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The traditional strategies of chemical catalysis and biocatalysis for producing octenyl succinic anhydride modified starch can only randomly graft hydrophobic groups on the surface of starch, resulting in unsatisfactory emulsification performance. In this work, a lipase-inorganic hybrid catalytic system with multi-scale flower like structure is designed and applied to spatially selective catalytic preparation of ocenyl succinic anhydride modified starch. With the appropriate floral morphology and petal density, lipases distributed in the "flower center" can selectively catalyze the grafting of hydrophobic groups in a spatial manner, the hydrophobic groups are concentrated on one side of starch particles. The obtaining OSA starch exhibits excellent emulsifying property, and the pickering emulsion has good protective effect on the embedded curcumin. This work provides a direction for the development of high-performance starch-based emulsifiers for the food and pharmaceutical industries, which is of great significance for improving the preparation and emulsification theory research of modified starch., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study focuses on the development a green synthesis of epoxy fatty acids (EFAs) which are commonly used as the plasticizer in polymer industries. The intracellular lipases of Candida catenulata cells as a whole-cell biocatalyst (WCB) were examined in the bio-epoxidation of free fatty acids (FFAs) with hydrogen peroxide. The FFAs in soybean soap stock, an industrial by-product of vegetable oil factories, was used as the feedstock of the process. To remove phosphates from soap stock a degumming process was tested before the bio-epoxidation reaction and results revealed that the EFAs yield was improved using the degummed fatty acids (DFAs). The attachments of magnetic Fe 3 O 4 nanoparticles to the surface of WCBs facilitated the recovery of the biocatalyst, and were improved stabilities. The activation energy for the magnetic whole-cell biocatalysts (MWCB) was 48.54 kJ mol -1 , which was lower than the WCB system (51.28 kJ mol -1 ). The EFA yield was about 47.1 % and 33.8 % after 3 h for the MWCBs and 2 h for the WCBs, respectively. The MWCBs displayed acceptable reusability in the repetitious bio-epoxidation reaction with maintaining 59 % of the original activity after 5 cycles whereas the performance of the WCBs was 5.9 % at the same conditions. The effects of influential factors such as reaction time, molar ratio of H 2 O 2 to CC, and batch and semi-batch operations were investigated for both biocatalyst systems. The quality of EFAs was characterized by FTIR and GC-MS analyses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, the initial focus was on exploring the simultaneous impact of the oil-based food matrix and the polarity of rosmarinic acid derivatives on the antioxidant properties. Rosmarinic acid (RA) showed remarkable DPPH, FRAP, and ABTS radical scavenging activities, followed by methyl rosmarinate (MR) and ethyl rosmarinate (ER). In bulk oil, both conjugated dienes and p-AnV values reached a peak in the following order after 30 days: ER > MR > RA = BHT > control (no antioxidant). In the oil structured using monoacylglycerol, MR was more effective than ER and RA. For ethyl cellulose oleogel, emulsion, and gelled emulsion systems, RA was more effective. Additionally, after confirming the importance of the food matrix on the antioxidant activity of RA derivatives, the lipophilization of RA with ethanol was optimized as a model with Lipozyme 435 in hexane. A conversion yield of as high as 85.59% for ER was achieved, as quantified by HPLC-UV and confirmed by HPLC-DAD-ESI-qTOFMS., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study aimed at Candida rugosa lipase immobilization on a low-cost and readily available support. Among agro-industrial crops, hemp tea waste was chosen as the carrier because it provides higher immobilization performance than hemp flower and leaf wastes. Support characterization by ATR-FTIR, SEM and elemental analysis and the optimization of the adsorption immobilization process were performed. The lipase adsorption immobilization was obtained by soaking the support with hexane under mild agitation for 2 h and a successively incubating the enzyme for 1 h at room temperature without removing the solvent. The esterification of oleic acid with n-decanol was tested in a solvent-free system by studying some parameters that influence the reaction, such as the substrates molar ratio, the lipase activity/oleic acid ratio, reaction temperature and the presence/absence of molecular sieves. The biocatalyst showed the ability to bring the esterification reaction to equilibrium under 60 min and good reusability (maintaining 60 % of its original activity after three successive esterification reactions) but low conversion (21 %) at the optimized conditions (40 °C, 1:2 substrates molar ratio, 0.56 lipase/oleic acid ratio, without sieves). Comparing the results with those obtained by free lipase form at the same activity (1 U) and experimental conditions, slightly higher conversion (%) appeared for the free lipase. All this highlighted that probably the source of lipase for its carbohydrate-binding pocket and lid structure affected the esterification of oleic acid but certainly, the immobilization didn't induce any lipase conformational change also allowing the reuse of the catalytic material., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This work evaluated structured lipids (SLs) through chemical and enzymatic interesterification (CSLs and ESLs). Blends of soybean oil and peanut oil 1:1 wt% were used, with gradual addition of fully hydrogenated crambe to obtain a final behenic acid concentration of 6, 12, 18, and 24 %. Chemical catalysis used sodium methoxide (0.4 wt%) at 100 °C for 30 min, while enzymatic catalysis used Lipozyme TL IM (5 wt%) at 60 °C for 6 h. Major fatty acids identified were C16:0, C18:0, and C22:0. It was observed that with gradual increase of hard fat, the CSLs showed high concentrations of reaction intermediates, indicating further a steric hindrance, unlike ESLs. Increased hard fat also altered crystallization profile and triacylglycerols composition and ESLs showed lower solid fat, unlike CSLs. Both methods effectively produced SLs as an alternative to trans and palm fats, view to potential future applications in food products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Lipase with unique regioselectivity is an attractive biocatalyst for elaborate lipid modification. However, the excavation of novel sn-2 regioselective lipases is difficult due to their scarcity in nature, with Candida antarctica lipase A (CALA) being the pronouncedly reported one. Here, we identified a novel CALA-like lipase from Cordyceps militaris (CACML7) via in silico mining. Through chiral-phase high-performance liquid chromatography, we determined that CACML7 displays sn-2 regioselectivity (>68 %) as does CALA, but exhibits distinctive chain length selectivity and bias against unsaturated fats. Notably, the curvature of the acyl-binding tunnel was expected to contribute to the 2.2-fold higher preference for cis-fatty acid (C18:1, cis-Δ 9 ) over trans-fatty acid (C18:1, trans-Δ 9 ) unlike trans-active CALA. Random pose docking of trioleoylglycerol (TOG) into the active site of a lid-truncated mutant of CACML7 revealed that TOG accepts a tuning fork conformation, of which the precise positioning of the reactive ester group towards the catalytic center was only favorable via sn-2 binding mode. The unique active site morphology, which we refer to as an "acyl-binding tunnel with a narrow entrance," may contribute to the sn-2 regioselectivity of CACML7. Our data provide an attractive model to better understand the mechanism underlying sn-2 regioselectivity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pahn-Shick Chang reports financial support was provided by the Rural Development Administration, Republic of Korea., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ultrasound technology has emerged as a promising tool for enhancing enzymatic biodiesel production, yet the cavitation effect induced can compromise enzyme stability. This study explored the efficiency of polyols in enhancing lipase stability under ultrasound conditions to further improve biodiesel yield. The incorporation of sorbitol resulted in the highest fatty acid methyl ester (FAME) content in the ultrasound-assisted biodiesel production catalyzed by Eversa® Transform 2.0 among the investigated polyols. Furthermore, sorbitol enhanced the stability of the lipase, allowing it to tolerate up to 100 % ultrasound amplitude, compared to 60 % amplitude in its absence. Enzyme activity assays revealed that sorbitol preserved 99 % of the lipase activity, in contrast to 84 % retention observed without sorbitol under an 80 % ultrasound amplitude. Circular dichroism (CD) and fluorescence spectroscopy analyses confirmed that sorbitol enhanced lipase rigidity and preserved its conformational structure under ultrasound exposure. Furthermore, employing a stepwise methanol addition strategy in ultrasound-assisted reactions with sorbitol achieved an 81.2 wt% FAME content in 8 h with only 0.2 wt% enzyme concentration. This promising result highlights the potential of sorbitol as a stabilizing agent in ultrasound-assisted enzymatic biodiesel production, offering a viable approach for enhancing biodiesel yield and enzyme stability in industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5 U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8 U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (T m ) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Peroxyacid synthesis is the first step in Prilezhaev epoxidation, which is an industrial method to form epoxides. Motivated by the development of a kinetic model as a tool for solvent selection, the effect of solvent type and acid chain length on the lipase-catalyzed peroxyacid synthesis was studied. A thermodynamic activity-based ping-pong kinetic expression was successfully applied to predict the effect of the reagent loadings in hexane. The activity-based reaction quotients provided a prediction of solvent-independent equilibrium constants. However, this strategy did not achieve satisfactory estimations of initial rates in solvents of higher polarity. The lack of compliance with some assumptions of this methodology could be confirmed through molecular dynamics calculations i.e. independent solvation energies and lack of solvent interaction with the active site. A novel approach is proposed combining the activity-based kinetic expression and the free binding energy of the solvent with the active site to predict kinetics upon solvent change. Di-isopropyl ether generated a strong interaction with the enzyme's active site, which was detrimental to kinetics. On the other hand, toluene or limonene gave moderate interaction with the active site rendering improved catalytic yield compared with less polar solvents, a finding sharpened when peroctanoic acid was produced., (© 2024 Wiley Periodicals LLC.)

