Free Enzyme Research Articles

Chitin membrane for efficient laccase immobilization and synergistic removal of 17α-ethynylestradiol from water-based solutions

A unique chitin–laccase membrane was fabricated as an environmentally friendly biocatalytic platform, utilizing 1-butyl-3-methylimidazolium acetate as the solvent for chitin. Observations using scanning electron microscopy showed that the chitin-laccase membrane possessed a uniform and densely packed structure. Based on the presence of FT-IR signals at 1020 cm−1 and changes in the intensity of signals at 1540 cm−1 and 1645 cm−1, the effectiveness of laccase immobilization was confirmed. FT-IR mapping revealed that the enzyme is evenly distributed on the surface of the membrane. The catalytic activity of the native enzyme and laccase immobilized using the membrane was determined based on a model reaction, and the retention of high activity was confirmed using real solutions. Laccase immobilized using the chitinous membrane retained over 60 % of its initial activity after 30 days of storage at 4 °C. By contrast, the free enzyme retained <40 % of its initial activity. Moreover, the activity of chitin-laccase system remained at 85 % after 5 cycles. This novel chitin–laccase combination was tested in the 17α-ethynylestradiol (EE2) removal from water-based solutions. It was found that EE2 underwent synergistic degradation through concurrent adsorption and biocatalytic transformation, with enzymatic conversion as the dominant mechanism.

Effects of fermented oyster extract supplementation on free fatty acid and liver enzymes in older women with obesity.

This study aimed to investigate the effects of a 12-week intake of fermented oyster extract on free fatty acids and liver enzymes in older women with obesity and to provide basic data for improving liver function in older individuals with obesity. A randomized, double-blind, placebo-controlled clinical trial aimed to confirm the effects of fermented oyster extract intake on free fatty acid (FFA) levels and liver function in older women with obesity. The study included 40 older women with obesity with a body mass index ≥ 25 kg/m2. Participants were divided into a fermented oyster intake group (n = 20) and control group (n = 20). Serum FFA, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) levels were measured at weeks 0 and 12. Our results showed an interaction effect between the two groups in terms of serum FFA levels (p<0.05), with a post-intervention decrease in the FSO group (p<0.05). AST, ALT, and GGT levels also showed an interaction effect between the two groups (p<0.05), with a significant postintervention decrease in the FSO group (p<0.05). The intake of fermented oyster extract significantly reduced FFA, ALT, AST, and GGT levels. These results suggested that the consumption of fermented oyster extract may improve liver function. However, the findings of this study were limited to elderly women with obesity, and the relatively short intake period and small sample size may limit the generalization of the results.

Evaluation of blue textile dye decolorization by immobilized polyphenol oxidase using pumice stone under optimum conditions

Industrial dyes are major pollutants in wastewater and river water with an initial visible concentration of 1 mg/L. Recent studies have shown the possibility of using polyphenol oxidase in catalytic biological treatment due to its ability to oxidize a large number of dyes and pollutants in wastewater and the flexibility to work in wide ranges of temperature, pH and salinity. It is easy availability as well as the low economic cost resulting from its use in biological treatments, this enzyme polyphenol oxidase was used. The findings in this study showed that the extraction of polyphenol oxidase (PPO) from potato peel was homogenized with potassium phosphate buffer (0.1 M, pH 7) at a ratio of 1:10 (weight: volume) for two min. The result of enzyme purification by ion exchange chromatography showed that the yield of this step was 64 % and the specific activity was 6541.67 U/mg. The polyphenol oxidase was immobilized covalently on the functionalized pumice stone (25.2 U/gm) compared with other methods. The characterization results demonstrated that the optimum pH for immobilized and free enzyme activity was 6.0 while the pH range of free and immobilized polyphenol oxidase stability was from 4.5 -7.0 and 3.5 - 8.5 respectively. The better temperature for free and immobilized polyphenol oxidase activity was 25 ºC, whereas the free and immobilized polyphenol oxidase was stable at 15-35 and 15-50 °C respectively. The outcomes showed that the decolorization efficiency of blue textile dye employing immobilized polyphenol oxidase reached 99.85 % after 24 hr. for 100 mg/L concentration while for other concentrations 200, 300, 400, 500 and 1000 mg/L the decolorization efficiencies were 85.96, 76.15, 72.54, 66.94 and 63.5 % respectively. Based on the results, the immobilized polyphenol oxidase on pumice stone is highly efficient in removing textile dyes at large concentrations and in different environmental conditions.

