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Candida rugosa lipase (CRL) was immobilized by the ionic gelling technique using alginate and chitosan as encapsulating agents. An immobilization yield of 99% and an immobilization efficiency of 51% were obtained. Maximum hydrolytic activity for free and immobilized CRL was detected at 40 °C and for synthesis activity at 35 °C. The optimum pH for immobilized and free CRL hydrolysis activity was 8.0. The Vmax obtained for the hydrolysis reaction was higher for free CRL (4121.4 μmol/min/g) compared to immobilized CRL (2359.13 μmol/min/g). A Vmax of 2.24 μmol/min/g was detected for the synthetic activity of free CRL. The Km obtained for the hydrolysis reaction was higher (660.02 μmol/L) for immobilized CRL than for free CRL (403.06 μmol/L). For the synthetic activity, a Km of 234.44 μmol/L was calculated. The conversion of β-sitosterol oleate ranged from 80.85 to 96.84% for free CRL, higher than the maximum found for immobilized CRL (32%). The scale-up (scale factor: 50) with the free CRL was successfully performed, achieving a high conversion value (92%) in a 500 mL bioreactor. This conversion value was within the range predicted by the mathematical model obtained using mini reactors. These mini reactors are good models to test several conditions of enzyme reactions that are intended for large scales.

Structured lipids (SL) represent a new generation of lipids, considered bioactive compounds. Medium-chain, oleic (18:1n-9), and medium-chain fatty acid (MCFA) structured lipids (MOM-SL) were produced by acidolysis reaction in solvent-free medium with capric (10:0) and lauric (12:0) free fatty acids (FFAs) and triolein or olive oil, using Yarrowia lipolytica lipase as biocatalyst. MCFAs were rapidly incorporated into sn-1,3 SL in acidolysis reactions with triolein and olive oil, up until 30% of incorporation efficiency of capric and lauric acids in SLs. The kinetics of MCFA incorporation in MOM-SL was influenced by the FFA:TAG molar ratio, and for reactions between triolein and lauric acid, increasing FFA:TAG from 2:1 to 4:1 enhanced MCFA incorporation in SL. Y. lipolytica lipase showed a strictly 1,3-regioselective profile in acidolysis reaction, confirmed by nuclear magnetic resonance spectroscopy. Immobilization of this lipase by microencapsulation in chitosan-alginate beads resulted in similar incorporation efficiency for lauric acid with olive oil TAG and this reaction could be performed for 5 cycles without catalytic activity loss. This lipase showed promising properties as a potential biocatalyst that may be effectively used in production of bioactive structured lipids, which might be applied for prevention of metabolic and inflammatory disorders related to obesity.

Magnetic nanoparticles (Fe3O4) were used for physical adsorption of lipase from Yarrowia lipolytica IMUFRJ 50682. The optimal adsorption conditions were obtained as follows: enzyme/support 19.3 mg/g and temperature of 20 °C for standard protein. High immobilization efficiency of 99% was obtained for 4 mL of crude lipase extract (containing 0.315 mg protein/mL) and 0.02 g of magnetic nanoparticles and this biocatalyst was recycled 30 times with 70% of lipase activity in the end. Purified lipase extracts were also efficiently immobilized and ultrafiltered lipase extract (ULE) and aqueous two-phase system lipase extract (ATPS_LE) when immobilized revealed higher hydrolytic activity in relation to CLE (2.8 and 4.0 times higher, respectively). Broad pH tolerance and high thermostability could be achieved by immobilization on magnetic nanoparticles, with 40% improvement in thermodynamic parameters at 60 °C. Kinetic parameters Vmax and Km were also better for ULE (Vmax: 2.3 times higher; Km 43% reduction) and ATPS_LE (Vmax: 3.0 times higher; Km: 38% reduction) immobilized on magnetic nanoparticles in relation to CLE. These results showed that the immobilization of lipase onto magnetic nanoparticles by physical adsorption is an efficient and simple way to obtain a great catalyst.

