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Herein, the manuscript presents a chemoenzymatic formal synthetic route of (+)-brazilin, a homoisoflavonoid natural product with a chroman skeleton cis-fused with a 2,3-dihydro-1H-indene unit, which is isolated from the traditional Chinese medicine, Caesalpinia sappan L. The key feature of the synthetic strategy includes an enzyme-mediated desymmetrization by employing lipase from Candida antarctica type B (CALB) and a one-pot SN2/hydrolysis reaction.

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Enzymes have been highly demanded in diverse applications such as in the food, pharmaceutical, and industrial fuel sectors. Thus, in silico bioprospecting emerges as an efficient strategy for discovering new enzyme candidates. A new program called ProspectBIO was developed for this purpose as it can find non-annotated sequences by searching for homologs of a model enzyme directly in genomes. Here we describe the ProspectBIO software methodology and the experimental validation by prospecting for novel lipases by sequence homology to Candida antarctica lipase B (CaLB) and conserved motifs. As expected, we observed that the new bioprospecting software could find more sequences (1672) than a conventional similarity-based search in a protein database (733). Additionally, the absence of patent protection was introduced as a criterion resulting in the final selection of a putative lipase-encoding gene from Ustilago hordei (UhL). Expression of UhL in Pichia pastoris resulted in the production of an enzyme with activity towards a tributyrin substrate. The recombinant enzyme activity levels were 4-fold improved when lowering the temperature and increasing methanol concentrations during the induction phase in shake-flask cultures. Protein sequence alignment and structural modeling showed that the recombinant enzyme has high similarity and capability of adjustment to the structure of CaLB. However, amino acid substitutions identified in the active pocket entrance may be responsible for the differences in the substrate specificities of the two enzymes. Thus, the ProspectBIO software allowed the finding of a new promising lipase for biotechnological application without the need for laborious and expensive conventional bioprospecting experimental steps.

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MCM-48 mesoporous support was synthesized with the ionic solid 1-tetradecyl-3-methylimidazolium chloride ([C14MI]Cl) as a structure-directing agent for in situ immobilization of Candida antarctica B (CALB). The MCM-48[C14MI]Cl support showed characteristics of mesoporous material of interest, with a pore size of 20.30 and 73.41 A for the support without and with the enzyme, respectively. The elongation of the carbonic chain of the ionic solid directly influenced the increase in the specific area and pore volume of the material. In addition, the decrease in the specific area and pore volume for support with the enzyme showed the effectiveness of immobilization in situ. It was possible to obtain the ideal levels for the best activities of esterification of the enzyme with optimization of a mathematical model. The optimized variables were 0.31 g of enzyme and 3.35% of ionic solid with a maximum esterification activity of 392.92 U/g and 688% of yield. The support showed residual activity above 50% when stored under refrigeration for 75 days. At 60 and 80 °C, the enzyme immobilized on the support retained more than 80 and 40% of its residual activity, respectively. In addition, the support presented the possibility of reuse for up to 10 cycles with residual activity of approximately 50%. The support synthesized in the present study presents a great industrial opportunity for the immobilization and use of the CALB enzyme.

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Sol-gel technique aiming enzymatic immobilization in situ with ionic liquids as additives is poorly studied. In this process, the addition of the enzyme is carried out in the synthesis of the support. The characteristics of ionic liquids, such as low vapor pressure, thermal stability, and non-flammability, make them strong candidates for use as immobilization additives. The objective of the present study was to immobilize the Candida antarctica B lipase by the sol-gel technique using ionic liquids as additives. The optimum points determined for ionic liquids 1-butyl-3-methylimidazolium chloride, 1-octyl-3-methylimidazolium bromide, and 1 hexadecyl-3-methylimimidazolium were 0.30, 0.27, and 0.22 g/mL of enzyme and 1.60, 1.52, and 1.52% of additive, respectively. The amount of enzyme and ionic liquids used in aerogel immobilization was the same as the optimized values in the xerogel immobilization process (for each ionic liquid). Ionic liquids proved to be good additives in the enzymatic immobilization process. Xerogel, regardless of the ionic liquid, presented a greater number of use cycles and better thermal stability compared to aerogel.

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MCM-41 and MCM-48 with niobium were successfully synthesized using 1-tetradecyl-3-methylimidazolium chloride ([C14MI]Cl) as a structure-directing agent. The best Si/Nb molar ratio was chosen (Si/Nb = 20) and the CALB enzyme was immobilized in situ in the synthesized Nb-MCM. SEM micrographs showed the formation of very regular spherical agglomerates with a diameter between 0.25 and 0.75 µm. The material presented a surface area of 954 and 704 m2/g and a pore volume of 0.321 and 0.286 cm3/g, for Nb-MCM-41 and Nb-MCM-48, respectively. Also, both materials showed a pore size of 2.261 nm. The number of recycles obtained for the CALB enzyme immobilized in Nb-MCM-41 and Nb-MCM-48 was 26 recycles with a residual activity of 49.62% and 16 recycles with a residual activity of 53.01%, respectively. For both materials, enzymatic activity remained stable for 5 months of storage at room temperature and refrigeration. The supports were able to catalyze the esterification reaction at 40, 60, and 80 °C, showing industrial application in reactions that require high temperatures. This methodology allows the preparation of new highly active and selective enzyme catalysts using niobium and [C14MI]Cl. Also, the new materials can provide greater viability in processes, ensuring a longer service life of catalysts. Graphical abstract.

