Carrier For Enzyme Immobilization Research Articles

A dual stable MOF constructed through ligand exchange for enzyme immobilization with improved performance in biodiesel production

The poor stability of metal-organic frameworks (MOFs) limits their practical applications in many fields. Zeolitic imidazolate frameworks (ZIF-8) has been widely studied as an enzyme immobilization carrier. In this paper, the stability of ZIF-8-immobilized lipase in methanolysis for biodiesel production was studied. Fatty acids as conventional substances involved in lipase-mediated natural acylation were found to destroy the structure of ZIF-8 through ligand exchange mechanism. A new MOF was successfully constructed by using phthalic acid (pKa much smaller than 2-methylimidazole) as the exchange ligand. This novel MOF (abbreviated as LeZIF-8-PAX, X represents reaction hours) had dual stability in water and even in pure oleic acid. Lipase immobilized on LeZIF-8-PA0.5 showed higher specific activity. FTIR analysis showed that enzyme molecule immobilized on Le-ZIF-8-PA0.5 had higher conformational flexibility than that with ZIF-8 as the carrier. When applied to methanolysis for biodiesel production, ET 2.0/LeZIF-8-PA0.5 maintained intact morphology while ET 2.0/ZIF-8 almost lost its original morphology, with enzyme activity of 81.5% and 60.2% retained respectively after 5 batch reactions. This work is anticipated to provide new ideas for constructing stable MOFs and promoting the application of MOFs-immobilized enzymes in many chemical and pharmaceutical industrial processes.

Mussel-Inspired Electro-oxidation-Modified Three-Dimensional Printed Carriers for a Versatile Enzyme Immobilization Approach

Conventional enzyme immobilization approaches can only immobilize certain specific enzymes with poor generality. Attempts to improve the universality of enzyme types tend to impart them with more enzymatic catalysis applications. Here, inspired by mussel adhesive proteins, we present a novel eco-friendly surface carrier that was 3D printed and modified by electro-oxidation for enzyme immobilization. The carrier was fabricated through 3D printing by transforming acrylonitrile butadiene styrene (ABS) material into a suitable structure (3DABS). Then, electro-oxidative modification was performed on the surface to form a polydopamine (PDA) coating (3DABS-PDA). The desired structures for the enzyme immobilization carriers were obtained through 3D printing technology, while electro-oxidation modification of the surface provided numerous and firmly covalent binding sites. Based on these features, we have demonstrated that 3D printed and electro-oxidation-modified carriers could be applied to immobilize different types of enzymes. The loading capacity of all immobilized enzymes (galV, EG5C-1, XynLK9, and kdcA) exceeded 25 mg·g–1 (37.7 mg·g–1 for galV), and after 10 reuse cycles, the substrate conversion rate of 3DABS-PDA@galV was still over 85%. The carriers can be reused after simple processing. These results indicate that 3DABS-PDA provides an efficient, sustainable, and versatile approach for enzyme immobilization and exhibits excellent value in various enzymatic catalysis applications.

Catechol-tetraethylenepentamine co-deposition modified cellulose filter paper for α-glucosidase immobilization and inhibitor screening from traditional Chinese medicine.

Cellulose filter paper (CFP) is expected to be an ideal carrier for enzyme immobilization due to its sustainability and biocompatibility. However, the interaction between the carrier and enzyme might change the spatial conformation of the enzyme and its microenvironment, and thus the flexibility of the enzyme molecule or the transport of the substrate to the active site would be hampered. In this work, a two-component system of catechol and tetraethylene pentamine was used to replace dopamine, and a polydopamine-like composite layer was deposited on the surface of CFP to introduce amino groups, which was similar to the self-polymerization-adhesion behavior of dopamine. Using polyethylene glycol diglycidyl ether with flexible spacer arms as the cross-linking agent, α-glucosidase was covalently bonded to amino-modified CFP through an epoxy ring-opening reaction. The immobilized α-glucosidase exhibited greater tolerance to pH and high temperature. After 10 repeated uses, the immobilized α-glucosidase maintained relatively high enzyme activity. Its kinetic behavior was investigated to illustrate the reliability for enzyme inhibitor screening. Finally, a screening method combining an immobilized enzyme and capillary electrophoresis analysis was proposed and applied to screening inhibitors from 11 kinds of traditional Chinese medicines, among which Chebulae Fructus, Phyllanthi Fructus and Terminaliae Relliricae Fructus exhibited strong enzyme inhibitory activities.

