Enzyme Aggregates Research Articles

PROTEIN INTERACTION STUDIES OF CROSS-LINKED ENDOLEVANASE AGGREGATES FROM BACILLUS LEHENSIS G1

The efficiency of cross-linked enzyme aggregates (CLEAs) is mainly affected by the strength and binding site of the formed linkages between the enzyme and cross-linker. Therefore, this study investigated the impact of different macromolecular cross-linkers on various functional groups, their binding energy, and intermolecular interaction in generating CLEAs of endolevanase from Bacillus lehensis G1 (rlevblg1), through the combination of computational and experimental analysis. Due to the distanced bonding of dextran from the active site, rlevblg1 cross-linked with dextran (rlevblg1-dex-CLEAs) exhibited the highest binding affinity (− 7.1 kcal/mol) and activity recovery compared to six other cross-linkers. Thus, the role of computational cross-linker screening is confirmed as a crucial step to predict strong attachment and construct efficient CLEAs.

Long chain capsaicin analogues synthetized by CALB-CLEAs show cytotoxicity on glioblastoma cell lines

Glioblastoma is one of the most lethal tumors, displaying striking cellular heterogeneity and drug resistance. The prognosis of patients suffering from glioblastoma after 5 years is only 5%. In the present work, capsaicin analogues bearing modifications on the acyl chain with long-chain fatty acids showed promising anti-tumoral activity by its cytotoxicity on U-87 and U-138 glioblastoma multiforme cells. The capsaicin analogues were enzymatically synthetized with cross-linked enzyme aggregates of lipase B from Candida antarctica (CALB). The catalytic performance of recombinant CALB-CLEAs was compared to their immobilized form on a hydrophobic support. After 72 h of reaction, the synthesis of capsaicin analogues from linoleic acid, docosahexaenoic acid, and punicic acid achieved a maximum conversion of 69.7, 8.3 and 30.3% with CALB-CLEAs, respectively. Similar values were obtained with commercial CALB, with conversion yields of 58.3, 24.2 and 22% for capsaicin analogues from linoleic acid, DHA and punicic acid, respectively. Olvanil and dohevanil had a significant cytotoxic effect on both U-87 and U-138 glioblastoma cells. Irrespective of the immobilization form, CALB is an efficient biocatalyst for the synthesis of anti-tumoral capsaicin derivatives.Key points• This is the first report concerning the enzymatic synthesis of capsaicin analogues from docosahexaenoic acid and punicic acid with CALB-CLEAs.• The viability U-87 and U-138 glioblastoma cells was significantly affected after incubation with olvanil and dohevanil.• Capsaicin analogues from fatty acids obtained by CALB-CLEAs are promising candidates for therapeutic use as cytotoxic agents in glioblastoma cancer cells.

An Overview of Crosslinked Enzyme Aggregates: Concept of Development and Trends of Applications.

Enzymes are commonly used as biocatalysts for various biological and chemical processes in industrial applications. However, their limited operational stability, catalytic efficiency, poor reusability, and high-cost hamper further industrial usage. Thus, crosslinked enzyme aggregates (CLEAs) are developed as a better enzyme immobilization tool to extend the enzymes' operational stability. This immobilization method is appealing because it is simpler due to the absence of ballast and permits the collective use of crude enzyme cocktails. CLEAs, so far, have been successfully developed using a variety of enzymes, viz., hydrolases, proteases, amidases, lipases, esterases, and oxidoreductase. Recent years have seen the emergence of novel strategies for preparing better CLEAs, which include the combi- and multi-CLEAs, magnetics CLEAs, and porous CLEAs for various industrial applications, viz., laundry detergents, organic synthesis, food industries, pharmaceutical applications, oils, and biodiesel production. To better understand the different strategies for CLEAs' development, this review explores these strategies and highlights the relevant concerns in designing innovative CLEAs. This article also details the challenges faced during CLEAs preparation and solutions for overcoming them. Finally, the trending strategies to improve the preparation of CLEAs alongside their industrial application trends are also discussed.

