Thermomyces Lanuginosus Lipase Research Articles

The effect of carbon chain length of cross-linking agent on the functionality of carrier- free immobilized Thermomyces lanuginosa lipase particles

The cross-linked enzyme (CLEs) of Thermomyces lanuginosa lipase (TLL) was prepared in an isocyanide-based multi-component reactions (ICMRs) platform by applying three di-acidic cross-linkers to unveil more factors contributing to the functional properties of CLEs. The linkers were 1,11-undecanedicarboxylic acid, azelaic acid, and adipic acid with 11, 7, and 4 carbon lengths, respectively, providing a proper tool to investigate the effect of linker length on the activity, stability, and selectivity of the resulting CLEs. The immobilization yields of 60–90 % and the specific activities of 168, 88.4 and 49 U/mg were obtained for the CLEs of 1,11-undecanedicarboxylic acid, azelaic acid, adipic acid, respectively. The lower activity of azelaic and adipic acid-mediated CLEs compared to the soluble TLL (110 U/mg) was explained by in silico calculations. The results revealed that as opposed to 1,11-undecanedicarboxylic acid, both linkers tended to penetrate the enzyme active site, thus resulting in a major inhibitory effect on the enzyme functionality. The thermal and co-solvent stability of the immobilized derivatives improved compared to those of free TLL. The selectivity of CLEs was also examined by catalytic release of main omega-3 fatty acids from fish oil, presenting the highest selectivity of 22 for the CLEs of azelaic acid.

One-Pot Synthesis of 1,3,5-Trisubstitued Pyrazoles via Immobilized Thermomyces lanuginosus Lipase (TLL) on a Metal-Organic Framework.

A regioselective enzyme-catalyzed system is selected for the synthesis of 1,3,5-trisubstituted pyrazole derivatives by adding phenyl hydrazines, nitroolefins, and benzaldehydes. The reaction is performed in a one-pot vessel with a yield ranging from 49 to 90%. TLL@MMI, immobilized Thermomyces lanuginosus lipase (TLL) on a multivariate of MOF-5/IRMOF-3 (MMI), showed good performance for the catalysis of this reaction. The prepared biocatalyst was characterized by FTIR, XRD, SEM, and EDX. The thermal and solvent stability of TLL@MMI was investigated in MeOH and EtOH after 24 h incubation. In the presence of 100% concentrations of EtOH, TLL@MMI has 80% activity.

Fabrication of Vascular Grafts Using Poly(ε-Caprolactone) and Collagen-Encapsuled ADSCs for Interposition Implantation of Abdominal Aorta in Rhesus Monkeys.

The production of small-diameter artificial vascular grafts continues to encounter numerous challenges, with concerns regarding the degradation rate and endothelialization being particularly critical. In this study, porous PCL scaffolds were prepared, and PCL vascular grafts were fabricated by 3D bioprinting of collagen materials containing adipose-derived mesenchymal stem cells (ADSCs) on the internal wall of the porous PCL scaffold. The PCL vascular grafts were then implanted in the abdominal aorta of Rhesus monkeys for up to 640 days to analyze the degradation of the scaffolds and regeneration of the aorta. Changes in surface morphology, mechanical properties, crystallization property, and molecular weight of porous PCL revealed a similar degradation process of PCL in PBS at pH 7.4 containing Thermomyces lanuginosus lipase and in situ in the abdominal aorta of rhesus monkeys. The contrast of in vitro and in vivo degradation provided valuable reference data for predicting in vivo degradation based on in vitro enzymatic degradation of PCL for further optimization of PCL vascular graft fabrication. Histological analysis through hematoxylin and eosin (HE) staining and fluorescence immunostaining demonstrated that the PCL vascular grafts successfully induced vascular regeneration in the abdominal aorta over the 640-day period. These findings provided valuable insights into the regeneration processes of the implanted vascular grafts. Overall, this study highlights the significant potential of PCL vascular grafts for the regeneration of small-diameter blood vessels.