Hydrogel-Based Multi-enzymatic System for Biosynthesis.

Biosynthesis involving multi-enzymatic reactions is usually an efficient and economic method to produce plentiful important molecules. To increase the product yield in biosynthesis, the involved enzymes can be immobilized to carriers for enhancing enzyme stability, increasing synthesis efficiency and improving enzyme recyclability. Hydrogels with three-dimensional porous structures and versatile functional groups are promising carriers for enzyme immobilization. Herein, we review the recent advances of the hydrogel-based multi-enzymatic system for biosynthesis. First, we introduce the strategies of enzyme immobilization in hydrogel, including the pros and cons of the strategies. Then we overview the recent applications of the multi-enzymatic system for biosynthesis, including cell-free protein synthesis (CFPS) and non-protein synthesis, especially high value-added molecules. In the last section, we discuss the future perspective of the hydrogel-based multi-enzymatic system for biosynthesis.

Synthesis of polymer-functionalized β-cyclodextrin, Mg2+ doped, coating magnetic Fe3O4 nanoparticle carriers for penicillin G acylase immobilization

Based on the beneficial effects of magnesium ions on biomolecules, it was first introduced into enzyme immobilization carriers to promote the comprehensive performance of immobilized enzymes in repeated use. Firstly, Fe3O4 nanoparticles (NPs) were prepared by the inverse microemulsion method, then, Mg2+ was doped in the process of coating β-cyclodextrin (β-CD) on the surface of Fe3O4 NPs. Afterward, through acetal reaction, glutaraldehyde (GA) was grafted to the material’s surface to obtain Mg2+-Fe3O4 @ β-CD-g-GA NPs, and covalent bond immobilization of Penicillin G acylase (PGA) was realized by the Schiff base reaction of the carriers’ aldehyde group and PGA’s amino group. After optimization of process conditions, the results show that the performances of the immobilized PGA achieve the best, when the concentration of the enzyme solution is 3.00 vol%, pH is 8.0, the immobilization time is 30 h, and the immobilization temperature is 37 °C. Its enzyme activity recovery (EAR), enzyme activity (EA) and enzyme loading capacity (ELC) are 95.6%, 31997 U/g, 110 mg/g, respectively. Lastly, the operational stability, reusability, and storage stability of Mg2+-Fe3O4 @ β-CD-g-GA-PGA NPs were researched. Relative to free PGA, immobilized PGA has better operation stability and storage stability. After 11 repeated uses, the immobilized PGA still has 56% of the initial vitality, and the carrier recovery (Re) is 86.6%.

Validation of Easy Fabrication Methods for PDMS-Based Microfluidic (Bio)Reactors

The common method for producing casting molds for the fabrication of polydimethylsiloxane (PDMS) chips is standard photolithography. This technique offers high resolution from hundreds of nanometers to a few micrometers. However, this mold fabrication method is costly, time-consuming, and might require clean room facilities. Additionally, there is a need for non-micromechanics experts, who do not have specialized equipment to easily and quickly prototype chips themselves. Simple, so-called, makerspace technologies are increasingly being explored as alternatives that have potential to enable anyone to fabricate microfluidic structures. We therefore tested simple fabrication methods for a PDMS-based microfluidic device. On the one hand, channels were replicated from capillaries and tape. On the other hand, different mold fabrication methods, namely laser cutting, fused layer 3D printing, stereolithographic 3D printing, and computer numerical control (CNC) milling, were validated in terms of machine accuracy and tightness. Most of these methods are already known, but the incorporation and retention of particles with sizes in the micrometer range have been less investigated. We therefore tested two different types of particles, which are actually common carriers for the immobilization of enzymes, so that the resulting reactor could ultimately be used as a microfluidic bioreactor. Furthermore, CNC milling provide the most reliable casting mold fabrication method. After some optimization steps with regard to manufacturing settings and post-processing polishing, the chips were tested for the retention of two different particle types (spherical and non-spherical particles). In this way, we successfully tested the obtained PDMS-based microfluidic chips for their potential applicability as (bio)reactors with enzyme immobilization carrier beads.