Background: Obesity is recognized as a lifestyle-related disease and the main risk factor for a series of pathological conditions, including cardiovascular diseases, hypertension and type 2 diabetes. Citrus limon is an important medicinal plant, and its fruits are rich in flavonoids investigated for their potential in managing obesity. In the present work, a green extraction applied to lemon squeezing waste (LSW) was optimized to recover pancreatic lipase (PL) inhibitors., Results: The microwave-assisted procedure yielded an extract with higher lipase inhibitory activity than those obtained by maceration and ultrasound. The main compounds present in the extract were identified by high-performance liquid chromatographic-mass spectrometric analysis, and hesperidin, eriocitrin and 4'-methyllucenin II were isolated. The three compounds were evaluated for in vitro PL inhibitory activity, and 4'-methyllucenin II resulted in the most promising inhibitor (IC 50  = 12.1 μmol L -1 ; K i  = 62.2 μmol L -1 ). Multispectroscopic approaches suggested the three flavonoids act as competitive inhibitors and the binding studies indicated a greater interaction between PL and 4'-methyllucenin II. Docking analysis indicated the significant interactions of the three flavonoids with the PL catalytic site., Conclusion: The present work highlights flavonoid glycosides as promising PL inhibitors and proposes LSW as a safe ingredient for the preparation of food supplements for managing obesity. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

With the industrialization and development of modern science, the application of enzymes as green and environmentally friendly biocatalysts in industry has been increased widely. Among them, lipase (EC. 3.1.1.3) is a very prominent biocatalyst, which has the ability to catalyze the hydrolysis and synthesis of ester compounds. Many lipases have been isolated from various sources, such as animals, plants and microorganisms, among which microbial lipase is the enzyme with the most diverse enzymatic properties and great industrial application potential. It therefore has promising applications in many industries, such as food and beverages, waste treatment, biofuels, leather, textiles, detergent formulations, ester synthesis, pharmaceuticals and medicine. Although many microbial lipases have been isolated and characterized, only some of them have been commercially exploited. In order to cope with the growing industrial demands and overcome these shortcomings to replace traditional chemical catalysts, the preparation of new lipases with thermal/acid-base stability, regioselectivity, organic solvent tolerance, high activity and yield, and reusability through excavation and modification has become a hot research topic.

The lipase (LA) and peroxidase (POD) activities, as well as morphological structure, physicochemical and digestion properties of sand rice flour (SRF) treated with superheated steam (SS), were investigated. SS treatment at 165 °C completely deactivated LA and resulted in a 98% deactivation of POD activities in SRF. This treatment also intensified gelatinization, induced noticeable color alterations, and decreased pasting viscosities. Furthermore, there was a moderate reduction in crystal structure, lamellar structure, and short-range ordered structure, with a pronounced reduction at temperatures exceeding 170 °C. These alterations significantly impacted SRF digestibility, leading to increased levels of rapidly digestible starch (RDS) and resistant starch (RS), with the highest RS content achieved at 165 °C. The effectiveness of SS treatment depends on temperature, with 165 °C being able to stabilize SRF with moderate changes in color and structure. These findings will provide a scientific foundation for SS applicated in SRF stabilization and modification., Competing Interests: Declaration of competing interest The authors hereby assert that they possess no discernible financial conflicts of interest or personal affiliations that may have exerted any perceived influence on the reports presented in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Conventional bulk protein structure determination methods are not suitable for understanding the distinct and diverse interactions of proteins with interfaces. Notably, interfacial activation is a feature common to many lipases involving movement of a helical "lid" region upon contact with a hydrophobic surface to expose the catalytic site. Here we use the surface specificity of vibrational sum frequency generation spectroscopy (VSFG) spectroscopy to directly probe the conformation of Thermomyces lanuginosus lipase (TLL) at hydrophobic interfaces. The TLL-catalyzed reaction at the air/water interface is monitored by VSFG spectroscopy, showing loss of ester carbonyl modes and appearance of carboxylate stretching modes of the fatty acid products. Furthermore, comparison of experimental and calculated VSFG spectra of the amide I band of TLL allows us to discern the subtle structural changes involved with lid-opening at a hydrophobic surface. Finally, we report a likely orientation of this lid-open state, which interacts with the surface through a loop region away from the lid and active site. This experimental framework for probing protein structure and function at interfaces addresses a significant problem in protein science that is not only impeding the design of better enzymes for biotechnology applications but also drug discovery targeting membrane associated proteins.

