Improve Enzyme Stability Research Articles

Antimicrobial effects of peptides from fenugreek and ginger proteins using Fe3O4@PDA-MWCNT conjugated trypsin by improving enzyme stability & applications

Trypsin was immobilized onto a newly formulated nanocomposite (NC) comprising magnetic (Fe3O4) multiwalled carbon nanotubes (MWCNTs) anchored with polydopamine (PDA). The fabricated NC and the NC-bound trypsin were subjected to comprehensive characterization using various biophysical techniques including Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence spectroscopy. The NC-bound trypsin exhibited significantly enhanced thermostability and increased tolerance to various organic solvents and denaturants. The enzymatic activity of trypsin was notably augmented through its coupling with the nano support, yielding an effectiveness factor (η) of 2.65. Fenugreek and ginger protein hydrolysates, prepared using both native and NC-bound enzyme, were evaluated for their antimicrobial activities. The analysis revealed that peptides generated by NC-bound trypsin showed higher antimicrobial activity (~ 10) in most cases compared to peptides obtained by using native trypsin. This strategy presents an innovative methodology for the production of potential biopeptides, with the prospect of their incorporation into pharmaceutical and therapeutic sectors through the utilization of NC-bound trypsin in protein hydrolysis.

Designing tailor-made steric matters to improve the immobilized ficin specificity for small versus large proteins

The development of strategies that can permit to adjust the size specificity of immobilized proteases by the generation of steric hindrances may enlarge its applicability. Using as a model ficin immobilized on glyoxyl agarose, two strategies were assayed to generate tailor made steric hindrances. First, ficin has been coimmobilized on supports coated with large proteins (hemoglobin or bovine serum albumin (BSA)). While coimmobilization of ficin with BSA presented no effect on the activity versus any of the assayed substrates, coimmobilization with hemoglobin permitted to improve the immobilized ficin specificity for casein versus hemoglobin, but still significant activity versus hemoglobin remained. Second, aldehyde-dextran has been employed to modify the immobilized ficin, trying to generate steric hindrances to avoid the entry of large proteins (hemoglobin) while enabling the entry of small ones (casein). This also increased the size specificity of ficin, but still did not suppress the activity versus hemoglobin. The combination of both strategies and the use of 37ºC during the proteolysis enabled to almost fully nullify the hydrolytic activity versus hemoglobin while preserving a high percentage of the activity versus casein. The modifications improved enzyme stability and the biocatalyst could be reused for 5 cycles without alteration of its properties.

Three birds with one stone: A nano zeolitic imidazolate framework-luciferase-antimicrobial peptide biocomposite-based biosensing platform for improving enzyme stability, detection sensitivity, and sterilization effect

Adenosine triphosphate-driven bioluminescence (ATP-BL) biosensors were widely employed for on-site screening of bacteria. However, unstable luciferase with strict storage conditions limited its field detection. Moreover, the traditional ATP-BL biosensors lacked the sterilization effect. In this study, a robust and multifunctional zeolitic imidazolate framework-90 decorated with luciferase and polyethylene glycol–antimicrobial peptides (ZIF-90@Luc-AMP) was prepared. It could not only enhance the activity and stability of luciferase at ambient temperatures (≥1 month) but also sensitively detect target bacteria via the bioluminescence (BL) method and kill them (three birds with one stone). During the detection process, the target pathogen Escherichia coli O157:H7 (E. coli O157:H7) was specifically captured on a phage-modified stir bar and formed a sandwich complex with ZIF-90@Luc-AMP. With the synergistic bacteriolysis of ZIF-90@Luc-AMP and the phage, the captured E. coli O157:H7 was decomposed and emitted adenosine triphosphate (ATP), achieving the sterilization effect. Meanwhile, the introduced luciferase catalyzed ATP to produce a BL signal, which was proportional to the concentration of E. coli O157:H7. Combining ZIF-90@Luc-AMP and a phage-labeled stir bar for target recognition and enrichment, the entire analytical process could be easily manipulated within 30 min with a low limit of detection (20 CFU/mL) through a portable ATP bioluminescent meter. The multifunctional ZIF-90@Luc-AMP biosensor with high stability, specificity, and sensitivity was also appropriate for rapid pathogen screening and antibacterial application in the wild.