Enhanced Catalytic Activity for Production of Biodiesel from Aloe vera Oil and the Removal of Methyl Violet Dye using Polyaniline-Fe3O4 Magnetic Nanocomposite

The aim of this work was to use a unique technique to maximize the efficiency and cost-effectiveness of biodiesel synthesis utilizing Aloe vera oil. Moreover, in order to facilitate the magnetic separation from the resultant mixture, lipase was immobilized on a magnetic nanocomposite consisting of polyaniline (PANI) and Fe3O4. Utilizing XRD, SEM, TGA and FTIR spectroscopy, the nanocomposite was thoroughly characterized. The results indicated that the immobilized lipase exhibited enhanced thermal stability and a modified optimum pH compared to its free form. Using immobilized lipase also significantly improved the biodiesel conversion yield from 27% with free enzyme to 60%. Remarkably, after five cycles of reutilization in the production of biodiesel, the immobilized lipase retained 90% activity. The catalytic effectiveness of lipase during biodiesel synthesis was significantly enhanced and its resistance to heat and pH fluctuations was strengthened by this novel immobilization approach. Beyond its potential usage in biodiesel production, the PANI-Fe3O4 magnetic nanocomposite also demonstrated photocatalytic removal and degradation of methyl violet dye from aqueous solution.

Combinatorial optimization of the hybrid cellulase complex structure designed from modular libraries.

Cellulase selectively recognizes cellulose surfaces and cleaves their β-1,4-glycosidic bonds. Combining hydrolysis using cellulase and fermentation can produce alternative fuels and chemical products. However, anaerobic bacteria produce only low levels of highly active cellulase complexes so-called cellulosomes. Therefore, we designed hybrid cellulase complexes from 49 biotinylated catalytic domain (CD) and 30 biotinylated cellulose-binding domain (CBD) libraries on streptavidin-conjugated nanoparticles to enhance cellulose hydrolysis by mimicking the cellulosome structure. The hybrid cellulase complex, incorporating both native CD and CBD, significantly improved reducing sugar production from cellulose compared to free native modular enzymes. The optimal CBD for each hybrid cellulase complex differed from that of the native enzyme. The most effective hybrid cellulase complex was observed with the combination of CD6-4 from Thermobifida fusca YX and CBD46 from the Bacillus halodurans C-125. The hybrid cellulase complex/CD6-4-CBD46 and -CD6-4-CBD2-5 combinations showed increased reducing sugar production. Similar results were also observed in microcrystalline cellulose degradation. Furthermore, clustering on nanoparticles enhanced enzyme thermostability. Our results demonstrate that hybrid cellulase complex structures improve enzyme function through synergistic effects and extend the lifespan of the enzyme.

Kinetics and Thermodynamic Study of Cellulase Embedded Metal Organic Frameworks

Cellulase is an important enzyme used for many purposes in different industrial sectors. Even though cellulase has so many applications, it easily denatures with a little change in pH and temperature, which causes low stability, usability, and activity. To enhance its activity and stability, immobilization of porous materials is the best way to enhance its activity, stability, and life span. For immobilization, a Metal-organic framework (MOF) is considered as a potential candidate. Cellulase@Zn- benzene 1-4 di-carboxylic acid (BDC) by hydrothermal method and Zn-cellulase-benzene 1-4 dicarboxylic acid (BDC) by de novo approach were prepared, and their activities were analyzed. Zn-cellulase-benzene 1-4 dicarboxylic acid (BDC) produced by the de novo approach, shows higher activity, stability, catalytic performance, and life span than the free enzyme, and cellulase@Zn- benzene 1-4 di-carboxylic acid (BDC) produced by the hydrothermal method.

