Over the last decades researchers have focused to enhance the electrocatalytic activity of the bi... more Over the last decades researchers have focused to enhance the electrocatalytic activity of the biosystems, increasing the energy generation and electronic conductivity into the biofilms. The use of enzymes immobilized onto the surface of electrodes as the main catalyst in biological fuel cells has been extensively reported. A biofuel cell provides a means to obtain clean, renewable energy and have great potential to be used as alternative energy source for low power devices. There still a lot of important issues in the development of these device, one of them, is to obtain the maximum energy from the fuel. In order to enhance the power density of enzymatic biofuel cell (EBFC) it is necessary to increase the degree of oxidation of the fuel, so that all the electrons from the fuel can be harvested. One approach is the immobilization of a group of enzymes to perform a cascade in order to maximize the fuel oxidation. Recently, we have concentrated our goal to the develop hybrid biofilms employing MWCNT in order to immobilize efficiently the enzymes. In this context we added organic catalyst (TEMPO (2,2,6,6- tetramethylpiperidin-1-yl) oxyl) or metallic nanoparticles (MWCNT/Pt, MWCNT/PtSn and MWCNT/Au) to the biofilms to create a hybrid catalytic system able to catalyze the complete electrooxidation of ethanol (ET) and ethylene glycol (EG) into CO2. In the case of ethanol extraction of 12 electrons per molecule at a single hybrid anode has been obtained with success. This simple methodology can replace the use of enzymatic cascade (7-8 enzymes) and may improve the application in biofuel cells. The electrochemical response of the enzyme system (MWCNT-COOH/LPEI/OxOx) at increasing concentrations of ethanol confirms the excellent activity of these hybrid bioanodes. Product analysis by HPLC techniques allowed us to correlate the increase in current with the formation of CO2or other more oxidable compounds as main product. In the case of EG the metallic catalyst was shifting the onset oxidation potential to lower potentials (200 mV) compared with TEMPO. The maximum power densities reached values around 351 µW cm-2 in the case of MWCNT/PtSn-OxOx for EG and 315 µW cm-2 for MWCNT-COOH/Pyrene-TEMPO/OxDc bioanode. Fig 1 show the products formed for EG. The modification of nanomaterials to obtain a large energy output form an enzymatic system will be highlighted. Therefore, the system open a new alternative of high performance hybrid-EBFC-based enabling the development of efficient EBFC based on alcohols. Acknowledge: FAPESP (grants # 2017/20431-7 and 2014/50945-4 ), CNPq (grant (465571/2014-0) and CAPES 001. Army Research Office MURI (W911NF-14-1-0263). Figure 1
Advances of Basic Science for Second Generation Bioethanol from Sugarcane, 2017
This chapter introduces the role of enzymes in the biomass degradation, namely sugarcane bagasse ... more This chapter introduces the role of enzymes in the biomass degradation, namely sugarcane bagasse and straw, leading to the formation of fermentable sugars and second-generation ethanol. The chapter begins with a retrospective of the structuring of the ethanol production chain and then presents current aspects where the deficit of production and its consequences in business can be seen. Subsequently, we list the role of enzymes involved in the biomass hydrolysis, the commercial cocktails, and the proposal of our laboratory in this context. On the other hand, the efficiency of enzymes on the biomass is increased when the bagasse and straw are pretreated. Thus, some technologies that may facilitate the enzymatic hydrolysis and the formation of fermentable sugars are described. Lastly, recent analytical methods that enable a better analysis of the composition and viewing of fiber in the sugarcane biomass are presented.
Plant materials represent a strategic energy source because they can give rise to sustainable bio... more Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60-80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternativ...
Amylases are enzymes with important biotechnological applications that are produced by several mi... more Amylases are enzymes with important biotechnological applications that are produced by several microorganisms in the form of filamentous fungi. Penicillium citrinum was recently isolated from the decomposed materials of Atlantic Forest, Bra-zil. It was identified by internal transcribed spacer (ITS) and beta-tubulin, and presented with excellent amylolytic levels. Thin layer chromatography showed that the amylase system was constituted by glucoamylase. This enzyme showed high activity at 60°C, pH 5.5, and an increment of 160% of the relative activity in the presence of 2 mM MgCl 2. The thermostabil-ity was increased after the immobilization on ionic supports. The enzyme immobilized on DEAE-sepharose retained 100% of its residual activity and when immobilized on MANAE-sepharose derivatives retained 72% at temperatures of 50°C and 60°C after 120 minutes of incubation. CNBr was used as a control. In contrast, the free enzyme only retained 50% of its residual activity after 90 minutes, ...
