Lipase‐Supported Metal–Organic Framework Bioreactor Catalyzes Warfarin Synthesis

A green and sustainable strategy synthesizes clinical medicine warfarin anticoagulant by using lipase‐supported metal–organic framework (MOF) bioreactors (see scheme). These findings may be beneficial for future studies in the industrial production of chemical, pharmaceutical, and agrochemical precursors.     

Zymography for Picogram Detection of Lipase and Esterase Activities

Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities, a simple and sensitive approach for picogram detection of lipolytic enzyme quantity is still highly desirable. Here we report a lipase detection assay which is 1000-fold more sensitive than previously reported methods. Our assay enables the detection of as low as 5 pg and 180 pg of lipolytic activity by direct spotting and zymography, respectively. Furthermore, we demonstrated that the detection sensitivity was adjustable by varying the buffering capacity, which allows for screening of both high and low abundance lipolytic enzymes. Coupled with liquid chromatography-mass spectrometry, our method provides a useful tool for sensitive detection and identification of lipolytic enzymes.     

铈基介孔金属有机骨架固定化酶的构建及催化性能

金属有机骨架(MOF)材料由于其孔隙率高、比表面积大以及具有发达的内联通孔道结构等优点,可以作为优良的生物分子固定化载体。通过表面活性自组装策略制备了铈基介孔MOF(Ce-MOF-F),表征结果表明,该材料有大的比表面积和呈辐射状的介孔孔道结构。以其为载体、南极假丝酵母脂肪酶B(CALB)为模型酶,通过物理吸附法制备了生物催化剂CALB@Ce-MOF-F,对该固定化酶的酶载量和催化性能进行了研究。在优化条件下,CALB的负载量为162.0mg/g载体,水解活性为899.1U/g蛋白。与游离CALB相比,CALB@Ce-MOF-F表现出对高温、酸碱和有机溶剂等有更强的耐受性;将Ce-MOF-F用于多种酶的固定化,研究其作为载体的普适性,结果表明,介孔Ce-MOF-F对洋葱伯克氏菌脂肪酶(BCL)和漆酶有良好的固定效果,可以作为良好载体,并能对酶起到较好的保护作用。     

Immobilization of Eversa® Transform via CLEA Technology Converts It in a Suitable Biocatalyst for Biolubricant Production Using Waste Cooking Oil

The performance of the previously optimized magnetic cross-linked enzyme aggregate of Eversa (Eversa-mCLEA) in the enzymatic synthesis of biolubricants by transesterification of waste cooking oil (WCO) with different alcohols has been evaluated. Eversa-mCLEA showed good activities using these alcohols, reaching a transesterification activity with isoamyl alcohol around 10-fold higher than with methanol. Yields of isoamyl fatty acid ester synthesis were similar using WCO or refined oil, confirming that this biocatalyst could be utilized to transform this residue into a valuable product. The effects of WCO/isoamyl alcohol molar ratio and enzyme load on the synthesis of biolubricant were also investigated. A maximum yield of around 90 wt.% was reached after 72 h of reaction using an enzyme load of 12 esterification units/g oil and a WCO/alcohol molar ratio of 1:6 in a solvent-free system. At the same conditions, the liquid Eversa yielded a maximum ester yield of only 34%. This study demonstrated the great changes in the enzyme properties that can be derived from a proper immobilization system. Moreover, it also shows the potential of WCO as a feedstock for the production of isoamyl fatty acid esters, which are potential candidates as biolubricants.     

Microwave-Assisted One-Pot Lipid Extraction and Glycolipid Production from Oleaginous Yeast Saitozyma podzolica in Sugar Alcohol-Based Media

Glycolipids are non-ionic surfactants occurring in numerous products of daily life. Due to their surface-activity, emulsifying properties, and foaming abilities, they can be applied in food, cosmetics, and pharmaceuticals. Enzymatic synthesis of glycolipids based on carbohydrates and free fatty acids or esters is often catalyzed using certain acyltransferases in reaction media of low water activity, e.g., organic solvents or notably Deep Eutectic Systems (DESs). Existing reports describing integrated processes for glycolipid production from renewables use many reaction steps, therefore this study aims at simplifying the procedure. By using microwave dielectric heating, DESs preparation was first accelerated considerably. A comparative study revealed a preparation time on average 16-fold faster than the conventional heating method in an incubator. Furthermore, lipids from robust oleaginous yeast biomass were successfully extracted up to 70% without using the pre-treatment method for cell disruption, limiting logically the energy input necessary for such process. Acidified DESs consisting of either xylitol or sorbitol and choline chloride mediated the one-pot process, allowing subsequent conversion of the lipids into mono-acylated palmitate, oleate, linoleate, and stearate sugar alcohol esters. Thus, we show strong evidence that addition of immobilized Candida antarctica Lipase B (Novozym 435^®), in acidified DES mixture, enables a simplified and fast glycolipid synthesis using directly oleaginous yeast biomass.     