A high-efficiency strategy for fruit preservation using green, natural raw materials

Straw is an abundant renewable biomass resource material. Lignin contained in straw is a unique natural aromatic compound in nature. At present, it is urgent to find ways to realize the higher value of natural lignin resources. In this study, alkali lignin was separated from rice straw by hydrothermal method in NaOH solution, which was prepared lignin nanoparticles by a simple green anti-solvent method. The obtained lignin nanoparticles had excellent anti-tyrosinase activity (IC50 = 0.329 mg mL−1) and anti-oxidation performance (IC50 = 0.0451 mg mL−1). Meanwhile, through the analysis of tyrosinase inhibition kinetics, it is concluded that the tyrosinase inhibition by lignin nanoparticles belongs to mixed inhibition. The affinity of lignin nanoparticles to the free enzyme is greater than that of enzyme and substrate complex. In addition, lignin nanoparticles were added to chitosan solution for compounding, then the composite films for fruit preservation were prepared by casting method. The experimental results show that the composite membrane can effectively extend the shelf life of fruits, which is expected to achieve a broader application in the field of fruit preservation and food packaging.

Design, Synthesis, and Xanthine Oxidase Inhibitory Activity of 4-(5-Aminosubstituted-4-cyanooxazol-2-yl)benzoic Acids.

Xanthine oxidase is a known therapeutic target for the treatment of hyperuricemia and related diseases. Despite the availability of current drugs such as allopurinol and febuxostat, the search for new compounds to effectively inhibit this enzyme remains relevant. In our study, 75 virtual structures of 4-(5-aminosubstituted-4-cyanooxazol-2-yl)benzoic acids with structural similarity to febuxostat were designed for evaluation of their potency against xanthine oxidase. After molecular docking simulations, eight compounds were selected for synthesis and in vitro testing. The synthesized compounds were found to exhibit in vitro xanthine oxidase inhibitory activity in the nanomolar concentration range. The most effective inhibitors with 4-benzylpiperidin-1-yl or 1,2,3,4-tetrahydroisoquinolin-2-yl substituents at position 5 of the oxazole ring had IC50 values close to that of febuxostat. The kinetic data suggest a mixed-type inhibition when the inhibitor binds preferentially to the free enzyme rather than to the enzyme-substrate complex. Molecular docking and molecular dynamic simulations were carried out to get insight into the key interactions of the inhibitors bound to the xanthine oxidase active site.

In Vitro In Silico Screening Strategy and Mechanism of Novel Tyrosinase Inhibitory Peptides from Nacre of Hyriopsis cumingii.

For thousands of years, pearl and nacre powders have been important traditional Chinese medicines known for their skin whitening effects. To prepare the enzymatic hydrolysates of Hyriopsis cumingii nacre powder (NP-HCH), complex enzymatic hydrolysis by pineapple protease and of neutral protease was carried out after the powder was pre-treated with a high-temperature and high-pressure method. The peptides were identified using LC-MS/MS and picked out through molecular docking and molecular dynamics simulations. Subsequently, the tyrosinase inhibitory and antioxidant properties of novel tyrosinase inhibitory peptides were investigated in vitro. In addition, the enzymatic activity of tyrosinase in B16F10 cells as well as melanin content and antioxidant enzyme levels were also examined. The results showed that a tyosinase inhibitory peptide (Tyr-Pro-Asn-Pro-Tyr, YPNPY) with an efficient IC50 value of 0.545 ± 0.028 mM was identified. The in vitro interaction results showed that YPNPY is a reversible competitive inhibitor of tyrosinase, suggesting that it binds to the free enzyme. The B16F10 cell whitening test revealed that YPNPY can reduce the melanin content of B16F10 cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that YPNPY could be widely used as a tyrosinase inhibitor in whitening foods and drugs.