Nano-fibrillated cellulose-based scaffolds for enzyme (co)-immobilization: Application to natural product glycosylation by Leloir glycosyltransferases

Polysaccharide-based scaffolds are promising carriers for enzyme immobilization. Here, we demonstrate a porous scaffold prepared by direct-ink-writing 3D printing of an ink consisting of nanofibrillated cellulose, carboxymethyl cellulose and citric acid for immobilization application. Negative surface charge introduced by the components made the scaffold amenable for an affinity-like immobilization via the cationic protein module Zbasic2. Zbasic2 fusions of two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Z-CGT; sucrose synthase, Z-SuSy) were immobilized individually, or co-immobilized, and applied to synthesize the natural C-glycoside nothofagin. The cascade reaction involved β-C-glycosylation of phloretin (10 mM, ~90 % conversion) from UDP-glucose, provided from sucrose and catalytic amounts of UDP (1.0 mM). Enzymes were co-immobilized at ~65 mg protein/g carrier to receive activities of 9.5 U/g (Z-CGT) and 4.5 U/g (Z-SuSy) in 22–33 % yield (protein) and an effectiveness of 23 % (Z-CGT) and 13 % (Z-SuSy). The scaffold-bound enzymes were recyclable for 5 consecutive reactions.

Immobilization of EreB on Acid-Modified Palygorskite for Highly Efficient Degradation of Erythromycin.

Erythromycin is one of the most commonly used macrolide antibiotics. However, its pollution of the ecosystem is a significant risk to human health worldwide. Currently, there are no effective and environmentally friendly methods to resolve this issue. Although erythromycin esterase B (EreB) specifically degrades erythromycin, its non-recyclability and fragility limit the large-scale application of this enzyme. In this work, palygorskite was selected as a carrier for enzyme immobilization. The enzyme was attached to palygorskite via a crosslinking reaction to construct an effective erythromycin-degradation material (i.e., EreB@modified palygorskite), which was characterized using FT-IR, SEM, XRD, and Brunauer–Emmett–Teller techniques. The results suggested the successful modification of the material and the loading of the enzyme. The immobilized enzyme had a higher stability over varying temperatures (25–65 °C) and pH values (6.5–10.0) than the free enzyme, and the maximum rate of reaction (Vmax) and the turnover number (kcat) of the enzyme increased to 0.01 mM min−1 and 169 min−1, respectively, according to the enzyme-kinetics measurements. The EreB@modified palygorskite maintained about 45% of its activity after 10 cycles, and degraded erythromycin in polluted water to 20 mg L−1 within 300 min. These results indicate that EreB could serve as an effective immobilizing carrier for erythromycin degradation at the industrial scale.

Preparation optimization and stability comparison study of alkali-modified biochar immobilized laccase under multi-immobilization methods

Alkali modified biochar has attracted many interests as a potential enzyme immobilization carrier. In this paper, the preparation of alkali-modified immobilized laccases under multi-immobilization methods (adsorption: A/lac@A-MB; adsorption-crosslinking: A-C/lac@A-MB; covalent bonding: CB/lac@A-MB) were optimized via Design-Expert for the first time, and the stability of alkali-modified immobilized laccases was compared with other literatures. In addition, SEM, BET, FT-IR and XRD analyses were used to characterize the prepared immobilized enzyme samples. The results showed that the three kinds of immobilized laccases have improved the stability to different degrees relative to free laccase. In the pH range of 3–7, temperature range of 25–55 °C, and storage for more than 30 days, after 5 reaction cycles, the relative activity of the laccase immobilized on three different supports can still be greater than 50%. In addition, CB/lac@A-MB obtained the highest Vmax (0.94 U/mg), specific activity (0.66 U/mg) and activity recovery (66.20%); A-C/lac@A-MB obtained the highest immobilization yield (67.40%) and enzyme loading (180.81 mg/g); and A/lac@A-MB obtained the lowest Km of 140 μM. The above results are comparable to other reports, aiming to provide a reference for practical application of different methods of immobilized laccase in the industrial field.

