Terreus MTCC Research Articles

Fermentative production, purification of inulinase from Aspergillus terreus MTCC 6324 and its application for hydrolysis of sucrose

Abstract The production of inulinase was carried out with Aspergillus terreus MTCC 6324. To enhance the production of inulinase, fermentation media was optimized by one factor at a time and response surface methodology (RSM), which increases the activity of inulinase by 3 folds. Inulinase was partially purified with ultrafiltration method with 67.7% yield. The purified inulinase was then immobilized by entrapment method in calcium alginate beads with retention of 42% of original activity and employed for hydrolysis of sucrose. After optimization of reaction conditions such as speed of agitation, catalyst loading and temperature, the conversion of sucrose hydrolysis reaction was increased up to 91.57%.

Highly Active and Stable Large Catalase Isolated from a Hydrocarbon Degrading Aspergillus terreus MTCC 6324.

A hydrocarbon degrading Aspergillus terreus MTCC 6324 produces a high level of extremely active and stable cellular large catalase (CAT) during growth on n-hexadecane to combat the oxidative stress caused by the hydrocarbon degrading metabolic machinery inside the cell. A 160-fold purification with specific activity of around 66 × 105 U mg−1 protein was achieved. The native protein molecular mass was 368 ± 5 kDa with subunit molecular mass of nearly 90 kDa, which indicates that the native CAT protein is a homotetramer. The isoelectric pH (pI) of the purified CAT was 4.2. BLAST aligned peptide mass fragments of CAT protein showed its highest similarity with the catalase B protein from other fungal sources. CAT was active in a broad range of pH 4 to 12 and temperature 25°C to 90°C. The catalytic efficiency (K cat/K m) of 4.7 × 108 M−1 s−1 within the studied substrate range and alkaline pH stability (half-life, t 1/2 at pH 12~15 months) of CAT are considerably higher than most of the extensively studied catalases from different sources. The storage stability (t 1/2) of CAT at physiological pH 7.5 and 4°C was nearly 30 months. The haem was identified as haem b by electrospray ionization tandem mass spectroscopy (ESI-MS/MS).

Valorization of Agricultural Residues for Compactin Production by Aspergillus terreus MTCC 279 in Mixed Substrate Solid State Fermentation

The present study was aimed to enhance the production of compactin by Aspergillus terreus MTCC 279 in mixed substrate solid state fermentation (SSF) using various solid substrates. Twenty solid substrates including agricultural residues and other solid substrates were tested for the compactin and lovastatin production by A. terreus. Among the twenty substrates tested, the three substrates which gave maximum compactin production was taken for further optimization in mixed substrate solid state fermentation in various combinations using response surface methodology. Green peas, millet and ragi were found to be suitable substrates which produced maximum compactin production in SSF. The combinations of the substrates with 1.5 g of green peas, 1.5 g of millet and 1.5 g of ragi gave maximum production of 389.34 mg/gds compactin and 1,467.12 mg/gds lovastatin. Response surface methodology was used to determine the optimal combination of each substrate. To the best of our knowledge this is the first report on enhancing compactin production in mixed substrate solid state fermentation.

OPTIMIZATION OF L-ASPARAGINASE PRODUCTION BY ASPERGILLUS TERREUS MTCC 1782 USING BAJRA SEED FLOUR UNDER SOLID STATE FERMENTATION

Enzymes are the biocatalysts synthesized by living cells. They are Complex protein molecules that brin g about chemical reactions concerned with life. They are protein in nature, co lloidal and thermolabile in character, and specific in their action. L-asparaginase (L-asparagine amido hydrolase, E.C.3.5.1.1) is an ecellular enzyme that has received considerable attention since it is used as an anticancer agent. The present work deals with production of extracell ular L-asparaginase from Aspergillus terreus MTCC 1using Bajra seed flour under solid state fermentation Pro cess parameters like Incubation time(96 h), Tempera ture (30 0 C), Moisture content (70% v/w), pH of the medium(8.0), Inoculum Age (5 dInoculum volume (1 ml), carbon source (1.5% w /v glucose), nitrogen source ( 2% w/v ammonium sulphate), and metal salts ( 0.1% w/v Magnesium sulphate) were optimized and giving an overall yield of 273.3 U/gds of maximum L-asparaginase activity afte r optimization. The observation made in this study hold great promise for scale up production of L-asparaginase from Aspergillus te rreus MTCC 1782 using Bajra seed flour as substrate under solid state fermentation.