Enhancing methanol tolerance of Thermomyces lanuginosus lipase by rational design and biodiesel production through one-step feeding of methanol

The addition of a significant quantity of methanol solution during the biodiesel production results in the denaturation of lipase. To address this issue, this study proposed a novel approach to the preparation process by designing a methanol-tolerant Thermomyces lanuginosus lipase (TLL) using a computer-aided strategy. In silico prediction yielded seven mutants, out of which two mutants, E129Y and E134W, exhibited varying degrees of improved methanol tolerance and catalytic efficiency. The methanol tolerance and stability of E129Y and E134W were observed to increase by 10–30 % and 17–35 %, respectively. Additionally, E134W exhibited a 1.13-fold increase in catalytic efficiency compared to the wild type TLL, while E129Y showed a reduction of 12.05 %. Following a one-shot addition of methanol (molar ratio of 3:1 methanol to Cornus wilsoniana oil) for 72 h in the transesterification reaction, the yields of fatty acid methyl ester (FAME) for E129Y and E134W were 69 % and 85 %, respectively, with the yield of E134W increasing by 22 %. Structural analysis indicated that the E129Y mutation formed a single hydrogen bonding interaction, while E134W produced hydrophobic interactions with neighboring amino acids, thereby augmenting the structural stability of the mutations under high methanol concentration. This study has the potential to advance the commercialization of lipases in the biodiesel production industry.

Enzymatic kinetic resolution of an intermediate alcohol of the drug Tegoprazan using lipase from Thermomyces lanuginosus

An enzymatic approach for the synthesis of intermediate alcohol of the drug tegoprazan in 99% ee at 20 °C in hexane has been developed utilizing commercially available immobilized Thermomyces lanuginosus lipase (TL lipase) as a biocatalyst and vinyl acetate as a acyl donor. This method involves the enantioselective esterification of (RS)-5,7-difluorochroman-4-ol with vinyl acetate using TL Lipase. This enzymatic approach provides the (S)-chiral alcohol in 44% yield with 99% enantiomeric excess (ee) and the (R)-acetate in 43% yield and >99% ee. It is a sustainable approach for the synthesis of enantiopure tegoprazan intermediate, contributing to the advancement of green chemistry. The recovered TL lipase was reused in 13 subsequent runs without loss of activity.

In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production

BackgroundBiodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement.ResultsIn this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. Thermomyces lanuginosus lipase (TLL) with high activity and thermostability was obtained using the Pichia pastoris expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt–TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, T5015\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$T_{50}^{15}$$\\end{document} (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt–TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt–TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil.ConclusionsAn integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.

Enzymatic synthesis of tyrosol esters in organic solvents and ionic liquids: Correlation between enzyme activity and solvent properties

Tyrosol is a principle phenolic compound present in olive oil and wine with great pharmacological potentials due to its strong antioxidant property. Its application can be highly extended by increasing its lipophilicity through esterification. In this study, tyrosol esters were synthesized by enzymatic transesterification of tyrosol with vinyl esters, with a commercial lipase from Novozymes (Novozym 435, Candida antarctica lipase B (CALB) immobilized on macroporous acrylic resin beads) as the catalyst, both in organic solvents and in ionic liquids. Methyl tert-butyl ether (MTBE) was the best solvent, offering complete conversions towards the synthesis of tyrosol esters with varying chain lengths (C2-C18) within 1 h. Taking the enzymatic transesterification between tyrosol and vinyl acetate as the model reaction, 18 organic solvents and 24 ionic liquids were screened, and their solvent properties (i.e. hydrophobicity, polarity, viscosity, water activity, and partition coefficients of substrate and product) were correlated to the enzyme activity. Upon enzyme screening, four new lipases were found to be highly efficient, relative to the best-performed Novozym 435 and Lipozyme TLIM (Thermomyces lanuginosus lipase (TLL) immobilized on silica gel), in tyrosol ester synthesis. This work provides enzymatic methods of esterifying tyrosol that are much more efficient than those reported in literature, and will shed light on re-consideration of the roles of solvents in nonaqueous enzymology.