The development of a green and facile method for the controlled synthesis of functional polypeptides is desired for sustainable material applications. In this study, the regioselective synthesis of poly(l-lysine) (polyLys) via enzyme-catalyzed aminolysis was achieved by bulk polymerization of l-lysine ethyl ester (Lys-OEt) using immobilized Candida antarctica lipase Novozym 435 (IM-lipase) or trypsin (IM-trypsin). Structural characterization of the obtained polyLys revealed that IM-lipase resulted solely in ε-linked amide bond formation, whereas IM-trypsin predominantly provided α-linked polyLys. Optimization of the conditions for the bulk polymerization using immobilized enzymes resulted in high monomer conversion and a high degree of polymerization, with excellent regioselectivity. Molecular docking simulations revealed different binding conformations of Lys-OEt to the catalytic pockets of lipase and trypsin, which putatively resulted in different amino moieties being used for amide bond formation. The immobilized enzymes were recovered and recycled for bulk polymerization, and the initial activity was maintained in the case of IM-trypsin. The obtained α- and ε-linked polyLys products exhibited different degradability against proteolysis, demonstrating the possibility of versatile applications as sustainable materials. This enzymatic regioregular control enabled the synthesis of well-defined polypeptide-based materials with a diverging structural variety.

The lipase from Prunus dulcis almonds was inactivated under different conditions. At pH 5 and 9, enzyme stability remained similar under the different studied buffers. However, when the inactivation was performed at pH 7, there were some clear differences on enzyme stability depending on the buffer used. The enzyme was more stable in Gly than when Tris was employed for inactivation. Then, the enzyme was immobilized on methacrylate beads coated with octadecyl groups at pH 7 in the presence of Gly, Tris, phosphate and HEPES. Its activity was assayed versus triacetin and S-methyl mandelate. The biocatalyst prepared in phosphate was more active versus S-methyl mandelate, while the other ones were more active versus triacetin. The immobilized enzyme stability at pH 7 depends on the buffer used for enzyme immobilization. The buffer used in the inactivation and the substrate used determined the activity. For example, glycine was the buffer that promoted the lowest or the highest stabilities depending on the substrate used to quantify the activities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The stimulus-responsive regulation of enzyme catalytic activity and selectivity provides a new opportunity to extend the functionality and efficiency of immobilized enzymes. This work aims to design and synthesize a thermo-switchable enzyme@MOF for size-selective biocatalysis and biosensing through the immobilization of Candida rugosa lipase (CRL) within ZIF-8 functionalized with thermally responsive polymer, poly( N -isopropylacrylamide) (PNIPAM) (CRL@ZIF-8-PNIPAM). Unlike free CRL, which does not demonstrate substrate selectivity, we can reversibly tune the pore size of the ZIF-8-PNIPAM nanostructures (open pores or blocked pores) through temperature stimulus and subsequently modulate the substrate selectivity of CRL@ZIF-8-PNIPAM. CRL@ZIF-8-PNIPAM had the highest hydrolytic activity for small molecules (12 mM p -nitrophenol/mg protein/min, 4-nitrophenyl butyrate ( p -NP Be)) and the lowest hydrolytic activity for large molecules (0.16 mM p-nitrophenol/mg protein/min, 4-nitrophenyl palmitate ( p -NP P)). In addition, CRL@ZIF-8-PNIPAM demonstrated thermo-switchable behavior for large molecules ( p -NP P). The p -NP P hydrolytic activity of CRL@ZIF-8-PNIPAM was significantly lower at 40 °C (blocked pores) than at 27 °C (open pores). However, the transition of blocked pores and open pores is a gradual process that resulted in a delay in the "thermo-switchable" catalytic behavior of CRL@ZIF-8-PNIPAM during thermal cycling. CRL@ZIF-8-PNIPAM was also successfully used for the fabrication of electrochemical biosensors for the selective biosensing of pesticides with different molecular sizes.