Utilization of whey powder as an alternate carbon source for production of hypocholesterolemic drug by Aspergillus terreus MTCC 1281

In this present study, we investigated the utilization of whey powder as an alternate carbon source and statistical optimization of medium components on lovastatin production by Aspergillus terreus MTCC 1281. Central composite design (CCD) of response surface methodology (RSM) was successfully employed to optimize the four medium variables whey powder, yeast extract, sodium chloride, and copper sulphate at five levels. Regression analysis explained that the linear and square effects of the variables were found significant with R2 value of 0.937. Maximum lovastatin production of 358.2 mg/L was obtained with the predicted optimal concentration of whey powder 1.5%, yeast extract 0.75%, sodium chloride 0.04%, and copper sulphate 0.019%.The results of this study indicated that statistical optimization approach leads to high lovastatin production by using whey powder as a carbon source.

Purification and biochemical characterization of feruloyl esterases fromAspergillus terreusMTCC 11096

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE-1, AtFAE-2, and AtFAE-3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH-dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5-8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca(2+), K(+), and Mg(2+) stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k(cat) /K(m)) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF-MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE-1 belonged to type A while AtFAE-2 and AtFAE-3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase.

Optimization of Culture Conditions and Bench-Scale Production of L-Asparaginase by Submerged Fermentation of Aspergillus terreus MTCC

Optimization of culture conditions for L-asparaginase production by submerged fermentation of Aspergillus terreus MTCC 1782 was studied using a 3-level central composite design of response surface methodology and artificial neural network linked genetic algorithm. The artificial neural network linked genetic algorithm was found to be more efficient than response surface methodology. The experimental L-asparaginase activity of 43.29 IU/ml was obtained at the optimum culture conditions of temperature 35 degrees C, initial pH 6.3, inoculum size 1% (v/v), agitation rate 140 rpm, and incubation time 58.5 h of the artificial neural network linked genetic algorithm, which was close to the predicted activity of 44.38 IU/ml. Characteristics of L-asparaginase production by A. terreus MTCC 1782 were studied in a 3 L bench-scale bioreactor.

Electrochemical investigations of fungal cytochrome P450

The electrochemical investigations of fungal ( Aspergillus terreus MTCC 6324) cytochrome P450 monooxygenase (CYP) (EC 1.14.14.1) immobilized on multi-walled carbon nanotubes–Nafion®–polyethyleneimine (MWCNT-NF/PEI) modified glassy carbon electrode (GCE) were carried out to understand the electrochemistry of the CYP and its application potential in electrochemical-based devices. In deoxygenated condition, the formal potential for Fe (III)/Fe (II) redox couple of CYP was about −0.53 V (vs. Ag/AgCl reference electrode, pH 8). A positive shift in the redox potential of the bioelectrode to −0.475 V was observed in the presence of oxygen and substrate. The electrocatalytic activity towards n-hexadecane and inhibition with piperonyl butoxide (PBO) confirmed the involvement of Fe (III/II) system of CYP in the redox process. The current response increased linearly from 1 μM to 100 μM of n-hexadecane with detection limit 0.1 μM. The IC 50 for PBO was 2.7 μM. The surface coverage of CYP immobilized on MWCNT-NF/PEI modified GCE was approximately 3.45 × 10 −10 mol cm −2. The electron transfer rate constant ( k s ) was 1.0 ± 0.2 s −1, indicating facilitation of the electron transfer between CYP and GCE.

Production of L-asparaginase from Natural Substrates by Aspergillus terreus MTCC 1782: Optimization of Carbon Source and Operating Conditions

In the present work, the effect of different carbon sources, namely glucose, sucrose, maltose, fructose and lactose, was studied for extracellular L-asparaginase production by Aspergillus terreus MTCC 1782 in submerged fermentation. The best carbon source and operating conditions such as initial pH, inoculum size, temperature and agitation rate were optimized. Glucose was found to be the best carbon source for L-asparaginase production using modified Czapek-Dox media containing L-proline as substrate. Sucrose was found to be the best carbon source for L-asparaginase production using modified Czapek-Dox media containing groundnut oil cake flour as substrate. Glucose was found to be the best carbon source for maximum L-asparaginase production using modified Czapek-Dox media containing soya bean meal flour as substrate. The soya bean meal flour was found to be the best natural substrate for maximum L-asparaginase activity of 35.3 IU/mL using 0.6% glucose as carbon source at the optimal culture conditions of initial pH 6, inoculum size 2%, temperature 35°C and agitation rate 160 rpm.

