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

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

Synthesis of Enantiopure (S)-Atenolol by Utilization of Lipase-Catalyzed Kinetic Resolution of a Key Intermediate.

(S)-Atenolol ((S)-2-(4-(2-Hydroxy-3-(isopropylamino)propoxy)phenyl)acetamide) has been synthesized in >99% enantiomeric excess (ee) with the use of Candida antarctica lipase B from Syncozymes (Shanghai, China), in a kinetic resolution of the corresponding racemic chlorohydrin. A catalytic amount of base was used in deprotonation of the phenol building block. The enantiopurity of the chlorohydrin building block remained unchanged upon subsequent amination to yield the final drug. All four steps in the synthesis protocol have been optimized compared to previously reported methods, which makes this new protocol more sustainable and in accordance with green chemistry principles. The overall yield of (S)-atenolol was 9.9%, which will be further optimized.

Activity and stability of lipase from Candida Antarctica after treatment in pressurized fluids.

Lipase B from Candida antarctica (CalB) is one of the biocatalysts most used in organic synthesis due to its ability to act in several medium, wide substrate specificity and enantioselectivity, tolerance to non-aqueous environment, and resistance to thermal deactivation. Thus, the objective of this work was to treat CalB in supercritical carbon dioxide (SC-CO2) and liquefied petroleum gas (LPG), and measure its activity before and after high-pressure treatment. Residual specific hydrolytic activities of 132% and 142% were observed when CalB was exposed to SC-CO2 at 35 â„ƒ, 75 bar and 1 h and to LPG at 65 â„ƒ, 30 bar and 1 h, respectively. Residual activity of the enzyme treated at high pressure was still above 100% until the 20th day of storage at low temperatures. There was no difference on the residual activity loss of CalB treated with LPG and stored at different temperatures over time. Greater difference was observed between CalB treated with CO2 and flash-frozen in liquid nitrogen (- 196 â„ƒ) followed by storage in freezer (- 10 â„ƒ) and CalB stored in freezer at - 10 â„ƒ. Such findings encourage deeper studies on CalB as well as other enzymes behavior under different types of pressurized fluids aiming at industrial application.

Amphiphilic Pentablock Copolymers Prepared from Pluronic and ε-Caprolactone by Enzymatic Ring Opening Polymerization.

Amphiphilic copolymers are appealing materials because of their interesting architecture and tunable properties. In view of their application in the biomedical field, the preparation of these materials should avoid the use of toxic compounds as catalysts. Therefore, enzymatic catalysis is a suitable alternative to common synthetic routes. Pentablock copolymers (CUC) were synthesized with high yields by ring-opening polymerization of ε-caprolactone (ε-CL) initiated by Pluronic (EPE) and catalyzed by Candida antarctica lipase B enzyme. The variables to study the structure-property relationship were EPEs' molecular weight and molar ratios between ε-CL monomer and EPE macro-initiator (M/In). The obtained copolymers were chemically characterized, the molecular weight determined, and morphologies evaluated. The results suggest an interaction between the reaction time and M/In variables. There was a correlation between the differential scanning calorimetry data with those of X-ray diffraction (WAXD). The length of the central block of CUC copolymers may have an important role in the crystal formation. WAXD analyses indicated that a micro-phase separation takes place in all the prepared copolymers. Preliminary cytotoxicity experiments on the extracts of the polymer confirmed that these materials are nontoxic.

Encapsulation of CALB by nucleotide/metal ions coordination nanoparticles: highly selective catalysis of esterification while poor performance in glycerolysis reaction.

BACKGROUND: Enzymatic esterification is attracting for particular high-acid oil deacidification. In this study, Candida antarctica lipase B (CALB) was encapsulated into a series of nucleotide-hybrid metal coordination polymers (CPs), which were constructed by guanosine 5'-monophosphate (GMP) and various metals. RESULTS: We here found that, most of the present CPs encapsulated CALB (CALB@CPs) samples were highly selective for esterification while poor in glycerolysis reaction. They exhibited quite poor performance in glycerolysis, with triacylglycerols (TAGs) conversion lower than 5%, despite this considerable enzymatic hydrolysis activities were observed. However, they (most of them) showed good performance in esterification of fatty acids and glycerol for TAG synthesis. In addition, the GMP/Tb (CPs constructed by GMP and Tb3+ ) encapsulated CALB (CALB@GMP/Tb) transformed over 98% of oleic acid into glycerides in the high-acid oil deacidification process, and TAG content from 87 to 89% was obtained. Moreover, the CALB@GMP/Tb showed good reusability in the esterification system. CONCLUSION: The present CALB@CPs samples are selective for esterification and suitable for high-acid oils deacidification. This work provides a new system for enzymatic selectivity improvement study. © 2021 Society of Chemical Industry.