Aims: Prospectingdifferentfilamentousfungiforhigh production ofamylasesinstandard conditionsforfu... more Aims: Prospectingdifferentfilamentousfungiforhigh production ofamylasesinstandard conditionsforfutureapplicationin biotechnologyindustries. Methodo logy:Sampleswerecollectedindifferentfield areasinthestateofBahia,Brazil, for isolating filamentous fungi .Fungiwere grown in Petridishes in a culture medium containing 4% QuakerAEoatmeal and 2% bacteriologicalagar.Fungi screening was carried outin liquid mediumcontaining1% starch at30∞C and pH6.0 under static conditions for4 days. Proteins and enzyme activities were de termined byBradford and DNS methods, respectively. A submerged fermentation was performed with different liquidmediainorder toobtainthebestgrowthcomposition andenzymeproduction forthe selected fungus. Several conditions such as time course of inactivation, pH, temperature, carbon and nitrogen sources were determined in the culture medium with the aim of
A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans ... more A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 • C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 • C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A-and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O-and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients.
Journal of industrial microbiology & biotechnology, 2014
Endophytic fungi, mostly belonging to the Ascomycota, are found in the intercellular spaces of th... more Endophytic fungi, mostly belonging to the Ascomycota, are found in the intercellular spaces of the aerial plant parts, particularly in leaf sheaths, sometimes even within the bark and root system without inducing any visual symptoms of their presence. These fungi appear to have a capacity to produce a wide range of enzymes and secondary metabolites exhibiting a variety of biological activities. However, they have been only barely exploited as sources of enzymes of industrial interest. This review emphasizes the suitability and possible advantages of including the endophytic fungi in the screening of new enzyme producing organisms as well as in studies aiming to optimize the production of enzymes through well-known culture processes. Apparently endophytic fungi possess the two types of extracellular enzymatic systems necessary to degrade the vegetal biomass: (1) the hydrolytic system responsible for polysaccharide degradation consisting mainly in xylanases and cellulases; and (2) the...
An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and s... more An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 • C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni 2+ , glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni 2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 • C. The half-life of glyoxyl 4% derivative at 60 • C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose.
Phytase hydrolyzes phytic acid from the plant components of animal feed, releasing inorganic phos... more Phytase hydrolyzes phytic acid from the plant components of animal feed, releasing inorganic phosphorus. The phytase production by Aspergillus japonicus was optimized using Plackett-Burman designs (PBD), composite central rotational designs (CCRD), and response surface methodology from standard Czapek medium. The enzyme was applied in broiler chicken and laying hen foods. Analysis from PBD showed that KH2 PO2, MgSO4 · 7H2O, and yeast extract had significant influences on phytase secretion (p < 0.05). The best results from the CCRD experiments were obtained using (A) 0.040% KH2 PO4, (B) 0.050% MgSO4 · 7H2O, and (C) 0.040% yeast extract, enhancing in 49-53 U mg(-1) protein. The determination coefficient (R(2)) was 0.92 and Fcalc was 7.48 times greater than Flisted . Thus, the reduced coded model: Y (U mg-1) = 50.29 + 4.30A - 3.35(A)2 - 4.80(B)2 + 5.62C - 4.26(C)2 was considered predictive and statistically significant (p < 0.05). The optimized culture medium increased the phytase yield in 250%. A. japonicus phytase released high levels of Pi from broiler chicken and laying hen food. A. japonicus is an excellent phytase producer in a culture medium using inexpensive components and agricultural wastes. Therefore, these results provide sound arguments for the formulation of a low cost culture medium for phytase production.
A glucoamylase activity produced by the thermophilic fungus Scytalidium thermophilum was purified... more A glucoamylase activity produced by the thermophilic fungus Scytalidium thermophilum was purified 6.8-fold by ion exchange chromatography. The protein exhibited a molecular mass of about 86 kDa (7% PAGE and SDS-PAGE), or 68.5 kDa (Bio Sil SEC-400 FPLC). The pI of the enzyme was 8.4. Optima of pH and temperature, with starch or maltose as substrates were, 6.5/60 qC and 5.0/55 qC, respectively. The enzyme had a half-life of 22 min at 55 qC with starch as substrate, and it was fully stable with maltose. Maltase activity was activated by 10 mM Ba ++ (7.8%); Mn ++ (12.4%) or Mg ++ (28%). The enzyme contained approximately 25.5% carbohydrate. K m and V max values for starch and maltose were 0.28 mg/ml, 67.2 U/mg protein and 1.40 mg/ml, 5.61 U/mg protein, respectively. The products of hydrolysis of starch, detected by thin layer chromatography, showed only glucose after 30 min, indicating a glucoamylase activity. The amino terminal sequence of the purified protein showed 93% homology with a glucoamylase activity purified from Humicola grisea var. thermoidea.
ABSTRACT Filamentous fungi isolated from soil samples were screened for extracellular lipase prod... more ABSTRACT Filamentous fungi isolated from soil samples were screened for extracellular lipase production. The best producer was Hypocrea pseudokoningii identified by taxonomical criteria, and by rDNA sequencing of the variable internal transcribed spacers (ITS I and II) and the intervening 5.8S gene. The fungus was grown in a complex medium supplemented with 1% Tween 80 and 0.2% yeast extract, for 4 days. The optimum pH for extracellular and intracellular lipases was 7.0 and 8.0, respectively. Both enzymes exhibited maximum activity at 40 degrees C. Extracellular and intracellular lipase activities were highly stable in the pH range 3.0-8.0 at room temperature. The intracellular lipase was thermostable up to 60 degrees C, for 15 min and the extracellular, for 107 min, at the same temperature. The intracellular lipase was stimulated by silver ions. Extracellular lipase was stable in organic solvents, such as DMSO, alcohols, acetone, and acetonitrile, for 24 hours. Lipase activity increased around 80% when detergents were added to the enzymatic assay, such as Tween 80, Triton X-100, and SDS.
Scytalidium thermophilum 15.8 produced two extracellular glucoamylases. Using a DEAE-Cellulose ch... more Scytalidium thermophilum 15.8 produced two extracellular glucoamylases. Using a DEAE-Cellulose chromatographic column glucoamylases form II (GAII) was separated and purified from glucoamylases form I (GAI) that was previously purified and characterised (Cereia et al., 2000) when the filtrate of the culture medium was applied to a DEAE-Cellulose chromatographic column. GAII bound to the DEAE-Cellulose and was eluted with a NaCl gradient, while GAI did not bind to the resin. GAII presented electrophoretic homogeneity in 6% denaturing and non-denaturing PAGE, separately, with a molecular mass of 83 kDa, after the second round DEAE-Cellulose purification step. The enzyme pI was 7.2. Optima pH and activity temperature were 5.5 and 55ºC respectively for starch and maltose as substrates, with a termostability of 2.5 min at 60ºC. Enzymatic activities were activated by 1 mM Na + , Mn 2+ and Mg 2+ or 10 mM NH 4+ , Ba 2+ and Mg 2+ . The carbohydrate content was 10%. The kinetic parameters Km and Vmax with starch and maltose as substrate were 0.2 and 1.5 mg/ml, and 22.3 and 4.39 U/mg of protein, respectively. The amino acid sequence of GAII had 92% homology with the glucoamylase of Humicola grisea var. thermoidea after 13 cycles. Generally, GAII had different properties compared with GAI (Cereia et al., 2000).
Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with sev... more Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with several applications in the food industry due to its great hydrolytic potential. In this work, the fungus produced high enzymatic levels when cultivated on inexpensive culture media consisting of flakes from different origins such as cassava flour, wheat fibre, crushed soybean, agro-industrial wastes, starch, glucose or maltose. Several enzymatic systems were produced from these carbon sources, but amylase was the most evident, followed by pectinase and xylanase. Traces of CMCases, avicelase, lipase, β-xylosidase, β-glucosidase and α-glucosidase activities were also detected. Amylases were produced on rye flakes, starch, oat flakes, corn flakes, cassava flour and wheat fibre. Significant amylolytic levels were produced in the culture medium with glucose or when this sugar was exhausted, suggesting an enzyme in the constitutive form. Cassava flour, rye, oats, barley and corn flakes were also...
This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe o... more This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and B...
Enzyme immobilization can promote several advantages for their industrial application. In this wo... more Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 • C, 60 • C and 70 • C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.
BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohyd... more BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen microorganisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS: In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION: Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages.
Statistical evidence pointing to the very soft change in the ionic composition on the surface of ... more Statistical evidence pointing to the very soft change in the ionic composition on the surface of the sugar cane bagasse is crucial to improve yields of sugars by hydrolytic saccharification. Removal of Li+ by pretreatments exposing -OH sites was the most important factor related to the increase of saccharification yields using enzyme cocktails. Steam Explosion and Microwave:H2SO4 pretreatments produced unrelated structural changes, but similar ionic distribution patterns. Both increased the saccharification yield 1.74-fold. NaOH produced structural changes related to Steam Explosion, but released surface-bounded Li+ obtaining 2.04-fold more reducing sugars than the control. In turn, the higher amounts in relative concentration and periodic structures of Li+ on the surface observed in the control or after the pretreatment with Ethanol:DMSO:Ammonium Oxalate, blocked -OH and O− available for ionic sputtering. These changes correlated to 1.90-fold decrease in saccharification yields. Li...
Fusarium verticillioides lipases were purified in a "cascade" method using octadecyl Sepabeads an... more Fusarium verticillioides lipases were purified in a "cascade" method using octadecyl Sepabeads and octyl Sepharose resins, which led to the isolation of two proteins with lipolytic activities. Lip 1 was purified after octyl Sepharose adsorption presenting 30.3 kDa and, Lip 2 presented 68.0 kDa after octadecyl adsorption. These immobilization processes resulted in an increase of 3-fold in activity of each immobilized enzyme. These enzymes presented optima of pH of 5.0 and 6.0, respectively and temperature at 40 • C. They were thermostable at 40 • C and both remained more than 50% of its activity at the pH range of 5.0 to 7.0, with 180 min of incubation. The sardine oil hydrolysis showed higher EPA/DHA ratio. Concerning the ethanolysis reaction, Lip 2 showed higher conversion (5.5%) and both lipases showed activity in the release of the S enantiomers from 2-O-butyryl-2-phenylacetic acid (mandelic butyrate acid) and HPBE hydrolysis. Lip 2 also demonstrated capacity of transesterification. These applications made these enzymes attractive for industrial application.
Over the last decades researchers have focused to enhance the electrocatalytic activity of the bi... more Over the last decades researchers have focused to enhance the electrocatalytic activity of the biosystems, increasing the energy generation and electronic conductivity into the biofilms. The use of enzymes immobilized onto the surface of electrodes as the main catalyst in biological fuel cells has been extensively reported. A biofuel cell provides a means to obtain clean, renewable energy and have great potential to be used as alternative energy source for low power devices. There still a lot of important issues in the development of these device, one of them, is to obtain the maximum energy from the fuel. In order to enhance the power density of enzymatic biofuel cell (EBFC) it is necessary to increase the degree of oxidation of the fuel, so that all the electrons from the fuel can be harvested. One approach is the immobilization of a group of enzymes to perform a cascade in order to maximize the fuel oxidation. Recently, we have concentrated our goal to the develop hybrid biofilms employing MWCNT in order to immobilize efficiently the enzymes. In this context we added organic catalyst (TEMPO (2,2,6,6- tetramethylpiperidin-1-yl) oxyl) or metallic nanoparticles (MWCNT/Pt, MWCNT/PtSn and MWCNT/Au) to the biofilms to create a hybrid catalytic system able to catalyze the complete electrooxidation of ethanol (ET) and ethylene glycol (EG) into CO2. In the case of ethanol extraction of 12 electrons per molecule at a single hybrid anode has been obtained with success. This simple methodology can replace the use of enzymatic cascade (7-8 enzymes) and may improve the application in biofuel cells. The electrochemical response of the enzyme system (MWCNT-COOH/LPEI/OxOx) at increasing concentrations of ethanol confirms the excellent activity of these hybrid bioanodes. Product analysis by HPLC techniques allowed us to correlate the increase in current with the formation of CO2or other more oxidable compounds as main product. In the case of EG the metallic catalyst was shifting the onset oxidation potential to lower potentials (200 mV) compared with TEMPO. The maximum power densities reached values around 351 µW cm-2 in the case of MWCNT/PtSn-OxOx for EG and 315 µW cm-2 for MWCNT-COOH/Pyrene-TEMPO/OxDc bioanode. Fig 1 show the products formed for EG. The modification of nanomaterials to obtain a large energy output form an enzymatic system will be highlighted. Therefore, the system open a new alternative of high performance hybrid-EBFC-based enabling the development of efficient EBFC based on alcohols. Acknowledge: FAPESP (grants # 2017/20431-7 and 2014/50945-4 ), CNPq (grant (465571/2014-0) and CAPES 001. Army Research Office MURI (W911NF-14-1-0263). Figure 1
Advances of Basic Science for Second Generation Bioethanol from Sugarcane, 2017
This chapter introduces the role of enzymes in the biomass degradation, namely sugarcane bagasse ... more This chapter introduces the role of enzymes in the biomass degradation, namely sugarcane bagasse and straw, leading to the formation of fermentable sugars and second-generation ethanol. The chapter begins with a retrospective of the structuring of the ethanol production chain and then presents current aspects where the deficit of production and its consequences in business can be seen. Subsequently, we list the role of enzymes involved in the biomass hydrolysis, the commercial cocktails, and the proposal of our laboratory in this context. On the other hand, the efficiency of enzymes on the biomass is increased when the bagasse and straw are pretreated. Thus, some technologies that may facilitate the enzymatic hydrolysis and the formation of fermentable sugars are described. Lastly, recent analytical methods that enable a better analysis of the composition and viewing of fiber in the sugarcane biomass are presented.
Plant materials represent a strategic energy source because they can give rise to sustainable bio... more Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60-80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternativ...
Amylases are enzymes with important biotechnological applications that are produced by several mi... more Amylases are enzymes with important biotechnological applications that are produced by several microorganisms in the form of filamentous fungi. Penicillium citrinum was recently isolated from the decomposed materials of Atlantic Forest, Bra-zil. It was identified by internal transcribed spacer (ITS) and beta-tubulin, and presented with excellent amylolytic levels. Thin layer chromatography showed that the amylase system was constituted by glucoamylase. This enzyme showed high activity at 60°C, pH 5.5, and an increment of 160% of the relative activity in the presence of 2 mM MgCl 2. The thermostabil-ity was increased after the immobilization on ionic supports. The enzyme immobilized on DEAE-sepharose retained 100% of its residual activity and when immobilized on MANAE-sepharose derivatives retained 72% at temperatures of 50°C and 60°C after 120 minutes of incubation. CNBr was used as a control. In contrast, the free enzyme only retained 50% of its residual activity after 90 minutes, ...
Aims: Prospectingdifferentfilamentousfungiforhigh production ofamylasesinstandard conditionsforfu... more Aims: Prospectingdifferentfilamentousfungiforhigh production ofamylasesinstandard conditionsforfutureapplicationin biotechnologyindustries. Methodo logy:Sampleswerecollectedindifferentfield areasinthestateofBahia,Brazil, for isolating filamentous fungi .Fungiwere grown in Petridishes in a culture medium containing 4% QuakerAEoatmeal and 2% bacteriologicalagar.Fungi screening was carried outin liquid mediumcontaining1% starch at30∞C and pH6.0 under static conditions for4 days. Proteins and enzyme activities were de termined byBradford and DNS methods, respectively. A submerged fermentation was performed with different liquidmediainorder toobtainthebestgrowthcomposition andenzymeproduction forthe selected fungus. Several conditions such as time course of inactivation, pH, temperature, carbon and nitrogen sources were determined in the culture medium with the aim of
A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans ... more A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 • C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 • C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A-and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O-and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients.
Journal of industrial microbiology & biotechnology, 2014
Endophytic fungi, mostly belonging to the Ascomycota, are found in the intercellular spaces of th... more Endophytic fungi, mostly belonging to the Ascomycota, are found in the intercellular spaces of the aerial plant parts, particularly in leaf sheaths, sometimes even within the bark and root system without inducing any visual symptoms of their presence. These fungi appear to have a capacity to produce a wide range of enzymes and secondary metabolites exhibiting a variety of biological activities. However, they have been only barely exploited as sources of enzymes of industrial interest. This review emphasizes the suitability and possible advantages of including the endophytic fungi in the screening of new enzyme producing organisms as well as in studies aiming to optimize the production of enzymes through well-known culture processes. Apparently endophytic fungi possess the two types of extracellular enzymatic systems necessary to degrade the vegetal biomass: (1) the hydrolytic system responsible for polysaccharide degradation consisting mainly in xylanases and cellulases; and (2) the...