A review on bioremediation by microbial immobilization-an effective alternative for wastewater treatment

<p>In this review, we describe recent developments and strategies involved in the utilization of solid supports for the management of wastewater by means of biological treatments. The origin of wastewater determines whether it is considered natural or industrial waste, and the source(s) singly or collectively contribute to increase water pollution. Pollution is a threat to aquatic and humans; thus, before the discharge of treated waters back into the environment, wastewater is put through a number of treatment processes to ensure its safety for human use. Biological treatment or bioremediation has become increasingly popular due to its positive impact on the ecosystem, high level of productivity, and process application cost-effectiveness. Bioremediation involving the use of microbial cell immobilization has demonstrated enhanced effectiveness compared to free cells. This constitutes a significant departure from traditional bioremediation practices (entrapment, adsorption, encapsulation), in addition to its ability to engage in covalent bonding and cross-linking. Thus, we took a comparative look at the existing and emerging immobilization methods and the related challenges, focusing on the future. Furthermore, our work stands out by highlighting emerging state-of-the-art tools that are bioinspired [enzymes, reactive permeable barriers linked to electrokinetic, magnetic cross-linked enzyme aggregates (CLEAs), bio-coated films, microbiocenosis], as well as the use of nanosized biochar and engineered cells or their bioproducts targeted at enhancing the removal efficiency of metals, carbonates, organic matter, and other toxicants and pollutants. The potential integration of 'omics' technologies for enhancing and revealing new insights into bioremediation via cell immobilization is also discussed.</p>

Magnetic Crosslinked Porcine Pancreatic Lipase Aggregates for Transesterification Process

Lipases have been used in industrial processes as biocatalysts for transesterification reactions. The synergism between enzymes and magnetic properties may be reached by using magnetic nanoparticles (MNPs) as support to immobilize them in aggregate structures, denominated by magnetic crosslinked enzyme aggregates (MCLEA). One of the advantages of such supports is the possibility of using magnetic separation for enzyme recovery, reducing costs and allowing reuse in continuous systems. Here, porcine pancreatic lipase (PPL) was immobilized onto functionalized magnetite support (Fe3O4-APTS) with a protein binding efficiency of 78.84%. Physical and chemical properties of the nanoparticles and immobilized lipase were characterized by X-ray diffraction (XDR), transmission electron microscopy (TEM), infrared spectroscopy (FTIR), dynamic light scattering (DLS), zeta potential, vibrating sample magnetometer measurements (VSM), and 57Fe Mössbauer spectroscopy. The immobilized lipase additionally exhibited improved stability across wide pH and temperature ranges compared with free lipase. The immobilized derivate also attained good reusability, maintaining 61.37% of its initial activity after 6 reaction cycles. Through magnetic behavior and also because of its surface modification to crosslinking the enzyme, the MCLEA produced in this work has enhanced the biocatalytic activities of PPL.

Characterization of Maltogenic Amylase Activity Recovery: A Potential Approach for Improving Immobilization

Cross-linked enzymes aggregate (CLEA) is a versatile carrier free-immobilization technique that has gained much attention in the development of biocatalyst technology. However, there is no precise and accurate method of using this technique that will lead to an expected outcome that meets the requirements of the industrial standard. Therefore, the objective of this study is to investigate the effect of a few methods of executing cross-linked enzyme aggregates approach using maltogenic amylase by measuring the activity recovery of the developed CLEA. Some factors that are considered in developing the methodologies are the interaction between cross-linkers and enzymes, size of the cross-linked enzyme aggregates and substrate diffusion. The addition of precipitant and the cross-linking agent steps has been manipulated and four different methodologies were developed. Based on the results, Method 2 showed the highest activity recovery (57.9%) whilst Method 4 gave the lowest activity recovery (15.7%). Method 2 is an improvised method that removed supernatant after centrifugation before proceeding to the cross-linking step. The characterization of cross-linked enzyme aggregates such as morphological characterization and Fourier Transform-Infrared Spectroscopy was also determined. In conclusion, the best and most productive preparation method was determined based on the highest activity recovery.