Ampullaviruses: From Extreme Environments to Biotechnological Innovation.

Ampullaviruses are unique among viruses. They live in extreme environments and have special bottle-shaped architecture. These features make them useful tools for biotechnology. These viruses have compact genomes. They encode a range of enzymes and proteins. Their natural environment highlights their suitability for industrial applications. Ongoing research explores ways in which these viruses can improve enzyme stability. They are also employed in the creation of new biosensors and the development of new bioremediation techniques. High coinfection rates and the ecology of ampullaviruses at larger scales can also reveal new viral vectors. They can also help improve phage therapy. Here, we have explored the structure and function of ampullaviruses. We have focused on their use in biotechnology. We have also identified their characteristics that could prove to be useful. We have also pointed out key knowledge gaps and bridging them could further extend the biotechnological uses.

Display of Bacterial Exochitanase on Bacillus subtilis Spores Improved Enzyme Stability and Recyclability.

Chitin is the second most prevalent polysaccharide found in nature, following cellulose. Amino-oligosaccharides, the byproducts of chitin degradation, exhibit favorable biological properties and potential for various uses. Chitinases play a crucial function in the breakdown of chitin, and their exceptionally effective production has garnered significant interest. Here, in this study, the exochitinase PbChiA, obtained from Paenibacillus barengoltzii, was recombinantly produced and immobilized using the CotG surface protein of Bacillus subtilis WB800N. The resulting strain Bacillus subtilis WB800N pHS-CotG-Chi exhibited exceptional heat stability and efficacy across various pH levels. The chitinolytic activity of the enzyme, which had been isolated and immobilized on the spore surface, was measured to be approximately 16.06 U/mL. Including Ni2+, Zn+2, and K+, and EDTA at various concentration levels in the reaction system, has significantly enhanced the activity of the immobilized enzyme. The immobilized exochitinase demonstrated a notable rate of recycling, as the recombinant spores sustained a relative enzyme activity of more than 70% after three cycles and 62.7% after four cycles. These findings established a basis for additional investigation into the role and practical use of the immobilized bacterial exochitinase in industry.

Enhancing the thermostability of lignin peroxidase: Heme as a keystone cofactor driving stability changes in heme enzymes

Heme-containing enzymes, critical across life's domains and promising for industrial use, face stability challenges. Despite the demand for robust industrial biocatalysts, the mechanisms underlying the thermal stability of heme enzymes remain poorly understood. Addressing this, our research utilizes a ‘keystone cofactor heme-interaction approach’ to enhance ligand binding and improve the stability of lignin peroxidase (LiP). We engineered mutants of the white-rot fungus PcLiP (Phanerochaete chrysosporium) to increase thermal stability by 8.66 °C and extend half-life by 29 times without losing catalytic efficiency at 60 °C, where typically, wild-type enzymes degrade. Molecular dynamics simulations reveal that an interlocked cofactor moiety contributes to enhanced structural stability in LiP variants. Additionally, a stability index developed from these simulations accurately predicts stabilizing mutations in other PcLiP isozymes. Using milled wood lignin, these mutants achieved triple the conversion yields at 40 °C compared to the wild type, offering insights for more sustainable white biotechnology through improved enzyme stability.

Exploring the Potential of Arginine to Increase Coelenterazine-Renilla Luciferase Affinity and Enzyme Stability: Kinetic and Molecular Dynamics Studies.

