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1
Zanco, Jasper J., Caroline Rodrigues Cardoso Rodrigues Cardoso, and Luíza Niehues. "Chromatographic’s image analysis in phytohomeopathies." International Journal of High Dilution Research - ISSN 1982-6206 24, no. 1 (2024): 25–26. https://doi.org/10.51910/ijhdr.v24i1.1491.
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Plants signal when establishing interaction with homeopathies in different succussions, however, the type of signaling is not always understandable. Recognizing symptomatic patterns in plants begins by understanding the behavior of homeopathy itself, through varied methods, widely disseminated in scientific literature¹. The present study proposes a new thin-layer chromatography (TLC) analysis method to identify patterns in different phytohomeopathic applications. CCDs were made on Sulfur 9Ch, Nux and Bell 11CH; Calc Ph 29CH; Bry and Cal Carb 30CH; Hyp 50CH; Mag Ph 200CH and Graph Nat 200CH. The analysis of the chromatographic stains was developed with ultraviolet light and the images were analyzed using the Fast Fourier Transform (FFT) method². The statistical design, ANOVA, consisted of 5 completely randomized, double-blind replications for each of the preparations. The results showed significant differences after a few minutes of chromatography, highlighting the order of signals between homeopathies: Bell < Sulph < Mag Ph < Graph Nat < Hyp < Bry < Calc Ph < Cal Carb < Nux. From these results, it is possible to identify phytohomeopathic’ signs and symptoms during the course of the treatment.
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Hati, AK Paital B* Sahoo AR Shankar U. "A CASE STUDY FOR SUCCESSFUL TREATMENT OF VITILIGO WITH A CONSTITUTIONAL HOMOEOPATHIC FORMULATION CALCAREA CARBONICA." INDO AMERICAN JOURNAL OF PHARMACEUTICAL SCIENCES 05, no. 01 (2018): 299–303. https://doi.org/10.5281/zenodo.1149335.
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Background: Vitiligo is alternatively known as leukoderma which is an autoimmune skin condition that arrives due to expression of low melanin pigment. Vitiligo is although is not very uncommonly seen, difficulties exist for its successful treatment. Objective: Treatment of Vitiligo with homeopathic medication. Case Reports: A female 7 years old child with Vitiligo around her both the eyes was considered as the subject for the present study. She was treated with some of homoeopathic medicines such as Arsenicum sulfuratum flavum-6X, Hydrocotyle-Ø and Psoralea cor- Ø were prescribed as empirical formulations; albeit a successful result was not achieved. Based on her the totality of symptom, a constitutional medicine Calc. carb- 0/1 to 0/8, was able to fully cure the Vitiligo spots within 4 months. Conclusions: In a 7 years girl with Vitiligo treated with individualized homeopathy, the cure was achieved. It is believed that homeopathy may be effective in treatment of Vitiligo with careful selection of medicine(s) as per the totality of the symptoms of the patient. Key words: Vitiligo, Homoeopathy, Calc. carb, Constitutional medicine
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Oliveira, Leyza Paloschi de, Simone Silmara Werner, Mari Inês Carissimi Boff, and Pedro Boff. "Homeopathy in the Rust Severity and Growth of Malva sylvestris L." Journal of Agricultural Science 13, no. 5 (2021): 69. http://dx.doi.org/10.5539/jas.v13n5p69.
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The production of medicinal plants which have an association with biotrophic fungi requires non-residual and favorable methods to the host with tolerance to the presence of phytopathogens. The objective of this work was to evaluate the effect of homeopathic preparations on the rust severity and the growth of Malva sylvestris plants. M. sylvestris seedlings were prepared in 600 ml containers with commercial substrate. The seedlings were arranged in pots at 26 days of age and outlined in two experiments. The treatments consisted of Amonnium carbonicum (Am. carb.), Atropa belladonna (Bell.), Calcarea carbonica (Calc. carb.), Silicea terra (Sil.) and Sulfur (Sulf.), all at 30CH (centesimal Hahnemannian dilution order). The last two dynamizations (29 and 30CH) were prepared in distilled water for all treatments. Control plants were treated with water. Natural inoculation of the plants with Puccinia malvacearum occurred in the first experiment, and the applications of homeopathic preparations were carried out every seven days for five weeks. Four evaluations of rust severity, diameter, height and number of leaves were conducted. Next, M. sylvestris seedlings were transplanted into pots with 5 liters of substrate in the second experiment and the growth curve of the plant was observed in relation to the diameter and height variables. Am. Carb. reduced 18.29% of the rust severity in relation to the control plants. Sil. 30CH contributed to an increase in stem diameter. There was no interference in the plants&rsquo; height by homeopathic preparations. The application of homeopathies in M. sylvestris can contribute to their production, reducing the rust intensity considered in the crop cycle and can assist in the plant growth without leaving residues which can harm pollinators and hyperparasites.
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Grijseels, Sietske, Carsten Pohl, Jens Christian Nielsen, et al. "Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin." Fungal Biology and Biotechnology 5, no. 1 (2018): 18. https://doi.org/10.1186/s40694-018-0063-4.
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<strong>Background: </strong>Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, <i>Aspergillus versicolor</i>-related species, and Penicillia. <i>Penicillium decumbens</i> produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol. <strong>Results: </strong>A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, <i>P. decumbens, A. aculeatus</i> and <i>A. versicolor</i>, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (<i>calA</i> to <i>calM</i>). Implementation of the CRISPR/Cas9 technology in <i>P. decumbens</i> allowed the targeted deletion of genes encoding a polyketide synthase (<i>calA</i>), a major facilitator pump (<i>calB</i>) and a binuclear zinc cluster transcription factor (<i>calC</i>). Detailed metabolic profiling, using UHPLC-MS, of the ∆<i>calA</i> (PKS) and ∆<i>calC</i> (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆<i>calB</i> (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B. <strong>Conclusion: </strong>Here we report the discovery of a gene cluster (<i>calA</i>-<i>M</i>) involved in the biosynthesis of the polyketide calbistrin in <i>P. decumbens</i>. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the <i>cal</i>-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.
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Gurvich, V. "To the question of replacing bismuth salts with white clay." Kazan medical journal 29, no. 7 (2022): 582–83. http://dx.doi.org/10.17816/kazmj89688.
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With the mention of the good action of Bolus alba, we meet with prof. Mayulov, who recommends them in large doses for intoxication (1 teaspoon of Bolus alba per 100.0 mucous broth), as well as for muco-bloody diarrhea in prolonged form, in the form of powders along with Bismuth, subnitric. tannalbin. Also recommends Bolus alba and prof. Birk for prolonged colitis. I began to use Bolus alba in practice 2 years ago on the advice of Professor Leonov (Minsk Children's Clinic). The last few months I have been working in a counseling and a mountain nursery. Orsha, made special observations on the effect of white clay so that it could be recommended to all pediatricians. Although the results of the previous 2-year observations were good enough, they need to be illustrated with case histories so that it would be convincing for others. As you can see from the excerpts from the medical history, I used Calc. carb. + Bolus alba aa 0.25, and recently Bolus alba 0.5 and 0.3.
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D’souza, Rosario Pascoal, and Sandesh Anandrao Kachare. "Scope of Homoeopathy in Peritonsillar Abscess." Advancements in Homeopathic Research 7, no. 3 (2022): 33–38. http://dx.doi.org/10.48165/ahr.2022.7.3.3.
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A Peritonsillar abscess is also known as Quinsy. It is an accumulation of pus behind the palatine tonsil due to an infection. Precisely, it is a collection of pus between the fibrous capsule of the tonsil, usually at the upper pole & the superior constrictor muscles of pharynx. The signs & symptoms of quinsy include fever, throat pain, trouble in opening mouth, redness, swelling and change in the voice. Pain is usually worse on one side. The complications may include blockage of the airway or aspiration pneumonitis. The conventional treatment is done for removing the pus by incision & drainage along with medications for swelling & pain management. The homeopathic line of treatment depends on the actual state of the condition. Homeopathic medicines can be used in mild to moderate cases of peritonsillar abscess successfully as see through this research study. In this study, 46 cases of peritonsillar abscess of age group between 5 to 80 years were included. After going through the results, it is observed that the peritonsillar abscess can be treated successfully by Homoeopathy. The scope of Homoeopathy in the treatment of peritonsillar abscess is excellent and shows high success rate of 44 out of 46 patients responding successfully to the Homoeopathic treatment with no signs of recurrence in near future. The outstanding Homoeopathic remedies in the treatment of peritonsillar abscess were found to be mostly Hepar Sulph & Merc Sol along with Calc Carb, Baryta Carb and Phytolacca. All that is required in the treatment of peritonsillar abscess is quick selection of right remedy and right dose for the instant relief rationally. Hence we can rightly claim that by proper Homoeopathic treatment we can make the world better for the future generation. This research paper will certainly help the health professionals to overcome such problems of surgical cases and consider the benefits of the facts in scope of Homoeopathy in peritonsillar abscess.
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Monteiro, Rodolpho R. C., Davino M. Andrade Neto, Pierre B. A. Fechine, et al. "Ethyl Butyrate Synthesis Catalyzed by Lipases A and B from Candida antarctica Immobilized onto Magnetic Nanoparticles. Improvement of Biocatalysts’ Performance under Ultrasonic Irradiation." International Journal of Molecular Sciences 20, no. 22 (2019): 5807. http://dx.doi.org/10.3390/ijms20225807.
