Synthesis of hexyl butyrate (apple and citrus aroma) by Candida rugosa lipase immobilized on Diaion HP-20 using the Box-Behnken design.

This study aims at the synthesis of hexyl butyrate by Candida rugosa lipase (CRL) immobilized on Diaion HP 20. The lipase load used was 28.7 ± 2.1 mg/g (mg of lipase/g of support), whose hydrolytic activity was 132.0 ± 2.5 U/g. To obtain the maximum production of hexyl butyrate, the Box-Behnken design statistical planning was used, having as independent variables; biocatalyst concentration, temperature and acid:alcohol molar ratio and ester conversion as a dependent variable at 60, 180 and 480 min. For 60 min, 90.8% conversion was obtained at 47.25 ºC, 1:1.4 molar ratio and 17.65% of biocatalyst; 180 min, 94.5% conversion at 59.5 ºC, 1:2 molar ratio and 15.8% biocatalyst; 480 min, 95.01% conversion at 47.0 ºC, 1:2 molar ratio and 16.9% biocatalyst. CRL-Diaion HP 20 retained 60% of its initial activity after ten cycles of reactions showing potential for industrial use. The ester produced was identified by gas chromatography analyses. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01200-1.

Application of a constrained mixture design for lipase production by Penicillium roqueforti ATCC 10110 under solid-state fermentation and using agro-industrial wastes as substrate.

Solid state fermentation (SSF) simulates the natural conditions fungal growth, where the amount of water in the reaction medium must be restricted, thus limiting the use of liquid substrate. An analytical strategy to deal with this limitation is the design of blending with constraints. Thus, the objective of the work was to optimize two constrained waste mixtures for the production of lipase by Penicillium roqueforti ATCC 10110 under SSF, using different substrates that combine solid and liquid waste. For this, the best fermentation time was determined through a fermentative profile, afterwards a restricted-mix design with lower and upper limits of the components of mixture I (cocoa residue, solid palm oil residue and liquid palm oil residue) and II (cocoa residue, mango residue and palm oil residue liquid palm) was applied. By means of Pareto and contour graphs, the maximum production points of lipase in mixtures I (6.67 ± 0.34 U g-1) and II (6.87 ± 0.35 U g-1) were obtained. The restricted mixture design proved to be a promising tool in the production of lipase by P. roqueforti ATCC 10110 under SSF since the use of restrictions is useful when intending to combine solid and liquid residues in fermentation processes.

Production of a fermented solid containing lipases from Penicillium roqueforti ATCC 10110 and its direct employment in organic medium in ethyl oleate synthesis.

The production and direct employment in organic medium in the ethyl-oleate synthesis of a fermented solid (FS) containing lipases by Penicillium roqueforti ATCC 10110 (PR10110) was investigated. For the production of this FS, the solid-state fermentation of different agroindustrial waste was used, such as: cocoa shell, sugarcane bagasse, sugarcane bagasse with cocoa shell, and cocoa shell with soybean oil and nutrient solution. The response surface methodology was used to study the effect of independent variables of initial moisture content and inductor concentration, as carbon source and inducer on lipase production. The characterization of the fermented solid in organic medium was also carried out. The highest lipase activity (53 ± 5 U g-1 ) was 16% higher than that obtained with the nonoptimized conditions. The characterization studies observed high stability of the FS in organic solvents for 5 h at 30°C, as well as at different temperatures, and the residual activity was measured against triolein. The FS was also able to catalyze ethyl-oleate synthesis maintaining high relative conversion over five reaction cycles of 96 h at 40°C in n-heptane. These results are promising and highlight the use of the FS containing PR10110 lipases for the first time in biocatalytic processes.

Candida rugosa lipase immobilized on hydrophobic support Accurel MP 1000 in the synthesis of emollient esters.

OBJECTIVES: To immobilize Candida rugosa lipase in Accurel MP 1000 (CRL-AMP) by physical adsorption in organic medium and apply in the synthesis of wax esters dodecanoyl octadecanoate 1 and hexadecanoyl octadecanoate 2 in a heptane medium, as well as evaluating the stability and recyclability of CRL-AMP in six reaction cycles. RESULTS: The specific activity (Asp) for CRL-AMP was 200 ± 20 U mg-1. Its catalytic activity was 1300 ± 100 U g-1. CRL-AMP was used in the synthesis of esters in heptane medium with a 1:1 acid:alcohol molar ratio at 45 °C and 200 rpm. In synthesis 1, conversion was 62.5 ± 3.9% in 30 min at 10% m v-1 and 56.9 ± 2.8% in 54 min at 5% m v-1; while in synthesis 2, conversion was 79.0 ± 3.9% in 24 min at 10% m v-1, and 46.0 ± 2.4% in 54 min at 5% m v-1. Reuse tests after six consecutive cycles of reaction showed that the biocatalyst retained approximately 50% of its original activity for both reaction systems. CONCLUSIONS: CRL-AMP showed a high potential in the production of wax esters, since it started from low enzymatic load and high specific activities and conversions were obtained, in addition to allowing an increase in stability and recyclability of the prepared biocatalyst.

High-throughput screening for distinguishing nitrilases from nitrile hydratases in Aspergillus and application of a Box-Behnken design for the optimization of nitrilase.

Nitrilases and nitrile hydratases/amidases hydrolyze nitriles into carboxylic acids and/or amides, which are used in industrial chemical processes. In the present study, 26 microorganisms, including yeasts and filamentous fungi, in a minimum solid mineral medium supplemented with glucose and phenylacetonitrile were screened to evaluate their biocatalytic potential. Of these microorganisms, five fungi of the genus Aspergillus were selected and subjected to colorimetry studies to evaluate the production and distinction of nitrilase and nitrile hydratase/amidase enzymes. Aspergillus parasiticus Speare 7967 and A. niger Tiegh. 8285 produced nitrilases and nitrile hydratase, respectively. Nitrilase optimization was performed using a Box-Behnken design (BBD) and fungus A. parasiticus Speare 7967 with phenylacetonitrile volume (µl), pH, and carbohydrate source (starch:glucose; g/g) as independent variables and nitrilase activity (U ml-1 ) as dependent variable. Maximum activity (2.97 × 10-3  U ml-1 ) was obtained at pH 5.5, 80 µl of phenylacetonitrile, and 15 g of glucose. A. parasiticus Speare 7967 showed promise in the biotransformation of nitriles to carboxylic acids.

Thermostable trypsin-like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting.

The increased demand for cheese and the limited availability of calf rennet justifies the search for milk-clotting enzymes from alternative sources. Trypsin-like protease by Penicillium roqueforti was produced by solid-state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert-type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10-12) and an optimum temperature of 80°C, being stable at temperatures above 60°C. Enzymatic activity was maximized in the presence of Na+ (192%), Co2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin-like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses.

Optimization of lipase production by Penicillium roqueforti ATCC 10110 through solid-state fermentation using agro-industrial residue based on a univariate analysis.

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are a class of enzymes with high industrial importance. An option for the production of this enzyme is through fungal growth via solid-state fermentation (SSF). Thus, this research presents a study of lipase production by Penicillium roqueforti ATCC 10110 through SSF using cocoa bran residues (Theobroma cacao) as a substrate. To achieve maximum lipase production, fermentation time (0 to 120 h) and palm oil (PO) percentage (0 to 50%) were optimized through analysis of one factor at a time (OFAT), with lipase activity as the response. The amount of cocoa was fixed (5 g), the incubation temperature was maintained at 27 °C, and the moisture content was established at 70%. For a 72 h incubation, the highest enzyme activity achieved using SSF without adding PO was 14.67 ± 1.47 U g-1, whereas with PO (30%), it was 33.33 ± 3.33 U g-1, thus demonstrating a 44% increase in enzyme activity. Through the OFAT methodology, it was possible to confirm that supplementation with palm residue was efficient and maximized the lipase of P. roqueforti ATCC 10110.

Application of lipase immobilized on a hydrophobic support for the synthesis of aromatic esters.

The present study aimed at preparing three biocatalysts via physical adsorption of lipases from Candida rugosa (CRL), Mucor javanicus, and Candida sp. on a hydrophobic and mesoporous support (Diaion HP-20). These biocatalysts were later applied to the synthesis of aromatic esters of apple peel and citrus (hexyl butyrate), apple and rose (geranyl butyrate), and apricot and pineapple (propyl butyrate). Scanning electron microscopy and gel electrophoresis confirmed a selective adsorption of lipases on Diaion, thus endorsing simultaneous immobilization and purification. Gibbs free energy (∆G) evinced the spontaneity of the process (-17.9 kJ/mol ≤ ∆G ≤ -5.1 kJ/mol). Maximum immobilized protein concentration of 30 mg/g support by CRL. This biocatalyst was the most active in olive oil hydrolysis (hydrolytic activity of 126.0 ± 2.0 U/g) and in the synthesis of aromatic esters. Maximum conversion yield of 89.1% was attained after 150 Min for the synthesis of hexyl butyrate, followed by the synthesis of geranyl butyrate (87.3% after 240 Min) and propyl butyrate (80.0% after 150 Min). CRL immobilized on Diaion retained around 93% of its original activity after six consecutive cycles of 150 Min for the synthesis of hexyl butyrate.

Development of amperometric biosensor in modified carbon paste with enzymatic preparation based on lactase immobilized on carbon nanotubes.

ABSTRACT: The variety of products derived from milk, without or with lactose, encourages the development of more effective analytical techniques that can be applied to the quality control of both the production line and the final products. Thus, in this work an efficient and minimally invasive method for the detection of lactose was proposed, using a biosensor containing the enzyme lactase (LAC) immobilised on carbon nanotubes (CNTs) that, when reacting with lactose, emit an electrochemical signal. This biosensor was connected to a potentiostat, and its electrochemical cell was composed of the following three electrodes: reference electrode (Ag/AgCl), auxiliary electrode (platinum wire), and working electrode (biosensor) on which graphite (carbon) paste (CP), CNTs, and LAC were deposited. The transmission electron microscopy and scanning electron microscopy were used in the characterisation of the composite morphology, indicating excellent interactions between the CNTs and LAC. The sensitivity of the CP/LAC/CNT biosensor was determined as 5.67 µA cm-2.mmol-1 L and detection limits around 100 × 10-6 mol L-1 (electrode area = 0.12 cm2) and an increase in the stability of the system was observed with the introduction of CNTs because, with about 12 h of use, there was no variation in the signal (current). The results indicate that the association between the CNTs and LAC favoured the electrochemical system.

Study of morphological properties and rheological parameters of cellulose nanofibrils of cocoa shell (Theobroma cacao L.).

Cocoa shell was evaluated as a precursor for cellulose nanofibrils (NFCs) using mechanical defibrillation. Its morphology was analysed using optical microscopy and scanning electron microscopy with field emission. Rheological and mechanical behaviour were evaluated through flow curves with a strain rate ranging from 0 to 300 s-1 at 25 °C and by means of oscillatory frequency sweeps (0.01 Hz-10 Hz) and shear stress (3 Pa). The thermal-mechanical behaviour was determined by a temperature sweep with a heating rate of 3 °C min-1 and a temperature range of 25 °C-100 °C. Micrographs identified the presence of protoxilem with a mean diameter of 23.34 nm. The flow curve showed the characteristic behaviour of a pseudoplastic fluid. The storage module (G') and the loss modulus (G″) were dependent on the frequency applied, indicating that the material exhibits a weak gel characteristic. The viscoelastic characteristics were influenced by temperature. Therefore, cocoa shell is a new alternative in the production of nanocellulose.