Renillaluciferase catalyzes the oxidation of coelenterazine to coelenteramide and results in the emission of a photon of light. Although Renilla luciferase has various applications in biotechnology, its low thermal stability limits the development of its applications. Arginine is a well-knownstabilizing amino acidthat plays a key role in protein stabilization against inactivation. However, its impact on enzyme properties is unpredictable. This study investigates the impact of arginine on the kinetics and thermal stability of Renilla luciferase. The enzyme's performance was significantly enhanced in the presence of arginine, with catalytic efficiency increasing by 3.31-fold and 3.08-fold when exposed to 0.2M and 0.3M arginine, respectively. Additionally, arginine improved the thermal stability of Renilla luciferase. Molecular dynamics simulation showed that the addition of 0.2M arginine reduced the binding of coelenteramide, the reaction product and an enzyme inhibitor, to the active site of the Renilla luciferase. Therefore, the release of the product was accelerated, and the affinity of Renilla luciferase for coelenterazine increased. Furthermore, Molecular dynamics studies indicated an increased network of water molecules surrounding Renilla luciferase in the presence of 0.2M arginine. This network potentially enhances the hydrophobic effect on the protein structure, ultimately improving enzyme stability. The findings of this study hold promise for the development of commercial kits incorporating Renilla luciferase.

Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation.

Enzyme-mediated polyethylene terephthalate (PET) depolymerization has recently emerged as a sustainable solution for PET recycling. Towards an industrial-scale implementation of this technology, various strategies are being explored to enhance PET depolymerization (PETase) activity and improve enzyme stability, expression, and purification processes. Recently, rational engineering of a known PET hydrolase (LCC-leaf compost cutinase) has resulted in the isolation of a variant harboring four-point mutations (LCC-ICCG), presenting increased PETase activity and thermal stability. Here, we revealed the enzyme's natural extracellular expression and used it to efficiently screen error-prone genetic libraries based on LCC-ICCG for enhanced activity toward consumer-grade PET. Following multiple rounds of mutagenesis and screening, we successfully isolated variants that exhibited up to a 60% increase in PETase activity. Among other mutations, the improved variants showed a histidine to tyrosine substitution at position 218, a residue known to be involved in substrate binding and stabilization. Introducing H218Y mutation on the background of LCC-ICCG (named here LCC-ICCG/H218Y) resulted in a similar level of activity improvement. Analysis of the solved structure of LCC-ICCG/H218Y compared to other known PETases featuring different amino acids at the equivalent position suggests that H218Y substitution promotes enhanced PETase activity. The expression and screening processes developed in this study can be further used to optimize additional enzymatic parameters crucial for efficient enzymatic degradation of consumer-grade PET.

Immobilization of Thiol-Modified Horseradish Peroxidase on Gold Nanoparticles Enhances Enzyme Stability and Prevents Proteolytic Digestion.

The specificity and efficiency of enzyme-mediated reactions have the potential to positively impact many biotechnologies; however, many enzymes are easily degraded. Immobilization on a solid support has recently been explored to improve enzyme stability. This study aims to gain insights and facilitate enzyme adsorption onto gold nanoparticles (AuNPs) to form a stable bioconjugate through the installation of thiol functional groups that alter the protein chemistry. In specific, the model enzyme, horseradish peroxidase (HRP), is thiolated via Traut's reagent to increase the robustness and enzymatic activity of the bioconjugate. This study compares HRP and its thiolated analog (THRP) to deduce the impact of thiolation and AuNP-immobilization on the enzyme activity and stability. HRP, THRP, and their corresponding bioconjugates, HRP-AuNP and THRP-AuNP, were analyzed via UV-vis spectrophotometry, circular dichroism, zeta potential, and enzyme-substrate kinetics assays. Our data show a 5-fold greater adsorption for THRP on the AuNP, in comparison to HRP, that translated to a 5-fold increase in the THRP-AuNP bioconjugate activity. The thiolated and immobilized HRP exhibited a substantial improvement in stability at elevated temperatures (50 °C) and storage times (1 month) relative to the native enzyme in solution. Moreover, HRP, THRP, and their bioconjugates were incubated with trypsin to assess the susceptibility to proteolytic digestion. Our results demonstrate that THRP-AuNP bioconjugates maintain full enzymatic activity after 18 h of incubation with trypsin, whereas free HRP, free THRP, and HRP-AuNP conjugates are rendered inactive by trypsin treatment. These results highlight the potential for protein modification and immobilization to substantially extend enzyme shelf life, resist protease digestion, and enhance biological function to realize enzyme-enabled biotechnologies.

