Enhanced keratinase production by Bacillus subtilis amr using experimental optimization tools to obtain feather protein lysate for industrial applications.

The poultry industry produces millions of tons of feathers waste that can be transformed into valuable products through bioprocess. The study describes the enhanced keratinase and feather hydrolysate production by Bacillus subtilis AMR. The metabolism of each microorganism is unique, so optimization tools are essential to determine the best fermentation parameters to obtain the best process performance. The evaluation of different propagation media indicated the constitutive production of two keratinases of approximately 80 kDa. The combination of Mn2+, Ca2+, and Mg2+ at 0.5 mM improved the keratinolytic activity and feather degradation 1.5-fold, while Cu2+ inhibited the enzymatic activity completely. Replace yeast extract for sucrose increased the feather hydrolysate production three times. The best feather concentration for hydrolysate production was 1.5% with an inoculum of 108 CFU/mL and incubation at 30 °C. None of the inorganic additional nitrogen sources tested increased hydrolysate production, although (NH4)2SO4 and KNO3 improved enzymatic activity. The optimization process improved keratinolytic activity from 205.4 to 418.7 U/mL, the protein concentration reached 10.1 mg/mL from an initial concentration of 3.9 mg/mL, and the feather degradation improved from 70 to 96%. This study characterized keratinase and feather hydrolysate production conditions offering valuable information for exploring and utilizing AMR keratinolytic strain for feather valorization. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03153-y.

Production, concentration and partial characterization of an enzymatic extract produced by an Aspergillus niger mutant in solid state fermentation.

An enzymatic extract from Aspergillus niger 3T5B8 was produced by Solid State Fermentation (SSF) in aerated columns, using wheat bran as substrate. A combination of extracts produced using three different process conditions varying temperature, pH and aeration formed the final extract (Mixture). The Mixture was concentrated by an ultrafiltration process that partially purified and provided an efficient recovery of the enzymatic activities of xylanase (88.89%), polygalacturonase (89.3%), ß-glucosidase (93.15%), protease (98.68%) and carboxymethylcellulase (CMCase) (98.93%). SDS-PAGE analysis showed 15 visible protein bands in the crude and concentrated Mixture with molecular weights ranging from 15.1 to 104.6 kDa. Thin layer chromatography confirmed the effective action of ß-glucosidase and xylanase hydrolysis activities over cellobiose and xylan, respectively. A central composite design (CCD) with two variables and four replicates at the center points was used to determine the optimal temperature and pH for CMCase and ß-glucosidase. The optimal temperature was 78.9 °C and pH 3.8 for CMCase and 52.8 °C and pH 4.8 for ß-glucosidase, respectively.

Lipase Production by Aspergillus niger C by Submerged Fermentation

Abstract This study aimed to evaluate the effects of variables on the process of lipases production by Aspergillus niger C by submerged fermentation (SmF). The production assays were performed in shake flasks for 72 hours at 150 rpm and 32°C. First, a fractional factorial design 25-1 (FFD) was carried out to evaluate the effect of the following process variables: sucrose, ammonium sulphate, soybean oil, yeast extract concentration and pH. After the selection of the variables that significantly influenced the lipase production, a central composite rotational design 22 (CCRD) was used, aiming to find the most favorable operational conditions. The selected assay condition (15.0 g.L-1 sucrose, 4.0 g.L-1 ammonium sulphate, 4.0 g.L-1 soybean oil and 1.0 g.L-1 yeast extract at pH 5.0) was the one that presented a lipase activity of 27.46 U.mL-1. It was very close to that best assay (30.76 U.mL-1), but using half of the inducer concentration, consequently reducing process cost. The kinetics of lipase production showed that the highest specific activity was 57.17 U.mg-1. The pH and temperature effects on lipase activity produced in this study was investigated. The optimum activity was found in a more acidic pH (5.0-6.0) and 55°C.

Cassava Pulp Enzymatic Hydrolysate as a Promising Feedstock for Ethanol Production

ABSTRACT The aim of this study was to produce bioethanol from enzymatic hydrolysates of cassava pulp, a by-product of cassava flour manufacturing, using an alcohol-tolerant Saccharomyces cerevisiae strain. First, the best operational condition of the starch hydrolysis process was determined through a complete factorial design (24), with triplicates at the central point. The independent variables evaluated were: the concentrations of α-amylase (Termamyl 2X) and glucoamylase (AMG 300L) and both liquefaction and saccharification times. The most favorable hydrolysis condition in the assay was achieved using 0.517 mL AMG.g starch-1 and 0.270 mL Termamyl.g starch-1, with liquefaction and saccharification times of 1 and 2 h, respectively. The broth obtained at this hydrolysis condition contained a high glucose concentration (160 g.L-1). Once the best reaction conditions were determined, fermentation tests were carried out in a 3 L bioreactor, in a batch system, at 30 °C, 100 rpm and pH 5.5, using 3 g.L-1 (dry biomass) of yeast as inoculum. After 24 h of fermentation, an ethanol concentration of 68 g.L-1 was obtained, with 0.48 ethanol yield and 2.83 g.L-1.h-1 productivity. These results indicate the potential use of cassava pulp, a by-product of cassava flour industries in Brazil, as a raw material for bioethanol production.