Immobilization of Thermomyces lanuginosus lipase in a novel polysaccharide-based hydrogel by a two-step crosslinking method and its use in the lauroylation of α-arbutin

The Thermomyces lanuginosus lipase (TLLs) was successfully immobilized within a novel hydrogel matrix through a two-step crosslinking method. TLLs were initially crosslinked through the Schiff base reaction by oxidized carboxymethyl cellulose (OCMC). The water-soluble OCMC@TLLs complex was subsequently crosslinked by carboxymethyl chitosan (CMCSH) in a microfluidic apparatus to form the CMCHS/OCMC@TLLs microspheres. The CD (Circular Dichroism, CD) and FT-IR (Fourier Transform infrared spectroscopy, FT-IR) spectra demonstrated that the crosslinking of TLLs with OCMC resulted in a less significant impact on their structure compared to that with glutaraldehyde. CMCHS/OCMC@TLLs showed decreased catalytic performance due to the mass transfer resistance, while its thermal stability was greatly improved. The CMCHS/OCMC@TLLs were used to catalyze the lauroylation of arbutin in tetrahydrofuran. After 12 h of reaction under optimal conditions, the yield of 6′-O-lauryl arbutin reached an impressive 92.12%. The prepared 6′-O-lauryl arbutin has high lipophilicity and exhibits similar tyrosinase inhibitory activity and higher antioxidant activity compared to its parent compound.Graphical

Improving the Thermostability of Thermomyces lanuginosus Lipase by Restricting the Flexibility of N-Terminus and C-Terminus Simultaneously via the 25-Loop Substitutions

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24–30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27…E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability.

Hyperactivation of lipases by immobilization on superhydrophobic graphene quantum dots inorganic hybrid nanoflower

Various nanoflowers are synthesized for enzyme immobilization. In order to increase the activity of nanoflowers, in this study, 3D flower-like structured organic–inorganic hybrid nanoflowers (hNFs) with various lipases Rhizomucor miehei lipase (RML), Candida antarctica lipase B (CALB), Humicola insolens lipase (HIL), Thermomyces lanuginosus lipase (TLL), Eversa® Transform 2.0 (ET) a genetically modified enzyme derived of TLL and graphene quantum dots (GQDs) were prepared and characterized.Lipase hNFs [lipase-(Cu/Co)3(PO4)2] and lipase@GQDs hNFs [lipase@GQDs-(Cu/Co)3(PO4)2] were straightforwardly prepared through mixing with metal ion (Cu2+or Co2+) aqueous solutions with or without GQDs. The ET@GQDs-(Cu)3(PO4)2 hNFs demonstrated 687 % higher activity than ET-(Cu)3(PO4)2 hNFs and 650 % higher activity than the free ET. Similar results were also observed with other lipase hybrid nanoflowers. For example, TLL@GQDs-(Cu)3(PO4)2 hNFs exhibited a 557 % higher activity than TLL-(Cu)3(PO4)2 hNFs and a 463 % higher activity than free TLL. Additionally, TLL@GQDs-(Co)3(PO4)2 hNFs showed a 141 % higher activity than TLL-(Co)3(PO4)2 hNFs and a 304 % higher activity than free TLL. Upon examining pH and thermal stability, it was revealed that lipase@GQDs hNFs exhibited higher activity compared to free lipase and other hNFs without GQDs. The effect of metal ions, enzyme concentrations and amount of GQDs on the morphology and enzyme activity of the lipase-hNFs was examined.

A betaine-modified whole-cell biocatalyst with surface display of Thermomyces lanuginosus lipase for biodiesel production from Jatropha curcas oil

Yeast surface displaying lipases are promising biocatalysts for biodiesel synthesis. Thermomyces lanuginosus lipase (TLL) was displayed on Pichia pastoris cell surface by screening proper anchor system, resulting in a whole-cell biocatalyst (termed D-TLL). The lipase activity of D-TLL is the highest activity to date for surface-displayed TLL, reaching 2274.7 U/g dry cell weight (DCW). The obtained biocatalyst was efficient in converting Jatropha curcas oil into biodiesel. The biodiesel yield peaked 92.6% after 12 h adopting only 3.3% (w/w) of D-TLL in the solvent-free system. As compared to free TLL, the whole-cell biocatalyst showed a dramatic reduction of 48 h in reaction time. The further treatment with betaine enabled enhanced thermostability, hydrophobicity and methanol tolerance of biocatalyst (named D-TLL-betaine), and thereby increasing the reusability of the whole-cell biocatalyst. These results exhibited that the new biocatalyst, D-TLL-betaine, has great potential for biodiesel production.