Lipases are versatile enzymes widely used in the pharmaceutical, cosmetic, and food industries. They are green biocatalysts with a high potential for industrial use compared to traditional chemical methods. In recent years, lipases have been used to synthesize a wide variety of molecules of industrial interest, and extraordinary results have been reported. In this sense, this review describes the important role of lipases in the synthesis of phytosterol esters, which have attracted the scientific community’s attention due to their beneficial effects on health. A systematic search for articles and patents published in the last 20 years with the terms “phytosterol AND esters AND lipase” was carried out using the Scopus, Web of Science, Scielo, and Google Scholar databases, and the results showed that Candida rugosa lipases are the most relevant biocatalysts for the production of phytosterol esters, being used in more than 50% of the studies. The optimal temperature and time for the enzymatic synthesis of phytosterol esters mainly ranged from 30 to 101 °C and from 1 to 72 h. The esterification yield was greater than 90% for most analyzed studies. Therefore, this manuscript presents the new technological approaches and the gaps that need to be filled by future studies so that the enzymatic synthesis of phytosterol esters is widely developed.

Enzymes are versatile biomolecules with broad applications. Since they are biological molecules, they can be easily destabilized when placed in adverse environmental conditions, such as variations in temperature, pH, or ionic strength. In this sense, the use of protective structures, as polymeric capsules, has been an excellent approach to maintain the catalytic stability of enzymes during their application. Thus, in this review, we report the use of polymeric materials as enzyme encapsulation agents, recent technological developments related to this subject, and characterization methodologies and possible applications of the formed bioactive structures. Our search detected that the most explored methods for enzyme encapsulation are ionotropic gelation, spray drying, freeze-drying, nanoprecipitation, and electrospinning. α-chymotrypsin, lysozyme, and β-galactosidase were the most used enzymes in encapsulations, with chitosan and sodium alginate being the main polymers. Furthermore, most studies reported high encapsulation efficiency, enzyme activity maintenance, and stability improvement at pH, temperature, and storage. Therefore, the information presented here shows a direction for the development of encapsulation systems capable of stabilizing different enzymes and obtaining better performance during application.

Extracellular lipase from Yarrowia lipolytica was immobilized by ionotropic gelation with alginate and chitosan as encapsulating agents. Photomicrographs revealed a collapsed and heterogeneous surface of these microcapsules due to freeze-drying process. The optimum reaction temperature for the microencapsulated lipase (40 °C) was higher than for free lipase (35 °C) as well as the optimum pH (8.0 and 7.5, respectively). The study of the reaction kinetics showed that a higher maximum reaction rate (Vmax) (221.1 U/mg) for the free lipase in comparison to the immobilized form (175.3 U/mg). A protective effect of the microcapsule was detected in the storage of the enzyme at room temperature, as after 75 days 35% of activity was maintained for the microcapsules, while no activity remained after 15 days with the free enzyme. Lower values for inactivation constant (kd) and increase in half-life for immobilized lipase showed that lipase microencapsulation favored the thermostability of this enzyme.

Palm oil production chain generates a greasy residue in the refining stage, the Palm Oil Deodorizer Distillate (PODD), mainly composed of free fatty acids. Palm oil is also used industrially to fry foods, generating a residual frying oil (RFO). In this paper, we aimed to produce lipase from palm agro-industrial wastes using an unconventional yeast. RFO_palm, from a known source, consisted of 0.11% MAG + FFA, 1.5% DAG, and 97.5 TAG, while RFO_commercial, from a commercial restaurant, contained 6.7% of DAG and 93.3% of TAG. All palm oil wastes were useful for extracellular lipase production, especially RFO_commercial that provided the highest activity (4.9 U/mL) and productivity (465 U/L.h) in 75 h of processing time. In 48 h of process, PODD presented 2.3 U/mL of lipase activity and 48.5 U/L.h of productivity. RFO_commercial also showed the highest values for lipase associated to cell debris (843 U/g). This naturally immobilized biocatalyst was tested on hydrolysis reactions to produce Lipolyzed Milk Fat and was quite efficient, with a hydrolysis yield of 13.1% and 3-cycle reuse. Therefore, oily palm residues seem a promising alternative to produce lipases by the non-pathogenic yeast Y. lipolytica and show great potential for industrial applications.