Reviewing research on the synthesis of CALB-catalyzed sugar esters incorporating systematic mapping principles.

Rigorous evidence reviews must follow specific guidelines designed to improve transparency, reproducibility, and to minimize biases to which traditional reviews are susceptible. While evidence synthesis methods, such as systematic reviews and maps, have been used in several research fields, the majority of reviews published in the realm of chemical engineering are nonsystematic. In this study, we incorporated principles of systematic mapping to conduct a literature review covering research on the synthesis of sugar fatty acid esters (SFAE) with Candida antarctica lipase B (CALB). Our results showed that the simple monosaccharides were the most cited sugars among studies we conducted. The direct use of renewable raw materials and frequently available resources to produce alternative sugar esters (SE) was scarcely reported in our data set. We found that free fatty acids (FFA) were the most commonly cited acyl donors amongst all publications, with lauric, oleic, and palmitic acids accounting for ∼43% of the occurrences. Tertiary alcohols (ter-butyl alcohol (T-but) and 2-methyl-2-butanol (2M2B)) and ionic liquids were the most used solvents to synthesize SE. The co-occurence analysis of keywords involving solvent terms showed that most of the papers evaluated different solvents as reaction media (mostly in the form of a bisolvent system), also investigating the impact of their choice on sugar ester productivities. Given the potential of reviews informing us of research decisions, this article reveals trends and spaces across CALB-catalyzed SE synthesis research, in addition to introducing a new methodological perspective for developing reviews in the field of chemical engineering.

Noninvasive, label-free, and quantitative monitoring of lipase kinetics using terahertz emission technology.

Enzymes catalyze chemical transformations of great importance in many fields, and analysis of the rate of these transformations is equally important. The latter are typically monitored using surrogate substrates that produce quantifiable optical signals, owing to limitations associated with "label-free" techniques that could be used to monitor the transformation of original substrate molecules. In this study, terahertz (THz) emission technology is used as a noninvasive and label-free technique to monitor the kinetics of lipase-induced hydrolysis of several substrate molecules (including the complex substrate whole cow's milk) and horseradish peroxidase-catalyzed oxidation of o-phenylenediamine in the presence of H2 O2 . This technique was found to be quantitative, and kinetic parameters are compared to those obtained by proton NMR spectroscopy or UV/Vis spectroscopy. This study sets the stage for investigating THz emission technology as a tool for research and development involving enzymes, and for monitoring industrial processes in the food, cosmetic, detergent, pharmaceutical, and biodiesel sectors.

Application of freeze-dried Yarrowia lipolytica biomass in the synthesis of lipophilic antioxidants.

OBJECTIVE: The aim of the study was to evaluate the possibility of using Y. lipolytica biomass as a whole-cell catalyst in the synthesis of lipophilic antioxidants, with the example of esterification of five phenolic acids with 1-butanol. RESULTS: Freeze-dried Y. lipolytica biomass was successfully applied as a biocatalyst in the synthesis of esters of phenylpropanoic acid derivatives with 75-98% conversion. However, in the case of phenylacetic acid derivatives, results below 10% were obtained. The biological activity of phenolic acid esters was strongly associated with their chemical structures. Butyl 3-(4-hydroxyphenyl)propanoate showed an IC50 value of 19 mg/ml (95 mM) and TEAC value of 0.427. Among the compounds tested, butyl esters of 3-(4-hydroxyphenyl)propanoic and 4-hydroxyphenylacetic acids exhibited the highest antifungal activity. CONCLUSIONS: Lipophilization of phenolic acids achieved by enzymatic esterification creates prospects for using these compounds as food additives with antioxidant properties in lipid-rich food matrices.

Microbial lipases and their industrial applications: a comprehensive review.