Use of spectroscopic and imaging techniques to evaluate pretreated sugarcane bagasse as a substrate for cellulase production under solid-state fermentation.

The enzymatic cocktail of cellulases is one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels and other chemicals. Here, the influence of liquid hot water, dilute acid, alkali, and combined acid/alkali pretreatments on sugarcane bagasse (SCB) used for cellulase production was investigated by means of spectroscopic and imaging techniques. Chemical composition and structural characteristics, such as crystallinity (determined by X-ray diffraction), functional groups (Fourier transform infrared spectroscopy), and microstructure (scanning electron microscopy), were used to correlate SCB pretreatments with enzymatic biosynthesis by a strain of the filamentous fungus Aspergillus niger under solid-state fermentation. The combined acid/alkali pretreatment resulted in a SCB with higher cellulose content (86.7%). However, the high crystallinity (74%) of the resulting biomass was detrimental to microbial uptake and enzyme production. SCB pretreated with liquid hot water yielded the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and xylanase activities (0.4, 14.9, and 26.1 U g(-1), respectively). The results showed that a suitable pretreatment for SCB to be used as a substrate for cellulase production should avoid severe conditions in order to preserve amorphous cellulose and to enhance the physical properties that assist microbial access.

Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains.

The viability of converting biomass into biofuels and chemicals still requires further development towards the reduction of the enzyme production costs. Thus, there is a growing demand for the development of efficient procedures for selection of cellulase-producing microorganisms. This work correlates qualitative screening using agar plate assays with quantitative measurements of cellulase production during cultivation under solid-state fermentation (SSF). The initial screening step consisted of observation of the growth of 78 preselected strains of the genus Trichoderma on plates, using microcrystalline cellulose as carbon source. The 49 strains that were able to grow on this substrate were then subjected to a second screening step using the Congo red test. From this test it was possible to select 10 strains that presented the highest enzymatic indices (EI), with values ranging from 1.51 to 1.90. SSF cultivations using sugarcane bagasse and wheat bran as substrates were performed using selected strains. The CG 104NH strain presented the highest EGase activity (25.93 UI·g(-1)). The EI results obtained in the screening procedure using plates were compared with cellulase production under SSF. A correlation coefficient (R(2)) of 0.977 was obtained between the Congo red test and SSF, demonstrating that the two methodologies were in good agreement.

Selection of cellulolytic fungi isolated from diverse substrates

The aim of the present work was to select filamentous fungi isolated from diverse substrates to obtain the strains with potential to produce the hydrolytic enzymes. From a total of 215 strains, seven strains from the soils, six from the plants and one from sugarcane bagasse were selected and identified as belonging to the Trichoderma, Penicillium and Aspergillus genera. The best hydrolytic activities obtained by semi-solid fermentation using these strains were approximately: 35; 1; 160; 170 and 120 U/gdm (CMCase, FPase, β-glucosidase, xylanase and polygalacturonase, respectively), demonstrating their potential to synthesize the enzymes compared with the results reported in the literature.

Production of Biomass-Degrading Multienzyme Complexes under Solid-State Fermentation of Soybean Meal Using a Bioreactor.

Biomass-degrading enzymes are one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels. This work evaluates the effects of operational conditions on biomass-degrading multienzyme production by a selected strain of Aspergillus niger. The fungus was cultivated under solid-state fermentation (SSF) of soybean meal, using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity, and initial substrate moisture content on multienzyme (FPase, endoglucanase, and xylanase) production were evaluated using a statistical design methodology. Highest production of FPase (0.55 IU/g), endoglucanase (35.1 IU/g), and xylanase (47.7 IU/g) was achieved using an initial substrate moisture content of 84%, an inlet air humidity of 70%, and a flow rate of 24 mL/min. The enzymatic complex was then used to hydrolyze a lignocellulosic biomass, releasing 4.4 g/L of glucose after 36 hours of saccharification of 50 g/L pretreated sugar cane bagasse. These results demonstrate the potential application of enzymes produced under SSF, thus contributing to generate the necessary technological advances to increase the efficiency of the use of biomass as a renewable energy source.

Keratinase Production by Three Bacillus spp. Using Feather Meal and Whole Feather as Substrate in a Submerged Fermentation.