A synthetic medium containing glucose, glycerol, yeast extract (YE), and ammonium sulfate (AS) was compared to several low-cost media in their ability to produce high emulsification index (EI). The goal was to reduce the production costs of an emulsifier with application in food oil-in-water emulsions. To this end, agro-industrial by-products were screened for bioemulsifier production from Yarrowia lipolytica. The statistical analysis showed that the EIs of media containing residual frying oil from palm oil (RFO_palm) or soybean oil (RFO_soy), residual liquid from butter production (butter whey, BWhey) or cheese production (cheese whey, CWhey), supplemented with YE and AS were similar to the EI of the synthetic medium. The replacement of YE by corn steep liquor (CSL) also resulted in similar EI, except for RFO_soy. BWhey was tested with CSL without AS and similar EI (66.8%) was detected in comparison to that of the same medium with AS (66.3%). The cell-free broth obtained after Y. lipolytica growth in BWhey+CSL was successfully used to obtain vegetable oil-in-water emulsions indicating its potential application in food products. RESUMO: O meio sintético contendo glicose, glicerol, extrato de levedura (YE) e sulfato de amônio (AS) foi comparado a meios de baixo custo para produzir alto índice de emulsificação (EI). Para este fim, os subprodutos agroindustriais foram rastreados quanto à produção de bioemulsificante por Yarrowia lipolytica. A análise estatística mostrou que EI de meios contendo óleo de fritura residual de óleo de palma (RFO_palm) ou óleo de soja (RFO_soy), líquido residual da produção de manteiga (soro de manteiga, BWhey) ou da produção de queijo (soro de queijo, CWhey), suplementado com YE e AS foram semelhantes ao EI do meio sintético. A substituição do YE por milhocina (CSL) também resultou em EI semelhante, exceto no RFO_soy. O BWhey foi testado com CSL sem AS e EI semelhante (66,8%) foi detectado em comparação com o mesmo meio com AS (66,3%). O meio isento de células obtido após o crescimento de Y. lipolytica no meio BWhey + CSL foi utilizado com sucesso para obter emulsões de óleo vegetal em água, indicando sua potencial aplicação em produtos alimentícios.

Palm oil production chain generates a greasy residue in the refining stage, the Palm Oil Deodorizer Distillate (PODD), mainly composed of free fatty acids. Palm oil is also used industrially to fry foods, generating a residual frying oil (RFO). In this paper, we aimed to produce lipase from palm agro-industrial wastes using an unconventional yeast. RFO_palm, from a known source, consisted of 0.11% MAG + FFA, 1.5% DAG, and 97.5 TAG, while RFO_commercial, from a commercial restaurant, contained 6.7% of DAG and 93.3% of TAG. All palm oil wastes were useful for extracellular lipase production, especially RFO_commercial that provided the highest activity (4.9 U/mL) and productivity (465 U/L.h) in 75 h of processing time. In 48 h of process, PODD presented 2.3 U/mL of lipase activity and 48.5 U/L.h of productivity. RFO_commercial also showed the highest values for lipase associated to cell debris (843 U/g). This naturally immobilized biocatalyst was tested on hydrolysis reactions to produce Lipolyzed Milk Fat and was quite efficient, with a hydrolysis yield of 13.1% and 3-cycle reuse. Therefore, oily palm residues seem a promising alternative to produce lipases by the non-pathogenic yeast Y. lipolytica and show great potential for industrial applications.

Fermentation of gases from biomass gasification, named syngas, is an important alternative process to obtain biofuels. Sequential experimental designs were used to increase cell growth and ethanol production during syngas fermentation by Clostridium carboxidivorans. Based on ATCC (American Type Culture Collection) 2713 medium composition, it was possible to propose a best medium composition for cell growth, herein called TYA (Tryptone-Yeast extract-Arginine) medium and another one for ethanol production herein called TPYGarg (Tryptone-Peptone-Yeast extract-Glucose-Arginine) medium. In comparison to ATCC&reg, 2713 medium, TYA increased cell growth by 77%, reducing 47% in cost and TPYGarg increased ethanol production more than four-times, and the cost was reduced by 31%. In 72 h of syngas fermentation in TPYGarg medium, 1.75-g/L of cells, 2.28 g/L of ethanol, and 0.74 g/L of butanol were achieved, increasing productivity for syngas fermentation.