Effect of the solid state fermentation of cocoa shell on the secondary metabolites, antioxidant activity, and fatty acids.

During cocoa (Theobroma cacao L.) processing, the accumulated cocoa shell can be used for bioconversion to obtain valuable compounds. Here, we evaluate the effect of solid-state fermentation of cacao flour with Penicillium roqueforti on secondary metabolite composition, phenol, carotenoid, anthocyanin, flavonol, and fatty acids contents, and antioxidant activity. We found that the total concentrations of anthocyanins and flavonols did not change significantly after fermentation and the phenolic compound and total carotenoid concentrations were higher. The fermentation process produced an increase in saponin concentration and antioxidant activity, as well as significant changes in the levels of oleic, linoleic, gamma-linolenic, and saturated fatty acids. Based on our findings, we propose that the reuse of food residues through solid state fermentation is viable and useful.

Enzymatic saccharification of lignocellulosic residues using cellulolytic enzyme extract produced by Penicillium roqueforti ATCC 10110 cultivated on residue of yellow mombin fruit.

The aim of this work was to enzymatic saccharification of food waste was performed by crude enzymatic cellulolytic extract produced by P. roqueforti cultivated in yellow mombin residue. The best yield of reducing sugars (259.45mgg-1) was achieved with sugarcane bagasse after 4h; the hydrolysis of corn cob, rice husk and peanut hull resulted in yields around 128-180mgg-1. The addition of 10mmolL-1 of Mn2+ potentiated the saccharification of sugarcane bagasse, in about 86%. The temperature and substrate (sugarcane bagasse) concentration parameters were optimized using a Doehlert Design and, a maximum sugar yield of 662.34±26.72mgg-1 was achieved at 62.40°C, 0.22% (w/v) of substrate, with the addition of Mn2+. Sugar yield was significantly high when compared to previous studies available in scientific literature, suggesting the use of crude cellulolytic supplemented with Mn2+ an alternative and promising process for saccharification of sugarcane bagasse.

Biotransformation of methylphenylacetonitriles by Brazilian marine fungal strain Aspergillus sydowii CBMAI 934: eco-friendly reactions.

This study reports the biotransformation of methylphenylacetonitriles by Brazilian marine filamentous fungus Aspergillus sydowii CBMAI 934 under eco-friendly reaction conditions. The phenylacetonitrile 1, 2-methylphenylacetonitrile 2, 3-methylphenylacetonitrile 3, and 4-methylphenylacetonitrile 4 were quantitatively biotransformed into 2-hydroxyphenylacetic 1a, 2-methylphenylacetic acid 2a, 3-methylphenylacetic acid 3a, and 4-methylphenylacetic acid 4a by enzymatic processes using whole cell as biocatalyst. The marine fungus A. sydowii CBMAI 934 is thus a promising biocatalyst for the preparation of important carboxylic acids under mild conditions (pH 7.5 and 32 °C) from nitrile compounds.

Biotransformation of phenylacetonitrile to 2-hydroxyphenylacetic acid by marine fungi.

Marine fungi belonging to the genera Aspergillus, Penicillium, Cladosporium, and Bionectria catalyzed the biotransformation of phenylacetonitrile to 2-hydroxyphenylacetic acid. Eight marine fungi, selected and cultured with phenylacetonitrile in liquid mineral medium, catalyzed it quantitative biotransformation to 2-hydroxyphenylacetic acid. In this study, the nitrile group was firstly hydrolysed, and then, the aromatic ring was hydroxylated, producing 2-hydroxyphenylacetic acid with 51 % yield isolated. In addition, the 4-fluorophenylacetonitrile was exclusively biotransformed to 4-fluorophenylacetic acid by Aspergillus sydowii Ce19 (yield = 51 %). The enzymatic biotransformation of nitriles is not trivial, and here, we describe an efficient method for production of phenylacetic acids in mild conditions.