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The synthesis of ethyl butyrate catalyzed by lipases A (CALA) or B (CALB) from Candida antarctica immobilized onto magnetic nanoparticles (MNP), CALA-MNP and CALB-MNP, respectively, is hereby reported. MNPs were prepared by co-precipitation, functionalized with 3-aminopropyltriethoxysilane, activated with glutaraldehyde, and then used as support to immobilize either CALA or CALB (immobilization yield: 100 ± 1.2% and 57.6 ± 3.8%; biocatalysts activities: 198.3 ± 2.7 Up-NPB/g and 52.9 ± 1.7 Up-NPB/g for CALA-MNP and CALB-MNP, respectively). X-ray diffraction and Raman spectroscopy analysis indicated the production of a magnetic nanomaterial with a diameter of 13.0 nm, whereas Fourier-transform infrared spectroscopy indicated functionalization, activation and enzyme immobilization. To determine the optimum conditions for the synthesis, a four-variable Central Composite Design (CCD) (biocatalyst content, molar ratio, temperature and time) was performed. Under optimized conditions (1:1, 45 °C and 6 h), it was possible to achieve 99.2 ± 0.3% of conversion for CALA-MNP (10 mg) and 97.5 ± 0.8% for CALB-MNP (12.5 mg), which retained approximately 80% of their activity after 10 consecutive cycles of esterification. Under ultrasonic irradiation, similar conversions were achieved but at 4 h of incubation, demonstrating the efficiency of ultrasound technology in the enzymatic synthesis of esters.
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Arana-Peña, Sara, Diego Carballares, Vicente Cortés Corberan, and Roberto Fernandez-Lafuente. "Multi-Combilipases: Co-Immobilizing Lipases with Very Different Stabilities Combining Immobilization via Interfacial Activation and Ion Exchange. The Reuse of the Most Stable Co-Immobilized Enzymes after Inactivation of the Least Stable Ones." Catalysts 10, no. 10 (2020): 1207. http://dx.doi.org/10.3390/catal10101207.
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The lipases A and B from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) or Rhizomucor miehei (RML), and the commercial and artificial phospholipase Lecitase ultra (LEU) may be co-immobilized on octyl agarose beads. However, LEU and RML became almost fully inactivated under conditions where CALA, CALB and TLL retained full activity. This means that, to have a five components co-immobilized combi-lipase, we should discard 3 fully active and immobilized enzymes when the other two enzymes are inactivated. To solve this situation, CALA, CALB and TLL have been co-immobilized on octyl-vinyl sulfone agarose beads, coated with polyethylenimine (PEI) and the least stable enzymes, RML and LEU have been co-immobilized over these immobilized enzymes. The coating with PEI is even favorable for the activity of the immobilized enzymes. It was checked that RML and LEU could be released from the enzyme-PEI coated biocatalyst, although this also produced some release of the PEI. That way, a protocol was developed to co-immobilize the five enzymes, in a way that the most stable could be reused after the inactivation of the least stable ones. After RML and LEU inactivation, the combi-biocatalysts were incubated in 0.5 M of ammonium sulfate to release the inactivated enzymes, incubated again with PEI and a new RML and LEU batch could be immobilized, maintaining the activity of the three most stable enzymes for at least five cycles of incubation at pH 7.0 and 60 °C for 3 h, incubation on ammonium sulfate, incubation in PEI and co-immobilization of new enzymes. The effect of the order of co-immobilization of the different enzymes on the co-immobilized biocatalyst activity was also investigated using different substrates, finding that when the most active enzyme versus one substrate was immobilized first (nearer to the surface of the particle), the activity was higher than when this enzyme was co-immobilized last (nearer to the particle core).
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Kaur, Sandeep, Ajay Kumar, Sharad Thakur, et al. "Antioxidant, Antiproliferative and Apoptosis-Inducing Efficacy of Fractions from Cassia fistula L. Leaves." Antioxidants 9, no. 2 (2020): 173. http://dx.doi.org/10.3390/antiox9020173.
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Cassia fistula L. is a highly admirable traditional medicinal plant used for the treatment of various diseases and disorders. The present study was performed to divulge the antioxidant, antiproliferative, and apoptosis-inducing efficacy of fractions from C. fistula leaves. The hexane (CaLH fraction), chloroform (CaLC fraction), ethyl acetate (CaLE fraction), n-butanol (CaLB fraction), and aqueous (CaLA fraction) were sequentially fractionated from 80% methanolic (CaLM extract) of C. fistula leaves. The CaLE fraction was fractionated using column chromatography to yield a pure compound, which was characterized as Epiafzelechin (CFL1) based on 1H, 13C, and DEPT135 NMR. Among these fractions, CaLE and isolated CFL1 fractions exhibited an effective antioxidant potential in Ferric ion reducing power, (2,2’-azino-bis (3-ethylbenzothiazoline -6-sulfonic acid)) cation radical scavenging, and nitric oxide radical scavenging assays. Epiafzelechin was investigated for its antiproliferative effects against MG-63 (osteosarcoma), IMR-32 (neuroblastoma), and PC-3 (prostate adenocarcinoma), and was found to inhibit cell proliferation with a GI50 value of 8.73, 9.15, and 11.8 μM respectively. MG-63 cells underwent apoptotic cell death on treatment with Epiafzelechin as the cells showed the formation of apoptotic bodies, enhanced reactive oxygen species (ROS) generation, mitochondrial membrane depolarization along with an increase in early apoptotic cell population analyzed using Annexin V-FITC/PI double staining assay. Cells showed cell cycle arrest at the G0/G1 phase accompanied by a downregulation in the expression levels of p-Akt (Protein kinase B), p-GSK-3β (Glycogen synthase kinase-3 beta), and Bcl-xl (B-cell lymphoma-extra large) proteins. RT-PCR (Real time-polymerase chain reaction) analysis revealed downregulation in the gene expression level of β-catenin and CDK2 (cyclin-dependent kinases-2) while it upregulated the expression level of caspase-8 and p53 genes in MG-63 cells.
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Arslan, Amine, Anders Rancke-Madsen, and Jesper Brask. "Enzymatic Synthesis of Estolides from Castor Oil." Catalysts 10, no. 8 (2020): 835. http://dx.doi.org/10.3390/catal10080835.
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Estolides are fatty acid polyesters with applications in both industry and consumer products. Recently, reports have emerged detailing lipase-catalyzed synthesis of estolides from free hydroxy fatty acids. In this paper, we describe a simple alternative enzymatic process, in which castor oil is directly converted to an estolide mixture by Candida antarctica lipase A (CALA) catalyzed transesterification. The reaction mixture is analyzed by NMR to determine the estolide number (EN) and MALDI MS to identify individual components, in addition to titration to determine the acid value (AV). Estolide trimers and tetramers (EN 2–3) were formed over 24 h in a system with 2:1 (v/v) castor oil–water. Further, utilizing different lipase specificities, addition of Thermomyces lanuginosus lipase (TLL), allowed the CALA product mixture to be cleaned up by hydrolyzing attached glycerol. In addition, a three-enzyme process is suggested, in which a simple alcohol is added and Candida antarctica lipase B (CALB) is used to esterify carboxylic acids to lower AV.
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Liu, Peng, Xinlong Liu, Na An, and Peng Wang. "Synthesis of Mesoporous Silica Nanowires and Their Application in Enzyme Immobilization." E3S Web of Conferences 245 (2021): 03006. http://dx.doi.org/10.1051/e3sconf/202124503006.
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Hydrophobic mesoporous silica nanowires were synthesis and then employed as support for immobilization of lipase from Candida antarctica via covalent bonding (CALB@MSW). The parameters were optimized and the optimum conditions were as follows: GA concentration 5.5 wt.%, activation time 60 min and CALB concentration 4 mg/mL. Under these conditions, the protein loading and specific activity of CALB@MSW were 138.3 mg/gsupport and 41.1 U/mgsupport, respectively. Compared with free CALB, CALB@MSW showed better thermal stability and pH stability. The maximum yield of biodiesel catalytic by CALB@MSW was 93.4 %. After reused 8 times, CALB@MSW still remained 95.75 % initial activity showing better stability than free CALB.
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Senthilkumar, B., D. Meshachpaul, and R. Rajasekaran. "Geometric Simulation Approach for Grading and Assessing the Thermostability of CALBs." Biochemistry Research International 2016 (2016): 1–7. http://dx.doi.org/10.1155/2016/4101059.
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Candida antarcticalipase B (CALB) is a known stable and highly active enzyme used widely in biodiesel synthesis. In this work, the stability of native (4K6G) and mutant (4K5Q) CALB was studied through various structural parameters using conformational sampling approach. The contours of polar surface area and surface area of mutant CALB were 11357.67 Å2and 30007.4 Å2, respectively, showing an enhanced stability compared to native CALB with a statistically significantPvalue of < 0.0001. Moreover, simulated thermal denaturation of CALB, a process involving dilution of hydrogen bond, significantly shielded against different intervals of energy application in mutant CALB revealing its augmentation of structural rigidity against native CALB. Finally, computational docking analysis showed an increase in the binding affinity of CALB and its substrate (triglyceride) in mutant CALB with Atomic Contact Energy (ACE) of −91.23 kcal/mol compared to native CALB (ACE of −70.3 kcal/mol). The computational observations proposed that the use of mutant CALB (4K5Q) could serve as a best template for production of biodiesel in the future. Additionally, it can also be used as a template to identify efficient thermostable lipases through further mutations.