Characterization of BioID tagging systems in budding yeast and exploring the interactome of the Ccr4-Not complex.

The modified E. coli biotin ligase BirA* was the first developed for proximity labeling of proteins (BioID). However, it has low activity at temperatures below 37˚C, which reduces its effectiveness in organisms growing at lower temperatures, such as budding yeast. Multiple derivatives of the enzymes have been engineered, but a thorough comparison of these variations of biotin ligases and the development of versatile tools for conducting these experiments in Saccharomyces cerevisiae would benefit the community. Here, we designed a suite of vectors to compare the activities of biotin ligase enzymes in yeast. We found that the newer TurboID versions were the most effective at labeling proteins, but they displayed low constitutive labeling of proteins even in the absence of exogenous biotin, due to biotin contained in the culture medium. We describe a simple strategy to express free BioID enzymes in cells that can be used as an appropriate control in BioID studies to account for the promiscuous labeling of proteins caused by random interactions between bait-BioID enzymes in cells. We also describe chemically-induced BioID systems exploiting the rapamycin-stabilized FRB-FKBP interaction. Finally, we used the TurboID version of the enzyme to explore the interactome of different subunits of the Ccr4-Not gene regulatory complex. We find that Ccr4-Not predominantly labeled cytoplasmic mRNA regulators, consistent with its function in mRNA decay and translation quality control in this cell compartment.

Entrapment of Cyanase from Thermomyces lanuginosus Using Biomimetic Silica and Its Application for Cyanate Bioremediation.

Cyanate, a toxic product from the chemical oxidation treatment of highly toxic cyanide, can be converted to harmless ammonia and carbon dioxide by cyanase (EC 4.2.1.104). Cyanase from Thermomyces lanuginosus was entrapped in biomimetic silica to improve stability and reusability. After entrapment, the enzyme's activity increased by two-fold, and the residual activity after 30-min of incubation at 60 °C also increased by two-fold, compared to the free enzyme. After being stored at room temperature for 28 days, the entrapped cyanase retained 79% of the initial activity, while the free form retained 61%. The immobilized cyanase was successfully applied to cyanate detoxification; the co-entrapment of carbonic anhydrase from Sulfurihydrogenibium azorense decreased the amount of bicarbonate necessary for cyanate detoxification by 50%. The cyanate degradation retained 53% of the initial value after the co-entrapped cyanate and carbonic anhydrase were reused five times.

Metagenomic and metatranscriptomic analyses of microbial genera and volatiles produced during the fermentation of doubanjiang meju

Fermented broad beans (Vicia faba L.), commonly known as meju, serve as a crucial raw material for producing Pixian Doubanjiang (DBJ), a traditional condiment in Chinese cuisine. However, there is limited information on the dynamics of fungal populations and the activity shifts of key enzymes during DBJ meju fermentation. This study aimed to elucidate the microbial composition, active genera, expressed genes and pathways during the DBJ meju fermentation. The general chemical components, free amino acids, enzymes and volatile compounds were also investigated; the correlations between active genera and physicochemical factors were analyzed, at different fermentation stages. The results demonstrated that protease was the predominant enzyme during meju fermentation. A total of 32 major volatile compounds were identified, with most alcohols and aldehydes showing a sharp increase from the early to the middle stages, followed by stabilization until the end of fermentation. Significant shifts in metatranscriptomic composition at the genus level were observed, with Aspergillus, Staphylococcus, and Tulasnella emerging as the core active genera in the process. Notably, cellulase activity was positively correlated with the presence of Tulasnella. Additionally, Aspergillus and Tulasnella were found to play a crucial role in developing the unique aroma of DBJ meju. Our findings on the succession of active genera and their correlation with physicochemical factors are expected to provide substantial evidence for potential quality control and enhancement of this renowned Chinese condiment.