Biocatalysts Based on Immobilized Lipases for the Production of Ethyl Esters of Fatty Acids including Bioactive Gamma-Linolenic Acid from Borage Oil

In the present work, borage oil (Borago officinalis) was used as the main source of gamma linolenic acid (GLA) to obtain ethyl esters by enzymatic ethanolysis using immobilized enzymes for its application in the food industry. Commercial Thermomyces lanuginosus lipase (TLL) was compared to chemical ethanolysis in alkaline medium. In addition, TLL was immobilized by adsorption on hydrophobic porous support (Octadecyl-Sepabeads®) to compare the results. Fatty acid ethyl ester (FAEE) yields of both reactions were compared under the same conditions (25 °C and 200 rpm) and analyzed by GC-MS. Moreover, the conversion yield for borage oil ethanolysis catalyzed by TLL immobilized on C18-Sepabeads® supports was similar to the chemical pathway (93.4% and 99.5%, respectively). When this biocatalyst was used in a solvent-free system (at 40 °C and 200 rpm), it was possible to obtain a high FAEE yield of 84.3% in the first 24 h of reaction. Furthermore, it was possible to re-use the immobilized biocatalyst for the performance of five reaction cycles maintaining 68% of its initial activity. Thus, the use of immobilized enzymes in solvent-free systems is an eco-friendly alternative to obtain GLA ethyl esters for its possible application in cosmetics and food.

Degradation of ochratoxins A and B by lipases: A kinetic study unraveled by molecular modeling

Mycotoxins are toxic substances produced by fungi and, frequently, different mycotoxins cooccur in food commodities. Ochratoxin A (OTA) and Ochratoxin B (OTB) may co-occur in a variety of foods, like red wines and wheat, presenting a significant risk of population exposure. In this study, we investigated the potential of five lipases (Candida rugosa Lipase, Candida antarctica B Lipase, Thermomyces lanuginosus Lipase, Amano Lipase A from Aspergillus niger (ANL) and Porcine Pancreas Lipase (PPL)) to hydrolyze OTA and OTB into non-hazardous products. Only ANL and PPL degraded both substrates, however, with varying degrees of efficiency. PPL completely degraded OTB (9 h), but only 43% of OTA (25 h). Molecular simulations indicated a high binding energy of OTA to PPL, that can be explained by the impact of the chlorine group, impairing hydrolysis. ANL was able to completely degrade both mycotoxins, OTA in 3 h and OTB in 10 h. The ANL enzyme showed also high specificity to OTA, however, the activity of this enzyme is not affected by chlorine and hydrolyzes OTA faster than OTB. These two enzymes were found to be able to detoxify co-occurring ochratoxins A and B, making isolated enzymes an alternative to the direct use of microorganisms for mycotoxin mitigation in food.

Improving the position specificity of Themomyces lanuginosus lipase based on semi-rational design

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.

Lipase-catalyzed preparation of deacidified Buah Merah (Pandanus conoideus) oil.

Buah Merah oil (BMO) is unrefined edible oil containing a high level of free fatty acids (FFA; ∼30% w/w). This study was aimed at preparing deacidified BMO from BMO via lipase-catalyzed esterification of FFA in BMO with added glycerol, using Duolite A568-immobilized Eversa Transform 2.0 (Thermomyces lanuginosus lipase) as biocatalyst. BMO containing 2.4% w/w FFA and 94.6% w/w triacylglycerol was obtained under optimal reaction conditions (temperature, 70°C; FFA-to-glycerol molar ratio, 3:1; enzyme loading based on the protein quantity, 3.75mg/g BMO, and reaction time, 48h). No significant difference was found in the contents of β-carotene, tocopherols, and phytosterols between raw and deacidified BMO. The induction period of oxidation was significantly longer in deacidified BMO (16.37h) than in raw BMO (0.03h). These results suggest that deacidified BMO could be enzymatically prepared without the loss of health-beneficial minor components while enhancing the oxidative stability. PRACTICAL APPLICATION: Although BMO has recently received much attention for its potential biological activities, the commercial use of BMO as a healthy oil has been limited due to its high FFA content. Unlike conventional alkali and steam refining, enzymatic deacidification of BMO employed in this study might help the commercialization of BMO, because this procedure enables the improvement of oil yield and the retaining of health-beneficial minor components.