Printing toner used as carrier for immobilization of laccase

The search for seeking novel immobilized enzyme carriers is a hot topic in the field of enzyme engineering . As a kind of common office consumable, magnetic printing toner has been terribly wasted, leading to potential harm to the environment. We try to use printing toner as carrier for enzyme immobilization to realize waste reuse. In this paper, the laccase extracted from Agaricus bisporus was successfully immobilized on chitosan coated magnetic printing toner by glutaraldehyde linking. The use of printing toner is proved to be feasible. The results show that the optimal temperature of immobilized laccase is 40 °C, and the optimal pH is 3.0. Compared with free laccase, the optimal temperature and optimal pH have been obviously changed after immobilization. And the pH tolerance has been significantly improved, which can remain 53% activity after 5 cycles. Because of the use of magnetic printing toner, it is more convenient to recycle the immobilized enzyme. In this study, printing toner was used as carrier for the immobilization of enzyme and the discarded printing toner recycling was realized. • The immobilized enzyme carrier with printing toner as the main body was successfully constructed. • Laccase was successfully immobilized with the new carrier. • The new carrier uses discarded print toner to reduce environmental pollution.

Fabrication of highly porous, functional cellulose-based microspheres for potential enzyme carriers

Here, we present highly porous, cellulose-based microspheres using (2,2,6,6-tetramethylpiperidine-1-oxyl) TEMPO-oxidized cellulose fibers (TOCFs) as starting materials. The TOCFs were first dissolved in a NaOH/urea solvent and transformed into microspheres via an emulsification method. The carboxyl groups on the surface of TOCFs were successfully carried on the cellulose-based microspheres, which provides them numerous reacting or binding sites, allowing them to be easily functionalized or immobilized with biomolecules for multi-functional applications. Furthermore, the introduction of magnetic nanoparticles awards these microspheres magnetic properties, allowing them to be attracted by a magnetic field. As a proof of concept, we demonstrate the application of using these carboxylate cellulose-based microspheres for enzyme immobilization. The cellulose-based microspheres can successfully create stable covalent bonds with enzymes after the activation of carboxyl groups. The enhanced pH tolerance, thermal stability, convenient recovery, and reusability position the emulsified microspheres as promising carriers for enzyme immobilization.

Magnetic nanomaterials assisted nanobiocatalysis systems and their applications in biofuels production

Sustainable generation of biofuels is gaining substantial attraction as an alternative for substitution of petrochemical-derived products, which allows to mitigate common problems caused by the overuse of fossil fuels, such as global warming and climate change. The integration of nanotechnology and biotechnology brings a bright future in the energy sector. By the application of nanobiocatalysis, it is possible to support efficient reactions with high stability, improving the overall fuel yields. Enzymatic biocatalysis is a highly efficient and widely employed procedure in biotechnological applications due to the biodegradable nature of enzymes. However, lack of stability, high cost of operability, hard recovery and non-reusable ability are among the most critical and challenging aspects, which can be solved by the immobilization of enzymes in nanostructured materials, leading towards the development of a nanobiocatalyst. As one of the most promising nanostructured materials as versatile carriers for enzyme immobilization, magnetic nanoparticles (MNPs) have gained special attention due to its important characteristics, including high specific area, elevated enzyme loading ability, controllable particle size, modifiable surface, and most importantly, easily, and rapid separation from solutions by applying and external magnetic field. It has been reported that surface coating or modification of MNPs with a variety of complex materials, such as polymers, silica, metallic-organic frameworks (MOFs), and carbon-based materials, might allow to improve some properties and provide potential industrial exploitability. Thus, the different steps to be considered at the time to design a proper magnetic nanobiocatalyst are reviewed, including routes of synthesis, surface modification and immobilization techniques. Moreover, this work will present the role of MNPs for enhancing biofuels production, such as biodiesel, bioethanol, biohydrogen and biogas; and how nanobiocatalysts may allow improving the performance of these bioprocesses.

Convenient Immobilization of α‐L‐Rhamnosidase on Cerium‐based Metal‐Organic Frameworks Nanoparticles for Enhanced Enzymatic Activity and Recyclability

AbstractThe α‐L‐Rhamnosidase (Rha) is a useful glycoside hydrolase for selectively hydrolyzing the terminal L‐rhamnose residues in flavonoids, being vital to food and pharmaceutical industries. However, Rha suffers from low recyclability and poor stability in harsh environments. Herein, we explored five typical metal‐organic frameworks (MOFs) as porous carriers to immobilize Rha, and the activities of the resultant Rha@MOF composites were compared with the free enzyme. The locations of the enzyme in MOFs were proved by a series of characterization techniques. It was found that Rha@Ce‐BTC (with enzyme immobilization efficiency of 23 % and enzyme loading content of 8.8 %) showed the highest enzymatic activity. The immobilized Rha@Ce‐BTC showed 80 % residual activity after five consecutive cycles, suggesting a limited leaching effect. Also, Rha@Ce‐BTC manifested markedly enhanced enzyme‐substrate affinity and catalytic efficiency compared to free Rha, as supported by Michaelis‐Menten kinetic studies. Accordingly, the Ce‐BTC would be an appealing carrier for enzyme immobilization, and the as‐designed enzyme/Ce‐BTC composites are promising candidates for industrial use with remarkably high activity, recyclability, and storage stability.