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Jang, Woo Young, Jung Hoon Sohn, and Jeong Ho Chang. "Thermally Stable and Reusable Silica and Nano-Fructosome Encapsulated CalB Enzyme Particles for Rapid Enzymatic Hydrolysis and Acylation." International Journal of Molecular Sciences 24, no. 12 (2023): 9838. http://dx.doi.org/10.3390/ijms24129838.
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This study reports the preparation of silica-coated and nano-fructosome encapsulated Candida antarctica lipase B particles (CalB@NF@SiO2) and a demonstration of their enzymatic hydrolysis and acylation. CalB@NF@SiO2 particles were prepared as a function of TEOS concentration (3–100 mM). Their mean particle size was 185 nm by TEM. Enzymatic hydrolysis was performed to compare catalytic efficiencies of CalB@NF and CalB@NF@SiO2. The catalytic constants (Km, Vmax, and Kcat) of CalB@NF and CalB@NF@SiO2 were calculated using the Michaelis–Menten equation and Lineweaver–Burk plot. Optimal stability of CalB@NF@SiO2 was found at pH 8 and a temperature of 35 °C. Moreover, CalB@NF@SiO2 particles were reused for seven cycles to evaluate their reusability. In addition, enzymatic synthesis of benzyl benzoate was demonstrated via an acylation reaction with benzoic anhydride. The efficiency of CalB@NF@SiO2 for converting benzoic anhydride to benzyl benzoate by the acylation reaction was 97%, indicating that benzoic anhydride was almost completely converted to benzyl benzoate. Consequently, CalB@NF@SiO2 particles are better than CalB@NF particles for enzymatic synthesis. In addition, they are reusable with high stability at optimal pH and temperature.
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Lu, Xinkun, Bin Chen, Xiaowei Shen, Ziheng Cui, and Biqiang Chen. "High-Level Expression and Engineering of Candida antarctica Lipase B in a Non-Methanol-Induced Pichia pastoris System." Catalysts 15, no. 1 (2024): 27. https://doi.org/10.3390/catal15010027.
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The efficient expression and excellent thermal stability of Candida antarctica lipase B (CALB) are crucial for its industrial production. In this study, through genetic engineering and rational design, while preserving the superior catalytic properties of CALB, we optimized the induction pathway using glycerol as the sole carbon source; moreover, the thermal stability sites of CALB were predicted and optimized. The results revealed that the level of CALB expression in this expression system reached 2.27 g/L under the condition of a 5 L fermenter. The Tm value of the CALB-Q231F increased by 10 °C. Moreover, after thermal inactivation at 80 °C for 1 h, the retention rate of esterification enzymatic activity over 24 h was 2.99 times that of wild-type (WT) CALB, whereas the retention rate of hydrolytic enzymatic activity was 2.23 times that of WT CALB. In this study, a non-methanol-induced Pichia pastoris expression system was successfully designed and constructed; a non-methanol-induced CALB-producing strain, X33-pGAPZ(Mα) A-CalB-Q231F, with high thermal stability and a high expression level was obtained, which can be used for the development of industrial enzymes.
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Zhang, Xia, Liting Wan, Lin Li, et al. "Effects of magnetic fields on the enzymatic synthesis of naringin palmitate." RSC Advances 8, no. 24 (2018): 13364–69. http://dx.doi.org/10.1039/c8ra01441h.
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Bodega, Francesca, Luciano Zocchi, and Emilio Agostoni. "Albumin transcytosis in mesothelium." American Journal of Physiology-Lung Cellular and Molecular Physiology 282, no. 1 (2002): L3—L11. http://dx.doi.org/10.1152/ajplung.00157.2001.
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Apparent permeability to albumin ( P alb) was measured with125I-albumin in specimens of rabbit parietal pericardium from lumen to interstitium (L-I) and from interstitium to lumen (I-L). With albumin concentration (Calb) 0.5%, P alb (× 10−5 cm/s) L-I at 37°C was 0.172 ± 0.019 SE; it decreased to 0.092 ± 0.022 I-L at 37°C, 0.089 ± 0.021 L-I at 12°C, and 0.084 ± 0.018 I-L at 12°C. These findings provide evidence for an active transport L-I, likely transcytosis. With Calb 2.5%, 0.05%, and 0.005%, P alb L-I at 37°C was 0.188 ± 0.023, 0.156 ± 0.021, and 0.090 ± 0.021, respectively; at 12°C it was 0.089 ± 0.017, 0.083 ± 0.019, and 0.087 ± 0.026, respectively. Hence, active albumin transport ceases with Calb 0.005%; P alb values I-L at 12°C and with Calb 0.005% are similar and provide diffusional permeability. With physiological Calb (∼1%), active albumin flux was ∼5 × 10−4μmol · h−1 · cm−2. Apparent permeability to FITC-dextran 70 ( P dx) was also measured. P dx (× 10−5 cm/s) L-I at 37°C with Calb 0.5% was 0.095 ± 0.018; it decreased to 0.026 ± 0.004 I-L (37°C, Calb 0.5%), 0.038 ± 0.007 at 12°C (L-I, Calb 0.5%), 0.030 ± 0.009 with Calb 0.005% (L-I, 37°C), and 0.032 ± 0.011 with nocodazole (L-I, 37°C, Calb 0.5%). These findings provide evidence for transcytosis and confirm conclusions drawn from P alb. Vesicular liquid flow, computed from vesicular dextran flux (fluid-phase only), was ∼3.5 μl · h−1 · cm−2. Transcytosis seems a relevant mechanism, removing protein and liquid from serous cavities.
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Shao, Wenyao, Ying Lin, and Yinghua Lu. "Study on the Extraction Technology of Candida antarctica Lipase B by Foam Separation." Processes 9, no. 1 (2020): 14. http://dx.doi.org/10.3390/pr9010014.
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Candida antarctica Lipase B (CALB) has a wide range of applications in many fields. In this study, Pichia pastoris was used to express CALB for fermentation tank culture. Sodium dodecyl sulfate (SDS) was used as a surfactant, and foam separation technology was used to explore the best experimental conditions for the harvest of CALB. The results showed that the optimal technological conditions for the foam separation and recovery of CALB were as follows: liquid volume was 150 mL, separating gas velocity was 600 mL/min, pH value was 7, and surfactant SDS concentration was 0.5 mg/mL. Under these conditions, the enrichment ratio of CALB was 0.95, and recovery rate R was 80.32%, respectively, indicating that the foam separation technology is feasible to extract lipase B.
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Imarah, Ali O., Fausto M. W. G. Silva, László Tuba, et al. "A Convenient U-Shape Microreactor for Continuous Flow Biocatalysis with Enzyme-Coated Magnetic Nanoparticles-Lipase-Catalyzed Enantiomer Selective Acylation of 4-(Morpholin-4-yl)butan-2-ol." Catalysts 12, no. 9 (2022): 1065. http://dx.doi.org/10.3390/catal12091065.
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This study implements a convenient microreactor for biocatalysis with enzymes immobilized on magnetic nanoparticles (MNPs). The enzyme immobilized onto MNPs by adsorption or by covalent bonds was lipase B from Candida antarctica (CaLB). The MNPs for adsorption were obtained by covering the magnetite core with a silica shell and later with hexadecyltrimethoxysilane, while for covalent immobilization, the silica-covered MNPs were functionalized by a layer forming from mixtures of hexadecyl- and 3-(2-aminoethylamino)propyldimethoxymethylsilanes in 16:1 molar ratio, which was further activated with neopentyl glycol diglycidyl ether (NGDE). The resulting CaLB-MNPs were tested in a convenient continuous flow system, created by 3D printing to hold six adjustable permanent magnets beneath a polytetrafluoroethylene tube (PTFE) to anchor the MNP biocatalyst inside the tube reactor. The anchored CaLB-MNPs formed reaction chambers in the tube for passing the fluid through and above the MNP biocatalysts, thus increasing the mixing during the fluid flow and resulting in enhanced activity of CaLB on MNPs. The enantiomer selective acylation of 4-(morpholin-4-yl)butan-2-ol (±)-1, being the chiral alcohol constituent of the mucolytic drug Fedrilate, was carried out by CaLB-MNPs in the U-shape reactor. The CaLB-MNPs in the U-shape reactor were compared in batch reactions to the lyophilized CaLB and to the CaLB-MNPs using the same reaction composition, and the same amounts of CaLB showed similar or higher activity in flow mode and superior activity as compared to the lyophilized powder form. The U-shape permanent magnet design represents a general and easy-to-access implementation of MNP-based flow microreactors, being useful for many biotransformations and reducing costly and time-consuming downstream processes.