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In this study, the modulation of enzymatic biocatalysts were developed by the use of lipase B from Candida antarctica covalently immobilized on an eco-friendly support, cashew apple bagasse, activated with 10% glycidol-ethylenediamine-glutaraldehyde (GEG) under different immobilization strategies (5 mM or 100 mM ionic strength and in absence or presence of 0.5% (v/v) Triton X-100). The biocatalysts were characterized for thermal and organic solvents stabilities and compared with the soluble enzyme. The biocatalysts were then applied to the hydrolysis of the rac-indanyl acetate (2:1 ratio enzyme/substrate) at pH 7.0 and 30 °C for 24 h. For all the strategies evaluated, GEG promoted kinetic resolution of rac-indanyl acetate with maximum conversion (50%) and led to (R)-indanol with excellent enantiomeric excess (97%), maintaining the maximum conversion for five consecutive cycles of hydrolysis. Therefore, the use of cashew apple bagasse has proved to be a promising eco-friendly support for enzyme immobilization, since it resulted in stable biocatalysts for enzymatic kinetic resolution.

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Despite the proven therapeutic role of capsaicin in human health, its usage is still hampered by its high pungency. In this sense, nonpungent capsaicin analogues as olvanil are a feasible alternative to the unpleasant sensations produced by capsaicin while maintaining a similar pharmacological profile. Olvanil can be obtained by a lipase-catalyzed chemoenzymatic process. In the present work, recombinant Candida antarctica lipase B (CALB) was expressed in Pichia pastoris and subsequently immobilized by cross-linked enzyme aggregate (CLEA) methodology for the synthesis of olvanil. The CALB-CLEAs were obtained directly from the fermentation broth of P. pastoris without any purification step in order to assess the role of the contaminant proteins of the crude extract as co-feeders. The CALB-CLEAs were also bioimprinted to enhance the catalytic performance in olvanil synthesis. When CALB was precipitated with isopropanol, the obtained CALB-CLEAs exhibited the highest activity in the synthesis of olvanil, regardless of the glutaraldehyde concentration. The maximum product synthesis was found at 72 hr obtaining 6.8 g L-1 of olvanil with a reaction yield of 16%. When CALB was bioimprinted with olvanil, the synthesis was enhanced 1.3 times, reaching 10.7 g L-1 of olvanil at 72 hr of reaction with a reaction yield of 25%. Scanning electron microscopy images indicated different morphologies of the CLEAs depending on the precipitating agent and the template used for bioimprinting. Recombinant CALB-CLEAs obtained directly from the fermentation broth are a suitable alternative to commercial enzymatic preparations for the synthesis of olvanil in organic medium.

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Magnetic biocatalysts offer enormous advantages over traditional ones. Their ability to be isolated by means of a magnet, in combination with their extensive reuse possibilities, makes them highly attractive and competitive from the commercial point of view. In this work, magnetic biocatalysts were prepared by immobilization of Candida antarctica Lipase B (E.C. 3.1.1.3, CALB) on magnetite-lysine nanoparticles. Two methodologies were explored tending to find the optimal biocatalyst in terms of its practical implementation: I-physical adsorption of CALB followed by cross-linking, and II-covalent coupling of the lipase on the nanoparticles surface. Both procedures involved the use of glutaraldehyde (GLUT) as cross-linker or coupling agent, respectively. A range of GLUT concentrations was evaluated in method I and the optimum one, in terms of efficiency and operational stability, was chosen to induce the covalent linkage CALB-support in method II. The chosen test reaction was solvent-free ethyl oleate synthesis. Method I produced operationally unstable catalysts that deactivated totally in four to six cycles. On the other hand, covalently attached CALB (method II) preserved 60% of its initial activity after eight cycles and also retained 90% of its initial activity along 6 weeks in storage. CALB immobilization by covalent linkage using controlled GLUT concentration appears as the optimum methodology to asses efficient and stable biocatalysts. The materials prepared within this work may be competitive with commercially available biocatalysts.

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Enzymes serve as biocatalysts for innumerable important reactions, however, their application has limitations, which can in many cases be overcome by using appropriate immobilization strategies. Here, a new support for immobilizing enzymes is proposed. This hybrid organic-inorganic support is composed of chitosan-a natural, nontoxic, biodegradable, and edible biopolymer-and sodium polyphosphate as the inorganic component. Lipase B from Candida antarctica (CALB) was immobilized on microspheres by encapsulation using these polymers. The characterization of the composites (by infrared spectroscopy, thermogravimetric analysis, and confocal Raman microscopy) confirmed the hybrid nature of the support, whose external part consisted of polyphosphate and core was composed of chitosan. The immobilized enzyme had the following advantages: possibility of enzyme reuse, easy biocatalyst recovery, increased resistance to variations in temperature (activity declined from 60 °C and the enzyme was inactivated at 80 °C), and increased catalytic activity in the transesterification reactions. The encapsulated enzymes were utilized as biocatalysts for transesterification reactions to produce the compound responsible for the aroma of jasmine.

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