermostability, increased proteolytic resistance, and the ability to be utilized as a recyclable biodiesel catalyst. These characteristics, along with its greatly simplified method of isolation, highlight the potential of Cry3Aa crystal-mediated enzyme entrapment for use in biocatalysis and other biotechnological applications.

Di‐functional magnetic nanoflowers: A highly efficient support for immobilizing penicillin G acylase

AbstractThe novel di‐functional magnetic nanoflowers (DMNF) which had both epoxy groups and hydrophilic catechol as well as phthaloquinone groups capable of covalently coupling of penicillin G acylase (PGA) were characterized by scanning electron microscopy, transmission electron microscope (TEM), vibrating sample magnetometer, N2 adsorption, and so on. The studies showed that DMNF possessed “hierarchical petal” structure of nanosheets had specific saturation magnetization of 39.7 emu/g and average pore diameter of 25.4 nm as well as specific surface area of 17.28 m2/g. For hydrolysis of penicillin G potassium catalyzed by the PGA immobilized on DMNF with enzyme loading of 106 mg/g‐support, its apparent activity reached 2,667 U/g, which benefited from the “hierarchical petal” and large pore structure of the magnetic DMNF leading to high enzyme loading and fast diffusion of substrate molecules to the immobilized PGA to reaction. The apparent activity of the immobilized PGA could keep 2,408 U/g (above 90% of its initial activity) after repeating use for 10 cycles. The magnetic immobilized PGA exhibited excellent operational stability due to covalently coupling of the enzyme molecules between the support by covalent interaction of the amino groups of PGA and the reactive groups of epoxy, catechol, and phthaloquinone groups on DMNF. Furthermore, the PGA displayed good acid and alkaline resistance as well as thermal stability by immobilization using DMNF.

Metal–organic frameworks (MOFs): a novel support platform for ASNase immobilization

Metal–organic frameworks (MOFs) have been recently studied for a variety of applications because of their huge surface area, large porosity, and tunable functionality. In this work, for the first time, the efficient immobilization of l-asparaginase (ASNase, EC 3.5.1.1) by using MOF as a simple and novel support is demonstrated. The functional groups, morphology, chemical composition, and crystal structure of the support and immobilized ASNase were investigated by using different methods, including Fourier transform infrared spectroscopy, scanning electron microscopy, energy-dispersive spectrometer, and X-ray diffraction. Afterward, the enzymatic activities and thermodynamic parameters of the immobilized l-ASNase (ASNase@ZIF-8) were compared with free one. After enzyme immobilization, the optimum temperature shifted from 50 to 60 °C, while the optimum pH remains unchanged at 9.0. However, the pH and thermal stability of the ASNase@ZIF-8 was significantly improved compared to the free one. The ASNase@ZIF-8 displayed an excellent long-term storage stability, which could protect more than 56% of the initial activity at 25 °C for 4 weeks. Besides, the ASNase@ZIF-8 had high reusability, which showed a high degree of activity (more than 45%) after 10 cycles. Km and Vmax values were 0.18 mM and 64.5 µmol/min for ASNase@ZIF-8 and those for free ASNase were 0.40 mM and 68.0 µmol/min, respectively. The proposed support based on ZIF-8 was superior in terms of high enzyme loading capacity (82.0%), high enzyme catalytic activity, and easy preparation process. Overall, newly developed support for ASNase may provide a new platform for its biotechnological applications.

Acid and Enzymatic Fractionation of Olive Stones for Ethanol Production Using Pachysolen tannophilus

Olive stones are an abundant lignocellulose material in the countries of the Mediterranean basin that could be transformed to bioethanol by biochemical pathways. In this work, olive stones were subjected to fractionation by means of a high-temperature dilute-acid pretreatment followed by enzymatic hydrolysis of the pretreated solids. The hydrolysates obtained in these steps were separately subjected to fermentation with the yeast Pachysolen tannophilus ATCC 32691. Response surface methodology with two independent variables (temperature and reaction time) was applied for optimizing D-xylose production from the raw material by dilute acid pretreatment with 0.01 M sulfuric acid. The highest D-xylose yield in the liquid fraction was obtained in the pretreatment at 201 °C for 5.2 min. The inclusion of a detoxification step of the acid prehydrolysate, by vacuum distillation, allowed the fermentation of the sugars into ethanol and xylitol. The enzymatic hydrolysis of the pretreated solids was solely effective when using high enzyme loadings, thus leading to easily fermentable hydrolysates into ethanol. The mass macroscopic balances of the overall process illustrated that the amount of inoculum used in the fermentation of the acid prehydrolysates strongly affected the ethanol and xylitol yields.

Laccase immobilisation on Poly(ethylene) terephthalate grafted with maleic anhydride (PET-g-MAH) nanofiber mat

A comparative study was carried out on three different enzyme immobilisation methods which were physical adsorptions (PA), covalent bonding (CV) and covalent bonding of cross-linked enzyme aggregates (CL) for laccase immobilisation on Poly(ethylene) Terephthalate (PET) grafted with Maleic Anhydride (MAH) nanofiber mats. The chemically inert PET was successfully grafted with MAH at temperature between 40 – 45 °C and used as the carrier to immobilised laccase in the form of electrospun nanofiber mats. The peaks from carbonyl (C=O) and alkene (C=C) groups appeared on the spectral subtraction between PET and PET-g-MAH nanofiber mats. These groups might be the potential group to form covalent bond between the amine groups of laccase enzyme. Laccase immobilised on the PET-g-MAH nanofiber mats using CL methods using glutaraldehyde as crosslinker gave the best performance with the highest enzyme loading and immobilisation yield which were 40.88 μg/mg and 48.37 %. On top of that, the immobilised laccase on PET-g-MAH nanofiber mats also managed to retain 69.01 % of its initial activity after 10 repeated cycles of 2, 2-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidations. These results demonstrate that PET-g-MAH nanofiber is a good material to be considered as laccase carrier.