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Characterization of clarified medium from submerse and semisolid cultivation of OF Aspergillus awamori NRRL3112 by size-exclusion chromatography

Abstract

In this study, a preparative size-exclusion chromatography of two different clarified media obtained from submerse and semisolid culture of the mold Aspergillus awamori was carried out. Characterization and comparison of the quantities of glucoamylase and contaminant proteins present in these media were possible. Glucoamylase is the protein with the higher molecular weight in both media analyzed, varying from 72 to 80kDa in the submerse culture and from 68 to 90kDa in the semisolid culture. Also, glucoamylase protein concentration is higher in the submerse culture than in the semisolid culture. The other proteins in the submerse culture presented molecular weights lower than 12kDa and in the semisolid culture their molecular weights varied from 21 to 37kDa and below 10kDa.

Glucoamylase; gel filtration chromatography; purification; submerse culture; semisolid culture


Characterization of clarified medium from submerse and semisolid cultivation of OF Aspergillus awamori NRRL3112 by size-exclusion chromatography

N.M. MINAMI1, A.C. LUCARINI2 and B.V. KILIKIAN1

1Departamento de Engenharia Química, Escola Politécnica da Universidade de São Paulo, (USP), C. P. Box 61548, 05424-970, São Paulo - SP, Brazil,

Fax: 55-11-2113020, E-mail: kilikian@usp.br

2Departamento de Engenharia Química, Faculdade de Engenharia Industrial (FEI),

CEP 09850-901, São Bernardo do Campo - SP, Brazil

(Received: January 19, 1999; Accepted: April 26, 1999)

Abstract - In this study, a preparative size-exclusion chromatography of two different clarified media obtained from submerse and semisolid culture of the mold Aspergillus awamori was carried out. Characterization and comparison of the quantities of glucoamylase and contaminant proteins present in these media were possible. Glucoamylase is the protein with the higher molecular weight in both media analyzed, varying from 72 to 80kDa in the submerse culture and from 68 to 90kDa in the semisolid culture. Also, glucoamylase protein concentration is higher in the submerse culture than in the semisolid culture. The other proteins in the submerse culture presented molecular weights lower than 12kDa and in the semisolid culture their molecular weights varied from 21 to 37kDa and below 10kDa.

Keywords: Glucoamylase, gel filtration chromatography, purification, submerse culture, semisolid culture.

INTRODUCTION

The microbial enzyme glucoamylase (1,4-a-D-Glucan glucohydrolase, E.C. 3.2.1.3) is important in the starch bioprocessing and brewing industry where the hydrolysis of starch is necessary. Currently, glucoamylase is mostly produced by the submerse culture of many molds, mainly Aspergillus sp., Rhizopus sp. and Endomyces sp. These glucoamylases are extracellular and can be recovered from culture filtrates (Saha and Zeikus, 1989). The molecular weight of glucoamylase can vary from 48 to 112 kDa depending on the producer microorganism. In addition to the presence of isoenzymes, enzyme concentration as well as the type of contaminants can be influenced by the composition of the culture medium, culture conditions and the microorganism (Miah and Ueda, 1977).

The aim of this work was the characterization of two clarified media originating in the submerse and semisolid culture of Aspergillus awamori NRRL 3112 in order to evaluate the type and concentration of contaminant proteins and glucoamylase. This type of characterization is important for glucoamylase purification studies where knowledge of sample composition is an essential preliminary step.

MATERIALS AND METHODS

Preparation of the Clarified Medium Containing Glucoamylase

Aspergillus awamori NRRL 3112 was cultivated in a submerse and a semisolid culture in order to obtain a liquid medium containing crude glucoamylase for purification.

The submerse culture was performed in a 15L bioreactor (LSL Biolafitte SA France) under the following conditions: reaction volume = 10L; inoculum fraction (v/v) = 10%; agitation rate = 700 rpm; air supply rate = 10L/min; head pressure = 0.2atm; pH = 4.0; temperature = 35°C. Cassava flour syrup supplied an initial total reducing sugar concentration (TRS0) of 80g/L. The composition of the medium and details of the culture procedure can be found elsewhere (Kilikian and Jurkiewicz, 1997). When the carbon source was exhausted, the submerse culture broth was centrifuged at 15.000xg for 20 minutes (Damon/Iec Division model B-20A centrifuge) to reduce the amount of mycelia and the resulting supernatant was then filtered (vacuum filtration with a SS5892 filter) to remove the residual mycelia obtaining the clarified medium.

The semisolid culture of the same microorganism was performed as described by Kapritchkoff (1996). The wheat bran mold containing glucoamylase was supplied by the Agrupamento de Biotecnologia at IPT-São Paulo. The proteins present in the semisolid culture were extracted by solubilization of 1g of the dry wheat bran mold in 15mL of 50mM citrate-phosphate buffer at pH 4.2 during 1 hour at 200rpm and 30°C. The mixture was vacuum filtrated (SS5892 filter) in order to obtain the clarified medium.

Preparative Size-Exclusion Chromatography

Fractionation of the samples was performed in a C16/70 column (1.6 x 70 cm Pharmacia) packed with Sephadex G-100 size-exclusion gel that had previously been equilibrated for 15 hours with 100mM phosphate buffer at pH 6, also used as the mobile phase. The fractionation range for globular proteins of Sephadex G-100 is from 4 to 150kDa. Samples of 5 mL of the clarified media, which had previously been filtered in a 0.45 mm membrane, were injected into the column and permeated with 0.5mL/min flow rate of mobile phase. During 5 hours, 5mL fractions were collected every ten minutes.

Analytical Methods

Each fraction of the Sephadex G-100 eluted medium was assayed for total protein concentration, glucoamylase activity and HPLC chromatography.

Glucoamylase activity was determined according to the procedure described by Schmidell and Menezes (1986). One mL of sample (previously diluted) was added to 25mL of starch solution (4%) at 60°C and pH 4.2. The amount of glucose produced after 60 min was measured by the GOD-POD method kit (Merck, Darmstadt, Germany) and absorbance was measured at 510nm. One glucoamylase activity unit (U) was defined as the amount of enzyme which produces 1g of glucose per hour under the standardised conditions.

The concentration of total protein was determined by the methods of Lowry et al. (1951) and Bradford (1976) using bovine serum albumin as the standard. Blank samples without protein were used as a reference.

HPLC chromatographic protein analysis was performed using a Waters 600E HPLC (Waters Associates) device equipped with a Waters 484 UV detector operating at 280nm. A size-exclusion GPC column (Protein-Pak 125/Waters, globular protein range from 2 to 80kDa) at room temperature with 0.5ml/min of mobile phase composed of 0.08M phosphate buffer at pH7, 0.3M NaCl and 20%v/v absolute ethanol (min. 99.5%), was employed. Molecular weights of glucoamylase fractions and other proteins present in the media were determined by means of a calibration curve made with the following standards: a-lactalbumin (14.2kDa), trypsinogen (24kDa), ovalbumin (45kDa) and bovine serum albumin (66kDa). All chemicals and reagents were of analytical reagent grade.

RESULTS AND DISCUSSION

Preparative Size-Exclusion Chromatography of the Submerse Clarified Culture

In Figure 1 , the concentration of total protein and glucoamylase activity of each fraction of the preparative size exclusion chromatography of the submerse clarified culture can be observed. In this chromatogram, there is one peak representing glucoamylase enzymatic activity as well as total protein when assayed by the Bradford method, in addition to the two peaks of total protein assayed by the Lowry method, in the same fractions analyzed. In size-exclusion chromatography the initial fractions represent substances of high molecular weight. Substances present in fractions 22 to 30 were not detected by the Bradford assay as Figure 1 shows, and these fractions represent molecules with molecular weights below 6kDa, close to the lower detection limit of this protein assay which is 5KDa (Córdova-López et al., 1996). These substances with molecular weight lower than that of glucoamylase are the main contaminants of this clarified medium and they confer a yellowish color to it.

Figure 1: Total protein concentration (Lowry and Bradford assay) and glucoamylase activity in the eluted fractions collected by the preparative size-exclusion chromatography (Sephadex G-100 column) of the clarified medium of the submerse culture.

Glucoamylase retention time in HPLC was determined by comparison of glucoamylase activity and HPLC chromatographic protein analysis of each fraction of the Sephadex G-100 size-exclusion chromatography. Fractions with glucoamylase activity and one peak in HPLC chromatograms were considered for identification of the retention time. Figure 2 presents a protein profile similar to that in Figure 1 but with area values from the HPLC chromatographic protein analysis.