Biochemical characterization of an alkaline and detergent-stable Lipase from Fusarium annulatum Bugnicourt strain CBS associated with olive tree dieback.

This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 μM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents.

Preparation of glycerides enriched with n-3 long-chain polyunsaturated fatty acids by Thermomyces lanuginosus lipase-catalyzed hydrolysis of tuna oil

n-3 Long-chain polyunsaturated fatty acids (LC-PUFAs) in glyceride form have attracted much attention due to their nutritional value and high bioavailability. In this work, Thermomyces lanuginosus lipase (TL 100 L) was used to selectively hydrolyze tuna oil for preparing glycerides enriched with n-3 LC-PUFAs. Effects of reaction variables on the hydrolysis reaction were optimized by response surface methodology, and the optimal conditions were as follows: reaction temperature 54 °C, enzyme load 3%, reaction time 6 h, mass ratio of water to tuna oil 1.5:1. Under these conditions, the resulting degree of hydrolysis was 55.01%, and the total content of eicosapentaenoic acid and docosahexaenoic acid in the glyceride fraction was found to be 46.39%. The final product was mainly composed of diacylglycerols (53.93%) and triacylglycerols (34.58%) after removal of the free fatty acids. This paper provides a simple and suitable method for industrial production of glycerides enriched with n-3 LC-PUFAs.

Evaluation the selectivity of three lipases in the synthesis of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol, an asymmetric triacylglycerol

1-Oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) is the most abundant triacylglycerol (TAG) molecule in Chinese human milk. The selectivity of three lipases including Rhizomucor miehei lipase (RML), Thermomyces lanuginose lipase (TL), and Candida antarctica lipase (CALB) in OPL synthesis were evaluated. The effects of different reaction conditions on selectivity were first investigated. Unexpectedly, the three sn-1,3 regioselectivity lipases, also have selectivity for the structure of product TAGs. Compared with symmetric TAGs, lipases had poor selectivity toward asymmetric OPL. Furthermore, molecular simulations were used to deeply understand the molecular factors affecting lipase selectivity. The results showed that hydrophobic interactions, lipases lid domain flexibility, and the stability of hydrogen bonds between lipases and TAGs were the main molecular factors responsible for the selectivity of lipases.

Solid‐Phase Lipase‐CuNPs Biohybrids as Catalysts for One‐Pot Parallel Synthesis of 2,3,4‐Triacetyl‐D‐Gluconic Acid

AbstractSolid‐phase lipase/metal nanobiohybrids, generated by growth of copper nanoparticles on enzyme matrixes immobilized on graphene, were used as heterogeneous catalysts with dual‐activity for the regioselective production of 2,3,4‐triacetyl‐D‐gluconic acid from α‐peracetylated‐glucose in a one‐pot parallel process combining a lipase‐mediated regioselective hydrolytic monodeprotection with a metal‐catalyzed oxidation in aqueous media. A novel synthetic strategy, based on the in situ fabrication of Cu nanoparticles induced by lipase molecules specifically immobilized on a multi‐layer graphene material by interfacial adsorption fixing them in the active open conformation, has been described. Thermomyces lanuginosus lipase was firstly used to prepare the functionalized multi‐layer graphene from graphite as a biographene preparation (Biographene, BIOG), support used to successfully immobilize Candida rugosa lipase (CRL). This immobilized form BIOG‐CRL was further used to create successful active bifunctional enzyme‐metal nanoarchitectures. Two different Cu‐lipase hybrids were synthesised, where Cu species and nanoparticles size were different depending on the methodology. Regioselectivity and stability of the hybrids were evaluated successfully in the production of monosaccharide building blocks, besides the robustness of the hybrids in recyclability experiments. These findings highlight the potential of these solid‐phase nanoarchitectures as useful tools in the synthesis of complex glycoderivatives for use in food, medicine, and cosmetics.

Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion.

The function of most lipases is controlled by the lid, which undergoes conformational changes at a water-lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases' function is important for designing improved variants. Lipases' function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes' diffusional behavior, to study Thermomyces lanuginosus lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent.