Calcium carbonate mediates higher lignin peroxidase activity in the culture supernatant of Streptomyces Viridosporus T7A

Calcium carbonate mediates higher lignin peroxidase activity in the culture supernatant of Streptomyces Viridosporus T7A

J. M. B. MACEDO^1,2, L. M. F. GOTTSCHALK² and E. P. S. BON2

1Instituto de Biologia Roberto Alcântara Gomes, Departamento de Bioquímica, (UERJ), Av. 28 de Setembro, 87 fundos, 4^° andar, Rio de Janeiro - RJ, 20551-013. E-mail: [e-mail] ²Centro de Tecnologia, Bloco A, Instituto de Química, Departamento de Bioquímica, (UFRJ), Ilha do Fundão - Rio de Janeiro, RJ, 21949-900.

(Received: January 19, 1999; Accepted: April 9, 1999)

Abstract - Lignin peroxidase (LiP) production has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by S. viridosporus T7A which have been studied include the improvement of yield and enzyme stabilization. In experiments performed in agitated flasks containing culture media composed of yeast extract as the source of nitrogen, mineral salts and different carbon sources, the use of glucose resulted in the highest values for LiP activity (350 U/L), specific LiP activity (450 U/g) and productivity (7 U/L/h). As the profile obtained with glucose-containing medium suggested enzyme instability, the effect of calcium carbonate was evaluated. The addition of CaCO₃ in two different concentrations, 0.5% and 5.0%, resulted in higher values of maximum LiP activity, 600 and 900 U/L, respectively. The presence of this salt also anticipated enzyme activity peaks and allowed the detection of higher enzyme activities in the extracellular medium for longer periods of time. These results indicate a positive effect of calcium carbonate on LiP production, which is extremely relevant for industrial processes.

Keywords: Streptomyces viridosporus, lignin peroxidase, glucose, calcium carbonate, enzyme stabilization.

INTRODUCTION

Lignin is one of the most abundant polymers in the biosphere, representing the biggest reservoir of aromatic compounds in the world. Biodegradation of this polymer is an oxidative process involving extracellular enzymes produced by certain species of microorganisms, including fungi and actinomycetes. Lignin peroxidase, a component of the ligninolytic complex, is responsible for lignin degradation in the presence of hydrogen peroxide (Odier and Artaud, 1992). Another valuable characteristic of this enzyme is its ability to oxidize a large number of aromatic substances, including highly polluting and recalcitrant compounds, such as azo dyes and pesticides (Goszczynski et al., 1994; Pasty-Grigsby et al., 1992). These properties make lignin peroxidase potentially useful in industries using lignocellulosic raw material such as the cellulose and pulp industries; in lignin conversion into products such as fuel, animal feeds and industrial feedstock; and in environmental pollution control (Gilbert et al., 1995).

For many years streptomycetes have received special attention because of their ability to produce a great variety of secondary metabolites. These products include different antibiotics and enzymes having industrial applications (Gilbert et al., 1995; Peczynska-Czoch and Mordaski, 1988). Several Streptomyces species, particularly S. viridosporus, also play an important role in the breakdown of lignocellulosic materials in nature producing considerable amounts of lignin peroxidase (Ramachandra et al., 1988). The major component of the ligninolytic system of S. viridosporus T7A, ALiP-P3, is a heme protein able to oxidize different substrates and the only isoform able to react with 2,4-dichlorophenol (2,4-DCP) (Ramachandra et al., 1988).

Since its first characterization by Ramachandra et al. (1988), several aspects of lignin peroxidase production by Streptomyces viridosporus T7A have been studied. Yeast extract proved to be the best nitrogen source (Ramachandra et al., 1988; Pasti et al., 1990). Different carbon sources, including some considered to be inducers, such as lignocellulose, cellulose and xylans, have been used in order to optimize LiP production. The use of glucose, however, allowed the highest value of LiP activity in spite of showing a repressive effect and causing enzyme deactivation by the end of fermentation (Pasti et al., 1990; Zerbini, 1994).

The structural role played by calcium ions in different peroxidases has been cited in the literature (Sundaramoorthy et al., 1994; Kunishima et al., 1994; Poulos et al., 1993). Thermal inactivation studies performed with fungal lignin peroxidase have proved the importance of these ions for the heme environment and consequently for enzyme activity (Nie and Aust, 1997a and 1997b). Furthermore, the culture medium containing calcium carbonate and starch as the carbon source resulted in the best yield when compared with the other media used for LiP production by S. viridosporus T7A (Yee et al., 1996).

In the present work LiP production using five different culture media was evaluated by determining specific LiP activity and productivity. The use of calcium carbonate was also investigated aiming at increasing enzyme productivity and/or avoiding enzyme inactivation during fermentation.

MATERIALS AND METHODS

Organism

Streptomyces viridosporus T7A (ATCC 39115) stock spores were maintained at -20°C in 20% glycerol (Hopwood et al., 1985) after growth at 37°C for 6 to 8 days on a medium composed of malt extract (3 g/L), yeast extract (3 g/l), peptone (5 g/L), glucose (10 g/L) and agar-agar (10 g/L).

Culture Media and Culture Conditions

The basal medium (YS) was composed of yeast extract (0.65%), mineral salts and trace metal in phosphate buffer (1.98 g/L KH₂PO₄ and 5.3 g/L Na₂HPO₄), as described by Zerbini (1994). Glu, Lac, Gal and Oil media consisted of YS medium supplemented with glucose (0.65%), lactose (0.62%), galactose (0.65%) and corn oil (0.5%), respectively, to give a C/N ratio of about 10. Phosphate buffer of the Glu medium was substituted by calcium carbonate in two different concentrations, 0.5 and 5.0%, constituting Glu0.5 and Glu5.0 media, respectively. Batch fermentations were performed in 500 mL Erlenmeyer flasks containing 100 mL culture medium inoculated with a spore suspension to give a final A_570nm of 0.01 cell density per mL. The cultures were incubated in a shaker (model Tecnal BTC 9090) at 200 rpm and 37°C for 105 hours. Samples of 2 mL taken at different time intervals were centrifuged at 4,000 rpm for 10 min. The pellet was used for determining cell growth, while the culture supernatant was used for the determination of pH, extracellular lignin peroxidase activity and glucose in those media containing this carbon source.

Cell Protein

Cell growth was monitored by determination of the protein content of the cellular material appropriately treated with 1N NaOH (Pasti et al., 1990) using the modified Folin-phenol method and the microassay procedure proposed by Peterson (1983). Protein concentration was estimated by using a standard curve of bovine serum albumin (2 to 40 mg) and was expressed as g of cell protein per L of culture.

pH

The pH values of the culture supernatants were determined in a pHmeter (model Digimed DMPH-2).

Lignin Peroxidase Activity

The extracellular peroxidase activity was determined by a method based on enzymatic oxidation of 2,4-dichlorophenol (2,4-DCP) in the presence of hydrogen peroxide (H₂O₂) and 4-aminoantypirene (4-AAP) (Pasti et al., 1991). A reaction mixture of 1.0 mL contained 50 mM of potassium phosphate (pH 7.0), 3 mM 2,4-DCP, 0.164 mM aminoantipyrine (Sigma), 4 mM hydrogen peroxide and 200 mL of the culture supernatant. The reaction was initiated by the addition of hydrogen peroxide and the increase in absorbance at 510 nm was monitored during 30 seconds at 25°C. One unit of LiP activity corresponded to an increase of 1.0 U of absorbance per minute and the absorbance values considered for this determination represented initial rates of reaction. Extracellular LiP activity was expressed as units of enzyme per L of culture. Specific LiP activity and productivity were expressed as units of enzyme per g of cell protein and units of enzyme per L of culture per hour, respectively.