Enhancing Chymotrypsin Activity and Stability of Capillary Immobilized Enzyme Microreactors Using Zeolitic Imidazolate Frameworks as Encapsulation Materials.

Open-tubular immobilized enzyme microreactors (OT-IMERs) are some of the most widely used enzyme reaction devices due to the advantages of simple preparation and fast sample processing. However, the traditional approaches for OT-IMERs preparation had some defects such as limited enzyme loading amount, susceptibility to complex sample interference, and less stability. Here, we report a strategy for the preparation of highly active and stable OT-IMERs, in which the single-stranded DNA-enzyme composites were immobilized in capillaries and then encapsulated in situ in the capillaries via zeolitic imidazolate frameworks (ZIF-L). The phosphate groups of the DNA adjusted the surface potential of the enzyme to negative values, which could attract cations, such as Zn2+, to promote the formation of ZIF-L for enzyme encapsulation. Using chymotrypsin (ChT) as a model enzyme, the prepared ChT@ZIF-L-IMER has higher activity and better affinity than the free enzyme and ChT-IMER. Moreover, the thermal stability, pH stability, and organic solvent stability of ChT@ZIF-L-IMER were much higher than those of free enzyme and ChT-IMER. Furthermore, the activity of ChT@ZIF-L-IMER was much higher than that of ChT-IMER after ten consecutive reactions. To demonstrate the versatility of this preparation method, we replaced ChT with glucose oxidase (GOx). The stability of GOx@ZIF-L-IMER was also experimentally demonstrated to be superior to that of GOx and GOx-IMER. Finally, ChT@ZIF-L-IMER was used for proteolytic digestion analysis. The results showed that ChT@ZIF-L-IMER had a short digestion time and high digestive efficiency compared with the free enzyme. The present study broadened the synthesis method of OT-IMERs, effectively integrating the advantages of metal-organic frameworks and IMER, and the prepared OT-IMERs significantly improved enzyme stability. All of the results indicated that the IMER prepared by this method had a broad application prospect in capillary electrophoresis-based high-performance enzyme analysis.

Endolysins: a new antimicrobial agent against antimicrobial resistance. Strategies and opportunities in overcoming the challenges of endolysins against Gram-negative bacteria.

Antibiotic-resistant bacteria are rapidly emerging, and the increasing prevalence of multidrug-resistant (MDR) Acinetobacter baumannii poses a severe threat to humans and healthcare organizations, due to the lack of innovative antibacterial drugs. Endolysins, which are peptidoglycan hydrolases encoded by a bacteriophage, are a promising new family of antimicrobials. Endolysins have been demonstrated as an effective therapeutic agent against bacterial infections of A. baumannii and many other Gram-positive and Gram-negative bacteria. Endolysin research has progressed from basic in vitro characterization to sophisticated protein engineering methodologies, including advanced preclinical and clinical testing. Endolysin are therapeutic agent that shows antimicrobial properties against bacterial infections caused by drug-resistant Gram-negative bacteria, there are still barriers to their implementation in clinical settings, such as safety concerns with outer membrane permeabilizers (OMP) use, low efficiency against stationary phase bacteria, and stability issues. The application of protein engineering and formulation techniques to improve enzyme stability, as well as combination therapy with other types of antibacterial drugs to optimize their medicinal value, have been reviewed as well. In this review, we summarize the clinical development of endolysin and its challenges and approaches for bringing endolysin therapies to the clinic. This review also discusses the different applications of endolysins.