An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and s... more An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 • C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni 2+ , glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni 2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 • C. The half-life of glyoxyl 4% derivative at 60 • C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose.
Phytase hydrolyzes phytic acid from the plant components of animal feed, releasing inorganic phos... more Phytase hydrolyzes phytic acid from the plant components of animal feed, releasing inorganic phosphorus. The phytase production by Aspergillus japonicus was optimized using Plackett-Burman designs (PBD), composite central rotational designs (CCRD), and response surface methodology from standard Czapek medium. The enzyme was applied in broiler chicken and laying hen foods. Analysis from PBD showed that KH2 PO2, MgSO4 · 7H2O, and yeast extract had significant influences on phytase secretion (p < 0.05). The best results from the CCRD experiments were obtained using (A) 0.040% KH2 PO4, (B) 0.050% MgSO4 · 7H2O, and (C) 0.040% yeast extract, enhancing in 49-53 U mg(-1) protein. The determination coefficient (R(2)) was 0.92 and Fcalc was 7.48 times greater than Flisted . Thus, the reduced coded model: Y (U mg-1) = 50.29 + 4.30A - 3.35(A)2 - 4.80(B)2 + 5.62C - 4.26(C)2 was considered predictive and statistically significant (p < 0.05). The optimized culture medium increased the phytase yield in 250%. A. japonicus phytase released high levels of Pi from broiler chicken and laying hen food. A. japonicus is an excellent phytase producer in a culture medium using inexpensive components and agricultural wastes. Therefore, these results provide sound arguments for the formulation of a low cost culture medium for phytase production.
A glucoamylase activity produced by the thermophilic fungus Scytalidium thermophilum was purified... more A glucoamylase activity produced by the thermophilic fungus Scytalidium thermophilum was purified 6.8-fold by ion exchange chromatography. The protein exhibited a molecular mass of about 86 kDa (7% PAGE and SDS-PAGE), or 68.5 kDa (Bio Sil SEC-400 FPLC). The pI of the enzyme was 8.4. Optima of pH and temperature, with starch or maltose as substrates were, 6.5/60 qC and 5.0/55 qC, respectively. The enzyme had a half-life of 22 min at 55 qC with starch as substrate, and it was fully stable with maltose. Maltase activity was activated by 10 mM Ba ++ (7.8%); Mn ++ (12.4%) or Mg ++ (28%). The enzyme contained approximately 25.5% carbohydrate. K m and V max values for starch and maltose were 0.28 mg/ml, 67.2 U/mg protein and 1.40 mg/ml, 5.61 U/mg protein, respectively. The products of hydrolysis of starch, detected by thin layer chromatography, showed only glucose after 30 min, indicating a glucoamylase activity. The amino terminal sequence of the purified protein showed 93% homology with a glucoamylase activity purified from Humicola grisea var. thermoidea.
ABSTRACT Filamentous fungi isolated from soil samples were screened for extracellular lipase prod... more ABSTRACT Filamentous fungi isolated from soil samples were screened for extracellular lipase production. The best producer was Hypocrea pseudokoningii identified by taxonomical criteria, and by rDNA sequencing of the variable internal transcribed spacers (ITS I and II) and the intervening 5.8S gene. The fungus was grown in a complex medium supplemented with 1% Tween 80 and 0.2% yeast extract, for 4 days. The optimum pH for extracellular and intracellular lipases was 7.0 and 8.0, respectively. Both enzymes exhibited maximum activity at 40 degrees C. Extracellular and intracellular lipase activities were highly stable in the pH range 3.0-8.0 at room temperature. The intracellular lipase was thermostable up to 60 degrees C, for 15 min and the extracellular, for 107 min, at the same temperature. The intracellular lipase was stimulated by silver ions. Extracellular lipase was stable in organic solvents, such as DMSO, alcohols, acetone, and acetonitrile, for 24 hours. Lipase activity increased around 80% when detergents were added to the enzymatic assay, such as Tween 80, Triton X-100, and SDS.