Hypoglycemic activity of immature persimmon (Diospyros kaki Thunb.) extracts and its inhibition mechanism for α-amylase and α-glucosidase

Persimmon tannins, particularly in immature persimmons, haven't yet received corresponding attention to research on therapy of diabetes mellitus in spite of high hypoglycemic activity. To accurately screening key hypoglycemic components, immature persimmon extracts were isolated and identified using enzyme affinity ultrafiltration and HRLC-ESI-MS/MS. Among them, Hederagenin (IC50 = 0.077 ± 0.003 mg/mL), Ursolic acid (IC50 = 0.001 ± 0.000 mg/mL) and Quercetin dehydrate (IC50 = 0.081 ± 0.001 mg/mL) exhibited the strongest inhibitory effect on α-amylase (HSA and PPA) and α-glucosidase, respectively. And their inhibition mechanisms were analyzed using multi-spectral analysis, atomic force microscope and molecular docking, indicating the bonding with starch digestion enzymes through hydrogen bonding and hydrophobic interaction, and generating the enzyme aggregation. In vivo starch-tolerance experiment further verified that these inhibitors could improve postprandial hyperglycemia (17.18 % ∼ 40.29 %), far more than acarbose. Suppressing, Hederagenin and Ursolic acid as triterpenoids appeared amazing potentiality to alleviate postprandial hyperglycemia, which suggested that IPE were comprehensive exploration values on prevention and treatment of hyperglycemia.

Biocatalytic membranes with crosslinked enzyme aggregates for micropollutant removal

The removal of micropollutants has drawn increasing attention due to the serious hazard even at low concentration. The construction of separation membranes with high micropollutant removal is highly desired. In this study, laccase cross-linked enzyme aggregates (CLEAs) were obtained with bovine serum albumin as a protective agent, followed by the preparation of biocatalytic membrane using glutaraldehyde as a crosslinking agent and preloaded polydopamine-modified UiO-66-NH2 as the intermediate layer. The membrane was systematically characterized. Under the optimal preparation conditions, the bisphenol A removal rate was 97.13% with high applicability at different feed pH and feed concentration with good storage stability and reusability. The multifunctional synergistic effect of the biocatalytic membrane for micropollutant removal by rejection, adsorption and catalysis was analyzed. The membrane also has good treatment effect on micropollutants like antibiotics and dye/salt mixture solution. Besides, the preparation method was expanded by replacing laccase with phosphotriesterase (PTE) to fabricate PTE-CLEAs biocatalytic membrane, which can have high removal rate of 94.04% for organophosphorus pesticide methyl paraoxon in water.

Crowded environments as modulators of oxidation reactions: Effects on the modification and aggregation of key metabolic and digestive enzymes

Crowded environments as modulators of oxidation reactions: Effects on the modification and aggregation of key metabolic and digestive enzymes

Enhanced Laccase Activity and Stability as Crosslinked Enzyme Aggregates on Magnetic Copper Ferrite Nanoparticles for Biotechnological Processes

AbstractHighly stable and reusable magnetic crosslinked enzyme aggregates (m‐CLEAS) of laccase are synthesized with simultaneous improved enzymatic activity. Magnetic copper ferrite nanoparticles (CFNPs) were synthesized by solvothermal procedure with an average size of ~8 nm. The nanometric m‐CLEAS were formed by co‐aggregation of enzyme with CFNPs and crosslinked using glutaraldehyde. Different mass ratios of CFNPs:Laccase were assayed (1 : 2, 1 : 3, and 1 : 6), where 1 : 6 resulted in the highest activity recovery (97 %). The m‐CLEAS showed an average size of ~239 nm, ~24 % enzyme immobilization efficiency, and loading as high as 1.75 g of protein per g of support. As expected, m‐CLEAS oxidized the substrate with a higher transformation rate (kcat) and catalytic efficiency (kcat/Km) than the free enzyme. m‐CLEAS showed superior storage and thermostability compared to free enzyme and non‐magnetic CLEAS. In particular, the m‐CLEAS showed ~150 % and ~100 % residual activity after 30 days of storage at 4 °C and room temperature, respectively. Furthermore, m‐CLEAS showed good recyclability, retaining ~78 % and ~54 % laccase activity after 5 and 10 cycles of reuse, respectively. This work highlights the facile and cost‐effective synthesis of nanometric m‐CLEAS with exceptional storage stability and simultaneously improved laccase activity, making them suitable for a range of green industrial processes.

Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation

Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering for biotechnology. Here we apply limited proteolysis-small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions shows that some metabolites interact in a species-specific manner. We estimate that approximately 35% of interacting metabolites affect enzyme activity in vitro, and the effect is often minor. Using LiP-SMap data as a guide, we find that the Calvin cycle intermediate glyceraldehyde-3-phosphate enhances activity of fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) from Synechocystis sp. PCC 6803 and Cupriavidus necator in reducing conditions, suggesting a convergent feed-forward activation of the cycle. In oxidizing conditions, glyceraldehyde-3-phosphate inhibits Synechocystis F/SBPase by promoting enzyme aggregation. In contrast, the glycolytic intermediate glucose-6-phosphate activates F/SBPase from Cupriavidus necator but not F/SBPase from Synechocystis. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated.

Fructose-aided cross-linked enzyme aggregates of laccase: An insight on its chemical and physical properties

Regardless of the wide biotechnological application of laccases, the critical limiting factors, namely: reusability, retrieval, and storage stability still prevail. These essential traits are rehabilitated by crosslinking laccases, albeit at the expense of their enzymatic activity. To overcome such constraints, we prepared crosslinked enzyme aggregates (CLEAs) of laccase from Trametes versicolor with the aid of sugars. The parameters such as, type of precipitant, crosslinking time, crosslinker and sugar concentration were optimised. The sugar CLEAs were compared with the classic BSA and GA laccase CLEAs concerning physico-chemical properties. The activity recovery for fructose-CLEA was ∼45%. Moreover, the Kcat/Km values of the CLEAs were two and three-fold higher than BSA-CLEA and GA-CLEA, respectively. The half-life (t1/2) deciphered by sugar-CLEA was higher than the t1/2 of GA-CLEAs and free enzyme at 50 °C, portraying more thermal stability. Besides, it demonstrated high pH stability, which was analogous to BSA-CLEA. The promising attributes of increased storage stability and recyclability (>80%) gives more edge to the sugar-CLEAs over conventional CLEAs of their corresponding free enzyme. Thus, sugar-CLEA prevails in furnishing the rudimentary properties required for a biocatalyst and holds many prospects.

Extended Biocatalytic Halogenation Cascades Involving a Single-Polypeptide Regeneration System for Diffusible FADH2.

Flavin-dependent halogenases have attracted increasing interest for aryl halogenation at unactivated C-H positions because they are characterised by high regioselectivity, while requiring only FADH2 , halide salts, and O2 . Their use in combined crosslinked enzyme aggregates (combiCLEAs) together with an NADH-dependent flavin reductase and an NADH-regeneration system for the preparative halogenation of tryptophan and indole derivatives has been previously described. However, multiple cultivations and protein purification steps are necessary for their production. We present a bifunctional regeneration enzyme for two-step catalytic flavin regeneration using phosphite as an inexpensive sacrificial substrate. This fusion protein proved amenable to co-expression with various flavin-dependent Trp-halogenases and enables carrier-free immobilisation as combiCLEAs from a single cultivation for protein production and the preparative synthesis of halotryptophan. The scalability of this system was demonstrated by fed-batch fermentation in bench-top bioreactors on a 2.5 L scale. Furthermore, the inclusion of a 6-halotryptophan-specific dioxygenase into the co-expression strain further converts the halogenation product to the kynurenine derivative. This reaction cascade enables the one-pot synthesis of l-4-Cl-kynurenine and its brominated analogue on a preparative scale.

Enzymatic hydrolysates of κ-carrageenan by κ-carrageenase-CLEA immobilized on amine-modified ZIF-8 confer hypolipidemic activity in HepG2 cells

κ-Carrageenase can degrade κ-carrageenan to produce bioactive κ-carrageenan oligosaccharides (KCOs) that have potential applications in pharmaceutical, food, agricultural, and cosmetics industries. Immobilized enzymes gain their popularity due to their good reusability, enhanced stability, and tunability. In this study, the previously characterized catalytic domain of Pseudoalteromonas purpurea κ-carrageenase was covalently immobilized on the synthesized amine-modified zeolitic imidazolate framework-8 nanoparticles with the formation of cross-linked enzyme aggregates, and the immobilized κ-carrageenase was further characterized. The immobilized κ-carrageenase demonstrated excellent pH stability and good reusability, and exhibited higher optimal reaction temperature, better thermostability, and extended storage stability compared with the free enzyme. The KCOs produced by the immobilized κ-carrageenase could significantly decrease the TC, TG, and LDL-C levels in HepG2 cells, increase the HDL-C level in HepG2 cells, and reduce the free fatty acids level in Caco-2 cells. Biochemical assays showed that the KCOs could activate AMPK activity, increase the ratios of p-AMPK/AMPK and p-ACC/ACC, and downregulate the expression of the lipid metabolism related proteins including SREBP1 and HMGCR in the hyperlipidemic HepG2 cells. This study provides a novel and effective method for immobilization of κ-carrageenase, and the KCOs produced by the immobilized enzyme could be a potential therapeutic agent to prevent hyperlipidemia.

Cross-Linked Enzyme Aggregate (CLEA) Preparation from Waste Activated Sludge.

Enzymes are used extensively as industrial bio-catalysts in various manufacturing and processing sectors. However, commercial enzymes are expensive in part due to the high cost of the nutrient medium for the biomass culture. Activated sludge (AS) is a waste product of biological wastewater treatment and consists of microbial biomass that degrades organic matter by producing substantial quantities of hydrolytic enzymes. Recovering enzymes from AS therefore offers a potential alternative to conventional production techniques. A carrier-free, cross-linked enzyme aggregate (CLEA) was produced from crude AS enzyme extract for the first time. A major advantage of the CLEA is the combined immobilization, purification, and stabilization of the crude enzymes into a single step, thereby avoiding large amounts of inert carriers in the final enzyme product. The AS CLEA contained a variety of hydrolytic enzymes and demonstrated high potential for the bio-conversion of complex organic substrates.

Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.

Enzyme immobilization technology as a tool to innovate in the production of biofuels: A special review of the Cross-Linked Enzyme Aggregates (CLEAs) strategy

This review emphasizes the crucial role of enzyme immobilization technology in advancing the production of two main biofuels, ethanol and biodiesel, with a specific focus on the Cross-linked Enzyme Aggregates (CLEAs) strategy. This method of immobilization has gained attention due to its simplicity and affordability, as it does not initially require a solid support. CLEAs synthesis protocol includes two steps: enzyme precipitation and cross-linking of aggregates using bifunctional agents. We conducted a thorough search for papers detailing the synthesis of CLEAs utilizing amylases, cellulases, and hemicellulases. These key enzymes are involved in breaking down starch or lignocellulosic materials to produce ethanol, both in first and second-generation processes. CLEAs of lipases were included as these enzymes play a crucial role in the enzymatic process of biodiesel production. However, when dealing with large or diverse substrates such as lignocellulosic materials for ethanol production and oils/fats for biodiesel production, the use of individual enzymes may not be the most efficient method. Instead, a system that utilizes a blend of enzymes may prove to be more effective. To innovate in the production of biofuels (ethanol and biodiesel), enzyme co-immobilization using different enzyme species to produce Combi-CLEAs is a promising trend.