Enzyme encapsulation in metal-organic frameworks using spray drying for enhanced stability and controlled release: A case study of phytase

Encapsulating enzymes in metal-organic frameworks is a common practice to improve enzyme stability against harsh conditions. However, the synthesis of enzyme@MOFs has been primarily limited to small-scale laboratory settings, hampering their industrial applications. Spray drying is a scalable and cost-effective technology, which has been frequently used in industry for large-scale productions. Despite these advantages, its potential for encapsulating enzymes in MOFs remains largely unexplored, due to challenges such as nozzle clogging from MOF particle formation, utilization of toxic organic solvents, controlled release of encapsulated enzymes, and high temperatures that could compromise enzyme activity. Herein, we present a novel approach for preparing phytase@MIL-88 A using solvent-free spray drying. This involves atomizing two MOF precursor solutions separately using a three-fluid nozzle, with enzyme release controlled by manipulating defects within the MOFs. The physicochemical properties of the spray dried particles are characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Leveraging the efficiency and scalability of spray drying in industrial production, this scalable encapsulation technique holds considerable promise for broad industrial applications.

Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery.

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts.

Enhanced thermostability of xylanase XynA via computationally designed assembly of multiple N-terminal disulfide bridges

Xylanase is one of the critical enzymes for the degradation of xylan. The poor thermostability of xylanase hinders its industrial application. Disulfide bonds are important in stabilizing protein structure. In this study, the disulfide bonds of xylanase XynA were predicted by Disulfide by Design 2, Discovery Studio 2020 and sequence alignment. Mutants Mut1, Mut2 and Mut3 were generated and successfully expressed via assembling the disulfide bonds A1CT30C, T26CT44C, S28CS40C and T39CA49C. The half-life of Mut1, Mut2 and Mut3 was significantly increased at both 60 °C and 80 °C compared to XynA, respectively, and the Tm of mutants were increased by 7–9 °C. Additionally, the specific activity of Mut1 was 1.24-fold that of XynA. Computer simulation showed that increased interactions and decreased net surface charge may be responsible for the increased thermostability, while the improved specific activity may be attributed to the flexibility of the "thumb" and more open cleft. To our knowledge, this is the first report of the simultaneous insertion of three disulfide bonds into the N-terminus of xylanase to improve enzyme stability while maintaining good activity, suggesting that inserting disulfide bonds at the suitable site of xylanase can achieve a balance between its thermal stability and specific activity.

Co-Immobilization of Laccase and Mediator into Fe-Doped ZIF-8 Significantly Enhances the Degradation of Organic Pollutants.

Co-immobilization of laccase and mediator 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) for wastewater treatment could simultaneously achieve the reusability of laccase and avoid secondary pollution caused by the toxic ABTS. Herein, Fe-induced mineralization was proposed to co-immobilize laccase and ABTS into a metal-organic framework (ZIF-8) within 30 min. Immobilized laccase (Lac@ZIF-8-Fe) prepared at a 1:1 mass ratio of Fe2+ to Zn2+ exhibited enhanced catalytic efficiency (2.6 times), thermal stability, acid tolerance, and reusability compared to free laccase. ABTS was then co-immobilized to form Lac+ABTS@ZIF-8-Fe (ABTS = 261.7 mg/g). Lac@ZIF-8-Fe exhibited significantly enhanced bisphenol A (BPA) removal performance over free laccase due to the local substrate enrichment effect and improved enzyme stability. Moreover, the Lac+ABTS@ZIF-8-Fe exhibited higher BPA removal efficiency than the free laccase+ABTS system, implying the presence of a proximity effect in Lac+ABTS@ZIF-8-Fe. In the successive malachite green (MG) removal, the MG degradation efficiency by Lac@ZIF-8-Fe was maintained at 96.6% at the fifth reuse with only an extra addition of 0.09 mM ABTS in each cycle. As for Lac+ABTS@ZIF-8-Fe, 58.5% of MG was degraded at the fifth cycle without an extra addition of ABTS. Taken together, this research has provided a novel strategy for the design of a co-immobilized laccase and ABTS system for the degradation of organic pollutants.

A silver cluster-assembled material as a matrix for enzyme immobilization towards a highly efficient biocatalyst.