Design of experiments and artificial neural network linked genetic algorithm for modeling and optimization of L-asparaginase production by Aspergillus terreus MTCC 1782

The sequential optimization strategy for design of an experimental and artificial neural network (ANN) linked genetic algorithm (GA) were applied to evaluate and optimize media component for L-asparaginase production by Aspergillus terreus MTCC 1782 in submerged fermentation. The significant media components identified by Plackett-Burman design (PBD) were fitted into a second order polynomial model (R2 = 0.910) and optimized for maximum L-asparaginase production using a five-level central composite design (CCD). A nonlinear model describing the effect of variables on L-asparaginase production was developed (R2 = 0.995) and optimized by a back propagation NN linked GA. Ground nut oil cake (GNOC) flour 3.99% (w/v), sodium nitrate (NaNO3) 1.04%, L-asparagine 1.84%, and sucrose 0.64% were found to be the optimum concentration with a maximum predicted L-asparaginase activity of 36.64 IU/mL using a back propagation NN linked GA. The experimental activity of 36.97 IU/mL obtained using the optimum concentration of media components is close to the predicted L-asparaginase activity of the ANN linked GA.

Statistical and evolutionary optimisation of operating conditions for enhanced production of fungal l-asparaginase

AbstractA three-level central composite design of the Response Surface Methodology and the Artificial Neural Network-linked Genetic Algorithm were applied to find the optimum operating conditions for enhanced production of l-asparaginase by the submerged fermentation of Aspergillus terreus MTCC 1782. The various effects of the operating conditions, including temperature, pH, inoculum concentration, agitation rate, and fermentation time on the experimental production of l-asparaginase, were fitted to a second-order polynomial model and non-linear models using Response Surface Methodology and the Artificial Neural Network, respectively. The Artificial Neural Network model fitted well, achieving a higher coefficient of determination (R 2 = 0.999) than the second-order polynomial model (R 2 = 0.962). The l-asparaginase activity (38.57 IU s mL−1) predicted under the optimum conditions of 32.08°C, pH of 5.85, inoculum concentration of 1 vol. %, agitation rate of 123.5 min−1, and fermentation time of 55.1 h was obtained using the Artificial Neural Networklinked Genetic Algorithm in very close agreement with the activity of 37.84 IU mL−1 achieved in confirmation experiments.

Optimization of L‐asparaginase production by Aspergillus terreus MTCC 1782 using response surface methodology and artificial neural network‐linked genetic algorithm

ABSTRACTThe sequential optimization strategy of design of experiments and back propagation algorithm of artificial neural network‐linked genetic algorithm were used to find the significant fermentation media components and optimum concentration for maximum L‐asparaginase production by Aspergillus terreus MTCC 1782 in submerged fermentation. Components such as L‐proline, sodium nitrate, L‐asparagine and glucose were identified as significant fermentation media components using Plackett–Burman design. The central composite design was used to fit the second‐order polynomial model describing the effect of significant media components on L‐asparaginase production with coefficient of determination (R2) 0.973. A nonlinear model was developed with high coefficient of determination (R2) 0.997 using incremental back propagation algorithm of neural network. The high value of coefficient of determination for artificial neural network model justified an excellent correlation between variables and L‐asparaginase activity and found to be more efficient than the second‐order polynomial model of central composite design. Hence, the optimum concentration of the significant media components was determined using artificial neural network‐linked genetic algorithm. The predicted optimum concentration of the media components was L‐proline 1.7% (w/v), sodium nitrate 1.99% (w/v), L‐asparagine 1.38% (w/v) and glucose 0.65% (w/v) with an experimentally confirmed L‐asparaginase activity of 40.86 IU mL−1. Copyright © 2010 Curtin University of Technology and John Wiley & Sons, Ltd.