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Wang, Lihui, Xinlong Liu, Yanjun Jiang, et al. "Silica Nanoflowers-Stabilized Pickering Emulsion as a Robust Biocatalysis Platform for Enzymatic Production of Biodiesel." Catalysts 9, no. 12 (2019): 1026. http://dx.doi.org/10.3390/catal9121026.
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Enzymatic production of biodiesel had attracted much attention due to its high efficiency, mild conditions and environmental protection. However, the high cost of enzyme, poor solubility of methanol in oil and adsorption of glycerol onto the enzyme limited the popularization of the process. To address these problems, we developed a silica nanoflowers-stabilized Pickering emulsion as a biocatalysis platform with Candida antarctica lipase B (CALB) as model lipase for biodiesel production. Silica nanoflowers (SNFs) were synthesized in microemulsion and served as a carrier for CALB immobilization and then used as an emulsifier for constructing Pickering emulsion. The structure of SNFs and the biocatalytic Pickering emulsion (CALB@SNFs-PE) were characterized in detail. Experimental data about the methanolysis of waste oil to biodiesel was evaluated by response surface methodology. The highest experimental yield of 98.5 ± 0.5% was obtained under the optimized conditions: methanol/oil ratio of 2.63:1, a temperature of 45.97 °C, CALB@SNFs dosage of 33.24 mg and time of 8.11 h, which was closed to the predicted value (100.00%). Reusability test showed that CALB@SNFs-PE could retain 76.68% of its initial biodiesel yield after 15 cycles, which was better than that of free CALB and N435.
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Song, Min, and Jeong-Ho Chang. "Thermally Stable and Reusable Ceramic Encapsulated and Cross-Linked CalB Enzyme Particles for Rapid Hydrolysis and Esterification." International Journal of Molecular Sciences 23, no. 5 (2022): 2459. http://dx.doi.org/10.3390/ijms23052459.
Full textAbstract:
Candida antarctica lipase B (CalB) enzyme was encapsulated and cross-linked by silica matrix to enhance its thermal stability and reusability, and demonstrated an enzymatic ability for rapid hydrolysis and esterification. Silica encapsulated CalB particles (Si-E-CPs) and silica cross-linked CalB particles (Si-CL-CPs) were prepared as a function of TEOS concentration. The particle size analysis, thermal stability, catalytic activity in different pHs, and reusability of Si-E-CPs and Si-CL-CPs were demonstrated. Furthermore, the determination of the CalB enzyme in Si-E-CPs and Si-CL-CPs was achieved by Bradford assay and TGA analysis. Enzymatic hydrolysis was performed against the p-nitrophenyl butyrate and the catalytic parameters (Km, Vmax, and Kcat) were calculated by the Michaelis–Menten equation and a Lineweaver–Burk plot. Moreover, enzymatic synthesis for benzyl benzoate was demonstrated by esterification with an acyl donor of benzoic acid and two acyl donors of benzoic anhydride. Although the conversion efficiency of Si-CL-CPs was not much higher than that of native CalB, it has an efficiency of 91% compared to native CalB and is expected to be very useful because it has high thermal and pH stability and excellent reusability.
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Jaito, Nongluck, Nattha Kaewsawat, Suthathip Phetlum, and Tanaporn Uengwetwanit. "Metagenomic discovery of lipases with predicted structural similarity to Candida antarctica lipase B." PLOS ONE 18, no. 12 (2023): e0295397. http://dx.doi.org/10.1371/journal.pone.0295397.
Full textAbstract:
Here we employed sequence-based and structure-based screening for prospecting lipases that have structural homolog to Candida antarctica lipase B (CalB). CalB, a widely used biocatalyst, was used as structural template reference because of its enzymatic properties. Structural homolog could aid in the discovery of novel wild-type enzymes with desirable features and serve as a scaffold for further biocatalyst design. The available metagenomic data isolated from various environments was leveraged as a source for bioprospecting. We identified two bacteria lipases that showed high structural similarity to CalB with <40% sequence identity. Partial purification was conducted. In comparison to CalB, the enzymatic characteristics of two potential lipases were examined. A candidate exhibited optimal pH of 8 and temperature of 50°C similar to CalB. The second lipase candidate demonstrated an optimal pH of 8 and a higher optimal temperature of 55°C. Notably, this candidate sustained considerable activity at extreme conditions, maintaining high activity at 70°C or pH 9, contrasting with the diminished activity of CalB under similar conditions. Further comprehensive experimentation is warranted to uncover and exploit these novel enzymatic properties for practical biotechnological purposes.
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Abellanas-Perez, Pedro, Diego Carballares, Javier Rocha-Martin, and Roberto Fernandez-Lafuente. "The Effects of Buffer Nature on Immobilized Lipase Stability Depend on Enzyme Support Loading." Catalysts 14, no. 2 (2024): 105. http://dx.doi.org/10.3390/catal14020105.
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The lipases from Thermomyces lanuginosus (TLL) and Candida antarctica (B) (CALB) were immobilized on octyl-agarose beads at 1 mg/g (a loading under the capacity of the support) and by overloading the support with the enzymes. These biocatalysts were compared in their stabilities in 10 mM of sodium phosphate, HEPES, and Tris-HCl at pH 7. Lowly loaded CALB was more stable than highly loaded CALB preparation, while with TLL this effect was smaller. Phosphate was very negative for the stability of the CALB biocatalyst and moderately negative using TLL at both loadings. The stability of the enzymes in HEPES and Tris-HCl presented a different response as a function of the enzyme loading (e.g., using lowly loaded CALB, the stabilities were similar in both buffers, but it was clearly smaller in HEPES using the highly loaded biocatalysts). Moreover, the specific activity of the immobilized enzymes versus p-nitrophenol butyrate, triacetin and R- or S-methyl mandelate depended on the buffer, enzyme loading, and interaction between them. In some cases, almost twice the expected activity could be obtained using highly loaded octyl-CALB, depending on the buffer. A co-interaction between the effects on enzyme activity and the specificity of support enzyme loading and buffer nature was detected.
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Zhang, Jia Chan, Chan Zhang, Lei Zhao, and Cheng Tao Wang. "Lipase-Catalyzed Synthesis of Sucrose Fatty Acid Ester and the Mechanism of Ultrasonic Promoting Esterification Reaction in Non-Aqueous Media." Advanced Materials Research 881-883 (January 2014): 35–41. http://dx.doi.org/10.4028/www.scientific.net/amr.881-883.35.
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The preparation of sucrose fatty acid ester (SFAE) by lipase-catalyzing reaction usingCandida antarcticalipase B (CalB) and its immobilized form Novozym 435 was reported in this work. The preparation was characterized in non-aqueous media with and without ultrasound irradiation treatment. A conversion rate of SFAE up to 49.60% was achieved using Novozym 435 under the optimal conditions (45.4°C; mole ratio of methyl oleate to sucrose = 6.0:1; 4.0 mL acetone; 4.0 mg/mL Novozym 435; and 24.6 h of reaction). Under optimal ultrasound conditions (50 kHz, 0.15 W/cm2, 166.55 min), reaction time decreased by 75% approximately, compared with the control without ultrasonic irradiation, but the ultrasound irradiation treatment did not affect the SFAE yield catalyzed by Novozym 435. In the CalB-catalyzed preparation of SFAE under the same optimal reaction conditions, ultrasonic irradiation enhanced the activity of CalB during early time points and inhibited its activity after a long period of treatment. Moreover, CalB was further examined using Far-UV circular dichroism (CD) spectroscopy and scanning electron microscopy (SEM) to study the conformation and micro-morphology of CalB structural variations in various ultrasound irradiation treatments. CD results indicated that α-helical regions were increased and random coil regions remained at a similar level of proportion. SEM images showed small holes appeared on the surface of irradiated CalB. Therefore, we conclude that proper ultrasound irradiation could change the secondary structure and the surface morphology of the CalB in molecular level, and could accelerate the esterification reaction process.
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Silva, Fausto M. W. G., József Szemes, Akan Mustashev, Orsolya Takács, Ali O. Imarah, and László Poppe. "Immobilization of Lipase B from Candida antarctica on Magnetic Nanoparticles Enhances Its Selectivity in Kinetic Resolutions of Chiral Amines with Several Acylating Agents." Life 13, no. 7 (2023): 1560. http://dx.doi.org/10.3390/life13071560.
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In lipase-catalyzed kinetic resolutions (KRs), the choice of immobilization support and acylating agents (AAs) is crucial. Lipase B from Candida antarctica immobilized onto magnetic nanoparticles (CaLB-MNPs) has been successfully used for diverse KRs of racemic compounds, but there is a lack of studies of the utilization of this potent biocatalyst in the KR of chiral amines, important pharmaceutical building blocks. Therefore, in this work, several racemic amines (heptane-2-amine, 1-methoxypropan-2-amine, 1-phenylethan-1-amine, and 4-phenylbutan-2-amine, (±)-1a–d, respectively) were studied in batch and continuous-flow mode utilizing different AAs, such as diisopropyl malonate 2A, isopropyl 2-cyanoacetate 2B, and isopropyl 2-ethoxyacetate 2C. The reactions performed with CaLB-MNPs were compared with Novozym 435 (N435) and the results in the literature. CaLB-MNPs were less active than N435, leading to lower conversion, but demonstrated a higher enantiomer selectivity, proving to be a good alternative to the commercial form. Compound 2C resulted in the best balance between conversion and enantiomer selectivity among the acylating agents. CaLB-MNPs proved to be efficient in the KR of chiral amines, having comparable or superior properties to other CaLB forms utilizing porous matrices for immobilization. An additional advantage of using CaLB-MNPs is that the purification and reuse processes are facilitated via magnetic retention/separation. In the continuous-flow mode, the usability and operational stability of CaLB-MNPs were reaffirmed, corroborating with previous studies, and the results overall improve our understanding of this potent biocatalyst and the convenient U-shape reactor used.
