Search Results (57)

Lipases are important industrial enzymes with a wide range of applications across various sectors. Cold-active lipases are particularly well suited for industrial processes that operate at low temperatures (such as food processing and environmental remediation) due to their high catalytic efficiency and energy-saving benefits. In this study, a novel lipase—LipU (GenBank accession: PV094892)—was heterologously expressed from Bacillus cereus U2 and characterized for its low-temperature adaptability and alkaline resistance. LipU belongs to the lipase Subfamily I.5 and shares the highest amino acid sequence identity (53.32%) with known homologs. Enzymatic assays revealed that LipU exhibits optimal activity at 20 °C and pH 11. It retained 95% of its initial activity after 24 h of incubation at 4 °C and pH 11.0. Furthermore, the activity of LipU was enhanced by Ca²⁺, Na⁺, Tween 20, and Tween 80, whereas it was inhibited by Cu²⁺, Zn²⁺, Mn²⁺, and sodium dodecyl sulfate (SDS). LipU demonstrated tolerance to various organic solvents of differing polarity; after 1 h of exposure to 15% (v/v) ethanol, n-butanol, isoamyl alcohol, dimethyl sulfoxide, or glycerol, it retained over 78.6% of its activity. These properties make LipU a promising candidate for industrial applications, including for leather degreasing, alkaline wastewater treatment, and low-temperature biocatalysis. Full article

Phenylalanine butyramide (FBA) is a novel butyric acid derivative with favorable sensory properties, which has broad prospects in medicine and feed processing. However, there is currently limited research on the enzymatic synthesis of FBA. As is well known, lipase plays a crucial role in amide bond synthesis, but it typically requires completely anhydrous conditions. The lipase from Sphingomonas sp. HXN-200 (SpL) is the only intracellular lipase identified to date, capable of catalyzing the ammonolysis of esters or acids in an aqueous phase. In this study, we developed a method for the synthesis of FBA catalyzed by SpL in a biphasic reaction system of water and n-hexane. SpL was successfully expressed in E. coli BL21, and the optimal induction conditions were 0.4 mM IPTG and 18 h. It was ascertained that the n-hexane system containing 2% water was conducive to the reaction. Under optimized reaction conditions, 0.89 mg/mL of FBA was obtained within 15 h at 30 °C. Additionally, we found that SpL also has the ability to hydrolyze amides in the reaction of SpL catalyzing the formation of amides, so we further analyzed its catalytic mechanism. Full article

Vitamin E succinate is a more mature vitamin E derivative, and its chemical stability and many effects have been improved compared with vitamin E, which can not only make up for the shortcomings of vitamin E application but also broaden the application field of vitamin E. At present, in developed countries such as Europe, America, and Japan, vitamin E succinate is widely used in health foods, and due to its good water solubility and stability, the vitamin E added to most nutritional supplements (tablets and hard capsules) is vitamin E succinate. At the same time, vitamin E succinate used in the food and pharmaceutical industries is mainly catalyzed by enzymatic catalysis. In this paper, Candida rugosa lipase (CRL) was studied. Chemical modification and immobilization were used to improve the enzymatic properties of CRL, and immobilized lipase with high stability and high activity was obtained. It was applied to the enzymatic synthesis of vitamin E succinate, and the reaction conditions were optimized to improve the yield and reduce the production cost. The review covered the research progress of the methods for enhancing the catalytic performance of immobilized enzymes and discussed its application in the synthesis of vitamin E succinate, providing new ideas and technical support for the catalytic performance enhancement of immobilized enzymes and its application in the synthesis of vitamin E succinate and promoting the production and application of vitamin E succinate. Full article

Lipases are enzymes that catalyze the hydrolysis of carboxylic esters at a lipid–water interface and are able to catalyze reactions such as alcoholysis, esterification, transesterification, and enantioselective synthesis in organic media. They are important biocatalysts for biotechnological and industrial applications—such as in the food and flavor industry—and in the production of biopharmaceuticals, biofuels, biopolymers, and detergents. A desirable property of lipases is stereoselectivity for the production of chemicals with high optical purity. In this work, we report the improvement of the enantioselective capabilities of the Geobacillus thermoleovorans CCR11 lipase. By means of a rational design and bioinformatic approaches, six amino acids of the catalytic cavity of the lipase LipTioCCR11 were substituted resulting in an increase in the optimum temperature of the enzyme and in the resistance to the presence of organic solvents in hydrolytic reactions, and in the promotion of the enantioselective recognition of R isomers of carboxylic acids with importance for the pharmaceutical and food industries. Full article

