Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides.

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and ß-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.

Substrates' and products' inhibition in fructanase production by a new Kluyveromyces marxianus CF15 from Agave tequilana fructan in a batch reactor.

This study focuses on fructanase production in a batch reactor by a new strain isolated from agave juice (K. marxianus var. drosophilarum) employing different Agave tequilana fructan (ATF) concentrations as substrate. The experimental data suggest that the fructanase production may be inhibited or repressed by high substrate (50 g/L) and ethanol (20.7 g/L) concentrations present in culture medium. To further analyze these phenomena an unstructured kinetic mathematical model taking into account substrate and products inhibition was proposed and fitted. The mathematical model considers six reaction kinetics and the ethanol evaporation, and predicts satisfactorily the biomass, fructan, glucose, fructose, ethanol, and fructanase behavior for different raw material initial concentrations. The proposed model is the first to satisfactorily describe the production of fructanase from branched ATF with a new strain of K. marxianus.

Recombinant expression, purification, and characterization of a cyclodextrinase from Massilia timonae.

Some microorganisms can produce cyclodextrin glycosyltransferases, which degrades starch by catalyzing cyclization and giving rise to cyclodextrin. Thus, to fully degrade starch, microorganisms can also synthesize cyclodextrinases, which hydrolyze cyclodextrins. In this work, a truncated gene, without the signal peptide coding sequence, encoding a cyclodextrinase from Massilia timonae was PCR amplified, cloned, and expressed in E. coli. The histidine-tagged recombinant enzyme was purified by immobilized metal ion affinity chromatography. The purified protein was found to be a tetramer of about 260 kDa, with monomers of about 65 kDa, as estimated by gel filtration and SDS-PAGE, respectively. The enzyme presented an optimum temperature of 40 °C, optimum pH of 7.0, and remained stable after 30 min of incubation at 45 °C, with a T50 of 48.45 °C. The enzyme showed a higher activity toward ß-cyclodextrin compared to that for maltodextrin and starch. KM for ß-cyclodextrin was 2.1 mM, Vmax was 0.084 µmol/min, kcat was 8326 min-1, and kcat/KM was 4.1 × 106 M-1min-1. Calcium acted as an activator and SDS, CTAB, several cations, and EDTA acted as strong inhibitors. The purified cyclodextrinase produced glucose and maltose as final products by hydrolysis of ß-cyclodextrin, maltotetraose, and maltoheptaose. This novel cyclodextrinase could be a promising alternative for the enzymatic hydrolysis of starch.

Production optimization, purification, expression, and characterization of a novel α-L-arabinofuranosidase from Paenibacillus polymyxa

Background: α-L-Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal α-L-1,2-, -1,3-, and -1,5- arabinofuranosyl residues in arabinose-containing polymers, and hence, it plays an important role in hemicellulose degradation. Herein, the bacterium Paenibacillus polymyxa, which secretes arabinofuranosidase with high activity, was selected for enzyme production, purification, and characterization. Results: Medium components and cultural conditions were optimized by the response surface method using shake flask cultures. Arabinofuranosidase production reached 25.2 U/mL under optimized conditions, which were pH 7.5, 28°C, and a basic medium supplemented with 1.5 g/L mannitol and 3.5 g/L soymeal. Furthermore, the arabinofuranosidase secreted by P. polymyxa, named as PpAFase-1, was partially purified from the supernatant using a DEAE Sepharose Fast Flow column and a hydroxyapatite column. The approximate molecular mass of the purified PpAFase-1 was determined as 56.8 kDa by SDS-PAGE. Protein identification by mass spectrometry analysis showed that the deduced amino acid sequence had significant similarity to the glycosyl hydrolase family 51. The deduced gene of 1515 bp was cloned and expressed in Escherichia coli BL21 (DE3) cells. Purified recombinant PpAFase-1 was active toward p-nitrophenyl-α-L-arabinofuranoside (pNPAraf). The Km and kcat values toward pNPAraf were 0.81 mM and 53.2 s−1 , respectively. When wheat arabinoxylan and oat spelt xylan were used as substrates, PpAFase-1 showed poor efficiency. However, a synergistic effect was observed when PpAFase-1 was combined with xylanase from Thermomyces lanuginosus. Conclusion: A novel GH51 enzyme PpAFase-1 was cloned from the genome of P. polymyxa and expressed in E. coli. This enzyme may be suitable for hemicellulose degradation on an industrial scale.