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Diaz-Vidal, Tania, Vicente Paúl Armenta-Pérez, Luis Carlos Rosales-Rivera, et al. "Long chain capsaicin analogues synthetized by CALB-CLEAs show cytotoxicity on glioblastoma cell lines." Applied Microbiology and Biotechnology 108, no. 1 (2024): 1–14. http://dx.doi.org/10.1007/s00253-023-12856-y.
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Abstract 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.
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Overhage, Jörg, Alexander Steinbüchel, and Horst Priefert. "Biotransformation of Eugenol to Ferulic Acid by a Recombinant Strain of Ralstonia eutropha H16." Applied and Environmental Microbiology 68, no. 9 (2002): 4315–21. http://dx.doi.org/10.1128/aem.68.9.4315-4321.2002.
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ABSTRACT The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16. A recombinant strain of R. eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of R. eutropha H16(pBBR1-JO2ehyABcalAcalB) from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.
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Wang, Lihui, Xinlong Liu, Yanjun Jiang, et al. "Biocatalytic Pickering Emulsions Stabilized by Lipase-Immobilized Carbon Nanotubes for Biodiesel Production." Catalysts 8, no. 12 (2018): 587. http://dx.doi.org/10.3390/catal8120587.
Full textAbstract:
Biodiesel is a promising renewable energy source that can replace fossil fuel, but its production is limited by a lack of high-efficiency catalysts for mass production and popularization. In this study, we developed a biocatalytic Pickering emulsion using multiwall carbon nanotube-immobilized Candida antarctica lipase B (CALB@PE) to produce biodiesel, with J. curcas L. seed oil and methanol as substrates. The morphology of CALB@PE was characterized in detail. A central composite design of the response surface methodology (CCD-RSM) was used to study the effects of the parameters on biodiesel yield, namely the amount of J. curcas L. seed oil (1.5 g), molar ratio of methanol to oil (1:1–7:1), CALB@PE dosage (20–140 mg), temperature (30–50 °C), and reaction time (0–24 h). The experimental responses were fitted with a quadratic polynomial equation, and the optimum reaction conditions were the methanol/oil molar ratio of 4.64:1, CALB@PE dosage of 106.87 mg, and temperature of 34.9 °C, with a reaction time of 11.06 h. A yield of 95.2%, which was basically consistent with the predicted value of 95.53%, was obtained. CALB@PE could be reused up to 10 times without a substantial loss of activity. CALB@PE exhibited better reusability than that of Novozym 435 in the process of biodiesel production.
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Losada-Garcia, Noelia, Alba Rodriguez-Otero, and Jose M. Palomo. "High Degradation of Trichloroethylene in Water by Nanostructured MeNPs@CALB Biohybrid Catalysts." Catalysts 10, no. 7 (2020): 753. http://dx.doi.org/10.3390/catal10070753.
Full textAbstract:
In this study, a methodology was developed for the rapid degradation of trichloroethylene (TCE) and 1,1-dichloroethylene (1,1-DCE) in distilled water and room temperature without the production of toxic chlorinated by-products. This process was carried out using bionanohybrids of different metals (Pd, Fe, Cu and Zn) obtained by enzyme–metal coordination called MeNPs@CALB, which present different metal species and nanoparticle sizes. The Cu2O@CALB biohybrid, which contained Cu2O nanoparticles, showed excellent catalytic performance in TCE degradation by removing 95% (>125 ppm) in 10 min using 1.5 g/L of catalyst. On the other hand, in the degradation reaction of 1,1-DCE, Cu2O@CALB eliminated 94% (93 ppm) in 1 min. Cu2O@CALB exhibited excellent stability and recyclability under sustainable conditions, maintaining its effectiveness in more than 90% for three cycles.
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de Moraes, Lanuza Ribeiro, Maria Eduarda Araújo Delicato, André da Silva Cruz, et al. "Methionine supplementing effects on intestine, liver and uterus morphology, and on positivity and expression of Calbindin-D28k and TRPV6 epithelial calcium carriers in laying quail in thermoneutral conditions and under thermal stress." PLOS ONE 16, no. 1 (2021): e0245615. http://dx.doi.org/10.1371/journal.pone.0245615.
Full textAbstract:
This study aimed to provide the performance, localization and expression of the epithelial calcium transporter channels Calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quail under heat stress (HS), and with methionine supplementation (MS). This study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels in the kidneys, intestine and uterus of 504 laying quails under different MS (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under HS (32°C) had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb, and increased positivity in proximal CTs. Deleterious effects of HS were minimized with MS for: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney (P≤0.05). Epithelial calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the MS, increasing egg production in HS and quality in termoneutrality (P≤0.05). MS further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity of Calb, and increase Calb and TRPV6 expression in the kidney (P≤0.001) under thermoneutrality. It was concluded that the use of MS (120%) is justifiable in order to partially reverse the deleterious effects of HS on the production, in the epithelial calcium carriers, and in the digestory and reproductive morphology of laying quail.
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Szelwicka, Anna, Karol Erfurt, Sebastian Jurczyk, Slawomir Boncel, and Anna Chrobok. "Outperformance in Acrylation: Supported D-Glucose-Based Ionic Liquid Phase on MWCNTs for Immobilized Lipase B from Candida antarctica as Catalytic System." Materials 14, no. 11 (2021): 3090. http://dx.doi.org/10.3390/ma14113090.
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This study presents a highly efficient method of a synthesis of n-butyl acrylate via esterification of acrylic acid and n-butanol in the presence of supported ionic liquid phase (SILP) biocatalyst consisting of the lipase B from Candida antarctica (CALB) and multi-walled carbon nanotubes (MWCNTs) modified by D-glucose-based ionic liquids. Favorable reaction conditions (acrylic acid: n-butanol molar ratio 1:2, cyclohexane as a solvent, biocatalyst 0.150 g per 1 mmol of acrylic acid, temperature 25 °C) allowed the achievement of a 99% yield of n-butyl acrylate in 24 h. Screening of various ionic liquids showed that the most promising result was obtained if N-(6-deoxy-1-O-methoxy-α-D-glucopyranosyl)-N,N,N-trimethylammonium bis-(trifluoromethylsulfonyl)imide ([N(CH3)3GlcOCH3][N(Tf)2]) was selected in order to modify the outer surface of MWCNTs. The final SILP biocatalyst–CNTs-[N(CH3)3GlcOCH3][N(Tf)2]-CALB contained 1.8 wt.% of IL and 4.2 wt.% of CALB. Application of the SILP biocatalyst led to the enhanced activity of CALB in comparison with the biocatalyst prepared via physical adsorption of CALB onto MWCNTs (CNTs-CALB), as well as with commercially available Novozyme 435. Thus, the crucial role of IL in the stabilization of biocatalysts was clearly demonstrated. In addition, a significant stability of the developed biocatalytic system was confirmed (three runs with a yield of ester over 90%).
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Xing, Xiu, Jun-Qi Jia, Jing-Fan Zhang, et al. "CALB Immobilized onto Magnetic Nanoparticles for Efficient Kinetic Resolution of Racemic Secondary Alcohols: Long-Term Stability and Reusability." Molecules 24, no. 3 (2019): 490. http://dx.doi.org/10.3390/molecules24030490.
Full textAbstract:
In this study, an immobilization strategy for magnetic cross-linking enzyme aggregates of lipase B from Candida antarctica (CALB) was developed and investigated. Magnetic particles were prepared by conventional co-precipitation. The magnetic nanoparticles were modified with 3-aminopropyltriethoxysilane (APTES) to obtain surface amino-functionalized magnetic nanoparticles (APTES–Fe3O4) as immobilization materials. Glutaraldehyde was used as a crosslinker to covalently bind CALB to APTES–Fe3O4. The optimal conditions of immobilization of lipase and resolution of racemic 1-phenylethanol were investigated. Under optimal conditions, esters could be obtained with conversion of 50%, enantiomeric excess of product (eep) > 99%, enantiomeric excess of substrate (ees) > 99%, and enantiomeric ratio (E) > 1000. The magnetic CALB CLEAs were successfully used for enzymatic kinetic resolution of fifteen secondary alcohols. Compared with Novozym 435, the magnetic CALB CLEAs exhibited a better enantioselectivity for most substrates. The conversion was still greater than 49% after the magnetic CALB CLEAs had been reused 10 times in a 48 h reaction cycle; both ees and eep were close to 99%. Furthermore, there was little decrease in catalytic activity and enantioselectivity after being stored at −20 °C for 90 days.