Here, we report a study of the effect of the blocking agent on the properties of the lipase from Thermomyces lanuginosus (TLL) immobilized on a heterofunctional support (Purolite C18-ethylnediamina (EDA)- vinyl sulfone (VS)-TLL-blocking agent) in different reactions. The performance of the biocatalysts was compared to those immobilized on standard hydrophobic support (Purolite C18-TLL) and the commercial one (TLL-IM). The nature of the blocking agent (Cys, Gly and Asp) altered the enzyme features. TLL-IM always gave a comparatively worse performance, with its specificity for the oil being very different to the Purolite biocatalysts. Under optimized conditions, Purolite C18-TLL yielded 97 % of hydrolysis conversion after 4 h using a water/waste cooking soybean oil (WCSO) mass ratio of 4.3, biocatalyst load of 6.5 wt% and a temperature of 44.2 °C (without buffer or emulsification agent). In esterification reactions of the purified free fatty acids (FFAs) obtained from WCSO, the best TLL biocatalysts depended on the utilized alcohol: linear amyl alcohol was preferred by Purolite C18-TLL and Purolite C18-EDA-VS-TLL-Gly, while higher activity was achieved utilizing isoamyl alcohol as nucleophile by Purolite C18-EDA-VS-TLL-Cys, Purolite C18-EDA-VS-TLL-Asp and IM-TLL as catalysts. All the results indicate the influence of the blocking step on the final biocatalyst features., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Heterogeneous biocatalysts were prepared by adsorbing T. lanuginosus lipase (TLL) onto uncalcined (SBAUC-TLL) and calcined (SBAC-TLL) SBA-15, using ammonium fluoride as a pore expander to facilitate TLL immobilization. At an enzyme load of 1 mg/g, high immobilization yields (>90 %) and recovered activities (>80 % for SBAUC-TLL and 70 % for SBAC-TLL) were achieved. When increasing the enzyme load to 5 mg/g, the immobilization yield of SBAUC-TLL was 80 %, and the recovered activity was 50 %, while SBAC-TLL had a yield of 100 % and a recovered activity of 36 %. Crosslinking with glutaraldehyde (GA) was conducted to improve stability (SBAUC-TLL-GA and SBAC-TLL-GA). Although SBAC-TLL-GA lost 25 % of initial activity after GA modifications, it exhibited the highest thermal (t 1/2  = 5.7 h at 65 °C), when compared to SBAC-TLL (t 1/2  = 12 min) and the soluble enzyme (t 1/2  = 36 min), and operational stability (retained 100 % activity after 5 cycles). Both biocatalysts presented high storage stability since they retained 100 % of initial activity for 30 days. These results highlight SBA-15's potential as an enzyme support and the protocol's efficacy in enhancing stability, with implications for industrial applications in the food, chemical, and pharmaceutical sectors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To investigate the effect of α3 and α5 helices on the biochemical characterization of Bacillus thermocatenulatus lipase (BTL2), both helices were deleted from native BTL2 lipase. After structural modeling and characterization, the truncated btl2 gene (Δbtl2) was cloned into E. coli BL21 under the control of the T7 promoter. After cultivation and induction of the recombinant bacteria, the Δα3α5 lipase was purified by Ni-NTA column chromatography. Next, the biochemical properties of the Δα3α5 lipase were compared with the previously expressed and purified native lipase. In the presence of the substrate tributyrin (C4), the maximum activity of native and Δα3α5 lipase was 9360 and 5000 U/mg, respectively. The deletion changed the substrate specificity from tributyrin (C4) to tricaprylin (C8) substrate. Native and Δα3α5 lipase showed similar activity patterns at all temperatures and pH values, with the activity of Δα3α5 lipase being approximately 20% lower than native lipase. Triton X100 increased the activity of native and Δα3α5 lipases by 2.1- and 2.5-fold, respectively., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Organic waste valorization is one of the principal goals of the circular economy. Bioprocesses offer a promising approach to achieve this goal by employing microorganisms to convert organic feedstocks into high value products through their metabolic activities. In this study, a fermentation process for yeast cultivation and extracellular lipase production was developed by utilizing food waste. Lipases are versatile enzymes that can be applied in a wide range of industrial fields, from detergent, leather, and biodiesel production to food and beverage manufacturing. Among several oleaginous yeast species screened, Saitozyma flava was found to exhibit the highest secreted lipase activity on pNP-butyrate, pNP-caproate, and pNP-caprylate. The production medium was composed of molasses, a by-product of the sugar industry, which provided nutrients for yeast biomass formation. At the same time, waste cooking oil was employed to induce and enhance extracellular lipase production. After 48 h of process, 20 g/L of yeast biomass and 150 mU/mg dw of lipase activity were achieved, with a productivity of 3 mU/mg dw /h. The purified lipase from S. flava showed optimal performances at temperature 28°C and pH 8.0, exhibiting a specific activity of 62 U/mg when using p-NPC as substrate., (© 2024 The Authors. Biotechnology and Applied Biochemistry published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

The catalytic performance of immobilized lipase is greatly influenced by functional support, which attracts growing interest for designing supports to achieve their promotive catalytic activity. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Herein, the behavioral differences of lipases with distinct lid structures on interfaces of varying hydrophobicity levels were firstly investigated by molecular simulations. It was found that a reasonable hydrophilic/hydrophobic surface could facilitate the lipase to undergo interfacial activation. Building on these findings, a novel "nest"-like superhydrophobic ZIFs (ZIFN) composed of hydrophobic ligands was prepared for the first time and used to immobilize lipase from Aspergillus oryzae (AOL@ZIFN). The AOL@ZIFN exhibited 2.0-folds higher activity than free lipase in the hydrolysis of p-Nitrophenyl palmitate (p-NPP). Especially, the modification of superhydrophobic ZIFN with an appropriate amount of hydrophilic tannic acid can significantly improve the activity of the immobilized lipase (AOL@ZIFN-TA). The AOL@ZIFN-TA exhibited 30-folds higher activity than free lipase, and still maintained 82% of its initial activity after 5 consecutive cycles, indicating good reusability. These results demonstrated that nanomaterials with rational arrangement of the hydrophilic/hydrophobic surface could facilitate the lipase to undergo interfacial activation and improve its activity, displaying the potential of the extensive application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Triacylglycerols (TAGs) are a primary energy source for marine mammals during lipid digestion. Walruses (Odobenus rosmarus divergens) consume prey with a high content of long-chain polyunsaturated fatty acids; however, their digestive physiology and lipid digestion remain poorly studied. The present study aims to model and characterize the gastric (PWGL) and pancreatic (PWPL) lipases of Pacific walruses using an in-silico approach. The confident 3D models of PWGL and PWPL were obtained via homology modeling and protein threading and displayed the structural features of lipases. Molecular docking analysis demonstrated substrate selectivity for long-chain TAG (Trieicosapentaenoin; TC20:5n-3) in PWGL and short-chain TAG (Trioctanoin; TC8:0) in PWPL. Molecular dynamics simulations demonstrate that PWGL bound to tridocosahexaenoin (TC22:6n-3), the protein is considerably stable at all three salinity conditions, but fluctuations are observed in the regions associated with catalytic sites and the lid, indicating the potential hydrolysis of the substrate. This is the first study to report on the digestion of TAGs in walruses, including modeling and lipases characterization and proposing a digestive tract for pinnipeds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Few reports exist on one-step enzymatic methods for the simultaneous production of biodiesel and eicosapentaenoic acid ethyl ester (EPA-EE), a high-value pharmaceutical compound. This study aimed to efficiently express Rhizomucor miehei lipase (pRML) in Pichia pastoris X-33 via propeptide mutation and high-copy strain screening. The mutated enzyme was then used to simultaneously catalyze the production of both biodiesel and EPA-EE. The P46N mutation in the propeptide (P46N-pRML) significantly boosted its production, with the four-copy strain increasing enzyme yield by 3.7-fold, reaching 3425 U/mL. Meanwhile, its optimal temperature increased to 45-50 °C, pH expanded to 7.0-8.0, specific activity doubled, K m reduced to one-third, and k cat / K m increased 7-fold. Notably, P46N-pRML efficiently converts Nannochloropsis gaditana oil's eicosapentaenoic acid (EPA). Under optimal conditions, it achieves up to 93% biodiesel and 92% EPA-EE yields in 9 h. Our study introduces a novel, efficient one-step green method to produce both biodiesel and EPA-EE using this advanced enzyme.