Figure 2: HPLC chromatographic protein analysis of the fractions collected by the preparative size-exclusion chromatography (Sephadex G-100 column) of the clarified medium of the submerse culture.

In submerse culture, there are possibly two glucoamylase isoenzymes due to the identification of two different retention times: 13.18 minutes – fraction with a molecular weight of 80KDa and 13.82 minutes – fraction with a molecular weight of 72 KDa. Only with the help of a more selective analytical device than size-exclusion chromatography could other isoenzymes in these fractions be identified.

The yellowish coloured fractions (fractions 24 to 27) presented high concentrations of a peptide with 23.02 minutes of retention time (molecular weight below 6KDa), but the protein profile in the HPLC chromatographic protein analysis of these fractions suggests the presence of different substances. Coloured biomolecules produced by molds are substances which usually have an affinity for iron ions. In this case, no sign of the presence of these substances was found in either of the media analysed, the submerse or the semisolid culture. A study on the production of yellow pigments by molds, conducted by Yongsmith et al. (1994) suggests that in the submerse culture of a Monascus sp. mutant that is also a producer of glucomaylase, this pigmented substance could be monascine or ankaflavine.

Preparative Size-Exclusion Chromatography of the SemiSolid Clarified Culture

The preparative size exclusion chromatography assay performed on the submerse clarified culture was also performed on the semisolid clarified culture. The most important and remarkable characteristic of this medium is the ratio of glucoamylase activity to total protein, which is lower than that in the submerse culture. This result indicates the presence of higher amounts of other proteins besides glucoamylase in the semisolid clarified culture in comparison with amounts in the submerse one. The clarified semisolid medium as well as the submerse clarified medium, presents a larger amount of proteins with lower molecular weights than glucoamylases; these are also nondetectable proteins by the Bradford method as can be observed in Figure 3 .

Figure 3: Total protein concentration (Lowry and Bradford assay) and glucoamylase activity in the fractions collected by the preparative size-exclusion chromatography (Sephadex G-100 column) of the clarified medium of the semisolid culture.

Preparative size-exclusion chromatography of semisolid clarified culture also indicated the presence of a larger variety of glucoamylase isoenzymes than seen in the submerse culture. Glucoamylase fractions of three different molecular weights were found: 90kDa (fractions 11 and 12), 85kDa (fraction 13) and fractions around 69kDa (fractions 14 to 23). In regards to the molecular weights of the other proteins, fraction 21 showed a low concentration of 37kDa protein, fraction 22, a 28kDa protein and fraction 23, a protein of 21kDa. But resolution of the chromatograms of these fractions was poor, indicating that possibly there are other proteins in this molecular weight range. The same occurred for fractions 24 to 27 with proteins from 10 to 4kDa and fractions 28 to 40 where peptides with molecular weights below 4kDa were found.


Figure 4: HPLC chromatographic protein analysis of the fractions collected by the preparative size-exclusion chromatography (Sephadex G-100 column) of the clarified medium of the semisolid culture.

Glucoamylase Protein Content in Both Clarified Media

Using the values from HPLC chromatographic protein analysis (area) and total protein concentrations determined by Lowry´s method (mg/L), a calibration curve for HPLC was determined for the calculation of glucoamylase protein concentration in the initial clarified sample. Fractions 11 to 17 of the preparative size-exclusion chromatography of the submerged culture were used as the standard for the calibration curve presented in Equation 1. These fractions were chosen due to their purity ¾ glucoamylase percentage area higher than 99% indicating pure fractions of the enzyme.

(1)

The percentage of glucoamylase protein content (%GA) in the initial clarified sample was determined by a mass/protein balance in the fractions that presented glucoamylase activity (fractions 9 to 18 of the submerse clarified medium size-exclusion chromatography and fractions 11 to 23 of the semisolid clarified culture medium). Table 1 Table 1: Glucoamylase percentage content (%GA) relative to the total protein concentration in the clarified media. presents the sum of glucoamylase areas in the HPLC analysis of each fraction described above and total protein concentration of the clarified initial medium measured before fractionation in the Sephadex G-100 column.

