Trends in the Use of Lipases: A Systematic Review and Bibliometric Analysis.

Scientific mapping using bibliometric data network analysis was applied to analyze research works related to lipases and their industrial applications, evaluating the current state of research, challenges, and opportunities in the use of these biocatalysts, based on the evaluation of a large number of publications on the topic, allowing a comprehensive systematic data analysis, which had not yet been conducted in relation to studies specifically covering lipases and their industrial applications. Thus, studies involving lipase enzymes published from 2018 to 2022 were accessed from the Web of Science database. The extracted records result in the analysis of terms of bibliographic compatibility among the articles, co-occurrence of keywords, and co-citation of journals using the VOSviewer algorithm in the construction of bibliometric maps. This systematic review analysis of 357 documents, including original and review articles, revealed studies inspired by lipase enzymes in the research period, showing that the development of research, together with different areas of knowledge, presents good results related to the applications of lipases, due to information synchronization. Furthermore, this review showed the main challenges in lipase applications regarding increased production and operational stability; establishing well-defined evaluation criteria, such as cultivation conditions, activity, biocatalyst stability, type of support and reactor; thermodynamic studies; reuse cycles; and it can assist in defining goals for the development of successful large-scale applications, showing several points for improvement of future studies on lipase enzymes.

Selection and validation of reference genes for RT-qPCR gene expression studies in Candida viswanathii cultivated under different grown conditions.

The properties presented by Candida viswanathii's lipases turn this specie into a promising producer of potentially applicable lipases in several industrial sectors, such as: food, textiles, in the oleochemical and paper industries, and also in different pharmaceutical applications. However, studies for elucidating growth and developmental processes at the molecular level in this species are still incipient. Performing such kinds of studies often rely on the use of the RT-qPCR, which is a highly sensitivity technique, but whose parameters must be carefully planned for achieving reliable data. Among the crucial parameters required for achieving reliable results through this technique, the use of appropriated and validated reference genes is one the most important, constituting a bottleneck, mainly in species where molecular studies are scarce. Thus, the aim of this study was to determine the best reference genes for RT-qPCR gene expression studies in C. viswanathii grown in culture media containing four different carbon sources (Olive oil, Triolein, Tributyrin, and Glucose). Eleven candidate reference genes (ACT, GPH1, AGL9, RPB2, SAP1, PGK1, TAF10, UBC13, TFC1, UBP6, and FBA1) were analyzed for their expression patterns and stability. Analysis of gene expression stability was performed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms, and validation of the results was performed through analyzing the expression of a lipase gene, CvLIP4. Analyzing the four treatments together, CvACT and CvRPB2 constituted the best reference gene pair. When treatments are analyzed individually, CvRPB2/CvACT, CvFBA1/CvAGL9, CvPGK1/CvAGL9 and CvACT/CvRPB2 were the best reference gene pairs for the culture media containing olive oil, triolein, tributyrin, and glucose as carbon sources, respectively. These results are essential and form the basis for the development of relative gene expression studies in C. viswanathii, since adequate reference genes are crucial for the reliability of RT-qPCR data.

Optimization of ß-Fructofuranosidase Production from Agrowaste by Aspergillus carbonarius and Its Application in the Production of Inverted Sugar.

RESEARCH BACKGROUND: Microbial ß-fructofuranosidases are widely employed in food industry to produce inverted sugar or fructooligosaccharides. In this study, a newly isolated Aspergillus carbonarius PC-4 strain was used to optimize the ß-fructofuranosidase production in a cost-effective process and the sucrose hydrolysis was evaluated to produce inverted sugars. EXPERIMENTAL APPROACH: Optimization of nutritional components of culture medium was carried out using simplex lattice mixture design for 72 and 120 h at 28 °C. One-factor-at-a-time methodology was used to optimize the physicochemical parameters. Crude enzyme was used for sucrose hydrolysis at different concentrations. RESULTS AND CONCLUSIONS: The optimized conditions of enzyme production were achieved from cultivations containing pineapple crown waste (1.3%, m/V) and yeast extract (0.3%, m/V) after 72 h with an enzyme activity of 9.4 U/mL, obtaining R2=91.85%, R2 adjusted=85.06%, highest F-value (13.52) and low p-value (0.003). One-factor-at-a-time used for optimizing the physicochemical conditions showed optimum temperature (20 °C), pH (5.5), agitation (180 rpm) and time course (72 h) with a 3-fold increase of enzyme production. The invertase-induced sucrose hydrolysis showed the maximum yield (3.45 mmol of reducing sugars) using 10% of initial sucrose concentration. Higher sucrose concentrations caused the inhibition of invertase activity, possibly due to the saturation of substrate or formation of sucrose aggregates, making it difficult for the enzyme to access sucrose molecules within the created clusters. Therefore, a cost-effective method was developed for the invertase production using agroindustrial waste and the produced enzyme can be used efficiently for inverted sugar production at high sucrose concentration. NOVELTY AND SCIENTIFIC CONTRIBUTION: This study presents an efficient utilization of pineapple crown waste to produce invertase by a newly isolated Aspergillus carbonarius PC-4 strain. This enzyme exhibited a good potential for inverted sugar production at high initial sucrose concentration, which is interesting for industrial applications.

Effect of co-encapsulation using a calcium alginate matrix and fructooligosaccharides with gelatin coating on the survival of Lactobacillus paracasei cells.

The demand for functional foods is increasing each year because consumers are gaining awareness about the importance of a healthy diet in the proper functioning of the body. Probiotics are among the most commonly known, commercialized, and studied foods. However, the loss of viability of probiotic products is observed during their formulation, processing, and storage. This study aimed to investigate the co-encapsulation of two Lactobacillus paracasei probiotic strains (LBC81 and ELBAL) with fructooligosaccharides (FOS) in a calcium alginate matrix using extrusion technology with gelatin as a coating material. The viability of the strains under gastrointestinal conditions and in storage at low temperature was also assessed. An immobilization yield of more than 59% was observed for both bacterial strains. Exposure to 2% biliary salts led to a decrease in the viability of free cells in the two L. paracasei strains, whereas the viability of microencapsulated cells increased up to 47%. After 35 days of storage at 4°C, the population of free cells was reduced, but microencapsulated cells remained stable after storage at low temperature. LBC81 bacteria microencapsulated with 1.5% FOS coated with gelatin were the most resistant to the stressful environments tested. Therefore, these results showed that co-encapsulation with FOS in a calcium alginate matrix coated with gelatin improved L. paracasei survival and may be useful for the development of more resistant probiotics and new functional foods.

Comparative assessment of three RNA extraction methods for obtaining high-quality RNA from Candida viswanathii biomass.

Isolating high quality RNA is a limiting factor in molecular analysis, since it is the base for transcriptional studies. The RNA extraction method can directly affect the RNA quality and quantity, as well as, its overall cost. The industrial importance of the yeast genus Candida in several sectors comes from their capacity to produce Lipases. These enzymes are one of the main metabolites produced by some Candida species, and it has been shown that Candida yeast can biodegrade petroleum hydrocarbons and diesel oil from biosurfactants that they can produce, a feature that turns these organisms into potential combatants for bioremediation techniques. Thus, this study aimed to determine an efficient method for isolating high quality RNA from Candida viswanathii biomass. To achieve this aim, three different RNA extraction methods, TRIzol, Hot Acid Phenol, and CTAB (Cetyltrimethylammonium Bromide), were tested. The three tested methods allowed the isolation of high-quality RNA from C. viswanathii biomass and yielded suitable RNA quantity for carrying out RT-qPCR studies. In addition, all methods displayed high sensitivity for the expression analysis of the CvGPH1 gene through RT-qPCR, with TRIzol and CTAB showing the best results and the CTAB method displaying the best cost-benefit ratio (US$0.35/sample).

Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides.

OBJECTIVE: Extracellular fructosyltransferase (FTase, E.C.2.4.1.9) from Aspergillus oryzae IPT-301 was immobilized on silica gel by adsorption and biochemically characterized aiming at its application in the transfructosylation reaction of sucrose for the production of fructooligossaccarides (FOS). RESULTS: The transfructosylation activity (AT) was maximized by the experimental design in function of the reaction pHs and temperatures. The AT of the immobilized enzyme showed the kinetics behavior described by the Hill model. The immobilized FTase showed reuse capacity for six consecutive reaction cycles and higher pH and thermal stability than the soluble enzyme. CONCLUSION: These results suggest a high potential of application of silica gel as support for FTase immobilization aiming at FOS production.

Polyhydroxyalkanoate Synthesis by Burkholderia glumae into a Sustainable Sugarcane Biorefinery Concept.

Polyhydroxyalkanoate (PHA) bioplastic was synthesized by Burkholderia glumae MA13 from carbon sources and industrial byproducts related to sugarcane biorefineries: sucrose, xylose, molasses, vinasse, bagasse hydrolysate, yeast extract, yeast autolysate, and inactivated dry yeast besides different inorganic nitrogen sources. Sugarcane molasses free of pre-treatment was the best carbon source, even compared to pure sucrose, with intracellular polymer accumulation values of 41.1-46.6% cell dry weight. Whereas, xylose and bagasse hydrolysate were poor inducers of microbial growth and polymer synthesis, the addition of 25% (v/v) sugarcane vinasse to the culture media containing molasses was not deleterious and resulted in a statistically similar maximum polymer content of 44.8% and a maximum PHA yield of 0.18 g/g, at 34°C and initial pH of 6.5, which is economic and ecologically interesting to save water required for the industrial processes and especially to offer a fermentative recycling for this final byproduct from bioethanol industry, as an alternative to its inappropriate disposal in water bodies and soil contamination. Ammonium sulfate was better even than tested organic nitrogen sources to trigger the PHA synthesis with polymer content ranging from 29.7 to 44.8%. GC-MS analysis showed a biopolymer constituted mainly of poly(3-hydroxybutyrate) although low fractions of 3-hydroxyvalerate monomer were achieved, which were not higher than 1.5 mol% free of copolymer precursors. B. glumae MA13 has been demonstrated to be adapted to synthesize bioplastics from different sugarcane feedstocks and corroborates to support a biorefinery concept with value-added green chemicals for the sugarcane productive chain with additional ecologic benefits into a sustainable model.

ß-Fructofuranosidase and ß -D-Fructosyltransferase from New Aspergillus carbonarius PC-4 Strain Isolated from Canned Peach Syrup: Effect of Carbon and Nitrogen Sources on Enzyme Production.

ß-fructofuranosidase (invertase) and ß-D-fructosyltransferase (FTase) are enzymes used in industrial processes to hydrolyze sucrose aiming to produce inverted sugar syrup or fructooligosaccharides. In this work, a black Aspergillus sp. PC-4 was selected among six filamentous fungi isolated from canned peach syrup which were initially screened for invertase production. Cultivations with pure carbon sources showed that invertase and FTase were produced from glucose and sucrose, but high levels were also obtained from raffinose and inulin. Pineapple crown was the best complex carbon source for invertase (6.71 U/mL after 3 days of cultivation) and FTase production (14.60 U/mL after 5 days of cultivation). Yeast extract and ammonium chloride nitrogen sources provided higher production of invertase (6.80 U/mL and 6.30 U/mL, respectively), whereas ammonium nitrate and soybean protein were the best nitrogen sources for FTase production (24.00 U/mL and 24.90 U/mL, respectively). Fermentation parameters for invertase using yeast extract were Y P/S = 536.85 U/g and P P = 1.49 U/g/h. FTase production showed values of Y P/S = 2,627.93 U/g and P P = 4.4 U/h using soybean protein. The screening for best culture conditions showed an increase of invertase production values by 5.10-fold after 96 h cultivation compared to initial experiments (fungi bioprospection), while FTase production increased by 14.60-fold (44.40 U/mL) after 168 h cultivation. A. carbonarius PC-4 is a new promising strain for invertase and FTase production from low cost carbon sources, whose synthesized enzymes are suitable for the production of inverted sugar, fructose syrups, and fructooligosaccharides.

Agroindustrial Wastes as Alternative for Lipase Production by Candida viswanathii under Solid-State Cultivation: Purification, Biochemical Properties, and Its Potential for Poultry Fat Hydrolysis.

The aims of this work were to establish improved conditions for lipase production by Candida viswanathii using agroindustrial wastes in solid-state cultivation and to purify and evaluate the application of this enzyme for poultry fat hydrolysis. Mixed wheat bran plus spent barley grain (1 : 1, w/w) supplemented with 25.0% (w/w) olive oil increased the lipase production to 322.4%, compared to the initial conditions. When olive oil was replaced by poultry fat, the highest lipase production found at 40% (w/w) was 31.43 U/gds. By selecting, yeast extract supplementation (3.5%, w/w), cultivation temperature (30°C), and substrate moisture (40%, w/v), lipase production reached 157.33 U/gds. Lipase was purified by hydrophobic interaction chromatography, presenting a molecular weight of 18.5 kDa as determined by SDS-PAGE. The crude and purified enzyme showed optimum activity at pH 5.0 and 50°C and at pH 5.5 and 45°C, respectively. The estimated half-life at 50°C was of 23.5 h for crude lipase and 6.7 h at 40°C for purified lipase. Lipase presented high activity and stability in many organic solvents. Poultry fat hydrolysis was maximum at pH 4.0, reaching initial hydrolysis rate of 33.17 mmol/L/min. Thus, C. viswanathii lipase can be successfully produced by an economic and sustainable process and advantageously applied for poultry fat hydrolysis without an additional acidification step to recover the released fatty acids.

Acid lipase from Candida viswanathii: production, biochemical properties, and potential application.

Influences of environmental variables and emulsifiers on lipase production of a Candida viswanathii strain were investigated. The highest lipase activity (101.1 U) was observed at 210 rpm, pH 6.0, and 27.5°C. Other fermentation parameters analyzed showed considerable rates of biomass yield (Y L/S = 1.381 g/g), lipase yield (Y L/S = 6.892 U/g), and biomass productivity (P X = 0.282 g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield (Y L/S ) of 10.061 U/g. Crude lipase presented optimal activity at acid pH of 3.5, suggesting a new lipolytic enzyme for this genus and yeast in general. In addition, crude lipase presented high stability in acid conditions and temperature between 40 and 45°C, after 24 h of incubation in these temperatures. Lipase remained active in the presence of organic solvents maintaining above 80% activity in DMSO, methanol, acetonitrile, ethanol, acetone, 1-propanol, isopropanol, and 2-propanol. Effectiveness for the hydrolysis of a wide range of natural triglycerides suggests that this new acid lipase has high potential application in the oleochemical and food industries for hydrolysis and/or modification of triacylglycerols to improve the nutritional properties.

Production, purification, and characterization of a major Penicillium glabrum xylanase using Brewer's spent grain as substrate.

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn(2+) and the reducing agents ß -mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg(2+), Zn(2+), and Cu(2+) as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.