Janus Separation-Sensing Membrane Hosted with Enzyme@MOF Nanoreactor for Real-Time Blood Sensing.

Plasma separation, rich in biomarkers crucial for diagnosis, is conventionally achieved via high-speed centrifugation, a method hindered by its blood usage, lengthy processes, and complex operations, which delays detection. We introduced a novel real-time blood sensing method based on a Janus membrane and enzymes @MOFs. Asymmetric driving of the janus membrane can realize spontaneous separation of plasma and prevent hemolysis during direct separation. Glucose oxidase (GOx), uric acid oxidase (UOx) and horseradish peroxidase (HRP) were encapsulated in a hydrophilic organometallic framework (MOFs) to construct an enzyme cascade nanoreactor. Embedding enzyme in hydrophilic MOFs not only retains the natural conformation of free enzyme but also improves the brittleness of enzyme, endows MOFs with new biological functions, and expands its sensing application. Using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen and a custom app for color interpretation, we achieved real-time visualization of glucose (Glu) and uric acid (UA) at a 50 μM limit. The system accurately analyzed serum samples, matching commercial kits and showing promise for portable, personalized diagnostics.

Single stage bi-substrate transglycosylation reaction for the synthesis of ascorbic acid 2 glucoside using immobilized α-glucosidase

Alpha glucosidases are multifunctional glycoside hydrolases with hydrolysis and transglycosylation ability. It can be utilized for the glycosidic bond synthesis or glycosylation of Ascorbic acid/Vitamin C to its stable analogue, Ascorbic acid 2 glucoside (AA2G), a compound with wide applications in cosmetics and pharma. The application of α-glucosidases for industrial scale transglycosylation is limited due to the low transglycosylation yield of free enzymes. Enzyme immobilization techniques could enable the development of efficient, reusable catalysts. Only a few glycoside hydrolases have been studied in immobilized form for transglycosylation reactions, and α-glucosidases are probably the least explored in this form. Transglycosylation activity of immobilized α-glucosidase from Aspergillus carbonarius BTCF 5 was studied for AA2G synthesis, where different immobilization techniques like calcium alginate encapsulation, adsorption on chitosan beads, covalent cross-linking on magnetic nanoparticles, and cross-linked enzyme aggregates (CLEA) were employed for the immobilization. The immobilization yield of calcium alginate encapsulated enzyme, enzyme immobilized on Fe-MNP support, enzyme immobilized on chitosan beads and as CLEA were 107%, 99%, 46% and 486%, respectively. CLEA was identified as the best immobilization technique for this bi-substrate reaction due to the high immobilization yield and activity retention (30% activity retained after 5 consecutive cycles). Enzyme immobilization increased the transglycosylation activity by 38%, yielding 118 mM AA2G against 72 mM by the free enzyme. This indicates the potential of immobilized α-glucosidase as a catalyst for synthesizing AA2G at an industrial scale.

Heavy metals altered the xenobiotic metabolism of rats by targeting the GST enzyme: An in vitro and in silico study