Phenylmethanesulfonyl fluoride pretreatment stabilizes plasma lipidome in lipidomic and metabolomic analysis

Though it is standard practice to test the stability of analytes in the matrix for routine bioanalytical method, stability evaluation is always impractical and skipped in untargeted lipidomic and metabolomic analysis because analytes in these studies are enormous, diverse and sometimes unknown. Lipidome represents a major class of plasma metabolome and shows great potential to be diagnostic and prognostic biomarkers. However, lipidome also faces stability problems because plasma contains kinds of lipid degradation enzyme. Here, using liquid chromatography time of flight mass spectrometry based lipidomic methodology, plasma levels of various lipids including triglyceride (TG), diglyceride (DG), free fatty acid (FFA), phosphatidylethanolamine (PE) phosphatidylcholine (PC), lyso-phosphatidylcholine (LPC), lyso-phosphatidylethanolamine (LPE), and sphingomyelin (SM) were dynamically determined within 4 h at ambient temperature. In mouse and rat plasma, the levels of most TG, DG, PC and PE species significantly decreased with respect to time, whereas those of LPC, LPE and FFA significantly increased with respect to time. However, such changes did not occur in human plasma, thus indicating hepatic lipase and esterase might involve in the species-specified degradation of lipid classes in plasma. Phenylmethanesulfonyl fluoride (PMSF) pretreatment prevented such lipidome instability in mouse plasma. The results suggested the instability of plasma lipidome should be highly concerned, and the enhancement of ex vivo stability of plasma lipidome could enable more reliable clinical translation of lipidomic data for biomarker discovery.     

A microconductometric biosensor based on lipase extracted from Candida rugosa for direct and rapid detection of organophosphate pesticides

Organophosphate pesticides (OPs) have been intensively used as insecticides in agriculture; after entering the aquatic environment, they may affect a wide range of organisms. A conductometric enzymatic biosensor based on lipase extracted from Candida rugosa (CRL) has therefore been developed for the direct and rapid quantitative detection of organophosphate pesticides: diazinon, methyl parathion and methyl paraoxon in water. The biosensor signal and response time were obtained under optimum conditions, the enzyme being immobilised in the presence of gold nanoparticles. Under these conditions, the enzymatic biosensor was able to measure concentrations as low as 60 µg/L of diazinon, 26 µg/L of methyl parathion and 25 µg/L of methyl paraoxon very rapidly (response time: 3 min). Moreover, this CRL biosensor was not sensitive to interferences such as carbamates. It presented good storage stability for 21 days when kept at 4°C and it was successfully applied to real samples.     

Ring‐Closing and Cross‐Metathesis with Artificial Metalloenzymes Created by Covalent Active Site‐Directed Hybridization of a Lipase

A series of Grubbs‐type catalysts that contain lipase‐inhibiting phosphoester functionalities have been synthesized and reacted with the lipase cutinase, which leads to artificial metalloenzymes for olefin metathesis. The resulting hybrids comprise the organometallic fragment that is covalently bound to the active amino acid residue of the enzyme host in an orthogonal orientation. Differences in reactivity as well as accessibility of the active site by the functionalized inhibitor became evident through variation of the anchoring motif and substituents on the N‐heterocyclic carbene ligand. Such observations led to the design of a hybrid that is active in the ring‐closing metathesis and the cross‐metathesis of N,N‐diallyl‐p‐toluenesulfonamide and allylbenzene, respectively, the latter being the first example of its kind in the field of artificial metalloenzymes.     

Enantioselective synthesis of (1S,4R)-N-(benzylcarbamoyl)-4-aminocyclopent-2-en-1-ol by Candida antarctica lipase B

An efficient biocatalytic process has been developed to obtain optically pure (1S,4R)-N-(benzylcarbamoyl)- 4-aminocyclopent-2-en-1-ol which can be used as the key intermediate of ticagrelor. In this research, several N-(benzylcarbamoyl)-4-aminocyclopent-2-en-1-ol derivatives have been investigated in which Candida antarctica lipase B (CALB) was used to catalyze the asymmetric hydrolysis reaction. As expected, some of these substrates successfully gave (1S,4R)-N-(benzylcarbamoyl)-4-aminocyclopent- 2-en-1-ol in >98% enantiomeric excess (ee) with conversion yields up to 45%.     

Biocatalysis for C–S bond formation: Porcine pancreatic lipase (PPL) catalysed thiolysis/hydrothiolation reactions in sole water

In this paper, a biocatalytic route is described wherein PPL, lipase from porcine pancreas, in conjunction with water on reaction with different thiophenols and styrene oxides undergo thiolysis with C-S bond formation without the use of any metal catalysts, oxidants, bases, additives or organic solvents towards formation of β-hydroxysulfides in good to excellent yields with high regioselectivity at room temperature. Furthermore, PPL also facilitates thiophenols to undergo hydrothiolation with styrenes or phenylacetylenes in sole water and thus forming linear thioethers or vinylsulfides respectively via C–S bond formation. In addition to the straightforward and atom-efficient protocol, a gram-scale synthesis of β-hydroxysulfide and recyclability for three consecutive cycles without decrease in efficiency of PPL make our biocatalytic protocol for constructing C–S bond highly valuable from both environmental and economic viewpoints than traditional chemical practices.