Robust cross-linked cyclodextrin glucanotransferase from Bacillus lehensis G1 aggregates using an improved cross-linker and a new co-aggregant for the production of cyclodextrins

One of the potentials of carrier-free cross-linked enzyme aggregates (CLEA) immobilization is the ability to be separated and reuse. Yet, it might be impeded by the poor mechanical stability resulting low recyclability. CLEA of CGTase from Bacillus lehensis G1 (CGTase G1-CLEA) using chitosan (CS) as a cross-linker demonstrated high activity recovery however, displayed poor reusability. Therefore, the relationship between mechanical strength and reusability is studied by enhancing the CS mechanical properties and applying a new co-aggregation approach. Herein, CS was chemically cross-linked with glutaraldehyde (GA) and GA was introduced as a co-aggregant (coGA). CGTase G1-CLEA developed using an improved synthesized chitosan-glutaraldehyde (CSGA) cross-linker and a new coGA technique showed to increase its mechanical stability which retained 63.4% and 52.2%, respectively compared to using CS that remained 33.1% of their initial activity after stirred at 500 rpm. The addition of GA impacted the morphology and interaction consequently stabilizing the CLEAs durability in production of cyclodextrins. As a result, the reusability of CGTase G1-CLEA with CSGA and coGA increased by 56.6% and 42.8%, respectively compared to previous CLEA after 5 cycles for 2 h of reaction. This verifies that the mechanical strength of immobilized enzyme influences the improvement of its operational stability.

Adsorption–precipitation–cross-linking immobilization of GDSL-type esterase from Aspergillus niger GZUF36 by polydopamine-modified magnetic clarity tetroxide nanocouplings and its enzymatic characterization

Recombinant INANE1 (rINANE1), a recombinant intracellular GDSL-type esterase from Aspergillus niger GZUF36, has high acetate substrate specificity. Here, rINANE1 was successfully immobilized on polydopamine (PDA)-modified magnetic ferric oxide nanoparticles (Fe3O4NPs) by adsorption–precipitation–cross-linking to obtain cross-linked enzyme aggregate (CLEA)–rINANE1–Fe3O4@PDA. Fe3O4, Fe3O4@PDA, and CLEA–rINANE1–Fe3O4@PDA were characterized by scanning electron microscopy, X-ray diffraction, vibrating-sample magnetometry, Fourier transform infrared (FTIR) spectroscopy, and zeta potential analysis. Upon immobilization, CLEA–rINANE1–Fe3O4@PDA, with a protein loading of 72.72 ± 1.01 mg/g, reached optimal activity recovery of 104.40 % ± 1.14 %. FTIR analysis showed that immobilization increased the relative content of β-folding in rINANE1 by 12.25 % and reduced irregular curl by 4.16 %, rendering the structure more orderly. Specifically, under an alkaline condition (pH 10), CLEA–rINANE1–Fe3O4@PDA performed over 100 % of initial activity. The optimum temperature increased by 5 °C, and over 55 % of the initial activity was observed after 12 h at 55 °C. CLEA–rINANE1–Fe3O4@PDA showed over 40 % of its relative activity, whereas free rINANE1 showed <10 % in acetonitrile. In addition, the relative activity of CLEA–rINANE1–Fe3O4@PDA was retained at about 80 % after eight cycles and maintained at 109 % after 45 days. The PDA-modified magnetic ferrite nanoparticles exhibited excellent stability and recyclability, providing a new avenue for developing industrial biocatalysts.

Fermentation, immobilization, and application of de-acylase for biosynthesis of anti-fungal intermediates

The enzymatic synthesis of antifungal intermediates is receiving huge attention due to global demand. A de-acylase (EC 3.5.1.98) was produced by recombinant Streptomyces lividans using minimal salt media under optimized conditions. The recombinant S. lividans was subjected to fermentation for 90 h followed by enzyme isolation by KCl extraction method. Further, it was concentrated by tangential flow filter (TFF). Produced de-acylase was immobilized in the form of cross-linked enzyme aggregates (CLEAs) to prepare a robust formulation for further applications. In order to make de-acylase CLEAs, de-acylase was precipitated by ammonium sulphate (100% saturation) and cross-linked by glutaraldehyde (0.5% v/v). The immobilized CLEAs were lyophilized, and was utilized for synthesizing micafungin precursor-2 (FR179642) from the micafungin precursor-1 (FR901379). It was found that the de-acylase CLEAs exhibited more than 96% molar yield which were more efficient than free enzyme (showed nearly 65% molar yield) after 12 h of reaction. In the end, the prepared CLEAs were effectively recycled for five cycles with the retention of 55% residual activity and stored for 24 days without observable loss of activity.