Silver cluster-assembled materials (SCAMs) epitomize well-defined extended crystalline frameworks that combine the ingenious designability at the atomic/molecular level and high structural robustness. They have captivated the interest of the scientific fraternity because of their modular construction which enables to systematically tailor their functions, and their capacity to not only inherit the characteristics of component building units but also introduce their uniqueness in endowing the final material with extraordinary properties. Herein, we demonstrate the synthesis of a novel (3,6)-connected two-dimensional (2D) SCAM [Ag12(StBu)6(CF3COO)6(THIT)6]n (described as TUS 5, THIT = 2,4,6-tri(1H-imidazol-1-yl)-1,3,5-triazine) composed of Ag12 cluster nodes and tritopic imidazolyl linkers. We have leveraged, for the first time, this precisely architected extended SCAM structure as a support matrix for enzyme immobilization. The electrostatic attraction between the negatively charged amano lipase PS and positively charged TUS 5 as well as the surface hydrophobicity of TUS 5 catered to great binding of lipase onto the TUS 5 matrix, in addition to boosting the activity of lipase via interfacial activation. Capitalizing on the cooperative benefits of organic and inorganic support matrices wherein organic supports impart with cost-efficiency, biocompatibility, and improved enzyme stability and reusability and inorganic supports confer high thermal, mechanical and microbial resistance, we have utilized the immobilized lipase on TUS 5 SCAM (lipase@TUS 5) for the kinetic resolution of (R,S)-1-phenylethanol by transesterification reaction. Importantly, lipase@TUS 5 could attain appreciably higher conversion into (R)-1-phenylethyl acetate, besides featuring superior thermal stability, solvent tolerance and recyclability, over the native lipase.

Enzyme Cascade Electrode Reactions with Nanomaterials and Their Applicability towards Biosensor and Biofuel Cells.

Nanomaterials, including carbon nanotubes, graphene oxide, metal-organic frameworks, metal nanoparticles, and porous carbon, play a crucial role as efficient carriers to enhance enzyme activity through substrate channeling while improving enzyme stability and reusability. However, there are significant debates surrounding aspects such as enzyme orientation, enzyme loading, retention of enzyme activity, and immobilization techniques. Consequently, these subjects have become the focus of intensive research in the realm of multi-enzyme cascade reactions. Researchers have undertaken the challenge of creating functional in vitro multi-enzyme systems, drawing inspiration from natural multi-enzyme processes within living organisms. Substantial progress has been achieved in designing multi-step reactions that harness the synthetic capabilities of various enzymes, particularly in applications such as biomarker detection (e.g., biosensors) and the development of biofuel cells. This review provides an overview of recent developments in concurrent and sequential approaches involving two or more enzymes in sequence. It delves into the intricacies of multi-enzyme cascade reactions conducted on nanostructured electrodes, addressing both the challenges encountered and the innovative solutions devised in this field.

Performance improvement of metagenomic laccase immobilized on nanocellulose-reinforced hydrogel nanocomposites for enhanced delignification and detoxification

Immobilization enhances the potential of laccases to degrade lignocellulosic waste, thereby providing an efficient approach for delignification and detoxification. In this study, a comparative analysis of the activity of free and immobilized metagenome-derived laccase (PersiLac2) on nanocellulose (NC)-reinforced hydrogel nanocomposites was performed, and the effectiveness of immobilized laccase in delignification, particularly in relation to rice straw degradation and phenol production, was evaluated. After 1 h of incubation at 90 °C, the immobilized laccase retained 80% of its activity, exhibiting 1.6-fold higher retention than that of the free enzyme. At alkaline pH (8.0–9.0) the free enzyme demonstrated enzymatic activity of 55–65%, while this value was 59–73% for the immobilized form. Immobilization also facilitated easy recovery and reuse of PersiLac2. After 15 cycles, 76.64% of enzyme activity was preserved, highlighting the improved reusability and stability of the immobilized enzyme. Furthermore, leaching tests revealed that the hydrogel-enzyme conjugate maintained a strong connection, with only approximately 20% of the enzyme being leached after 240 min of incubation at 60 °C. This indicates a strong connection between the hydrogel and enzyme structure. Additionally, our study also employed SEM and FTIR analyses, demonstrating the superior performance of immobilized PersiLac2 compared to its free form in the treatment of rice straw. Furthermore, the immobilized enzyme reduced soluble phenols (77.45%) in the slurry of acid-pretreated rice straw. Overall, our findings highlight the potential of immobilizing PersiLac2 on hydrogel nanocomposites to improve enzyme stability, activity, and reusability. This study demonstrates a promising pathway for efficient delignification, phenolic compound removal, and enhanced biomass processing and waste management.