Three Bacillus species (B. subtilis LFB-FIOCRUZ 1270, B. subtilis LFB-FIOCRUZ 1273, and B. licheniformis LFB-FIOCRUZ 1274), isolated from the poultry industry, were evaluated for keratinase production using feathers or feather meal as the sole carbon and nitrogen sources in a submerged fermentation. The three Bacillus spp. produced extracellular keratinases and peptidases after 7 days. Feather meal was the best substrate for keratinase and peptidase production in B. subtilis 1273, with 412 U/mL and 463 U/ml. The three strains were able to degrade feather meal (62-75%) and feather (40-95%) producing 3.9-4.4 mg/ml of soluble protein in feather meal medium and 1.9-3.3 mg/ml when feather medium was used. The three strains produced serine peptidases with keratinase and gelatinase activity. B. subtilis 1273 was the strain which exhibited the highest enzymatic activity.

Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.

In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers completely after 7 days at room temperature and produced the highest levels of keratinase (446 U ml(-1)). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15-140 kDa range, and B. cereus produced a keratinase of ~200 kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40-50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50-70°C and pH 7.0-11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate.

Finding stable cellulase and xylanase: evaluation of the synergistic effect of pH and temperature.

Ethanol from lignocellulosic biomass has been recognized as one of the most promising alternatives for the production of renewable and sustainable energy. However, one of the major bottlenecks holding back its commercialization is the high costs of the enzymes needed for biomass conversion. In this work, we studied the enzymes produced from a selected strain of Aspergillus niger under solid state fermentation. The cellulase and xylanase enzymatic cocktail was characterized in terms of pH and temperature by using response surface methodology. Thermostability and kinetic parameters were also determined. The statistical analysis of pH and temperature effects on enzymatic activity showed a synergistic interaction of these two variables, thus enabling to find a pH and temperature range in which the enzymes have a higher activity. The results obtained allowed the construction of mathematical models used to predict endoglucanase, ß-glucosidase and xylanase activities under different pH and temperature conditions. Optimum temperature values for all three enzymes were found to be in the range between 35°C and 60°C, and the optimum pH range was found between 4 and 5.5. The methodology employed here was very effective in estimating enzyme behavior under different process conditions.

Purification and characterisation of an extracellular phytase from Aspergillus niger 11T53A9

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3 percent referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55ºC. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K M = 54 µmol l-1 and k cat = 190 sec-1 at pH 5.0 and 37ºC. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 to finally Ins(2)P.

Purification and characterisation of an extracellular phytase from Aspergillus niger 11T53A9.

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3% referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be KM = 54 µmol l(-1) and kcat = 190 sec(-1) at pH 5.0 and 37°C. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 to finally Ins(2)P.

Utilization of agroindustrial residues for lipase production by solid-state fermentation / Utilização de resíduos agroindustriais para produção de lipase por fermentação em estado sólido

The aim of this work was to produce lipases by solid-state fermentation (SSF) using, as substrate, agroindustrial residue supplemented with by-products from corn oil refining process or olive oil. For a group of ten fungi strains selected in the first steps, the lipase activity obtained by SSF varied from 7.7 to 58.6 U/g of dry substrate (gds). Among the evaluated strains, the Aspergillus niger mutant 11T53A14 was selected by presenting the best enzymatic production. For the fermentation tests, two substrates were also investigated: wheat bran and corn cob, both supplemented with olive oil. The best results were obtained with wheat bran. Additionally, three industrial by-products from corn oil refining (soapstock, stearin and fatty acids) were evaluated as substitutes to the olive oil in the function of lipases production inducer. Among them, soapstock and stearin were the best inducers, whereas fatty acids presented an inhibitor effect. The highest lipase activities using soapstock, stearin and fatty acids were 62.7 U/gds, 37.7 U/gds and 4.1 U/gds, respectively.

O objetivo deste trabalho foi produzir lipases por fermentação em estado sólido (FES) utilizando, como substrato, resíduo agroindustrial enriquecido com subprodutos do processo de refino do óleo de milho ou óleo de oliva. Para um conjunto de dez linhagens de fungos selecionadas nas primeiras etapas, a atividade lipásica obtida por FES variou de 7,7 a 58,6 U/g de substrato seco (gss). Dentre as linhagens avaliadas, o mutante Aspergillusniger 11T53A14 foi selecionado por apresentar a melhor produção enzimática.Para os testes de fermentação, dois substratos foram investigados: farelo de trigo e sabugo de milho, ambos enriquecidos com óleo de oliva. Nestes testes, os melhores resultados foram obtidos com farelo de trigo. Adicionalmente, três subprodutos industriais do refino do óleo de milho (borra, estearina e ácidos graxos) foram avaliados como substitutos do óleo de oliva na função de indutor para a produção de lipases. Dentre eles, borra e estearina demonstraram ser melhores indutores, enquanto ácidos graxos apresentaram um efeito inibidor. As mais altas atividades lipásicas utilizando borra, estearina e ácidos graxos foram 62,7 U/gss, 37,7 U/gss e 4,1 U/gss, respectivamente.