Natural deep eutectic solvents (NADESs) are a sustainable, green option for extraction and reaction media in biorefineries and various chemical and biotechnological applications. Particularly, enzymatic reactions profit from NADES applications, as these solvents help to maintain high substrate solubility while improving both enzyme stability and efficiency. Recent studies confirmed that NADESs can perform multiple functions simultaneously, as reaction media for biocatalytic conversions, but also as substrates and catalysts for reactions, fulfilling the role of a reactive solvent. This study reports the beneficial effect of designed reactive natural deep eutectic solvents (R-NADESs) on the esterification activity and thermal stability of free and immobilized lipases in the synthesis of polyol- and carbohydrate-based biosurfactants. We manufactured and characterized 16 binary and ternary R-NADES systems with choline chloride (ChCl) as the hydrogen bond acceptor (HBA) and carbohydrate polyols; mono-, di-, and oligosaccharides; urea (U); N-methyl urea (MU); and water as the hydrogen bond donors (HBDs), in different combinations and molar ratios, most of which are reported for the first time in this paper. We determined their physicochemical, thermal, and molecular properties, including among others viscosity, polarizability, and the number of hydrogen bonds, and we showed that these properties are controlled by composition, molar ratio, molecular properties, temperature, and water content. Many lipases, both native and immobilized, showed high stability and remarkable catalytic performance in R-NADESs during esterification reactions. Full article

The efficient expression and excellent thermal stability of Candida antarctica lipase B (CALB) are crucial for its industrial production. In this study, through genetic engineering and rational design, while preserving the superior catalytic properties of CALB, we optimized the induction pathway using glycerol as the sole carbon source; moreover, the thermal stability sites of CALB were predicted and optimized. The results revealed that the level of CALB expression in this expression system reached 2.27 g/L under the condition of a 5 L fermenter. The Tm value of the CALB-Q231F increased by 10 °C. Moreover, after thermal inactivation at 80 °C for 1 h, the retention rate of esterification enzymatic activity over 24 h was 2.99 times that of wild-type (WT) CALB, whereas the retention rate of hydrolytic enzymatic activity was 2.23 times that of WT CALB. In this study, a non-methanol-induced Pichia pastoris expression system was successfully designed and constructed; a non-methanol-induced CALB-producing strain, X33-pGAPZ(Mα) A-CalB-Q231F, with high thermal stability and a high expression level was obtained, which can be used for the development of industrial enzymes. Full article

Lipase, a type of enzyme that decomposes and synthesizes triglycerides, plays an important role in lipid processing. In this study, a heat-resisting lipase gene (lip4) from Thermomyces lanuginosus was subcloned into the pPICZαA vector and then transformed into Pichia pastoris X33. The recombinant yeast cell concentration reached the maximum (119.5 g/L) at 144 h, and the lipase (Lip4) activity reached the maximum (3900 U/mL) at 168 h in 10 L bioreactor. Through bioinformatics analysis, S168, as the key site of Lip4, participated in the formation of the catalytic triads S168-D223-H280 and G166-H167-S168-L169-G170. Furthermore, S168 and seven conserved amino acids of G104/288, S105, A195, P196, V225 and I287 constitute the active center of Lip4. Specifically, the structure modeling showed two α-helices of the lid domain, outside the active pocket domain, controlling the entry of the substrate on Lip4. The potential glycosylation of Asn-33 may be involved in exhibiting the high stable temperature for lipase activity. Therefore, the eukaryotic system was constructed to express Lip4 efficiently, and the amino acid sites related to the catalytic efficiency of Lip4 were clarified, providing a new way for its subsequent property research and industrial application. Full article