Recent Advances in Enzyme Immobilisation Strategies: An Overview of Techniques and Composite Carriers

For over a century, enzyme immobilisation has been proven to be a superior strategy to improve catalytic activity and reusability and ensure easy separation, easy operation, and reduced cost. Enzyme immobilisation allows for an easier separation of the enzyme from the reaction mixture, thus simplifying downstream processing. This technology protects the enzyme from degradation or inactivation by harsh reaction conditions, making it more robust and suitable to be used in various applications. Recent strategies of immobilisation methods, such as adsorption, cross-linking, entrapment or encapsulation, and covalent bonding, were critically reviewed. These strategies have shown promising results in improving enzyme stability, activity, and reusability in various applications. A recent development in enzyme immobilisation in nanomaterials and agrowaste renewable carriers is underlined in the current review. Furthermore, the use of nanomaterials and agrowaste carriers in enzyme immobilisation has gained significant attention due to their unique properties, such as high surface area, high mass transfer, biocompatibility, and sustainability. These materials offer promising outcomes for developing more efficient and sustainable immobilised enzymes. This state-of-the-art strategy allows for better control over enzyme reactions and enhances their reusability, leading to more cost-effective and environmentally friendly processes. The use of renewable materials also helps to reduce waste generation and promote the utilisation of renewable resources, further contributing to the development of a circular economy.

Optimization of IspSib stability through directed evolution to improve isoprene production.

Enzyme stability is often a limiting factor in the microbial production of high-value-added chemicals and commercial enzymes. A previous study by our research group revealed that the unstable isoprene synthase from Ipomoea batatas (IspSib) critically limits isoprene production in engineered Escherichia coli. Directed evolution was, therefore, performed in the present study to improve the thermostability of IspSib. First, a tripartite protein folding system designated as lac'-IspSib-'lac, which could couple the stability of IspSib to antibiotic ampicillin resistance, was successfully constructed for the high-throughput screening of variants. Directed evolution of IspSib was then performed through two rounds of random mutation and site-saturation mutation, which produced three variants with higher stability: IspSibN397V A476V, IspSibN397V A476T, and IspSibN397V A476C. The subsequent in vitro thermostability test confirmed the increased protein stability. The melting temperatures of the screened variants IspSibN397V A476V, IspSibN397V A476T, and IspSibN397V A476C were 45.1 ± 0.9°C, 46.1 ± 0.7°C, and 47.2 ± 0.3°C, respectively, each of which was higher than the melting temperature of wild-type IspSib (41.5 ± 0.4°C). The production of isoprene at the shake-flask fermentation level was increased by 1.94-folds, to 1,335 mg/L, when using IspSibN397V A476T. These findings provide insights into the optimization of the thermostability of terpene synthases, which are key enzymes for isoprenoid production in engineered microorganisms. In addition, the present study would serve as a successful example of improving enzyme stability without requiring detailed structural information or catalytic reaction mechanisms.IMPORTANCEThe poor thermostability of IspSib critically limits isoprene production in engineered Escherichia coli. A tripartite protein folding system designated as lac'-IspSib-'lac, which could couple the stability of IspSib to antibiotic ampicillin resistance, was successfully constructed for the first time. In order to improve the enzyme stability of IspSib, the directed evolution of IspSib was performed through error-PCR, and high-throughput screening was realized using the lac'-IspSib-'lac system. Three positive variants with increased thermostability were obtained. The thermostability test and the melting temperature analysis confirmed the increased stability of the enzyme. The production of isoprene was increased by 1.94-folds, to 1,335 mg/L, using IspSibN397V A476T. The directed evolution process reported here is also applicable to other terpene synthases key to isoprenoid production.