Overexpression of a Cellobiose-Glucose-Halotolerant Endoglucanase from Scytalidium thermophilum.

Enzyme reaction products and by-products from pretreatment steps can inhibit endoglucanases and are major factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis. The gene encoding the endoglucanase from Scytalidium thermophilum (egst) was cloned and expressed as a soluble protein in Pichia pastoris GS115. The recombinant enzyme (Egst) was monomeric (66 kDa) and showed an estimated carbohydrate content of 53.3% (w/w). The optimum temperature and pH of catalysis were 60-70 °C and pH of 5.5, respectively. The enzyme was highly stable at pH 3.0-8.0 with a half-life in water of 100 min at 65 °C. The Egst presented good halotolerance, retaining 84.1 and 71.4% of the control activity in the presence of 0.5 and 2.0 mol L-1 NaCl, respectively. Hydrolysis of medium viscosity carboxymethylcellulose (CMC) by Egst was stimulated 1.77-, 1.84-, 1.64-, and 1.8-fold by dithiothreitol, ß-mercaptoethanol, cysteine, and manganese at 10, 10, 10, and 5 mmol L-1 concentration, respectively. The enzyme hydrolyzed CMC with maximal velocity and an apparent affinity constant of 432.10 ± 16.76 and 10.5 ± 2.53 mg mL-1, respectively. Furthermore, the Egst was tolerant to reaction products and able to act on pretreated fractions sugarcane bagasse demonstrating excellent properties for application in the hydrolysis of lignocellulosic biomass.

Biomass sorghum as a novel substrate in solid-state fermentation for the production of hemicellulases and cellulases by Aspergillus niger and A. fumigatus.

AIMS: We investigated the role of carbon and nitrogen sources in the production of cellulase and hemicellulase by Aspergillus strains. METHODS AND RESULTS: The strains Aspergillus niger SCBM1 and Aspergillus fumigatus SCBM6 were cultivated under solid-state fermentation (SSF), with biomass sorghum (BS) and wheat bran (WB) as lignocellulosic substrates, in different proportions, along with variable nitrogen sources. The best SSF condition for the induction of such enzymes was observed employing A. niger SCBM1 in BS supplemented with peptone; maximum production levels were achieved as follows: 72 h of fermentation for xylanase and exoglucanase (300·07 and 30·64 U g-1 respectively), 120 h for ß-glucosidase and endoglucanase (54·90 and 41·47 U g-1 respectively) and 144 h for ß-xylosidase (64·88 U g-1 ). CONCLUSIONS: This work demonstrated the viability of the use of BS for the production of hemi- and cellulolytic enzymes; the high concentration of celluloses in BS could be associated with the significant production of cellulases, mainly exoglucanase. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study which presents the promising use of biomass sorghum (genetically modified sorghum to increase its biomass content) as an alternative carbon source for the production of enzymes by SSF.

Potential of Mangrove-Associated Endophytic Fungi for Production of Carbohydrolases with High Saccharification Efficiency.

The endophytic fungi represent a potential source of microorganisms for enzyme production. However, there have been only few studies exploiting their potential for the production of enzymes of industrial interest, such as the (hemi)cellulolytic enzymatic cocktail required in the hydrolysis of lignocellulosic biomass. Here, a collection of endophytic fungi isolated from mangrove tropical forests was evaluated for the production of carbohydrolases and performance on the hydrolysis of cellulose. For that, 41 endophytic strains were initially screened using a plate assay containing crystalline cellulose as the sole carbon source and the selected strains were cultivated under solid-state fermentation for endoglucanase, ß-glucosidase, and xylanase enzyme quantification. The hydrolysis of a cellulosic material with the enzymes from endophytic strains of the Aspergillus genus resulted in glucose and conversion values more than twofold higher than the reference strains (Aspergillus niger F12 and Trichoderma reesei Rut-C30). Particularly, the enzymes from strains A. niger 56 (3) and A. awamori 82 (4) showed a distinguished saccharification performance, reaching cellulose conversion values of about 35% after 24 h. Linking hydrolysis performance to the screening steps played an important role towards finding potential fungal strains for producing enzymatic cocktails with high saccharification efficiency. These results indicate the potential of mangrove-associated endophytic fungi for production of carbohydrolases with efficient performance in the hydrolysis of biomass, thus contributing to the implementation of future biorefineries.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM)

Abstract The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30 °C, 6% (v/v), inoculum size and 150 rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM).

The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30°C, 6% (v/v), inoculum size and 150rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.

Penicillium purpurogenum produces a highly stable endo-ß-(1,4)-galactanase.

The polysaccharides of galactose present in the pectin of the plant cell wall are degraded by endo-ß-1,4-galactanases. The filamentous fungus Penicillium purpurogenum, which grows on a number of natural carbon sources, among them sugar beet pulp which contains pectin, has a gene (ppgal1) coding an endo-ß-1,4-galactanase (PpGAL1). This enzyme was expressed heterologously in Pichia pastoris. It has a molecular mass of 38 kDa, a pH optimum of 4-4.5, and an optimal temperature of 60 °C. It is 100 % stable for up to 24 h at pH 4-4.5 and 40 °C. These stability properties, which exceed those from other endo-ß-1,4-galactanases reported to date, make it particularly suitable for industrial processes requiring acidic conditions and temperatures up to 40 °C. PpGAL1 is, therefore, a potentially effective tool in the food industry and in other biotechnological applications.

Enhancement of Penicillium echinulatum glycoside hydrolase enzyme complex.

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and ß-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and ß-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance ß-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and ß-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance ß-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed ß-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-ß-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with ß-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.

Aspergillus fumigatus Produces Two Arabinofuranosidases From Glycosyl Hydrolase Family 62: Comparative Properties of the Recombinant Enzymes.

The genes of two α-L-arabinofuranosidases (AbfI and II) from family GH 62 have been identified in the genome of Aspergillus fumigatus wmo. Both genes have been expressed in Pichia pastoris and the enzymes have been purified and characterized. AbfI is composed of 999 bp, does not contain introns and codes for a protein (ABFI) of 332 amino acid residues. abfII has 1246 bp, including an intron of 51 bp; the protein ABFII has 396 amino acid residues; it includes a family 1 carbohydrate-binding module (CBM) in the N-terminal region, followed by a catalytic module. The sequence of ABFI and the catalytic module of ABFII show a 79 % identity. Both enzymes are active on p-nitrophenyl α-L-arabinofuranoside (pNPAra) with KM of 94.2 and 3.9 mM for ABFI and II, respectively. Optimal temperature for ABFI is 37 °C and for ABFII 42 °C, while the pH optimum is about 4.5 to 5 for both enzymes. ABFII shows a higher thermostability. When assayed using natural substrates, both show higher activity over rye arabinoxylan as compared to wheat arabinoxylan. ABFII only is active on sugar beet pulp arabinan and both are inactive towards debranched arabinan. The higher thermostability, higher affinity for pNPAra and wider activity over natural substrates shown by ABFII may be related to the presence of a CBM. The availability of the recombinant enzymes may be useful in biotechnological applications for the production of arabinose.

Comparative study of fungal strains for thermostable inulinase production.

Fructose and fructo-oligosaccharides (FOS) are important ingredients in the food industry. Fructose is considered an alternative sweetener to sucrose because it has higher sweetening capacity and increases iron absorption in children, and FOS's are a source of dietary fiber with a bifidogenic effect. Both compounds can be obtained by enzymatic hydrolysis of inulin. However, inulin presents limited solubility at room temperature, thus, fructose and FOS production is carried out at 60°C. Therefore, there is a growing interest to isolate and characterize thermostable inulinases. The aim of this work was to evaluate the capacity of different fungal strains to produce potential thermostable inulinases. A total of 27 fungal strains belonging to the genera Aspergillus, Penicillium, Rhizopus, Rhizomucor and Thermomyces were evaluated for production of inulinase under submerged culture using Czapek Dox medium with inulin as a sole carbon source. Strains were incubated at 37°C and 200 rpm for 96 h. Crude enzyme extract was obtained to evaluate inulinase and invertase activity. In order to select the fungal strain with the highest thermostable inulinase production, a selection criterion was established. It was possible to determine the highest inulinase activity for Rhizopus microsporus 13aIV (10.71 U/mL) at 36 h with an optimum temperature of inulinase of 70°C. After 6 h at 60°C, the enzyme did not show any significant loss of activity and retained about 87% activity, while it only retains 57% activity at 70°C. According to hydrolysis products, R. microsporus produced endo and exo-inulinase.

High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC).

Recent advances in DNA sequencing techniques have led to an explosion in the amount of available genome sequencing data and this provided an inexhaustible source of uncharacterized glycoside hydrolases (GH) to be studied both structurally and enzymatically. Ligation-Independent Cloning (LIC), an interesting alternative to traditional, restriction enzyme-based cloning, and commercial recombinatorial cloning, was adopted and optimized successfully for a high throughput cloning, expression and purification pipeline. Using this platform, 130 genes encoding mainly uncharacterized glycoside hydrolases from 13 different organisms were cloned and submitted to a semi-automated protein expression and solubility screening in Escherichia coli, resulting in 73 soluble targets. The high throughput approach proved to be a powerful tool for production of recombinant glycoside hydrolases for further structural and biochemical characterization and confirmed that thioredoxin fusion tag (TRX) is a better choice to increase solubility of recombinant glycoside hydrolases expressed in E. coli, when compared to His-tag alone.

Production and characterization of ß-glucanase secreted by the yeast Kluyveromyces marxianus.

An extracellular ß-glucanase secreted by Kluyveromyces marxianus was identified for the first time. The optimal conditions for the production of this enzyme were evaluated by response surface methodology. The optimal conditions to produce ß-glucanase were a glucose concentration of 4% (w/v), a pH of 5.5, and an incubation temperature of 35 °C. Response surface methodology was also used to determine the pH and temperature required for the optimal enzymatic activity. The highest enzyme activity was obtained at a pH of 5.5 and a temperature of 55 °C. Furthermore, the enzyme was partially purified and sequenced, and its specificity for different substrates was evaluated. The results suggest that the enzyme is an endo-ß-1,3(4)-glucanase. After optimizing the conditions for ß-glucanase production, the culture supernatant was found to be effective in digesting the cell wall of the yeast Saccharomyces cerevisiae, showing the great potential of ß-glucanase in the biotechnological production of soluble ß-glucan.

Screening and xylanase production by Streptomyces sp. grown on lignocellulosic wastes.