In the era of the blue bio-economy, which promotes the sustainable utilization and exploitation of marine resources for economic growth and development, the fisheries and aquaculture industries still face huge sustainability issues. One of the major challenges of these industries is associated with the generation and management of wastes, which pose a serious threat to human health and the environment if not properly treated. In the best-case scenario, fishery and aquaculture waste is processed into low-value commodities such as fishmeal and fish oil. However, this renewable organic biomass contains a number of highly valuable bioproducts, including enzymes, bioactive peptides, as well as functional proteins and polysaccharides. Marine-derived enzymes are known to have unique physical, chemical and catalytic characteristics and are reported to be superior to those from plant and animal origins. Moreover, it has been established that enzymes from marine species possess cold-adapted properties, which makes them interesting from technological, economic and sustainability points of view. Therefore, this review centers around enzymes from fishery and aquaculture waste, with a special focus on proteases, lipases, carbohydrases, chitinases and transglutaminases. Additionally, the use of fishery and aquaculture waste as a substrate for the production of industrially relevant microbial enzymes is discussed. The application of emerging technologies (i.e., artificial intelligence and machine learning) in microbial enzyme production is also presented. Full article

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase. Full article

Hydrolases are the most popular enzymes, and among the most valuable in biotechnological applications. Some hydrolases, such as lipases, esterases, proteases, cellulases and amylases, are used in the food industry and the production of biopharmaceuticals, biofuels, biopolymers and detergents. Of special interest are those obtained from thermophilic microorganisms. Although there is great microbial diversity in extreme environments, the investigations aimed at detecting and isolating enzymes with potential for polyester degradation such as polyethylene terephthalate (PET) are limited. In this work, we explored the metagenomic library of an oil-enriched soil sample from the “Los Humeros” geothermal field by means of in silico probes in search for enzymes potentially able to degrade polyesters. Using conserved motifs and activity-relevant sites of reported polyester hydrolases, we designed probes that allowed us to identify 6 potential polyester hydrolases in the metagenome. Three-dimensional structure prediction revealed a canonical α/β fold and a cap covering the active site of the enzymes. The catalytic triads were composed of Ser, His and Asp. Structural comparison, substrate binding site analysis and molecular docking suggested their potential as polyester hydrolases, particularly cutinases and PETases. An enzyme, REC98271, was cloned, expressed and characterized, showing thermophilic properties and preference for short-chain substrates. These findings contribute to our understanding of enzyme diversity in “Los Humeros” metagenome and their potential applications in biodegradation and recycling processes. Full article

Biodiesel is a mixture of fatty acid alkyl esters (FAAEs) mainly produced via transesterification reactions among triglycerides and short-chain alcohols catalyzed by chemical catalysts (e.g., KOH, NaOH). Lipase-assisted enzymatic transesterification has been proposed to overcome the drawbacks of chemical synthesis, such as high energy consumption, expensive separation of the catalyst from the reaction mixture and production of large amounts of wastewater during product separation and purification. However, one of the main drawbacks of this process is the enzyme cost. In recent years, nano-immobilized lipases have received extensive attention in the design of robust industrial biocatalysts for biodiesel production. To improve lipase catalytic efficiency, magnetic nanoparticles (MNPs) have attracted growing interest as versatile lipase carriers, owing to their unique properties, such as high surface-to-volume ratio and high enzyme loading capacity, low cost and inertness against chemical and microbial degradation, biocompatibility and eco-friendliness, standard synthetic methods for large-scale production and, most importantly, magnetic properties, which provide the possibility for the immobilized lipase to be easily separated at the end of the process by applying an external magnetic field. For the preparation of such effective magnetic nano-supports, various surface functionalization approaches have been developed to immobilize a broad range of industrially important lipases. Immobilization generally improves lipase chemical-thermal stability in a wide pH and temperature range and may also modify its catalytic performance. Additionally, different lipases can be co-immobilized onto the same nano-carrier, which is a highly effective strategy to enhance biodiesel yield, specifically for those feedstocks containing heterogeneous free fatty acids (FFAs). This review will present an update on the use of magnetic iron oxide nanostructures (MNPs) for lipase immobilization to catalyze transesterification reactions for biodiesel production. The following aspects will be covered: (1) common organic modifiers for magnetic nanoparticle support and (2) recent studies on modified MNPs-lipase catalysts for biodiesel production. Aspects concerning immobilization procedures and surface functionalization of the nano-supports will be highlighted. Additionally, the main features that characterize these nano-biocatalysts, such as enzymatic activity, reusability, resistance to heat and pH, will be discussed. Perspectives and key considerations for optimizing biodiesel production in terms of sustainability are also provided for future studies. Full article