Pancreatic lipase (PL) and cholesterol esterase (CE) are vital digestive enzymes that regulate lipid digestion. Three bioactive peptides (LFCMH, RIPAGSPF, YFRPR), possessing enzyme inhibitory activities, were identified in the seed proteins of R. roxburghii. It is hypothesized that these peptides could inhibit the activities of these enzymes by binding to their active sites or altering their conformation. The results showed that LFCMH exhibited superior inhibitory activity against these enzymes compared to the other peptides. The inhibition mechanisms of the three peptides were identified as either competitive or mixed, according to inhibition models. Further studies have shown that peptides could bind to the active sites of enzymes, thus affecting their spatial conformation and restricting substrate entry into the active site. Molecular simulation further proved that hydrogen bonds and hydrophobic interactions played a vital role in the binding of peptides to enzymes. This study enriches our understanding of interaction mechanisms of peptides on PL and CE., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

This study aimed to explore the capacity of immobilized lipases on the acetylation of six aglycon flavonoids, namely myricetin, quercetin, luteolin, naringenin, fisetin and morin. For this purpose, lipase B from Candida antarctica (CaLB) and lipase from Thermomyces lanuginosus (TLL) were immobilized onto the surface of ZnOFe nanoparticles derived from an aqueous olive leaf extract. Various factors affecting the conversion of substrates and the formation of monoesterified and diesterified products, such as the amount of biocatalyst and the molar ratio of the substrates and reaction solvents were investigated. Both CaLB and TLL-ZnOFe achieved 100% conversion yield of naringenin to naringenin acetate after 72 h of reaction time, while TLL-ZnOFe achieved higher conversion yields of quercetin, morin and fisetin (73, 85 and 72% respectively). Notably, CaLB-ZnOFe displayed significantly lower conversion yields for morin compared with TLL-ZnOFe. Molecular docking analysis was used to elucidate this discrepancy, and it was revealed that the position of the hydroxyl groups of the B ring on morin introduced hindrances on the active site of CaLB. Finally, selected flavonoid esters showed significantly higher antimicrobial activity compared with the original compound. This work indicated that these lipase-based nanobiocatalysts can be successfully applied to produce lipophilic derivatives of aglycon flavonoids with improved antimicrobial activity.

Controlling physicochemical processes that drive changes in supramolecular aggregates is an important objective toward creating artificial soft micro- and nanomachines. Previous research explored the morphology control of membrane-based materials subjected to externally imposed chemical stimuli. Here, we modulate the microscale morphology of pH-responsive assemblies by using biocatalysis to internally generate changes in global pH. Catalytic reactions offer flexibility in the mechanism and rate at which stimuli are introduced to responsive assemblies, ultimately enabling precision and control over size and morphology. We observed, by dynamic light scattering and fluorescence microscopy, substantial microscale differences between assemblies subjected to manually titrated pH changes compared to biocatalytically activated pH changes, including the growth of giant vesicles from micelles. Coarse-grained molecular dynamics simulations of these metastable self-assembled structures provided insight into the thermodynamics and kinetics of the preferred structures. These results demonstrate the feasibility of using biocatalytic reactions to modulate the size and morphology of supramolecular assemblies, from micelles to giant vesicles.

Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg -1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency ( k cat / K m ) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v / v ethanol and 70% v / v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.

Laccase is an oxidase of great industrial interest due to its ability to catalyze oxidation processes of phenols and persistent organic pollutants. However, it is susceptible to denaturation at high temperatures, sensitive to pH, and unstable in the presence of high concentrations of solvents, which is a issue for industrial use. To solve this problem, this work develops the synthesis in an aqueous medium of a new Mn metalloenzyme with laccase oxidase mimetic catalytic activity. Geobacillus thermocatenulatus lipase (GTL) was used as a scaffold enzyme, mixed with a manganese salt at 50 °C in an aqueous medium. This leads to the in situ formation of manganese(IV) oxide nanowires that interact with the enzyme, yielding a GTL-Mn bionanohybrid. On the other hand, its oxidative activity was evaluated using the ABTS assay, obtaining a catalytic efficiency 300 times higher than that of Trametes versicolor laccase. This new Mn metalloenzyme was 2 times more stable at 40 °C, 3 times more stable in the presence of 10% acetonitrile, and 10 times more stable in 20% acetonitrile than Novozym 51003 laccase. Furthermore, the site-selective immobilized GTL-Mn showed a much higher stability than the soluble form. The oxidase-like activity of this Mn metalloenzyme was successfully demonstrated against other substrates, such as l-DOPA or phloridzin, in oligomerization reactions.

In this research, we utilized an efficient approach to synthesize superparamagnetic graphene oxide (SPGO) rapidly in a one-pot method using microwave irradiation of graphene oxide (GO), urea, and Fe(III) ion. Tannic acid (TA) was introduced to the surface of SPGO through a straightforward and eco-friendly process. Methods were devised to furnish GO nanosheets and modify their surfaces with TA in an environmentally friendly manner. Two series of nanosheets, namely, SPGO/TA-COOH and SPGO/TA-IM, were engineered on the surface and used for immobilizing lipase enzyme. Through various analytical tools, the unique biocatalysts SPGO/TA-COOH/L and SPGO/TA-IM/L were confirmed. These biocatalysts exhibited enhanced stability at high temperatures and pH levels compared with free lipase. They also demonstrated prolonged storage stability and reusability over four months and seven cycles, respectively. Furthermore, the catalytic activity of immobilized lipase showed minimal impairment based on kinetic behavior analysis. The kinetic constants of SPGO/TA-IM/L were determined as V max = 0.24 mM min -1 , K m = 0.224 mM, and k cat = 0.8 s -1 . Additionally, the efficiency of biocatalysts for biodiesel production from palmitic acid was studied, focusing on various reaction parameters, such as temperature, alcohol to palmitic acid molar ratio, water content, and lipase quantity. The esterification reaction of palmitic acid with methanol, ethanol, and isopropanol was tested in the presence of SPGO/TA-COOH/L and SPGO/TA-IM/L, and the corresponding esters were obtained with a yield of 30.6-91.6%.

This study presents the potential role of deep eutectic solvents (DESs) in a lipase-catalyzed hydrolysis reaction as a co-solvent in an aqueous solution given by a phosphate buffer. Ammonium salts, such as choline chloride, were paired with hydrogen bond donors, such as urea, 1,2,3-propanetriol, and 1,2 propanediol. The hydrolysis of p- nitrophenyl laureate was carried out with the lipase Candida antarctica Lipase B (CALB) as a reaction model to evaluate the solvent effect and tested in different DES/buffer phosphate mixtures at different % w/w. The results showed that two mixtures of different DES at 25 % w/w were the most promising solvents, as this percentage enhanced the activities of CALB, as evidenced by its higher catalytic efficiency (kcatKM). The solvent analysis shows that the enzymatic reaction requires a reaction media rich in water molecules to enable hydrogen-bond formation from the reaction media toward the enzymatic reaction, suggesting a better interaction between the substrate and the enzyme-active site. This interaction could be attributed to high degrees of freedom influencing the enzyme conformation given by the reaction media, suggesting that CALB acquires a more restrictive structure in the presence of DES or the stabilized network given by the hydrogen bond from water molecules in the mixture improves the enzymatic activity, conferring conformational stability by solvent effects. This study offers a promising approach for applications and further perspectives on genuinely green industrial solvents.