Lipases are very versatile enzymes, and produced the attention of the several industrial processes. Lipase can be achieved from several sources, animal, vegetable, and microbiological. The uses of microbial lipase market is estimated to be USD 425.0 Million in 2018 and it is projected to reach USD 590.2 Million by 2023, growing at a CAGR of 6.8% from 2018. Microbial lipases (EC 3.1.1.3) catalyze the hydrolysis of long chain triglycerides. The microbial origins of lipase enzymes are logically dynamic and proficient also have an extensive range of industrial uses with the manufacturing of altered molecules. The unique lipase (triacylglycerol acyl hydrolase) enzymes catalyzed the hydrolysis, esterification and alcoholysis reactions. Immobilization has made the use of microbial lipases accomplish its best performance and hence suitable for several reactions and need to enhance aroma to the immobilization processes. Immobilized enzymes depend on the immobilization technique and the carrier type. The choice of the carrier concerns usually the biocompatibility, chemical and thermal stability, and insolubility under reaction conditions, capability of easy rejuvenation and reusability, as well as cost proficiency. Bacillus spp., Achromobacter spp., Alcaligenes spp., Arthrobacter spp., Pseudomonos spp., of bacteria and Penicillium spp., Fusarium spp., Aspergillus spp., of fungi are screened large scale for lipase production. Lipases as multipurpose biological catalyst has given a favorable vision in meeting the needs for several industries such as biodiesel, foods and drinks, leather, textile, detergents, pharmaceuticals and medicals. This review represents a discussion on microbial sources of lipases, immobilization methods increased productivity at market profitability and reduce logistical liability on the environment and user.

PEGylation of Fluorescein by Enzyme-Catalyzed "Click" Michael Addition.

This paper reports the first "Click" Michael addition catalyzed by Candida antarctica lipase B (CALB) between fluorescein o-acrylate and thiol-functionalized poly(ethylene glycol)s (HS-PEG-SH, Mn = 1200 g mol-1 , D = 1.14, and Mn = 2200 g mol-1 , D = 1.09). The progress of the reactions is monitored with 1 H-NMR spectroscopy. In the absence of CALB, the reaction does not go to completion even after 18 h but completes in less than 2 min when CALB is added. Similarly, the reaction with HS-PEG-SH having Mn = 2200 g mol-1 and D = 1.09 completes in less than 2 min by CALB catalysis. The structures of the products are also confirmed by 13 C-NMR. This enzyme-catalyzed "Click" Michael addition is found to be a powerful tool to synthesize fluorescein-based polymeric conjugates for a wide variety of applications.

Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators.

Controlling gene expression is often the foremost goal of most biological endeavors like the production of industrial enzymes and expression of heterologous metabolic pathway genes. The components of the entire "expression cassette" exert control on net protein output. This control is primarily achieved through altering the promoter driving expression and by changing the copy number of the gene. However, there are only a few recent studies on terminators. Terminators are essential components in expression cassettes that influence the 3' end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. In Pichia pastoris (Komagataella phaffi), little attention has been paid to the selection of terminator regions in efforts to increase heterologous gene expression. To explore the potential application of the terminator regions on increased secretory production of Candida antarctica lipase B (CALB), we assessed the ability of three different classes of terminator regions: (1) terminator regions of methanol oxidation pathway genes of P. pastoris; (2) terminator regions of well-expressed and housekeeping genes of P. pastoris; and (3) terminators of other yeast genes like Saccharomyces cerevisiae. The terminator of dihydroxyacetone synthase (DHAS TT), a high expressing gene in the methanol utilization pathway, shows inducible CALB expression levels similar to the AOX1 terminator (AOX1 TT) under the control of AOX1 promoter and threefold higher in constitutive expression of CALB under the control of GAP promoter. The Calb transcript abundance was also found to correlate with protein expression. Furthermore, mRNA half-life determination showed a direct correlation between the stability of transcripts and increased transcription rate. Together, our results emphasize that enhancing transcript stability using the correct choice of transcription terminators (TT) will help in developing robust production strains suitable for scale-up.Key points• Influence of transcription terminators on Calb gene expression• Modulation of gene expression by enhancing transcript stability.

The Immobilization of Candida antarctica lipase B by ZIF-8 encapsulation and macroporous resin adsorption: preparation and characterizations.

A novel enzyme immobilization method employing metal-organic framework (MOF) encapsulation and macroporous resin adsorption was developed in this study. Candida antarctica lipase B (CALB) was firstly encapsulated onto metal-organic frame structures (Zeolitic imidazole framework-8, ZIF-8) and further bonded to macroporous resin by physical adsorption. Under optimized immobilization conditions, the activity of the prepared immobilized lipase (CALB-ZIF-8@D101) determined via the methyl esterification of oleic acid was 38.4 U/mg. Compared with free lipase, the immobilized lipase exhibited improved thermal and operational stability and organic solvent tolerance. These results demonstrate that the immobilization method of ZIF-8 encapsulation and macroporous resin adsorption enhanced enzyme properties at a superior level.

Screening of solvents for favoring hydrolytic activity of Candida antarctica Lipase B.

Lipases are a group of enzymes of considerable significance in organic synthesis, among which Candida antarctica lipase B (CALB) is one of the most widely studied enzymes. The activity of the biocatalyst has been intensively characterized in many organic media, but this paper aimed to compare the effect of 20 different solvents on the activity of CALB in the hydrolysis of p-nitrophenyl laurate. Nonpolar, polar aprotic, and polar protic solvents were used for enzyme pretreatment and then entered the composition of mixed solvents reaction medium. An impact of solvents on solvation processes affecting the catalysis steps, protein denaturation, and changes of its conformation was discussed. Moreover the hydrolytic activity of CALB with partition coefficient (logP) of the solvent used was correlated. It was emphasized that the substrate solubility plays an important role in solvent selection. In the presence of hydrophobic solvents, hydration layer becomes more hydrophobic facilitating the substrate access to the enzyme surface. In turn, polar compounds are good solvents for organic substrates facilitating the penetration of the aqueous layer that surrounds the surface of the enzyme. Two variants proved to be favorable for ester hydrolysis reaction: isooctane or polar solvent such as acetone, tert -butyl methyl ether, tert-butanol or acetonitrile.

Enzymatic Synthesis of Glucose Monodecanoate in a Hydrophobic Deep Eutectic Solvent.

Environmentally friendly and biodegradable reaction media are an important part of a sustainable glycolipid production in the transition to green chemistry. Deep eutectic solvents (DESs) are an ecofriendly alternative to organic solvents. So far, only hydrophilic DESs were considered for enzymatic glycolipid synthesis. In this study, a hydrophobic DES consisting of (-)-menthol and decanoic acid is presented for the first time as an alternative to hydrophilic DES. The yields in the newly introduced hydrophobic DES are significantly higher than in hydrophilic DESs. Different reaction parameters were investigated to optimize the synthesis further. Twenty milligrams per milliliter iCalB and 0.5 M glucose resulted in the highest initial reaction velocity for the esterification reaction, while the highest initial reaction velocity was achieved with 1.5 M glucose in the transesterification reaction. The enzyme was proven to be reusable for at least five cycles without significant loss of activity.

Rational enhancement of enzyme-catalyzed enantioselective reaction by construction of recombinant enzymes based on additive strategy.

A rational enhancement of kinetic resolution process for producing (S)-N-(2-ethyl-6-methylphenyl) alanine from racemic methyl ester using lipase B from Candida antarctica (CalB) was investigated. With the benefit results that lipase CalB-catalyzed reactions can be effectively regulated using amino acids (such as histidine and lysine) as additives, CalBs modified (mCalBs) by n-histidines at the N terminal and n-lysines at the C terminal were constructed and expressed. The results show that both soluble and precipitated mCalBs can effectively catalyze the hydrolysis reaction without adding any extra additives. The enantioselective ratio (E value) of soluble and precipitated mCalBs could be improved from 12.1 to 20.3, which were higher than that (E value was only 10.2) of commercial Novozym 435 (immobilized CalB). The study indicated that the amino acid-rich molecules introduced on lipase CalB can produce positive effects on enantioselectivity of enzyme. It provides unusual ideas for reasonable regulation of enzyme-catalyzed reactions.

Parametric optimization of polycaprolactone synthesis catalysed by Candida antarctica lipase B using response surface methodology.

A statistical approach with D-optimal design was used to optimize the process parameters for polycaprolactone (PCL) synthesis. The variables selected were temperature (50°C-110°C), time (1-7 h), mixing speed (50-500 rpm) and monomer/solvent ratio (1:1-1:6). Molecular weight was chosen as response and was determined using matrix-assisted laser desorption/ionization time of flight (MALDI TOF). Using the D-optimal method in design of experiments, the interactions between parameters and responses were analysed and validated. The results show a good agreement with a minimum error between the actual and predicted values.

Efficient access to both enantiomers of 3-(1-hydroxyethyl)phenol by regioselective and enantioselective CAL-B-catalyzed hydrolysis of diacetate in organic media by sodium carbonate.

In the present paper, we describe several pathways employing immobilized lipase from Candida antarctica B (CAL-B) as biocatalyst to prepare easily both enantiomers of 3-(1-hydroxyethyl)phenol. We have applied hydrolysis with Na2 CO3 in organic media under mild conditions. The reaction parameters solvent effect, amount of lipase, and Na2 CO3 were examined with 3-(1-acetoxyethyl)phenyl acetate as substrate. In alkaline hydrolysis, (R)-3-(1-hydroxyethyl)phenol was obtained with ee = 99% and (S)-(-)-3-(1-acetoxyethyl)phenol with ee = 98% at optimal conversion (c = 50%) and high selectivity (E > 200). Two other deacylation reactions were compared: alcoholysis with MeOH and with NEt3 . The acylation of 3-(1-hydroxyethyl)phenol with vinyl acetate was also examined. Alkaline hydrolysis gave the best results, while good regioselectivity and enantioselectivity were observed in alcoholysis and acylation reactions. Finally, (S)- and (R)-3-(1-hydroxyethyl)phenol (ee > 98%), key intermediates for the synthesis of important drugs, were prepared from the corresponding racemic diacetate through alkaline hydrolysis.

Enzymatic Synthesis of Amino Acids Endcapped Polycaprolactone: A Green Route Towards Functional Polyesters.

ε-caprolactone (CL) has been enzymatically polymerized using α-amino acids based on sulfur (methionine and cysteine) as (co-)initiators and immobilized lipase B of Candida antarctica (CALB) as biocatalyst. In-depth characterizations allowed determining the corresponding involved mechanisms and the polymers thermal properties. Two synthetic strategies were tested, a first one with direct polymerization of CL with the native amino acids and a second one involving the use of an amino acid with protected functional groups. The first route showed that mainly polycaprolactone (PCL) homopolymer could be obtained and highlighted the lack of reactivity of the unmodified amino acids due to poor solubility and affinity with the lipase active site. The second strategy based on protected cysteine showed higher monomer conversion, with the amino acids acting as (co-)initiators, but their insertion along the PCL chains remained limited to chain endcapping. These results thus showed the possibility to synthesize enzymatically polycaprolactone-based chains bearing amino acids units. Such cysteine endcapped PCL materials could then find application in the biomedical field. Indeed, subsequent functionalization of these polyesters with drugs or bioactive molecules can be obtained, by derivatization of the amino acids, after removal of the protecting group.

Effect of supercritical carbon dioxide on the enzymatic production of biodiesel from waste animal fat using immobilized Candida antarctica lipase B variant.

BACKGROUND: Waste animal fat is a promising feedstock to replace vegetable oil that widely used in commercial biodiesel process, however the high content of free fatty acid in waste fat makes it unfeasible to be processed with commercial base-catalytic process. Enzymatic process is preferable to convert waste fat into biodiesel since enzyme can catalyze both esterification of free fatty acid and transesterification of triglyceride. However, enzymatic reaction still has some drawbacks such as lower reaction rates than base-catalyzed transesterification and the limitation of reactant concentration due to the enzyme inhibition of methanol. Supercritical CO2 is a promising reaction media for enzyme-catalyzed transesterification to overcome those drawbacks. RESULT: The transesterification of waste animal fat was carried out in supercritical CO2 with varied concentration of feedstock and methanol in CO2. The CO2 to feedstock mass ratio of 10:1 showed the highest yield compared to other ratios, and the highest FAME yield obtained from waste animal fat was 78%. The methanol concentration effect was also observed with variation 12%, 14%, and 16% of methanol to feedstock ratio. The best yield was 87% obtained at the CO2 to feedstock ratio of 10: 1 and at the methanol to feedstock ratio of 14% after 6 h of reaction. CONCLUSION: Enzymatic transesterification to produce biodiesel from waste animal fat in supercritical fluid media is a potential method for commercialization since it could enhance enzyme activity due to supercritical fluid properties to remove mass transfer limitation. The high yield of FAME when using high mass ratio of CO2 to oil showed that supercritical CO2 could increase the reaction and mass transfer rate while reducing methanol toxicity to enzyme activity. The increase of methanol concentration also increased the FAME yield because it might shift the reaction equilibrium to FAME production. This finding describes that the application of supercritical CO2 in the enzymatic reaction enables the application of simple process such as a packed-bed reactor.

No Strain, No Gain? Enzymatic Ring-Opening Polymerization of Strainless Aliphatic Macrolactones.

Starting from readily available oleic and erucic acid, macrocyclic nonadecalactone (C19 ) and tricosalactone (C23 ) can be synthesized in polymerization grade purity in a four-step reaction sequence. Ring-opening polymerization (ROP) of these strainless macrolactones can be performed utilizing an enzyme as a catalyst. Despite the missing ring-strain as key driving force for smaller (strained) lactones, high molar masses (Mn ≈ 105 g mol-1 ) can be accessed in an entropically driven ROP. Polyester-19 and polyester-23 prepared feature melting temperatures well above 100 °C. Further analysis of the mechanical properties of these materials displays the resemblance to polyethylene. For example, Young's moduli on the order of 600 MPa are observed as a result of the high crystallinity of the polymer.