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Ismail, Hilda, Evi Lande Setiyani, Dwi Titus Indriyawati, and B. S. Ari Sudarmanto. "Employing lipase of candida antarctica (calb) as catalyst in the acetylation of para-aminophenol in aqueous and water-free medium." Jurnal Teknosains 11, no. 1 (2021): 66. http://dx.doi.org/10.22146/teknosains.69113.
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Candida antarctica lipase B (CaLB) is one of lipase classes enzymes that has many advantages to be used in the process of synthesizing organic compounds. In this study, some experiments were conducted to examine the ability of CaLB as a catalyst in the para-aminophenol (PAP) acetylation to produce paracetamol as the result. Two types of research have been carried out, the first one is to utilize CaLB to catalyze acetylation of PAP in a water-free reaction medium, and the second one is to use CaLB as catalyst in aqueous medium through oxidative amidation reaction. Reaction in water free system was held in ethyl catalyst acetate as solvent that also act as the acyl donor, while in the aqueous medium, acetylacetone was used as acyl donor and ethyl acetate as source to produce peracid that will be used as oxidator. Analysis was done by HPLC and TLC densitometric to follow the amount of paracetamol produced. The results of CaLB-catalyzed acylation in water free system showed that the enzyme could accept PAF and ethyl acetate as a substrate in a nucleophilic substitution reaction, resulting in paracetamol as a product. However, the yield from the acylation of PAP is still not satisfactory. In the reaction in aqueous medium, CaLB has been proven to show its activity to catalyze the acylation of PAP with acetylacetone, as well as the reaction of peracid formation from ethyl acetate. The results show that this strategy can work well and give better yields than the other reaction in water-free medium.
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Barrera Valderrama, Daniel Iván, Markus Doerr, and Martha Cecilia Daza Espinosa. "Función de los confórmeros de ataque cercano en la acilación enantioselectiva del (R,S)-propranolol catalizada por lipasa B de Candida antarctica." Revista Colombiana de Biotecnología 20, no. 1 (2018): 16–30. http://dx.doi.org/10.15446/rev.colomb.biote.v20n1.73652.
Full textAbstract:
La lipasa B de Candida antarctica (CalB) se ha utilizado en la acilación quimio- y enantioselectiva del racemato (R,S)-propranolol. CalB tiene enantioselectividad moderada (E=63) por el R-propranolol. La enantioselectividad, se origina en la reacción de transferencia del grupo acilo desde la serina catalítica, acilada, al propranolol. La fase inicial de esta reacción involucra la formación de complejos de Michaelis y posteriormente conformaciones de ataque cercano. El análisis de las conformaciones de ataque cercano ha permitido en varios casos explicar el origen de la catálisis o reproducir el efecto catalítico. En este trabajo se profundiza en la comprensión la función de las conformaciones de ataque cercano en la enantioselectividad de la acilación del (R,S)-propranolol catalizada por CalB. Para lo anterior se realizó un estudio detallado de los complejos de Michaelis y de las conformaciones de ataque cercano del paso enantioselectivo de la reacción de acilación del (R,S)-propranolol utilizando un protocolo de dinámica molecular QM/MM (SCCDFTB/CHARMM) utilizando 6 distribuciones de velocidades iniciales y simulaciones de 2,5 ns. Se estudiaron 7 complejos CalB-propranolol. Los enlaces de hidrógeno del sitio activo de CalB acilada relevantes para la actividad catalítica fueron estables en todas las simulaciones. Las poblaciones de los complejos de Michaelis y de las conformaciones de ataque cercano son dependientes de la distribución de las velocidades iniciales de la dinámica molecular. La enantioselectividad moderada de CalB acilada, encontrada experimentalmente, puede ser parcialmente atribuida a la alta población de conformaciones de ataque cercano observada para el S-propranolol.
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Xu, Chuanbang, Yan Sun, Yuanyuan Sun, Ruiyun Cai, and Shengmiao Zhang. "High Internal Phase Pickering Emulsion Stabilized by Lipase-Coated ZIF-8 Nanoparticles towards Recyclable Biphasic Biocatalyst." Catalysts 13, no. 2 (2023): 383. http://dx.doi.org/10.3390/catal13020383.
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High internal phase Pickering emulsion (Pickering HIPE) stabilized by enzyme-decorated metal-organic frameworks (MOFs) nanoparticles is developed for biphasic biocatalysts to enhance lipase catalysis and recycling. Specifically, enzyme decorated nanoparticles are prepared via ZIF-8 physisorption of a model lipase Candida antarctica Lipase B (CALB), named ZIF-8@CALB, to be both Pickering stabilizer and catalytic sites. An oil-in-water (o/w) Pickering HIPE with oil/water volume ratio of 3 could then be fabricated by homogenizing p-nitrophenyl palmitate (p-NPP) n-heptane solution into the ZIF-8@CALB aqueous dispersion. The biocatalytic hydrolysis of p-NPP is conducted by just standing the biphasic system at room temperature. The Pickering HIPE system achieves a product conversion of up to 48.9% within 0.5 h, whereas the p-NPP n-heptane solution system containing free CALB only achieves a stable product conversion of 6.8% for the same time. Moreover, the ZIF@CALB could be recovered by a simple centrifugation at 800 rpm, and then reused in the next cycle. The hydrolysis equilibrium conversion rate of p-NPP keeps over 40% for all 8 cycles, reflecting the high catalytic efficiency and recyclability of the Pickering HIPE. This study provides a new opportunity in designing Enzyme-MOFs-based Pickering interfacial biocatalyst for practical applications.
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Prošková, A., Z. Kopicová, J. Kučera, and L. Škarková. "Lipase-catalyzed transesterification of rendering plant fat – Short Communication." Research in Agricultural Engineering 56, No. 3 (2010): 122–25. http://dx.doi.org/10.17221/40/2009-rae.
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Soluble lipase (Lipozyme CALB L) was immobilized by covalent bond to chitosan pellets prepared from Aspergillus niger mycelium. This immobilized enzyme was compared with commercial immobilized lipase of the same origin (Novozym 435). Novozym 435 is also lipase CALB L commercially immobilized by sorption on poly-(methyl acrylate). Novozym 435 shows much higher conversion of rendering plant fat in methanol under optimum conditions, having, at the same time, lower optimum temperature and lower stability at higher temperature. Lipozyme CALB L immobilized on chitosan leads to a low conversion, regardless its higher thermal stability. Novozym 435 gives conversion of about 50% of theoretical value, which is in good accordance with basically catalyzed transesterification of rendering plant fat described elsewhere. Lipozyme CALB L immobilized on chitosan gives conversion of about 10% of theoretical value only. The use of Novozym 435 in two-step system (enzyme-acid) seems to be more convenient compared with traditional two-step system (base-acid)
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Arana-Peña, Sara, Yuliya Lokha, and Roberto Fernández-Lafuente. "Immobilization of Eversa Lipase on Octyl Agarose Beads and Preliminary Characterization of Stability and Activity Features." Catalysts 8, no. 11 (2018): 511. http://dx.doi.org/10.3390/catal8110511.
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Eversa is an enzyme recently launched by Novozymes to be used in a free form as biocatalyst in biodiesel production. This paper shows for first time the immobilization of Eversa (a commercial lipase) on octyl and aminated agarose beads and the comparison of the enzyme properties to those of the most used lipase, the isoform B from Candida antarctica (CALB) immobilized on octyl agarose beads. Immobilization on octyl and aminated supports of Eversa has not had a significant effect on enzyme activity versus p-nitrophenyl butyrate (pNPB) under standard conditions (pH 7), but immobilization on octyl agarose beads greatly enhanced the stability of the enzyme under all studied conditions, much more than immobilization on aminated support. Octyl-Eversa was much more stable than octyl-CALB at pH 9, but it was less stable at pH 5. In the presence of 90% acetonitrile or dioxane, octyl-Eversa maintained the activity (even increased the activity) after 45 days of incubation in a similar way to octyl-CALB, but in 90% of methanol, results are much worse, and octyl-CALB became much more stable than Eversa. Coating with PEI has not a clear effect on octyl-Eversa stability, although it affected enzyme specificity and activity response to the changes in the pH. Eversa immobilized octyl supports was more active than CALB versus triacetin or pNPB, but much less active versus methyl mandelate esters. On the other hand, Eversa specificity and response to changes in the medium were greatly modulated by the immobilization protocol or by the coating of the immobilized enzyme with PEI. Thus, Eversa may be a promising biocatalyst for many processes different to the biodiesel production and its properties may be greatly improved following a suitable immobilization protocol, and in some cases is more stable and active than CALB.
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Szelwicka, Anna, Agnieszka Siewniak, Anna Kolanowska, Sławomir Boncel, and Anna Chrobok. "PTFE-Carbon Nanotubes and Lipase B from Candida antarctica—Long-Lasting Marriage for Ultra-Fast and Fully Selective Synthesis of Levulinate Esters." Materials 14, no. 6 (2021): 1518. http://dx.doi.org/10.3390/ma14061518.