1 GA = glucoamylase

2 calculated using Equation (1)

3 assayed with Lowry method in the clarified medium

CONCLUSIONS

The greater variety of molecules found in the semisolid culture, as compared with the submerse culture, indicates idea semisolid cultures produce a larger amount and a greater variety of biomolecules that cause more difficulties in the purification process. In addition, these results show that the amount of isoenzymes produced in the semisolid culture is higher than that produced in the submerse culture; however, alternative techniques are still required to confirm this.

Determination of the glucoamylase percentile content in the initial clarified medium shows that 51.5% of the protein concentration in the submerse culture is in the form of glucoamylase fractions, while in the semisolid culture, glucoamylase represents 25.1% of the total protein content. In the submerse culture, contaminants are at lower concentrations than in the semisolid culture and present a great difference in molecular weight compared with the target enzyme, which allows an easy purification of glucoamylase using low resolution techniques such as precipitation or aqueous two-phase extraction.

ACKNOWLEDGEMENTS

Support for this research by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) is gratefully acknowledged.

REFERENCES

Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analyt. Biochem.,72, 248-54 (1976).

Córdova-López, J., Gutierréz-Rojas, M., Huerta, S., Saucedo-Castaneda, G. and Favela-Torres, E., Biomass estimation of Aspergillus niger growing on real and model supports in solid state fermentation, Biotechnol. Techniq., 10, 1, 1-6 (1996).

Kapritchkoff, F.M., Estudo comparativo dos processos de produção de pectinase por fermentação semi-sólida e submersa, Master's thesis, Instituto Butantan, Instituto de Pesquisas Tecnológicas da Universidade de São Paulo, São Paulo (1996).

Kilikian, B.V. and Jurkiewicz, C.H., The gas balance technique and the RQ variability in cultures of Aspergillus awamori NRRL3112, Brazilian J. Chem. Eng., 14, 2, 113-118 (1997).

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, Analyt. Biochem., 193, 265-75, (1951).

Miah, M.N.N. and Ueda, S., Multiplicity of glucoamylase of Aspergillus oryzae. Part 1. Separation and purification of three forms of glucoamylase, Die Stärke, 29, 6, 191-96 (1977).

Saha, B.C. and Zeikus, J.G., Microbial glucoamylases: biochemical and biotechnological Features, Starch, 41, 2, 57-64 (1989).

Schmidell, W. and Menezes, J.R.G., Influência da glicose na determinação da atividade da amiloglicosidase, Rev. Microbiol., 17, 3, 194-200 (1986).

Yongsmith, B., Krairak, S. and Bavavoda, R., Production of yellow pigments in submerged culture of a mutant of Monascus spp., J. Ferment. Bioeng., 78, 223-8 (1994).

  • Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analyt. Biochem.,72, 248-54 (1976).
  • Córdova-López, J., Gutierréz-Rojas, M., Huerta, S., Saucedo-Castaneda, G. and Favela-Torres, E., Biomass estimation of Aspergillus niger growing on real and model supports in solid state fermentation, Biotechnol. Techniq., 10, 1, 1-6 (1996).
  • Kapritchkoff, F.M., Estudo comparativo dos processos de produçăo de pectinase por fermentaçăo semi-sólida e submersa, Master's thesis, Instituto Butantan, Instituto de Pesquisas Tecnológicas da Universidade de Săo Paulo, Săo Paulo (1996).
  • Kilikian, B.V. and Jurkiewicz, C.H., The gas balance technique and the RQ variability in cultures of Aspergillus awamori NRRL3112, Brazilian J. Chem. Eng., 14, 2, 113-118 (1997).
  • Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, Analyt. Biochem., 193, 265-75, (1951).
  • Miah, M.N.N. and Ueda, S., Multiplicity of glucoamylase of Aspergillus oryzae Part 1. Separation and purification of three forms of glucoamylase, Die Stärke, 29, 6, 191-96 (1977).
  • Saha, B.C. and Zeikus, J.G., Microbial glucoamylases: biochemical and biotechnological Features, Starch, 41, 2, 57-64 (1989).
  • Schmidell, W. and Menezes, J.R.G., Influęncia da glicose na determinaçăo da atividade da amiloglicosidase, Rev. Microbiol., 17, 3, 194-200 (1986).
  • Table 1: Glucoamylase percentage content (%GA) relative to the total protein concentration in the clarified media.
  • Publication Dates

    • Publication in this collection
      15 Sept 1999
    • Date of issue
      June 1999

    History

    • Accepted
      26 Apr 1999
    • Received
      19 Jan 1999
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