Scytalidium thermophilum 15.8 produced two extracellular glucoamylases. Using a DEAE-Cellulose ch... more Scytalidium thermophilum 15.8 produced two extracellular glucoamylases. Using a DEAE-Cellulose chromatographic column glucoamylases form II (GAII) was separated and purified from glucoamylases form I (GAI) that was previously purified and characterised (Cereia et al., 2000) when the filtrate of the culture medium was applied to a DEAE-Cellulose chromatographic column. GAII bound to the DEAE-Cellulose and was eluted with a NaCl gradient, while GAI did not bind to the resin. GAII presented electrophoretic homogeneity in 6% denaturing and non-denaturing PAGE, separately, with a molecular mass of 83 kDa, after the second round DEAE-Cellulose purification step. The enzyme pI was 7.2. Optima pH and activity temperature were 5.5 and 55ºC respectively for starch and maltose as substrates, with a termostability of 2.5 min at 60ºC. Enzymatic activities were activated by 1 mM Na + , Mn 2+ and Mg 2+ or 10 mM NH 4+ , Ba 2+ and Mg 2+ . The carbohydrate content was 10%. The kinetic parameters Km and Vmax with starch and maltose as substrate were 0.2 and 1.5 mg/ml, and 22.3 and 4.39 U/mg of protein, respectively. The amino acid sequence of GAII had 92% homology with the glucoamylase of Humicola grisea var. thermoidea after 13 cycles. Generally, GAII had different properties compared with GAI (Cereia et al., 2000).
Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with sev... more Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with several applications in the food industry due to its great hydrolytic potential. In this work, the fungus produced high enzymatic levels when cultivated on inexpensive culture media consisting of flakes from different origins such as cassava flour, wheat fibre, crushed soybean, agro-industrial wastes, starch, glucose or maltose. Several enzymatic systems were produced from these carbon sources, but amylase was the most evident, followed by pectinase and xylanase. Traces of CMCases, avicelase, lipase, β-xylosidase, β-glucosidase and α-glucosidase activities were also detected. Amylases were produced on rye flakes, starch, oat flakes, corn flakes, cassava flour and wheat fibre. Significant amylolytic levels were produced in the culture medium with glucose or when this sugar was exhausted, suggesting an enzyme in the constitutive form. Cassava flour, rye, oats, barley and corn flakes were also...
This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe o... more This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and B...
Enzyme immobilization can promote several advantages for their industrial application. In this wo... more Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 • C, 60 • C and 70 • C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.
BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohyd... more BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen microorganisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS: In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION: Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages.
Statistical evidence pointing to the very soft change in the ionic composition on the surface of ... more Statistical evidence pointing to the very soft change in the ionic composition on the surface of the sugar cane bagasse is crucial to improve yields of sugars by hydrolytic saccharification. Removal of Li+ by pretreatments exposing -OH sites was the most important factor related to the increase of saccharification yields using enzyme cocktails. Steam Explosion and Microwave:H2SO4 pretreatments produced unrelated structural changes, but similar ionic distribution patterns. Both increased the saccharification yield 1.74-fold. NaOH produced structural changes related to Steam Explosion, but released surface-bounded Li+ obtaining 2.04-fold more reducing sugars than the control. In turn, the higher amounts in relative concentration and periodic structures of Li+ on the surface observed in the control or after the pretreatment with Ethanol:DMSO:Ammonium Oxalate, blocked -OH and O− available for ionic sputtering. These changes correlated to 1.90-fold decrease in saccharification yields. Li...
Fusarium verticillioides lipases were purified in a "cascade" method using octadecyl Sepabeads an... more Fusarium verticillioides lipases were purified in a "cascade" method using octadecyl Sepabeads and octyl Sepharose resins, which led to the isolation of two proteins with lipolytic activities. Lip 1 was purified after octyl Sepharose adsorption presenting 30.3 kDa and, Lip 2 presented 68.0 kDa after octadecyl adsorption. These immobilization processes resulted in an increase of 3-fold in activity of each immobilized enzyme. These enzymes presented optima of pH of 5.0 and 6.0, respectively and temperature at 40 • C. They were thermostable at 40 • C and both remained more than 50% of its activity at the pH range of 5.0 to 7.0, with 180 min of incubation. The sardine oil hydrolysis showed higher EPA/DHA ratio. Concerning the ethanolysis reaction, Lip 2 showed higher conversion (5.5%) and both lipases showed activity in the release of the S enantiomers from 2-O-butyryl-2-phenylacetic acid (mandelic butyrate acid) and HPBE hydrolysis. Lip 2 also demonstrated capacity of transesterification. These applications made these enzymes attractive for industrial application.
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