Xylanases have raised interest because of their potential applications in various industrial fields, including the pulp and paper industries, bioethanol production, and the feed industry. In bioethanol production from lignocellulosic compounds, xylanase can improve the hydrolysis of cellulose into fermentable sugars, since the xylan restricts the cellulases from acting efficiently. In this work, a new thermophilic Streptomyces sp. was selected for its ability to produce xylanase. Carbon source selection is an important factor in the production of hemicellulases. The highest activity was obtained when Streptomyces sp. I3 was grown in the presence of wheat bran. Xylanase activity was partially characterized concerning the effect of pH and temperature on activity and thermostability, and the effects of different metal ions were also tested. The pH and temperature profile showed optimal activity at pH 6.0/70 °C. Zymogram analysis showed multiple xylanases (39, 21, 18, and 17 kDa). Xylanases studied in this work are thermophilic, thermostable, and active in a wide pH range; they have potential to be used in the development of new processes of biotechnological interest.

Bioconversion of Agave tequilana fructans by exo-inulinases from indigenous Aspergillus niger CH-A-2010 enhances ethanol production from raw Agave tequilana juice.

Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme(®) (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.

Experimental mixture design as a tool to enhance glycosyl hydrolases production by a new Trichoderma harzianum P49P11 strain cultivated under controlled bioreactor submerged fermentation.

This work investigates the glycosyl hydrolase (GH) profile of a new Trichoderma harzianum strain cultivated under controlled bioreactor submerged fermentation. The influence of different medium components (delignified steam-exploded sugarcane bagasse, sucrose, and soybean flour) on GH biosynthesis was assessed using experimental mixture design (EMD). Additionally, the effect of increased component concentrations in culture media selected from the EMD was studied. It was found that that a mixed culture medium could significantly maximize GH biosynthesis rate, especially for xylanase enzymes which achieved a 2-fold increment. Overall, it was demonstrated that T. harzianumP49P11 enzymes have a great potential to be used in the deconstruction of biomass.

Effect of extrinsic and intrinsic parameters on inulinase production by Aspergillus niger ATCC 20611

Background: Inulinase is a versatile enzyme from glycoside hydrolase family which targets the beta-2, 1 linkage of fructopolymers. In the present study, the effect of medium composition and culture conditions on inulinase production by Aspergillus niger ATCC 20611 was investigated in shake-flasks. Results: The highest extracellular inulinase (3199 U/ ml) was obtained in the presence of 25 percent (w/v) sucrose, 0.5 percent (w/v) meat extract, 1.5 percent (w/v) NaNO3 and 2.5 mM (v/v) Zn2+, at initial pH of 6.5, temperature 35ºC and 6 percent (v/v) of spores suspension in the agitation speed of 100 rpm. Surfactants showed an inhibitory effect on enzyme production. The optimum temperature for inulinase activity was found to be 50ºC. TLC analysis showed the presence of both exo- and endo-inulinase. Conclusion: Sucrose, Zn2+, and aeration were found to be the most effective elements in inulinase production by A. niger ATCC 20611. TLC analysis also showed that the crude enzyme contained both endo and exo-inulinases. The strain is suggested as a potential candidate for industrial enzymatic production of fructose from inulin.

Production, characterization and application of inulinase from fungal endophyte CCMB 328.

Inulinase (ß-2,1-D- fructan fructanohydrolase), EC 3.2.1.7, targets the ß-2,1 linkage of inulin, a polyfructan consisting of linear ß-2,1 linked fructose, and hydrolyzes it into fructose. This use provides an alternative to produce fructose syrup through the hydrolysis of inulin. The objective of this work was to study the production, characterization and applications of inulinases from the fungal endophyte CCMB 328 isolated from the Brazilian semi-arid region. Response Surface Methodology (RSM) was employed to evaluate the effect of variables (concentration of glucose and yeast extract), on secreted inulinase activities detected in the culture medium and also in the inulin hydrolysis. The results showed that the best conditions for inulinase production by CCMB 328 are 9.89 g / L for glucose and 1.09 g / L for yeast extract. The concentration of 0.20 mol/L of NaCl and KCl increased the activity of inulinase from CCMB 328 by approximately 63% and 37%, respectively. The results also showed that the inulinase has potential for inulin hydrolysis, whose conversion yields roughly 72.48 % for an initial concentration of inulin at 1% (w/v).