Extracellular β-D-fructofuranosidase from Aspergillus parasiticus: Optimization of the production under submerged fermentation and biochemical characterization

The β-D-fructofuranosidases are enzymes with biotechnological potential that can be used in different industrial sectors as food and beverage. In this context, microorganisms are important producers of these biomolecules, especially filamentous fungi. The production of extracellular β-Dfructofuranosidase from Aspergillus parasiticus using sugarcane bagasse as a carbon source under submerged fermentation was optimized by factorial design and high levels of enzyme were obtained in 24 h-old cultures at 30°C using 1.5% sugarcane bagasse under agitation. The extracellular β-Dfructofuranosidase was purified 119-fold using diethyl aminoethyl (DEAE)-cellulose and Sephacryl S-200 chromatographic columns with recovery of 16%. The native molecular mass was estimated as 136 kDa with two subunits of 63 kDa determined by 7% sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and 64% carbohydrate content. The purified enzyme showed optimal temperature of activity from 38-56°C and optimum pH from 4.5 to 6.2 determined by experimental design (CCRD), with half-life of 25 min at 50°C. It was stable from pH 5.0-10.0. The extracellular enzyme activity was stimulated by Ba2+ and Mg2+, and it was not affected by urea, silver and ethylenediaminetetraacetic acid (EDTA). The K0.5 and Vmax values were 10 mM and 1565 U/mg of protein, and 19 mM and 1965 U/mg of protein for sucrose and raffinose, respectively.Keywords: Invertase, fructofuranosidase, Aspergillus parasiticus, sugarcane bagasse, factorial design.African Journal of Biotechnology Vol. 12(38), pp. 5678-568

Evidence of high production levels of thermostable dextrinizing and saccharogenic amylases by Aspergillus niveus

The aim of this work was to analyze the effect of several nutritional and environmental parameters on amylase production by a novel, isolated from the thermotolerant filamentous fungus Aspergillus niveus. This strain produced high levels of amylolytic activity in Khanna liquid medium supplemented with commercial starch, initial pH 6.5, under static conditions for 72 h. Among the tested carbon sources, milled corn, oatmeal, soluble potato starch and maisena were the best inducers of enzymatic secretion (220, 180, 170 and 150 U/mL), respectively. The main products of hydrolysis analyzed by thin layer chromatography were glucose, maltose and traces of maltooligosaccharides, suggesting the presence of α-amylase and glucoamylase activities in the crude extract. The optimal pH were 4.5 and 5.5 and the optimum temperature was 65°C. The enzymes were fully stable up to 1 h at 55°C. It was possible to verify the presence of three bands with amylolytic activity in non-denaturing polyacrylamide gel electrophoresis (PAGE). These aspects and other properties suggested that the amylases produced by A. niveus might be suitable for biotechnological applications.KKeywords: Starch, α-amylase, glucoamylase, Aspergillus niveus, submerged fermentation, thermostabilityAfrican Journal of Biotechnology Vol. 12(15), pp. 1874-188

Biotechnological Potential of Agro-Industrial Wastes as a Carbon Source to Thermostable Polygalacturonase Production in Aspergillus niveus

Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. The aim of this work was to study polygalacturonase (PG) production by Aspergillus niveus cultured on liquid or solid media supplemented with agro-industrial wastes. Submerged fermentation (SbmF) was tested using Czapeck media supplemented with 28 different carbon sources. Among these, orange peel was the best PG inducer. On the other hand, for solid state fermentation (SSF), lemon peel was the best inducer. By comparing SbmF with SSF, both supplemented with lemon peel, it was observed that PG levels were 4.4-fold higher under SSF. Maximum PG activity was observed at 55°C and pH 4.0. The enzyme was stable at 60°C for 90 min and at pH 3.0–5.0. The properties of this enzyme, produced on inexpensive fermentation substrates, were interesting and suggested several biotechnological applications

Biotechnological potential of alternative carbon sources for production of pectinases by Rhizopus microsporus var. rhizopodiformis