Medium- and long-chain triacylglycerol (MLCT), as a novel functional lipid, is valuable due to its special nutritional properties. Its low content in natural resources and inefficient synthesis during preparation have limited its practical applications. In this study, we developed an effective Pickering emulsion interfacial catalysis system (PE system) for the enzymatic synthesis of MLCT by trans-esterification. Lipase NS 40086 served simultaneously as a catalyst and a solid emulsifier to stabilize the Pickering emulsion. Benefitting from the sufficient oil–water interface, the obtained PE system exhibited outstanding catalytic efficiency, achieving 77.5% of MLCT content within 30 min, 26% higher than that of a water-free system. The K_m value (0.259 mM) and activation energy (14.45 kJ mol⁻¹) were 6.8-fold and 1.6-fold lower than those of the water-free system, respectively. The kinetic parameters as well as the molecular dynamics simulation and the tunnel analysis implied that the oil–water interface enhanced the binding between substrate and lipase and thus boosted catalytic efficiency. The conformational changes in the lipase were further explored by FT-IR. This method could give a novel strategy for enhancing lipase activity and the design of efficient catalytic systems to produce added-value lipids. This work will open a new methodology for the enzymatic synthesis of structured lipids. Full article

To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain. Full article

An easy and versatile method was designed and applied successfully to obtain access to lipase-based cross-linked-enzyme aggregate-like copolymers (CLEA-LCs) using one-pot, consecutive cross-linking steps using two types of homobifunctional cross-linkers (glutaraldehyde and putrescine), mediated with amine activation through pH alteration (pH jump) as a key step in the process. Six lipases were utilised in order to assess the effectiveness of the technique, in terms of immobilization yields, hydrolytic activities, thermal stability and application in kinetic resolution. A good retention of catalytic properties was found for all cases, together with an important thermal and storage stability improvement. Particularly, the CLEA-LCs derived from Candida rugosa lipase showed an outstanding behaviour in terms of thermostability and capability for catalysing the enantioselective hydrolysis of racemic ibuprofen ethyl ester, furnishing the eutomer (S)-ibuprofen with very high conversion and enantioselectivity. Full article

Pseudomonas sp. D01, capable of growing in tributyrin medium, was isolated from the gut microbiota of yellow mealworm. By using in silico analyses, we discovered a hypothesized esterase encoding gene in the D01 bacterium, and its encoded protein, EstD04, was classified as a bacterial hormone-sensitive lipase (bHSL) of the type IV lipase family. The study revealed that the recombinant EstD04-His(6x) protein exhibited esterase activity and broad substrate specificity, as it was capable of hydrolyzing p-nitrophenyl derivatives with different acyl chain lengths. By using the most favorable substrate p-nitrophenyl butyrate (C₄), we defined the optimal temperature and pH value for EstD04 esterase activity as 40 °C and pH 8, respectively, with a catalytic efficiency (k_cat/K_m) of 6.17 × 10³ mM⁻¹ s⁻¹ at 40 °C. EstD04 demonstrated high stability between pH 8 and 10, and thus, it might be capably used as an alkaline esterase in industrial applications. The addition of Mg²⁺ and NH₄⁺, as well as DMSO, could stimulate EstD04 enzyme activity. Based on bioinformatic motif analyses and tertiary structural simulation, we determined EstD04 to be a typical bHSL protein with highly conserved motifs, including a triad catalytic center (Ser¹⁶⁰, Glu²⁵³, and His²⁸³), two cap regions, hinge sites, and an oxyanion hole, which are important for the type IV enzyme activity. Moreover, the sequence analysis suggested that the two unique discrete cap regions of EstD04 may contribute to its alkali mesophilic nature, allowing EstD04 to exhibit extremely distinct physiological properties from its evolutionarily closest esterase. Full article