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An effective method for levulinic acid esters synthesis by the enzymatic Fischer esterification of levulinic acid using a lipase B from Candida antarctica (CALB) immobilized on the advanced material consisting of multi-wall carbon nanotubes (MWCNTs) and a hydrophobic polymer—polytetrafluoroethylene (Teflon, PTFE)—as a heterogeneous biocatalyst, was developed. An active phase of the biocatalyst was obtained by immobilization via interfacial activation on the surface of the hybrid material MWCNTs/PTFE (immobilization yield: 6%, activity of CALB: 5000 U∙L∙kg−1, enzyme loading: 22.5 wt.%). The catalytic activity of the obtained biocatalyst and the effects of the selected reaction parameters, including the agitation speed, the amount of PTFE in the CALB/MWCNT-PTFE biocatalyst, the amount of CALB/MWCNT-PTFE, the type of organic solvent, n-butanol excess, were tested in the esterification of levulinic acid by n-butanol. The results showed that the use of a two-fold excess of levulinic acid to n-butanol, 22.5 wt.% of CALB on MWCNT-PTFE (0.10 wt.%) and cyclohexane as a solvent at 20 °C allowed one to obtain n-butyl levulinate with a high yield (99%) and selectivity (>99%) after 45 min. The catalyst retained its activity and stability after three cycles, and then started to lose activity until dropping to a 69% yield of ester in the sixth reaction run. The presented method has opened the new possibilities for environmentally friendly synthesis of levulinate esters.
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Giraldo, Liliana, Fernando Gómez-Granados, and Juan Carlos Moreno-Piraján. "Biodiesel Production Using Palm Oil with a MOF-Lipase B Biocatalyst from Candida Antarctica: A Kinetic and Thermodynamic Study." International Journal of Molecular Sciences 24, no. 13 (2023): 10741. http://dx.doi.org/10.3390/ijms241310741.
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This research presents the results of the immobilization of Candida Antarctica Lipase B (CALB) on MOF-199 and ZIF-8 and its use in the production of biodiesel through the transesterification reaction using African Palm Oil (APO). The results show that the highest adsorption capacity, the 26.9 mg·g−1 Lipase, was achieved using ZIF-8 at 45 °C and an initial protein concentration of 1.20 mg·mL−1. The results obtained for the adsorption equilibrium studies allow us to infer that CALB was physically adsorbed on ZIF-8 while chemically adsorbed with MOF-199. It was determined that the adsorption between Lipase and the MOFs under study better fit the Sips isotherm model. The results of the kinetic studies show that adsorption kinetics follow the Elovich model for the two synthesized biocatalysts. This research shows that under the experimental conditions in which the studies were carried out, the adsorption processes are a function of the intraparticle and film diffusion models. According to the results, the prepared biocatalysts showed a high efficiency in the transesterification reaction to produce biodiesel, with methanol as a co-solvent medium. In this work, the catalytic studies for the imidazolate, ZIF-8, presented more catalytic activity when used with CALB. This system presented 95% biodiesel conversion, while the biocatalyst formed by MOF-199 and CALB generated a catalytic conversion percentage of 90%. Although both percentages are high, it should be noted that CALB-MOF-199 presented better reusability, which is due to chemical interactions.
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Merrell, D. Scott, and Andrew Camilli. "Regulation of Vibrio cholerae Genes Required for Acid Tolerance by a Member of the “ToxR-Like” Family of Transcriptional Regulators." Journal of Bacteriology 182, no. 19 (2000): 5342–50. http://dx.doi.org/10.1128/jb.182.19.5342-5350.2000.
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ABSTRACT The ability of the intestinal pathogen Vibrio choleraeto undergo an adaptive stress response, known as the acid tolerance response (ATR), was previously shown to enhance virulence. An essential component of the ATR is CadA-mediated lysine decarboxylation. CadA is encoded by the acid- and infection-induced gene cadA. Herein, cadA is shown to be the second gene in an operon with cadB, encoding a lysine/cadaverine antiporter.cadC, which is 5′ of cadB, encodes an acid-responsive, positive transcriptional regulator ofcadBA. Unlike in Escherichia coli, V. cholerae cadB and cadA are also transcribed monocistronically. Of note, bicistronic cadBA is transcribed at low constitutive levels in an acid- and CadC-independent manner. CadC represents a new member of the “ToxR-like” family of transcriptional regulators in V. cholerae and, in addition, exhibits extensive amino acid and functional similarity to E. coli CadC. The amino-terminal, putative DNA binding domains of ToxR and CadC are highly conserved, as are the putative promoter elements recognized by these transcription factors.
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Siódmiak, Joanna, Jacek Dulęba, Natalia Kocot, et al. "A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives." International Journal of Molecular Sciences 25, no. 10 (2024): 5084. http://dx.doi.org/10.3390/ijms25105084.
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The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base—pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.
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Arora, Benu. "Highly Efficient Synthesis of Glucose Fatty Acid Esters Catalyzed by High Performance Lipase Preparations." Asian Journal of Chemistry 33, no. 10 (2021): 2489–97. http://dx.doi.org/10.14233/ajchem.2021.23391.
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Glucose fatty acid esters were synthesized using cross-linked enzyme aggregates (CLEAs), protein coated microcrystals (PCMCs) and cross-linked protein coated microcrystals (CLPCMCs) of Candida antarctica lipase B (CALB) as biocatalyst designs in single and mixed solvent systems. Up to 90% conversion and more than 99% regioselectivity were obtained using vinyl acetate as the acyl donor in a solvent system composed of 2-methyl-2-butanol (2M2B) and 30% (v/v) DMSO, with CALB CLEAs within 45 min. Similar results were obtained with CALB CLPCMCs as the biocatalyst under the same reaction conditions. This approach was then extended to the synthesis of glucose esters with higher acyl chain length. The synthetic strategy used in this work can potentially be extended for the fast and regioselective esterification/transesterification of other sugars as well.
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Fabbri, Filippo, Federico A. Bertolini, Georg M. Guebitz, and Alessandro Pellis. "Biocatalyzed Synthesis of Flavor Esters and Polyesters: A Design of Experiments (DoE) Approach." International Journal of Molecular Sciences 22, no. 16 (2021): 8493. http://dx.doi.org/10.3390/ijms22168493.
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In the present work, different hydrolases were adsorbed onto polypropylene beads to investigate their activity both in short-esters and polyesters synthesis. The software MODDE® Pro 13 (Sartorius) was used to develop a full-factorial design of experiments (DoE) to analyse the thermostability and selectivity of the immobilized enzyme towards alcohols and acids with different chain lengths in short-esters synthesis reactions. The temperature optima of Candida antarctica lipase B (CaLB), Humicola insolens cutinase (HiC), and Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) were 85 °C, 70 °C, and 50 °C. CaLB and HiC preferred long-chain alcohols and acids as substrate in contrast to Thc_Cut1, which was more active on short-chain monomers. Polymerization of different esters as building blocks was carried out to confirm the applicability of the obtained model on larger macromolecules. The selectivity of both CaLB and HiC was investigated and best results were obtained for dimethyl sebacate (DMSe), leading to polyesters with a Mw of 18 kDa and 6 kDa. For the polymerization of dimethyl adipate (DMA) with BDO and ODO, higher molecular masses were obtained when using CaLB onto polypropylene beads (CaLB_PP) as compared with CaLB immobilized on macroporous acrylic resin beads (i.e., Novozym 435). Namely, for BDO the Mn were 7500 and 4300 Da and for ODO 8100 and 5000 Da for CaLB_PP and for the commercial enzymes, respectively. Thc_Cut1 led to polymers with lower molecular masses, with Mn < 1 kDa. This enzyme showed a temperature optimum of 50 °C with 63% of DMA and BDO when compared to 54% and 27%, at 70 °C and at 85 °C, respectively.
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Gkini, Olga A., Panagiota-Yiolanda Stergiou, Athanasios Foukis, Panayiotis V. Ioannou, and Emmanuel M. Papamichael. "Kinetic Analysis of the Lipase-Catalyzed Hydrolysis of Erythritol and Pentaerythritol Fatty Acid Esters: A Biotechnological Application for Making Low-Calorie Healthy Food Alternatives." Catalysts 10, no. 9 (2020): 965. http://dx.doi.org/10.3390/catal10090965.
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Contemporary consumers demand healthier and more nourishing food, and thus, alternative foods that are low-calorie in fats and/or sugars are preferred. These desired properties may be attained by substituting the fatty acid esters of erythritol and pentaerythritol due to their antioxidant action and low toxicity for humans. In this work, the catalyzed hydrolysis of five fatty acid tetraesters of erythritol and/or pentaerythritol by both porcine pancreas type VI-s lipase (PPL) and Candida antarctica lipase-B (CALB) were studied kinetically. In all cases, except the hydrolysis of pentaerythritol tetrastearate by CALB, Michaelis–Menten kinetics were observed. In addition, the pKa values of the fatty acids released due to the catalyzed hydrolysis of the studied tetraesters by CALB were estimated. In the course of the aforementioned procedures, it was found that the CALB-catalyzed hydrolysis was incomplete to various degrees among four of the five studied tetraesters (excluding erythritol tetraoleate), and one or more estimated apparent pKa values were obtained. These results are novel, and by means of applied methodology, they reveal that erythritol and/or pentaerythritol tetraesters of medium- and long-chain fatty acids are suitable candidates for use as beneficial alternatives to butter and/or sweeteners.