Oxytetracycline removal by biological/chemical activated mesoporous carbon

The presence of emerging pollutants in water matrices is an increasing concern; therefore, the development of sustainable technologies that enable the removal of these pollutants is required. The goal of the present study was to propose a methodology to prepare a sustainable catalyst using functionalized mesoporous activated carbon and an enzyme for the removal of oxytetracycline. Four strategies of laccase immobilization were evaluated using a 32 experimental design to evaluate the enzyme immobilization efficiency. The activated carbon was functionalized with hydrochloric acid, glutaraldehyde, or carbodiimide, resulting in four different enzyme immobilization carriers. The response variables evaluated were the protein-specific load capacity and specific activity of immobilized laccase. All developed carriers successfully immobilized the enzyme; among these, the carriers treated with hydrochloric acid and glutaraldehyde had significant protein-specific capacities with yields of 90%, while the carbodiimide carrier had a yield below 40%. In addition, the hydrochloric acid-treated carrier achieved the highest specific activity (168 ± 52 U/g) and specific protein load (13.48 ± 0.90 mg/g). The antibiotic removal assays that employed this catalyst showed better performance (100%) than the free enzyme, which was used as a reference. However, the apparent antibiotic removal mechanism was found to be pollutant adsorption instead of degradation, which was ascribed to the high affinity of the hydrochloric acid-treated carrier for the studied pollutant.

The Simple Method of Preparation of Highly Carboxylated Bacterial Cellulose with Ni- and Mg-Ferrite-Based Versatile Magnetic Carrier for Enzyme Immobilization.

The bacterial cellulose (BC) is a versatile biopolymer of microbial origin characterized by high purity and unusual water and material properties. However, the native BC contains a low number of functional groups, which significantly limits its further application. The main goal of its effective modification is to use methods that allow the unusual properties of BC to be retained and the desired functional group to be efficiently introduced. In the present study, the new magnetic carrier based on functionalized citric acid (CA) bacterial cellulose was developed and tested to support critical industrial enzymes such as lipase B from Candida antarctica and phospholipase A from Aspergillus oryzae. The applied method allowed BC to be effectively modified by citric acid and a sufficient number of carboxylic groups to be introduced, up to 3.6 mmol of COOH per gram of dry mass of the prepared carrier. The DSC and TGA analyses revealed carrier stability at operational temperatures in the range of 20 °C to 100 °C and substantially influenced the amount of the introduced carboxyl groups on carrier properties. Both enzymes’ immobilization significantly improves their thermal stability at 60 °C without a significant thermal and pH optima effect. The analyzed enzymes showed good operational stability with a significant residual activity after ten cycles of repeated uses. The new magnetic carrier based on highly carboxylated bacterial cellulose has a high application capability as matrix for immobilization the various enzymes of industrial interest.

Chitosan derivative functionalized carbon nanotubes as carriers for enzyme immobilization to improve synthetic efficiency of ethyl caproate

To acquire stable non-covalent functionalized carbon nanotubes with good dispersion and biocompatibility, cinnamaldehyde-chitosan conjugates (CA-CS conjugates) were synthesized to modify carbon nanotubes. The functionalized carbon nanotubes (CA–CS–CNTs) were characterized by FTIR, TGA and TEM. CA–CS–CNTs could be dispersed well in water, and the loading of CA-CS conjugates on carbon nanotubes was about 23%. CA–CS–CNTs were further extended to immobilize porcine pancreatic lipase (PPL), and the successful attachment of PPL onto CA–CS–CNTs was revealed by the result of FTIR. The lipase loading and relative lipase hydrolysis activity of immobilized PPL (CA–CS–CNTs-PPL) reached 248 mg/g and 8064 U/g, respectively. CA–CS–CNTs-PPL possessed good thermostability, and maintained 69% of enzyme activity after reusing 7 times. Additionally, characteristic flavor of strong aromatic liquors, ethyl caproate, was prepared using CA–CS–CNTs-PPL as biocatalysts, and the highest yield of 88% was observed. CA–CS–CNTs-PPL possessed positive properties, which could be expected to become low-cost and useful biocatalysts.