Herein we present the biocatalysed preparation of a mono- N -carbamate-protected precursor of antitumoral Nutlin-3a through enantioselective alkoxycarbonylation of meso -1,2-disubstituted-1,2-diaminoethane using enzyme lipases and dialkyl carbonates as acylating agents. A series of supported or free lipase enzymes were screened in combination with commercially available diallyl, diethyl and dimethyl carbonates. The reactions were conducted at different temperatures, for different reaction times and with variable co-solvent systems to evaluate the effects on the enzyme catalytic activity. The best results in terms of conversion, enantiomeric excess and yield were obtained when lipase from Candida antarctica B (CAL-B) was used with diallyl carbonate (DAC) when conducting the reaction solventless at 75 °C., Competing Interests: The authors declare no conflicts of interest.

Over the past decade, the preparation of novel materials by enzyme-embedding into biopolyesters has been proposed as a straightforward method to produce self-degrading polymers. This paper reports the preparation and enzymatic degradation of extruded self-degradable films of three different biopolyesters: poly(lactic acid) (PLA), poly(butylene adipate- co -terephthalate) (PBAT), and poly(butylene succinate) (PBS), as well as three binary/ternary blends. Candida antarctica lipase B (CalB) has been employed for the enzyme-embedding procedure, and to the best of our knowledge, the use of this approach in biopolyester blends has not been reported before. The three homopolymers exhibited differentiated degradation and suggested a preferential attack of CalB on PBS films over PBAT and PLA. Moreover, the self-degradable films obtained from the blends showed slow degradation, probably due to the higher content in PLA and PBAT. These observations pave the way for exploring enzymes capable of degrading all blend components or an enzymatic mixture for blend degradation.

Lipase from Rhizopus oryzae (ROL) exhibits remarkable sn -1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of 1,3-dioleoyl-2-palmitoyl glycerol (OPO, a high-quality substitute for human milk fat). In this work, a semirational method was proposed to engineer the thermostability and catalytic activity of 4M (ROL mutant in our previous study). First, a computer-aided design is performed using 4M as a template, and N -glycosylation mutants are then recombinantly expressed and screened in Pichia pastoris , the optimal mutant N227 exhibited a half-life of 298.8 h at 45 °C, which is 7.23-folds longer than that of 4M. Its catalytic activity also reached 1043.80 ± 61.98 U/mg, representing a 29.2% increase compared to 4M (808.02 ± 47.02 U/mg). Molecular dynamics simulations of N227 suggested that the introduction of glycan enhanced the protein rigidity, while the strong hydrogen bonds formed between the glycan and the protein stabilized the lipase structure, thereby improving its thermostability. The acidolysis reaction between oleic acid (OA) and glycerol tripalmitate (PPP) was successfully carried out using immobilized N227, achieving a molar conversion rate of 90.2% for PPP. This engineering strategy guides the modification of lipases, while the glycomutants obtained in this study have potential applications in the biosynthesis of OPO.

Polyethylene (PE) exhibits high resistance to degradation, contributing to plastic pollution. PE discarded into the environment is photo-oxidized by sunlight and oxygen. In this study, a key enzyme capable of degrading oxidized PE is reported for the first time. Twenty different enzymes from various lipase families were evaluated for hydrolytic activity using substrates mimicking oxidized PE. Among them, Pelosinus fermentans lipase 1 (PFL1) specifically cleaved the ester bonds within the oxidized carbon-carbon backbone. Moreover, PFL1 (6 μM) degraded oxidized PE film, reducing the weight average and number average molecular weights by 44.6 and 11.3 %, respectively, within five days. Finally, structural analysis and molecular docking simulations were performed to elucidate the degradation mechanism of PFL1. The oxidized PE-degrading enzyme reported here will provide the groundwork for advancing PE waste treatment technology and for engineering microbes to repurpose PE waste into valuable chemicals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A strategy based on artificial antibody-antigen recognition was proposed for the specific directed immobilization of lipase. The artificial antibody was synthesized using catechol as a template, α-methacrylic acid as a functional monomer, and Fe 3 O 4 as the matrix material. Lipase was modified with 3,4-dihydroxybenzaldehyde as an artificial antigen. The artificial antibody can specifically recognize catechol fragment in the enzyme structure to achieve the immobilization of lipase. The immobilization amount, yield, specific activity, and immobilized enzyme activity were 13.2 ± 0.2 mg/g, 78.9 ± 0.4 %, 7.9 ± 0.2 U/mg protein , and 104.6 ± 1.7 U/g carrier , respectively. Moreover, the immobilized lipase exhibited strong reusability and regeneration ability. Additionally, the immobilized lipase successfully catalyzed the synthesis of benzyl acetate and demonstrated robust continuous catalytic activity. These results fully demonstrate the feasibility of the proposed artificial antibody-antigen-directed immobilization of lipase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory., Results: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL -1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL -1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca 2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate., Conclusion: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

The study illustrated here aims on an organic solvent tolerant lipase from Staphylococcus capitis (SCL). The gene part, encoding the mature lipase, was cloned and sequenced. The concluded polypeptide sequence, equivalent to the protein, consist of 388 amino acid residues with a molecular mass of about 45 kDa. A structure-based alignment of the SCL amino acid sequence shows high identities with those many staphylococcal lipases. From this alignment of sequences, the catalytic triad (Ser 117, Asp 308 and His 347) of SCL could be identified. The mature part of the SCL was expressed in Escherichia coli and the recombinant lipase (r-SCL) was purified to homogeneity. The purified r-SCL presented a quite interesting stability at low temperatures (< 30 °C) and the enzyme was found to be highly stable in polar organic solvent and at a pH ranging from 3 to 12. After that, we have demonstrated that the recombinant enzyme may be implicated in the biodegradability of oily wastewater from effluents of fast-food restaurants; the maximum conversion yield into fatty acids obtained at 30 °C, was 65%.