Application of latin square design for the evaluation and screening of supplementary nitrogen source for L-asparaginase production by Aspergillus terreus MTCC 1782

The effect of ammonium chloride, urea and sodium nitrate as supplementary nitrogen source on the production of extracellular L-asparaginase using Aspergillus terreus MTCC 1782 was investigated using 5- level Latin square design. The statistical reliability and significance of the variables was studied by performing ANOVA for experimental L-asparaginase activity using Dataplot software. Among the supplementary nitrogen sources studied, ammonium chloride was found to be the best for maximum L-asparaginase activity (confidence level of 96.61%) with less biomass formation (confidence level of 21.93%). The maximum L-asparaginase activity obtained was of 26.47 IU/mL by A. terreus MTCC 1782 using groundnut oil cake powder as natural substrate in submerged fermentation.

Production of L-Asparaginase from Natural Substrates by Aspergillus terreus MTCC 1782: Effect of Substrate, Supplementary Nitrogen Source and L-Asparagine

In the present work, the fungal species Aspergillus terreus MTCC 1782 was used for the production of L-asparaginase using natural substrates like groundnut oil cake, cottonseed oil cake and corn flour in modified Czepak-Dox media and compared the L-asparaginase production using synthetic L-proline. The modified Czepak-Dox media with 2% L-proline, supplemented with 1% sodium nitrate and 1% L-asparagine has shown maximum L-asparaginase activity of 34.98 IU/mL on the third day of production. The modified Czepak-Dox media with 2% groundnut oil cake, 1% sodium nitrate and 1.2% L-asparagine showed maximum L-asparaginase activity of 30.35 IU/mL on the fourth day. These results are comparatively higher than the maximum L-asparaginase production reported in the literature (19.5 U/mL), by isolated Aspergillus sp. using Czepak-Dox media containing 2% (w/v) L-asparagine as the sole substrate along with 1% (w/v) ammonium sulfate as an additional nitrogen source. It is observed that the addition of L-proline or ground nut oil cake increases the L-asparaginase production. The groundnut oil cake is found as the potential natural and cheaper substrate for L-asparaginase production by Aspergillus terreus MTCC1782.

Enhanced production of xylanase by a newly isolated Aspergillus terreus under solid state fermentation using palm industrial waste: A statistical optimization

Xylanase production by a newly isolated Aspergillus terreus MTCC 8661 was optimized using palm fiber in solid state fermentation (SSF). Different fermentation parameters such as incubation temperature, moisture content, medium pH, particle size, incubation time, inoculum, xylose and sodium nitrate concentrations were investigated at the individual and interactive level by the Taguchi methodology. All selected fermentation parameters influenced xylanase production. Moisture content, incubation time and inoculum concentration were the major [∼85%] influential parameters on xylanase production at the individual level. At the interactive level, inoculum concentration was important and accounted for more than 50% of the severity index with particle size and incubation temperature. Xylanase production improved from 41,000 to 115,000 U/g indicating 227% improvement after optimization suggesting that this fungal strain, A. terreus MTCC 8661, has the commercial potential for hemicellulosic enzyme production.

Statistical screening of process variables for the production of L-asparaginase from cornflour by Aspergillus terreus MTCC 1782 in submerged fermentation

L-asparaginase has been used as anti-tumor agent for the effective treatment of acute lymphoblastic leukemia and as food processing aid to reduce the formation of acrylamide during frying of starchy foods at high temperature. Objective of the present work was to study the effect of process variables such as cornflour (as natural substrate), urea (as supplementary nitrogen source), L-asparagine, glucose, di-potassium hydrogen phosphate, potassium chloride, inoculum size and pH on the production of extracellular L-asparaginase by Aspergillus terreus MTCC 1782 in shake culture fermentation. The effect and significance of these variables was studied using 2-level Plackett-Burman statistical design. The experimental L-asparaginase activity was subjected to statistical analysis using MINITAB 14 software. It was found that L-asparagine, cornflour and urea significantly influenced as most important factors on L-asparaginase production. Keywords: Extracellular L-asparaginase, acute lymphoblastic leukemia, food processing aid, PlackettBurman design, fermentation.