Among all the heavy metals, Pb, Cd, and As are the most harmful pollutants in the environment. They reach into the organisms via various levels of food chains i.e. air and water. Glutathione-s-transferase (GST, E.C. 2.5.1.18), a key enzyme of xenobiotics metabolism, plays an important role in the removal of several toxicants. The present study aimed to evaluate any inhibitory action of these heavy metals on the GST enzyme isolated from the hepatic tissues of rats. A 10 % (w/v) homogenate of rat liver was prepared in cold and centrifuged at 4 °C at 9000xg for 30 min. The supernatant was collected and kept frozen at −20 °C or used fresh for carrying out different experiments. The activity of GST was monitored spectrophotometrically at 340 nm using 220 μg of soluble protein with varying equal substrate concentrations (0.125–2 mM) in phosphate buffer (50 mM, pH 6.5). To assess the impact of heavy metals on the enzyme activity, different concentrations of Cd (0–0.6 mM) and Pb (0–2 mM) were added to the reaction mixture followed by monitoring the residual activity. The optimum temperature and pH of rat liver GST were found to be 37 °C and 6.5, respectively. The Km value for GST was 0.69 mM and the Vmax was found to be 78.67 U/mg. The Cd and Pb significantly altered the kinetic behaviour of the enzyme. The Vmax and Kcat/Km parameters of GST were recorded to be decreased after interaction with Cd and Pb individually and showed a mixed type of inhibition pattern suggesting that these inhibitors may have a greater binding affinity either for the free enzyme or the substrate-enzyme complex. These metals showed a time-dependent enzyme inhibition profile. Cd was found to be the most potent inhibitor when compared to other treated metals; the order of inhibitory effect of metal ions was Cd>Pb>As. The in silico ion docking analysis for determining the probable interactions of Cd and Pb with fragmented GST validated that Cd exhibited higher inhibition potential for the enzyme as compared to Pb. The results of the present study indicated that exposure of both the Cd and Pb may cause significant inhibition of hepatic GST; the former with higher inhibitory potential than the later. However, As proved to be least effective against the enzyme under the aforesaid experimental conditions.

Innovative approaches in invertase immobilization: Utilizing green synthesized zinc oxide nanoparticles to improve biochemical properties

Invertase enzyme can effectively improve the taste, color, and durability of these products. Various methods have been proposed to increase the stability and efficiency of enzymes. One of the most important is enzyme immobilization, which can be implemented on different materials. The purpose of this study was to immobilize the invertase enzyme on the surface of green synthesized zinc oxide nanoparticles and to investigate its biochemical properties. The enzyme immobilization was confirmed by SEM and Raman spectroscopy. Then, the biochemical characteristics, such as optimal pH and temperature, thermal stability, and storage stability of free and immobilized enzymes, were determined. The results of SEM showed that the diameter of synthesized nanoparticles was about 60 ± 5 nm. FTIR of immobilized invertase confirmed the immobilization process. The immobilization efficiency was determined to be 72 %. Immobilized enzyme showed higher thermal stability at 40 and 50 °C. Immobilized enzyme could be used 8 times in optimum condition. Also, an Examination of the kinetic parameters of the immobilized enzyme compared with those of the free enzyme showed a decrease in the maximum velocity of the enzyme. It seems that the immobilized invertase has improved characteristics for application in different industries.

Entrapment of glucose oxidase from Aspergillus niger ISL-09 in poly(acrylamide-co-acrylic acid) hydrogels for improved stability and catalytic efficiency towards industrial applications

The present study highlights the true potential of Aspergillus niger ISL-09 to produce glucose oxidase (GOx) and focuses on improving the catalytic properties of GOx by entrapping it in poly acrylamide-co-acrylic acid (AAm-co-AAc) hydrogels, fabricated by free-radical polymerization method. For this purpose, the optimum conditions for GOx activity were found to be: 15 g soybean meal (SBM) substrate, 20 mL moisture content, 96 h incubation period, and 1.5 mL of fungal spore suspension. The purified extracellular GOx was observed to have 32.4% yield, with 0.02 U/mg specific activity and approximately 80 kDa subunit molecular weight. The GOx was most active at pH 5.5 and 40 °C. The immobilization of GOx in poly (AAm-co-AAc) hydrogels led towards improved stability and catalytic efficiency, resulting in a 21.7% increase in activity compared to free enzyme. The study also examined the potential of GOx in the pharmaceutical and textile industries as Ca-gluconate producer and bleaching agent, respectively. The study concludes that A. niger ISL-09 is a promising source for GOx production under optimal conditions. Furthermore, the immobilization of GOx in poly (AAm-co-AAc) hydrogels can significantly improve its catalytic properties, making it suitable for different industrial applications. However, further scaling up is required for the better implementation in industry.