Lipases are remarkable biocatalysts, adept at catalyzing the breakdown of diverse compounds into glycerol, fatty acids, and mono- and di-glycerides via hydrolysis. Beyond this, they facilitate esterification, transesterification, alcoholysis, acidolysis, and more, making them versatile in industrial applications. In industrial processes, lipases that exhibit high stability are favored as they can withstand harsh conditions. However, most native lipases are unable to endure adverse conditions, making them unsuitable for industrial use. Protein engineering proves to be a potent technology in the development of lipases that can function effectively under challenging conditions and fulfill criteria for various industrial processes. This review concentrated on new trends in protein engineering to enhance the diversity of lipase genes and employed in silico methods for predicting and comprehensively analyzing target mutations in lipases. Additionally, key molecular factors associated with industrial characteristics of lipases, including thermostability, solvent tolerance, catalytic activity, and substrate preference have been elucidated. The present review delved into how industrial traits can be enhanced through directed evolution (epPCR, gene shuffling), rational design (FRESCO, ASR), combined engineering strategies (i.e. CAST, ISM, and FRISM) as protein engineering methodologies in contexts of biodiesel production, food processing, and applications of detergent, pharmaceutics, and plastic degradation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Covalent organic frameworks (COFs) with large specific surface areas, high porosity, good stability, and designable structure are promising carriers for immobilized enzymes. It is important to explore lipase inhibitors from natural foods as lipase inhibitors are closely related to the treatment of obesity. In this work, a carboxyl functionalized covalent organic framework (TpBD-3COOH) was prepared by solvothermal method for covalent immobilization of porcine pancreatic lipase (PPL) and obtained the enzyme-decorated COF (PPL@COF). The immobilized lipase showed wider pH and temperature tolerance with the same optimal pH and temperature of 7.5 and 50 ℃ compared to free lipase. After 6 successive reuses, the PPL@COF maintained 53.0% of its original activity. Immobilized lipase also displayed enhanced storage stability (55.4% after 14 days at 4 ℃). When p-nitrophenyl acetate was applied as the substrate, the calculated Michaelis constant was 3.57 mM and the half maximal inhibitory concentration of orlistat was 3.20 μM. Finally, the PPL@COF was used for enzyme inhibitors screening from natural foods combined with UV spectrophotometry, and Hawthorn was screened for excellent lipase inhibitory activity., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A novel approach to ultrasound-assisted Pickering interfacial biocatalysis (PIB) has been proposed and implemented for the efficient enzymatic transesterification production of vitamin A fatty acid esters. This is the first instance of exploiting the synergistic effect of ultrasound and the bifunctional modification of enzyme supports to accelerate biocatalytic performance in PIB systems. The optimal conditions were determined to be ultrasound power of 70 W, on/off time of 5 s/5 s, substrate molar ratio of 1:1, enzyme addition of 2 %, and a volume ratio of n-hexane to PBS of 3:1, a temperature of 40 °C, and a time of 30 min. The application of ultrasound technology not only improved lipase activity but also allowed for a reduction in emulsion droplet size to enhance interfacial mass transfer.Bifunctional modification of silica-based supports enhanced stability of immobilized enzymes by increasing hydrogen bonding while maintaining the active interface microenvironment. Compared with a non-ultrasound-assisted PIB system stabilized by mono-modified immobilized enzyme particles, the catalytic efficacy (CE) of the novel system reached 8.18 mmol g -1 min -1 , which was enhanced by 3.33-fold, while the interfacial area was found to have increased by 17.5-fold. The results facilitated the conversion of vitamin A palmitate (VAP), vitamin A oleate (VAO), vitamin A linoleate (VAL), and vitamin A linolenate (VALn), with conversion rates of approximately 98.2 %, 97.4 %, 96.1 %, and 94.7 %, respectively. This represents the most efficient example that has been reported to our knowledge. Furthermore, the system demonstrated improved reusability, with a conversion rate of 62.1 % maintained even after 10 cycles. The findings presented in this paper provide valuable insights into an efficient and conveniently promising protocol for the development of PIB systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The detailed characterization of the structural features of peptides targeting cholesterol esterase (CEase) or pancreatic lipase (PPL) will benefit the management of hyperlipidemia and obesity. This study employed the Glide SP (standard precision)-peptide method to predict the binding modes of 20 2 dipeptides and 20 3 tripeptides to these targets, correlating residue composition and position with binding energy. Strong preferences for Trp, Phe, and Tyr were observed at all positions of potential inhibitory peptides, whereas negatively charged residues Glu and Asp were disfavored. Notably, Arg and aromatic rings significantly influenced the peptide conformation at the active site. Tripeptide IWR demonstrated the high efficacy, with IC 50 values of 0.214 mg/mL for CEase and 0.230 mg/mL for PPL. Five novel IWR scaffold-tetrapeptides exhibited promising inhibitory activity. Non-covalent interactions and energy contributions dominated the formation of stable complexes. Our results provide insights for the development of new sequences or peptide-like molecules with enhanced inhibitory activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Enzyme is an important class of catalyst. However, the efficiency of enzyme-catalyzed reactions is constrained by the limited contact between the enzyme and its substrate. In this study, to overcome this challenge, lipase-loaded microcapsules were prepared from natural shellac and nanoparticles using the emulsion template method. These microcapsules can perform dual roles as stabilizers and enzyme carriers to construct a water-in-oil Pickering interfacial biocatalytic system. The results showed that the hydrolytic conversion of the microcapsules could reach 90% within 20 min, which was significantly higher than that of the traditional biphasic system. The catalytic activity was influenced by the oil-to-water volume ratio and the microcapsule content. The microcapsules remained highly catalytic efficiency even after storage for three months or seven cycles of reuse. These microcapsules were prepared without the use of any cross-linkers or harsh solvents. This green and efficient catalytic system has great application prospects in the food industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Three-liquid-phase systems (TLPSs) are novel interfacial enzymatic reaction systems that have been successfully applied in many valuable reactions. However, these systems are suitable only for hydrolysis reactions and not for more widely used esterification reactions. Surprisingly, our recent research revealed that two water-insoluble substrates (β-sitosterol and conjugated linoleic acid) could be rapidly esterified in this system. The initial rate of the esterification reaction in the TLPS based on sodium citrate was enhanced by approximately 10-fold relative to that in a traditional water/n-hexane system. The special emulsion structure (S/W 1 /W 2 emulsion) formed may be vital because it not only provides a larger reaction interface but also spontaneously generates a middle phase that might regulate water activity to facilitate esterification. Furthermore, the lipase-enriched phase could be reused at least 8 times without significant loss of catalytic efficiency. Therefore, this TLPS is an ideal enzymatic esterification platform for ester synthesis because it is efficient, convenient to use, and cost-effective., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Docosahexaenoic acid (DHA) was concentrated successfully in the glyceride fractions from tuna oil via a two-step enzyme reaction involving hydrolysis and ethanolysis. In the first step, Candida rugosa lipase-catalyzed hydrolysis was carried out to concentrate DHA in the glyceride fractions. The DHA content in the glyceride fraction after hydrolysis increased from 30% in the initial tuna oil to 46%. In the second step, Lipozyme RM IM-catalyzed ethanolysis was conducted with the reaction mixture from the first step to further concentrate DHA in the glyceride fraction. In this step, the reaction mixture obtained from the first step was employed directly in Lipozyme RM IM-catalyzed ethanolysis without additional steps needed to remove free fatty acid. Finally, DHA was concentrated from an initial content of 30% in the tuna oil to 68.4% in the glyceride fractions via a novel two-step enzyme reaction strategy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The present study found for the first time that phenolic glycosides were an important material basis for mulberry leaves to inhibit lipase. The corresponding IC 50 for hyperoside, rutin, astragalin and quercetin were 68, 252, 385 and 815 μg/mL respectively. The inhibitory effect was ranked as monoglycosides > phenolic hydroxyl groups > disaccharides on the benzone ring. Hyperoside bound to lipase in competitive inhibition type with one binding site, while the others bound to lipase in a mixed inhibition type by two similar sites. All four compounds altered the microenvironment and secondary conformation of lipase through static quenching. The docking score, stability, and binding energy were consistent with the compound inhibitory activity. The main binding between compounds and lipase amino acid residues were spontaneously though hydrophobic interactions and hydrogen bonding. The strong hydrogen bonds formed with SER-152 inside the lipase pocket, might be important for the strong inhibitory activity of hyperoside., Competing Interests: Declaration of competing interest The authors declare that they have no financial interest or personal relationships which influenced the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