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Wang, Honghai, Wenda Yue, Shuling Zhang, Yu Zhang, Chunli Li, and Weiyi Su. "Modification of Silica Xerogels with Polydopamine for Lipase B from Candida antarctica Immobilization." Catalysts 11, no. 12 (2021): 1463. http://dx.doi.org/10.3390/catal11121463.
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Silica xerogels have been proposed as a potential support to immobilize enzymes. Improving xerogels’ interactions with such enzymes and their mechanical strengths is critical to their practical applications. Herein, based on the mussel-inspired chemistry, we demonstrated a simple and highly effective strategy for stabilizing enzymes embedded inside silica xerogels by a polydopamine (PDA) coating through in-situ polymerization. The modified silica xerogels were characterized by scanning and transmission electron microscopy, Fourier tranform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy and pore structure analyses. When the PDA-modified silica xerogels were used to immobilize enzymes of Candida antarctica lipase B (CALB), they exhibited a high loading ability of 45.6 mg/gsupport, which was higher than that of immobilized CALB in silica xerogels (28.5 mg/gsupport). The immobilized CALB of the PDA-modified silica xerogels retained 71.4% of their initial activities after 90 days of storage, whereas the free CALB retained only 30.2%. Moreover, compared with the immobilization of enzymes in silica xerogels, the mechanical properties, thermal stability and reusability of enzymes immobilized in PDA-modified silica xerogels were also improved significantly. These advantages indicate that the new hybrid material can be used as a low-cost and effective immobilized-enzyme support.
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Papanikolaou, Angelos, Alexandra V. Chatzikonstantinou, Renia Fotiadou, et al. "A Study on the Regioselective Acetylation of Flavonoid Aglycons Catalyzed by Immobilized Lipases." Biomolecules 14, no. 8 (2024): 897. http://dx.doi.org/10.3390/biom14080897.
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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.
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Patterson, C. E., R. A. Rhoades, and J. G. Garcia. "Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung." Journal of Applied Physiology 72, no. 3 (1992): 865–73. http://dx.doi.org/10.1152/jappl.1992.72.3.865.
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Determination of protein transfer across the endothelial barrier or the entire alveolar capillary membrane is critical for investigation of mechanisms leading to pulmonary edema. The purpose of this study was to evaluate Evans blue dye for determination of protein clearance across cultured bovine pulmonary artery endothelial cell monolayers and as a quantitative marker for albumin leakage to the air spaces in isolated perfused rat lungs. Evans blue dye bound tightly to albumin (EBA) as determined by lack of transfer through dialysis membranes and specific elution with albumin from a molecular exclusion column. EBA was equivalent to 125I-labeled albumin for calculation of albumin clearance rates (Calb) across intact and challenged monolayers [Calb (+ vehicle) = 0.12 microliters/min; Calb (+10 nM alpha-thrombin) = 0.47 microliters/min; Calb (+5 mg/ml trypsin) = 1.29 microliters/min]. Transfer of EBA was linear with time in both the endothelial cell monolayer model and the perfused lung. EBA was a sensitive marker for early edema in the perfused lung (before detectable weight gain) as well as for severe edema in the oxidant-injured lung (marked EBA accumulation in lavage fluid) and was a more specific marker for protein transfer than lavage fluid protein. EBA transfer is a convenient, reproducible, and accurate means to assess alterations in vascular permeability.
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Pospiech, Doris, Renata Choińska, Daniel Flugrat, et al. "Enzymatic Synthesis of Poly(alkylene succinate)s: Influence of Reaction Conditions." Processes 9, no. 3 (2021): 411. http://dx.doi.org/10.3390/pr9030411.
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Application of lipases (preferentially Candida antarctica Lipase B, CALB) for melt polycondensation of aliphatic polyesters by transesterification of activated dicarboxylic acids with diols allows to displace toxic metal and metal oxide catalysts. Immobilization of the enzyme enhances the activity and the temperature range of use. The possibility to use enzyme-catalyzed polycondensation in melt is studied and compared to results of polycondensations in solution. The experiments show that CALB successfully catalyzes polycondensation of both, divinyladipate and dimethylsuccinate, respectively, with 1,4-butanediol. NMR spectroscopy, relative molar masses obtained by size exclusion chromatography, MALDI-TOF MS and wide-angle X-ray scattering are employed to compare the influence of synthesis conditions for poly(butylene adipate) (PBA) and poly(butylene succinate) (PBS). It is shown that the enzymatic activity of immobilized CALB deviates and influences the molar mass. CALB-catalyzed polycondensation of PBA in solution for 24 h at 70 °C achieves molar masses of up to Mw~60,000 g/mol, higher than reported previously and comparable to conventional PBA, while melt polycondensation resulted in a moderate decrease of molar mass to Mw~31,000. Enzymatically catalyzed melt polycondensation of PBS yields Mw~23,400 g/mol vs. Mw~40,000 g/mol with titanium(IV)n-butoxide. Melt polycondensation with enzyme catalysis allows to reduce the reaction time from days to 3–4 h.
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Gawler, D. J., L. J. W. Zhang, and M. F. Moran. "Mutation-deletion analysis of a Ca2+-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras." Biochemical Journal 307, no. 2 (1995): 487–91. http://dx.doi.org/10.1042/bj3070487.
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p120 GAP is a GTPase activating protein for p21 ras. It is a multidomain protein which exhibits sequence similarity with other GTPase-activating proteins, src, pleckstrin and a central portion of the protein kinase C conserved region 2 domain known as CaLB (Ca(2+)-dependent phospholipid-binding). The presence of this CaLB motif has led to the speculation that p120 GAP may be a member of a family of structurally related proteins containing a Ca(2+)-dependent membrane/lipid-binding domain. Here we have studied the in vitro Ca(2+)-dependent phospholipid-binding properties of the isolated proposed CaLB sequence in human GAP and deduce that a phospholipid-binding sequence is indeed located between amino acids 606 and 648. Binding of phosphatidylserine and phosphatidylinositol, but not phosphatidylcholine, within this sequence is Ca(2+)-dependent, with an estimated EC50 for Ca2+ of approx. 1 microM. Using deletion-mutation analysis we have further defined the minimal boundaries for this in vitro phospholipid-binding activity. p120 GAP amino acids 612-643 exhibit full phospholipid-binding activity, but further deletion of either amino acids 612-617 or amino acids 633-648 significantly decreased or abolished phospholipid binding. These studies establish that amino acids 612-643 of p120 GAP indeed constitute a functional CaLB domain and thereby imply a role for Ca2+ in the regulation of p120 GAP association with cellular (membrane) phospholipids.
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Pauli, Oliver, Achim Ecker, Alvaro Cruz-Izquierdo, Alessandra Basso, and Simona Serban. "Visualizing Hydrophobic and Hydrophilic Enzyme Interactions during Immobilization by Means of Infrared Microscopy." Catalysts 12, no. 9 (2022): 989. http://dx.doi.org/10.3390/catal12090989.
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A novel Fourier transform infrared (FT-IR) microscopy method was developed and used to analyze the diffusion of lipase CalB in two different resins during immobilization. The method consisted of a streamlined sample preparation process and an automated transmission FT-IR microscopic measurement using a commercial benchtop device. The immobilization of CalB was performed on a hydrophobic resin containing aromatic groups (ECR1030M based on divinylbenzene) and on a hydrophilic resin containing ester groups and thus oxygen (ECR8204M based on methacrylate) and FT-IR revealed that the kinetic of immobilization and the distribution of the enzyme on the two resins were completely different. Furthermore, the technique revealed that CalB was immobilized on the external surface only in the case of the hydrophobic ECR1030M in a layer of about 50–70 µm, whereas when immobilized on the hydrophilic carrier ECR8204M the interaction of the enzyme with the carrier was uniform over the full diameter of the polymer bead. The enzyme activity however was higher on the hydrophobic support ECR1030M.
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Marruchella, Giuseppe, Ciriaco Ligios, Valeria Albanese, et al. "Enteroglial and neuronal involvement without apparent neuron loss in ileal enteric nervous system plexuses from scrapie-affected sheep." Journal of General Virology 88, no. 10 (2007): 2899–904. http://dx.doi.org/10.1099/vir.0.82907-0.
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The enteric nervous system (ENS) probably plays a dominant role in sheep scrapie pathogenesis, but little is known about the cell types involved. We investigated the ileal myenteric and submucosal plexuses of four naturally and four orally experimentally scrapie-affected ARQ/ARQ Sarda sheep, as well as those of 12 healthy-control Sarda sheep carrying different PrP genotypes. All scrapie-affected animals, euthanized at clinical-disease end stage, showed PrPd deposition within enteric glial cells (EGCs) and calbindin-immunoreactive (CALB-IR) and neuronal nitric oxide synthase (nNOS)-IR neurons. Whole-mount investigations revealed no significant differences between the densities of total, CALB-IR and nNOS-IR neurons in scrapie-affected versus healthy sheep, irrespective of PrP genotype. Our results suggest that EGCs and CALB-IR and nNOS-IR neurons are probably involved in the pathogenesis of natural and oral experimental sheep scrapie. Furthermore, the infectious agent may be less pathogenic towards ENS neurons than it is towards central nervous system neurons.