Immobilization of recombinant serine protease from Virgobacillus natechei FarDT on amino graphene-chitosan biocompatible nanohybrid for enhancing pH and thermal stability

The serine protease gene was heterologously expressed in Escherichia coli BL21 (DE3) using the PET 28a vector. The purified enzyme was immobilized on a nanohybrid of amino graphene and chitosan. The characterization of synthesized nanohybrids and immobilized enzymes was confirmed by Fourier transform infrared (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and field emission scanning electron microscopy (FE-SEM). Immobilization increased the temperature optimum from 60 to 70 °C for both free and immobilized enzymes, while the optimal pH of the enzymes did not change post-immobilization (pH 8). The immobilized biocatalyst significantly enhanced thermal stability, as well as enzyme stability at significant pH ranges. After 30 days of storage, the immobilized enzymes exhibited approximately 83 % of their relative activity, while the free protease retained only 56 % of its initial activity. Stabilization also altered the kinetic parameters (increasing Km, decreasing Kcat/Km, and Vmax) and thermodynamic parameters (increasing enzyme half-life and activation energy). The study's outcomes represent a significant advancement in the realm of enzyme synthesis and its stabilization using several combined technologies, including enzyme production with recombinant DNA technology based on gene synthesis, and its stabilization using a hybrid substrate synthesized from nanomaterials. Based on these findings, the immobilized recombinant enzyme has high potential for industrial use as an efficient and stable biocatalyst.

Hyperactivation of crosslinked lipases in elastic hydroxyapatite microgel and their properties

Effective enzyme stabilization through immobilization is essential for the functional usage of enzymatic reactions. We propose a new method for synthesizing elastic hydroxyapatite microgel (E-HAp-M) materials and immobilizing lipase using this mesoporous mineral via the ship-in-a-bottle-neck strategy. The physicochemical parameters of E-HAp-M were thoroughly studied, revealing that E-HAp-M provides efficient space for enzyme immobilization. As a model enzyme, lipase (LP) was entrapped and then cross-linked enzyme structure, preventing leaching from mesopores, resulting in highly active and stable LP/E-HAp-M composites. By comparing LP activity under different temperature and pH conditions, it was observed that the cross-linked LP exhibited improved thermal stability and pH resistance compared to the free enzyme. In addition, they demonstrated a 156% increase in catalytic activity compared with free LP in hydrolysis reactions at room temperature. The immobilized LP maintained 45% of its initial activity after 10 cycles of recycling and remained stable for over 160 days. This report presents the first demonstration of a stabilized cross-linked LP in E-HAp-M, suggesting its potential application in enzyme-catalyzed processes within biocatalysis technology.

Strategy in Synthesizing Longer-Chain Levan-Type Fructooligosaccharides by Selective Dextran Macromolecular Cross-Linked Bacillus lehensis G1 Endolevanase Aggregate Immobilization

The formation of cross-linked enzyme aggregates (CLEAs) using macromolecular cross-linkers improves substrate accessibility and enhances enzyme retention. However, there have been few studies exploring the use of macromolecular cross-linkers due to the challenges related to cross-linker screening. In compliance with our previous computational and experimental screening, dextran is the optimal macromolecular cross-linker to develop CLEAs of endolevanase from Bacillus lehensis G1 (rlevblg1-dex-CLEA) for levan-type-fructooligosaccharides (L-FOS) production. In this study, rlevblg1-dex-CLEAs was optimized, and the activity recovery continued to increase and reached 90.5%. Subsequently, the rlevblg1-dex-CLEAs were characterized and they displayed higher thermal stability after 1 h of incubation in comparison to the free enzyme. Moreover, the rlevblg1-dex-CLEAs were reusable for five cycles and exhibited greater storage stability over 180 days at 4 °C (60.9%) than that of free rlevblg1. In addition, the rlevblg1-dex-CLEAs demonstrated similar catalytic efficiency as the free enzyme and generated a substantial amount of L-FOS with a longer degree of polymerization, which is more beneficial for industrial use.