Purification and properties of a novel broad substrate specific alcohol oxidase from Aspergillus terreus MTCC 6324

An alcohol oxidase was isolated from the microsome of n-hexadecane grown Aspergillus terreus and purified by ion exchange chromatography. The oxidase was found to act on short chain-, long chain-, secondary-, and aromatic-alcohol substrates with highest affinity for n-heptanol ( K M = 0.498 mM, K cat = 2.7 × 10 2 s − 1 ). The native protein molecular mass was 269 ± 5 kDa and the subunit molecular masses were 85-, 63-, 43-, 27-, and 13-kDa. The isoelectric point of the proteins was within 8.3–8.5. High aggregating property of the protein was demonstrated by AFM, DLS and TEM analyses. Chemical analysis showed the presence of oleic acid and palmitic acid at a ratio of 2:1 in the purified protein. This lipoidic nature of the protein particles was correlated to the high aggregating property. In this flavoenzyme, flavin was non-covalently but avidly associated. Peptide mass fingerprinting studies showed the presence of two FAD binding domains in 63 kDa protein. Among these two FAD binding domain sequences only the YPVIDHEYDAVVV GA GGA GLR peptide shows 45–50% sequence homology with the reported N-terminal sequences of other known alcohol oxidases. Non-redundant database search of 63- and 43-kDa subunits peptide sequences showed no sequence similarity with the other alcohol oxidase protein reported till now.

Broad substrate Cytochrome P450 monooxygenase activity in the cells of Aspergillus terreus MTCC 6324

Cytochrome P450 (CYP) monooxygenase activities with different category of substrates namely, alkanes, alkane derivatives, alcohols, aromatic compounds, organic solvents, and steroids were detected in the cells of Aspergillus terreus. High CYP specific activity was observed when methanol (5.6 ± 0.017 U mg −1), acetone (7.76 ± 0.02 U mg −1), dimethylsulphoxide (DMSO) (9.70 ± 0.005 U mg −1), n-hexadecane (4.39 ± 0.02 U mg −1), or n-octadecane (4.23 ± 0.01 U mg −1) were used as substrates. Significant CYP specific activity was also detected when naphthalene (3.80 ± 0.002 U mg −1) was used as substrate. The CYP catalysis of n-hexadecane had followed both terminal and sub terminal oxidations. The activity was localized in the cytosol of n-hexadecane grown cells, while, it was apparently distributed in light mitochondrial fraction and microsomal fraction of glucose grown cells. The substrate specificities of CYP present in all the locations were similar irrespective of the substrates used for the growth. Heme staining of the microsomal fraction containing CYP and other proteins in SDS–PAGE showed single heme protein band with corresponding molecular weight of 110 kDa.

Functional characterization of alcohol oxidases from Aspergillus terreus MTCC 6324

Short chain alcohol oxidase (SCAO), long chain alcohol oxidase (LCAO), secondary alcohol oxidase (SAO), and aryl alcohol oxidase (AAO) activities were localized in the microsome of Aspergillus terreus during growth of the fungi on n-hexadecane. Zymogram analysis of the microsomes of n-hexadecane-grown cells in polyacrylamide gel electrophoresis showed distinct bands, H4, H3, H2, and H1, in a sequence of their molecular weight (Mr) from high to low. The Mr of the isozymes corresponding to the bands H4, H3, and H2 were close to each other and were higher than 272 kDa. While, the Mr of the isozyme H1 was found to be approximately 48 kDa. H1 gave activity only as SCAO. Although the substrates for other bands were varied, strong (S), medium (M), and weak (W) activity for the bands were as follows: H2: SAO (S), AAO (S), LCAO (M), SCAO (S); H3: LCAO (S), SCAO (S); H4: SCAO (S), LCAO (W), SAO (W). The pH and temperature optima of these isozymes were found to be 8.5+/-0.5 and 30+/-1 degrees C, respectively. The stability of the isozymes was drastically decreased beyond 30 degrees C. The SAO showed 33% enantiomeric excess for the R(-)2-octanol over S(+)2-octanol, which may be correlated with the lower Michaelis-Menten constant (K (M)) values of the enzyme for the R(-)2-octanol than the S(+)2-octanol. The fluorescence emission spectra of the chromatographically purified SCAO at 443 nm excitation were similar to that obtained with authentic flavin adenine dinucleotide.