Nickel-Carnosine complex: A new carrier for enzymes immobilization by affinity adsorption

Immobilization is an effective method to promote the application of enzyme industry for improving the stability and realizing recovery of enzyme. To some extent, the performance of immobilized enzyme depends on the choice of carrier material. Therefore, the development of new carrier materials has been one of the key issues concerned by enzyme immobilization researchers. In this work, a novel organic–inorganic hybrid material, nickel-carnosine complex (NiCar), was synthesized for the first time by solvothermal method. The obtained NiCar exhibits spherical morphology, hierarchical porosity and abundant unsaturated coordination nickel ions, which provide excellent anchoring sites for the immobilization of proteins. His-tagged organophosphate-degrading enzyme (OpdA) and ω-transaminase (ω-TA) were used as model enzymes to evaluate the performance of NiCar as a carrier. By a simple adsorption process, the enzyme molecules can be fixed on the particles of NiCar, and the stability and reusability are significantly improved. The analysis of protein adsorption on NiCar verified that the affinity adsorption between the imidazole functional group on the protein and the unsaturated coordination nickel ions on NiCar was the main force in the immobilization process, which provided an idea way for the development of new enzyme immobilization carriers.

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Preparation of Functionalized Mesoporous Silica as a Novel Carrier and Immobilization of Laccase.

Amino-modified mesoporous silica (SBA-15-NH2) was prepared by hydrothermal method, which is a kind of excellent carrier for enzyme immobilization. The structure of SBA-15 was characterized by SEM and FTIR, which proved that amino group was successfully attached to the surface of SBA-15. The carrier had good mesoporous structure proved by nitrogen adsorption and desorption test. Using SBA-15-NH2 as the carrier, the optimal conditions of laccase immobilization by two different cross-linking methods were explored. At the same time, the properties of the immobilized enzyme and free enzyme were compared. The results showed that the activity of immobilized laccase by two-step method (2977.5 U/g) was much higher than that by one-step method (239.5 U/g). The optimal conditions were as follows: free laccase (35°C, pH=4.5), two-step immobilized laccase (40°C, pH=4.0), one-step immobilized laccase (35°C, pH=4.0). The two-step method was more adaptable to temperature. The pH adaptation range of the immobilized enzyme is wider, and the thermal stability is greatly enhanced. After five cycles of repeated reaction, the residual enzyme activity of two-step and one-step methods was 56% and 43% of the original. The treatment of simulated wastewater containing 2,4-dichlorophenol (2,4-DCP) by immobilized laccase was also studied. Under optimum conditions (40°C, pH=5.0, 20 mg/L), the removal of 2,4-DCP reached 89.06%. The immobilized laccase is really effective for treatment of 2,4-DCP-containing wastewater.

Conversion of Lignocellulosic Biomass to Reducing Sugars in High Pressure and Supercritical Fluids: Greener Alternative for Biorefining of Renewables

AbstractSupercritical fluids offer great potential to be employed in lignocellulosic biomass (LCB) fractionation in biorefinery. Supercritical carbon dioxide and water are greener alternatives compared with conventional reagents and have been investigated for the pretreatment and hydrolysis of lignocellulosic biomass. This review is focused on examining the fundamentals that govern the function of supercritical fluids in the pretreatment stage, as well as in the main hydrolysis reaction. Sub/supercritical carbon dioxide is used in pretreatment and sub/supercritical water has been the solvent of choice in hydrolysis of LCB. Significant research has gone into understanding the effect of process parameters such as temperature, pressure, cosolvent, and use of external catalyst on the sugar yield in biorefining of the LCB in supercritical fluids. It is shown that processes with reduced environmental impact and energy consumption can significantly enhance biorefining of LCB at commercial scale. Enzymatic hydrolysis of LCB in supercritical carbon dioxide is a promising approach that can accommodate mild reaction conditions. Developing an understanding of the performance of enzymes in high pressure systems and designing carriers for enzyme immobilization and further recycling is expected to enable one pot pretreatment and hydrolysis and is an important milestone in processing renewable resources for deriving biofuel and value‐added chemicals.