Fungi collected from Brazilian soil and decomposing plants were screened for pectinase production. R. microsporus var. rhizopodiformis was the best producer and was selected to evaluate the pectic enzyme production under several nutritional and environmental conditions. The pectinase production was studied at 40ºC, under 28 carbon sources-supplemented medium. The inducer effect of several agro-industrial residues such as sugar cane bagasse, wheat flour and corncob on polygalacturonase (PG) activity was 4-, 3- and 2-fold higher than the control (pectin). In glucose-medium, a constitutive pectin lyase (PL) activity was detected. The results demonstrated that R. microsporus produced high levels of PG (57.7 U/mg) and PL (88.6 U/mg) in lemon peel-medium. PG had optimum temperature at 65 ºC and was totally stable at 55 ºC for 90 min. Half-life at 70 ºC was 68 min. These results suggested that the versatility of waste carbon sources utilization by R. microsporus, produce pectic enzymes, which could be useful to reduce production costs and environmental impacts related to the waste disposal.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)(CNPq) Conselho de Desenvolvimento Científico e Tecnológic

Characterization of a multi-tolerant tannin acyl hydrolase II from Aspergillus carbonarius produced under solid-state fermentation

Background: Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results: The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20–60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion: A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application

Production of Fructooligosaccharides by Aspergillus phoenicis Biofilm on Polyethylene as Inert Support

Za proizvodnju fruktooligosaharida (FOS) u podlozi s 25 % (m/V) saharoze kao izvora ugljika upotrijebljen je biofilm Aspergillus phoenicis na inertnoj podlozi od polietilena. Maksimalna koncentracija ukupnih FOS od 122 mg/mL, od toga 68 % 1-kestoze i 32 % nistoze, postignuta je primjenom Khanna podloge pri 30 °C tijekom 48 h uzgoja uz miješanje (100 rpm). Veća je koncentracija FOS dobivena u podlozi s više (30 %, m/V), nego u onoj s manje saharoze (5 %, m/V). Također je veća koncentracija FOS od 242 mg/L dobivena pomoću biofilma u acetatnom puferu s velikom koncentracijom saharoze (50 %, m/V) tijekom 10 h uzgoja. Ponovnim korištenjem osušenog biofilma u svježoj podlozi dobiveno je otprilike 13,7 % FOS (od toga 10 % 1-kestoze) nakon 72 h uzgoja pri 30 °C. Pomoću skenirajućeg elektronskog mikroskopa ispitana je morfologija biofilma, te otkrivena nekompaktna struktura micelija, s praznim prostorima i kanalima između hifa. Rezultati ovog istraživanja pokazuju da se biofilm A. phoenicis može upotrijebiti za proizvodnju FOS jeftinom i učinkovitom jednostupanjskom fermentacijom, pri čemu se biofilm može ponovno iskoristiti.fructooligosaccharides (FOS) in media containing 25 % (m/V) of sucrose as a carbon source. The maximum production of total FOS (122 mg/mL), with 68 % of 1-kestose and 32 % of nystose, was obtained in Khanna medium maintained at 30 °C for 48 h under orbital agitation (100 rpm). At high concentrations of sucrose (30 %, m/V), the recovery of FOS was higher than that observed at a low concentration (5 %, m/V). High levels of FOS (242 mg/mL) were also recovered when using the biofilm in sodium acetate buffer with high sucrose concentration (50 %, m/V) for 10 h. When the dried biofilm was reused in a fresh culture medium, there was a recovery of approx. 13.7 % of total FOS after 72 h of cultivation at 30 °C, and 10 % corresponded to 1-kestose. The biofilm morphology, analyzed by scanning electron microscope, revealed a noncompact mycelium structure, with unfilled spaces and channels present among the hyphae. The results obtained in this study show that A. phoenicis biofilms may find application for FOS production in a single-step fermentation process, which is cost-effective in terms of reusability, downstream processing and efficiency