Lipase production in solid-state fermentation monitoring biomass growth of aspergillus niger using digital image processing.

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.

Utilization of agroindustrial residues for lipase production by solid-state fermentation.

The aim of this work was to produce lipases by solid-state fermentation (SSF) using, as substrate, agroindustrial residue supplemented with by-products from corn oil refining process or olive oil. For a group of ten fungi strains selected in the first steps, the lipase activity obtained by SSF varied from 7.7 to 58.6 U/g of dry substrate (gds). Among the evaluated strains, the Aspergillus niger mutant 11T53A14 was selected by presenting the best enzymatic production. For the fermentation tests, two substrates were also investigated: wheat bran and corn cob, both supplemented with olive oil. The best results were obtained with wheat bran. Additionally, three industrial by-products from corn oil refining (soapstock, stearin and fatty acids) were evaluated as substitutes to the olive oil in the function of lipases production inducer. Among them, soapstock and stearin were the best inducers, whereas fatty acids presented an inhibitor effect. The highest lipase activities using soapstock, stearin and fatty acids were 62.7 U/gds, 37.7 U/gds and 4.1 U/gds, respectively.

Tanase: conceitos, produção e aplicação / Tannase: concepts, production and application

O objetivo deste trabalho foi efetuar revisão do conhecimento sobre tanase, apontando necessidades e perspectivas de utilização, em especial para a área de alimentos. Foram enfatizados tópicos como importância fisiológica da tanase e seu mecanismo de ação, sua produção por microorganismos, os métodos analíticos para sua determinação, a sua produção por fermentãção, a regulação da biosíntese, suas características e propriedades, a imobilização e suas aplicações. A principal aplicação dessa enzima encontra-se na produção de chás instantâneos, porém outros campos potenciais podem ser vislumbrados como sua utilização para elaboração de rações animais com maior valor nutricional, produção de compostos antioxidantes e produçaõ de ácido gálico

Influence of metal ions on pellet morphology and polygalacturonase synthesis by Aspergillus niger 3T5B8

The effects of cations addition on pellet morphology and polygalacturonase (PG) synthesis by Aspergillus niger 3T5B8 were studied and compared with a control system. Fe(II), Cu(II), Zn(II) and Mn(II) were added to the fermentation medium separately, and also as combined groups of cations. The addition of Fe2+ and/or Zn2+ ions was significantly positive to the enzyme production. A positive effect in the biomass content, however, was only obtained when the same metal ions were added separately. On the other hand, Cu2+ and Mn2+ ions had almost no effect on these parameters. The morphology of the pellets was studied by image processing techniques. Small pellets with small cores were usually obtained when Fe2+ and Zn2+ ions were individually or collectively added to the medium. The pellets produced in media containing Fe2+ or Zn2+ ions were compact, while the ones produced in a medium containing both cations were considered diffuse. Autolysis of the core was observed for large control pellets, due to the deficient nutrient transfer to the interior of the pellet. The pellets obtained in a medium containing both Fe2+ and Zn2+ ions were high enzyme producers, probably due to a loose morphology, induced by the presence of combined groups of metal ions in the medium, favoring the nutrient transfer.

Comparaçäo entre os tratamentos com tanase e com gelatina para clarificaçäo do suco de caju (Anacardium occidentale L) / Comparison of the cashew apple (Anacardium occidentale L) juice clarification with tannase and gelatin

Comparou-se o tratamento da polpa do caju com tanase e com gelatina, visando à reduçäo da turbidez e do teor de taninos. Os resultados indicaram que a reduçäo das concentraçöes de taninos totais, hidroslisáveis, proantocianidinas e turbidez ficaram em 30 por cento, 50 por cento, 32 por cento e 94 por cento para sucos tratados com gelatina e 46 por cento 88 port cento e 2 por cento e 88 por cento para a bolpa tratada com tanase, respectivamente. Deste modo, o tratamento com gelatina foi eficiente para remoçäo de proantocianidinas, enquanto que o tratamento enzimático foi mais eficiente na reduçäo de taninos hidrolisáveis. Os produtos obtidos näo apresentaram diferenças vissuais. Estudo comparativo de pré-vialidadeeconömica mostrou que o investimento de capital para obtençäo do suco de caju clarificado com enzima é menor que para o suco clarificado com gelatina. Entretanto, o custo de produçäo do suco de caju clarificado com gelatina é menor devido ao preço elevado da enzima tanase