We propose a co-immobilized chemo-enzyme cascade system to mitigate random intermediate diffusion from the mixture of individual immobilized catalysts and achieve a one-pot reaction of multi-enzyme and reductant. Catalyzed by lipase and lipoxygenase, unsaturated lipid hydroperoxides (HPOs) were synthesized. 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid (13-HPODE), one compound of HPOs, was subsequently reduced to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) by cysteine. Upon the optimized conditions, 75.28 mg of 13-HPODE and 4.01 mg of 13-HODE were produced from per milliliter of oil. The co-immobilized catalysts exhibited improved yield compared to the mixture of individually immobilized catalysts. Moreover, it demonstrated satisfactory durability and recyclability, maintaining a relative HPOs yield of 78.5% after 5 cycles. This work has achieved the co-immobilization of lipase, lipoxygenase and the reductant cysteine for the first time, successfully applying it to the conversion of soybean oil into 13-HODE. It offers a technological platform for transforming various oils into high-value products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Efficient, green and stable catalysis has always been the core concept of enzyme catalysis in industrial processes for manufacturing. Therefore, we construct a new strategy with photothermal interfacial molecular transfer for green and efficient biodiesel catalysis. We encapsulate Candida albicans lipase B (CalB) in a γ-cyclodextrin metal-organic framework (γ-CD-MOF) loading with Ti 3 C 2 T X by in situ growth and electrostatic assembly. The γ-CD-MOF not only protects the fragile enzyme, but also enhances the catalytic performance through the synergistic effects of porous adsorption (MOF pore structure) and interfacial enrichment (cyclodextrins host-guest assembly structure) for accelerating substrate transfer (642.6 %). The CalB@γ-CD-MOF/MXene-i activity can be regulated up to 274.6 % by exposure to near-infrared (NIR). Importantly, CalB@γ-CD-MOF/MXene-i achieves 93.3 % biodiesel conversion under NIR and maintained 86.9 % activity after 6 cycles. Meanwhile, the MXene after the CalB@γ-CD-MOF/MXene catalytic cycle can be almost completely recovered. We verify the mechanism of high catalytic activity of γ-CD-MOF and rationalize the mechanism of CD molecular channel by DFT. Therefore, this highly selective enzyme catalytic platform offers new possibilities for green and efficient preparation of bioenergy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The aim of the presented study was to evaluate the release of the enzymatically initiated production of hexanal from double emulsion electrospun bio-active membranes at a temperature of fruit storage. Among different formulations of water-in-oil (W1/O) primary emulsions, the emulsion composed of 12% w/v Tween20 and 0.1 M NaCl in water (W1) and 6% of poly(glycerol) poly(ricinoleate) dissolved in sunflower oil (O) using W1/O ratio of 80/20 (w/w) (Tween20-NaCl/6% PGPR) was selected, for further incorporation of enzymes, based on the lowest average droplet size (391.0 ± 15.6 nm), low polydispersity index (0.255 ± 0.07), and good gravitational stability also after 14 days. Both enzymes, lipase and lipoxygenase are needed to produce hexanal (up to 58 mg/L). Additionally, double emulsions were prepared with sufficient conductivity and viscosity using different W1/O to W2 ratios for electrospinning. From the selected electrospun membrane, up to 4.5 mg/L of hexanal was released even after 92 days., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The limited availability of phospholipase A 1 (PLA 1 ) has posed significant challenges in enzymatic degumming. In this study, a novel PLA1 (UM2) was introduced to address this limitation, which had a unique thermo-responsive ability to switch phospholipase and lipase activities in response to temperature variations. Remarkably, UM2 displayed an unprecedented selectivity under optimized conditions, preferentially hydrolyzing phospholipids over triacylglycerols-a specificity superior to that of commercial PLA 1 . Moreover, UM2 demonstrated high efficiency in hydrolyzing phospholipids with a predilection for phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A practical application of UM2 on crude flaxseed oil led to a dramatic reduction in phosphorus content, plummeting from an initial 384.06 mg/kg to 4.38 mg/kg. Broadening its industrial applicability, UM2 effectively performed enzymatic degumming for other distinct crude vegetable oils with a unique phospholipid composition. Collectively, these results highlighted the promising application of UM2 in the field of oil degumming., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Hypothesis: Understanding the digestion of lipid-based pharmaceutical formulations and food systems is necessary for optimising drug and nutrient delivery and has been extensively studied in bulk emulsion systems using the pH-stat method [1]. However, this approach is not suitable for investigation of individual lipid droplets, in particular the interface where the lipase acts. Microfluidic approaches to study digestion at lipid-water interfaces using droplet trapping have been proposed, however the aqueous phase in that case washes over the interface presenting uncertainty over the stoichiometry of interactions [2]. The internal interface of a Janus-like droplet, containing distinct aqueous and lipid compartments, mimics the interface of a lipid droplet in aqueous solution with controlled stoichiometry [3]. Hence, it was hypothesised that the internal interface of Janus droplets can offer a precise way to study the enzymatic digestion of lipids formulations., Experiments: Using microfluidic methods, Janus-like droplets were formed by coalescing emulsion droplets containing lipid formulation and pancreatic lipase. Polarised light microscopy (PLM) and in-situ small-angle X-ray scattering (SAXS) were used to investigate the droplets., Findings: PLM revealed the growth of an aligned inverse hexagonal phase (H 2 ), and with SAXS showed that this phase transformation and alignment resulted from enzymatic digestion. A subsequent partial transformation from H 2 to inverse bicontinuous cubic phase occurred when simulated intestinal fluid was used instead of Tris buffer. Suggesting that phospholipids and bile salts could diffuse across the internal interface to locally affect their surroundings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)