The irregular disposal of urban waste oils (UWO) from homes and commercial establishments directly in sewers has become a major environmental concern nowadays. In the last years, intensive research has been carried out to seek alternatives to reduce disposal and to properly use UWO. In a search using the Scopus database with “waste cooking oil” OR “waste oil” OR “waste frying oil” OR “residual cooking oil” OR “residual frying oil” OR “residual oil” as queries (title, abstract, and key-words) from 2020 to Jully 2021, the production of biodiesel using UWO was the most frequent topic of manuscripts related to the use of this waste (43.8%). Beside this, a wide range of bioproducts with industrial interest can also be obtained using super-processed oils, such as emulsifiers, esters, bioplastics, biolubricants, among others. This review focus on the production of lipase and biosurfactants, which are bioproducts with great industrial application potential. According to the Scopus search, performed including “enzyme production” as query, lipase is the most prominent enzyme produced from UWO, and the yeast species Y. lipolytica is mainly used in those processes. One to five percent of UWO titer is used in lipase production medium, and many investigations are focused on developing co-production processes to obtain other bioproducts simultaneously. Most of the biosurfactants produced from UWO were identified as glycolipids, mainly rhamnolipids, and they are generally applied to remove oil from soil and water contamination sites.

Lipase immobilized on Yarrowia lipolytica cell debris after sonication of yeast cells (LipImDebri) was used in hydrolysis reaction as a novel strategy to produce lipolyzed milk fat (LMF). Extracellular (4732.1 U/L), intracellular (130.0 U/g), and cell debris (181.0 U/g) lipases were obtained in a 4 L bioreactor using residual frying oil as inducer in 24 h fermentation process. LipImDebri showed a good operational stability retaining 70% of lipolytic activity after the second cycle and 40% after the fourth. The highest degree of hydrolysis (28%) was obtained with 500 mg LipImDebri for 6 h of lipolysis of anhydrous milk fat. LMF produced with LipImDebri presented high contents of oleic (35.2%), palmitic (25.0%), and stearic (15.4%) acids and considerable amounts of odor-active short and medium chain fatty acids (C:4&ndash, C:10) (8.13%).

Microencapsulation of lipase from Yarrowia lipolytica IMUFRJ 50682 was performed by ionotropic gelation with sodium alginate. Sodium alginate, calcium chloride and chitosan concentrations as well as complexation time were evaluated through experimental designs to increase immobilization yield (IY) and immobilized lipase activity (ImLipA) using p-nitrophenyl laurate as substrate. To adjust both parameters (IY and ImLipA), the desirability function showed that microcapsule formation with 3.1%(w/v) sodium alginate, 0.19%(w/v) chitosan, 0.14 M calcium chloride, and 1 min complexation time are ideal for maximal immobilization yield and immobilized lipase activity. A nearly twofold enhancement in Immobilization yield and an increase up to 280 U/g of the lipase activity of the microcapsules were achieved using the experimental design optimization tool. Chitosan was vital for the high activity of this new biocatalyst, which could be reused a second time with about 50% of initial activity and for four more times with about 20% of activity.

Microencapsulation of lipase from Yarrowia lipolytica IMUFRJ 50682 was performed by ionotropic gelation with sodium alginate. Sodium alginate, calcium chloride and chitosan concentrations as well as complexation time were evaluated through experimental designs to increase immobilization yield (IY) and immobilized lipase activity (ImLipA) using p-nitrophenyl laurate as substrate. To adjust both parameters (IY and ImLipA), the desirability function showed that microcapsule formation with 3.1%(w/v) sodium alginate, 0.19%(w/v) chitosan, 0.14 M calcium chloride, and 1 min complexation time are ideal for maximal immobilization yield and immobilized lipase activity. A nearly twofold enhancement in Immobilization yield and an increase up to 280 U/g of the lipase activity of the microcapsules were achieved using the experimental design optimization tool. Chitosan was vital for the high activity of this new biocatalyst, which could be reused a second time with about 50% of initial activity and for four more times with about 20% of activity.

The α-amylase enzyme was produced by solid state fermentation (SSF) and its partition behavior in Aqueous Two-Phase Systems (ATPS) was determined using systems consisting of polyethylene glycol (PEG) and potassium phosphate buffer. To determine the best conditions for α-amylase production, a central composite design was constructed for two independent variables: fermentation time and moisture content, and for the partition study, a face-centered central composite design (CCF) with four independent variables: PEG molecular weight, pH, temperature and partition time. It was indicated that the best enzyme production conditions were a fermentation time of 35.0 hours and 45.7% moisture content. From the results obtained, it was found that increasing the polymer molecular weight promotes a reduction of the enzyme concentration in the upper phase, increasing its concentration in the lower phase. It was also observed that the partition coefficients increased with increasing pH values and a reduction in temperature.