Glucose

Glucose consumption was monitored by automatic determination of glucose concentration in the culture supernatant using a glucose analyzer (BECKMAN Glucose Analyzer 2 model). Glucose concentration was expressed as g of glucose per L of culture.

RESULTS AND DISCUSSION

Figure ¹ shows the comparison of three parameters obtained in S. viridosporus T7A fermentations using culture media containing different carbon sources. The Glu medium allowed the highest value of maximum LiP activity (350 U/L), specific LiP activity (450 U/g) and productivity (7 U/L/h) when compared with the other four media. In spite of favoring enzyme production, the Glu medium caused enzyme deactivation as the values of extracellular LiP activity decreased after 48 hours of fermentation. Similar behavior was observed in all media containing carbohydrate as the carbon source. A different profile however was observed in medium oil, suggesting that the presence of corn oil prevented enzyme deactivation probably by preventing cell lysis.

Figure 1: Comparison of extracellular lignin peroxidase activity (a), specific lignin peroxidase activity (b) and productivity (c) of batch fermentations of S. viridosporus T7A using the YS, Glu, Lac, Gal and Oil media. Data represent the average of duplicate 100 mL cultures grown at 37°C and 200 rpm.

The use of calcium carbonate favored LiP production by mediating higher values of LiP activity in the culture supernatant of S. viridosporus T7A (Figures ², ³ and ⁴). The Glu0.5 and Glu5.0 media resulted in enzyme activity peaks of 600 and 900 U/L, respectively. Moreover high values of LiP activity were maintained for longer periods of time with regards to the Glu medium(Figures ³ and ⁴). As mentioned previously, calcium ions play important structural roles in peroxidases of different organisms, being required for the integrity of the active site (Nie and Aust, 1997a and 1997b). In the present work it was also observed that calcium carbonate had a great effect on pellet size. The reduction in pellet size might have been relevant for the mechanisms of mass transfer, facilitating nutrient uptake and enzyme release into the culture supernatant. This might be related to the earlier detection of high values of extracellular enzyme activity in the Glu0.5 and Glu5.0 media, which corresponded to a more rapid glucose consumption (Figures ³ and ⁴).

Figure 2: Cell growth, pH variation, glucose consumption and extracellular lignin peroxidase activity profiles of S. viridosporus T7A grown in the Glu culture medium at 37°C and 200 rpm. The data represent the average of duplicate 100 mL cultures.

Figure 3: Cell growth, pH variation, glucose consumption and extracellular lignin peroxidase activity profiles of S. viridosporus T7A grown in the Glu0.5 culture medium at 37°C and 200 rpm. The data represent the average of duplicate 100 mL cultures.

Figure 4: Cell growth, pH variation, glucose consumption and extracellular lignin peroxidase activity profiles of S. viridosporus T7A grown in the Glu5.0 culture medium at 37°C and 200 rpm. The data represent the average of duplicate 100 mL cultures grown.

The maximum values of specific LiP activity were 450, 1400 and 2100 U/g in the Glu, Glu0.5 and Glu5.0 media, respectively (Figure ^5a). The maximum value in the Glu5.0 medium was obtained in 48 hours of fermentation, while those in the other two media were obtained later (in 57 hours).

The maximum productivities of 7.0, 15.0 and 23.5 U/L/h were obtained using the Glu, Glu0.5 and Glu5.0 media, respectively (Figure ^5b). The use of calcium carbonate resulted in maximum productivity in 33 hours of fermentation, while in the medium without this salt the maximum value was obtained later (in 48 hours).

(a)

(b)

Figure 5: Comparison of specific lignin peroxidase activity (a) and productivity (b) of batch fermentations of S. viridosporus T7A using the Glu, Glu0.5 and Glu5.0 media. Data represent the average of duplicate 100 mL cultures grown at 37°C and 200 rpm.

The maximum values of LiP activity and productivity were summarily increased 2.6- and 3.4-fold, respectively. These data corroborate the positive effect of calcium carbonate on lignin peroxidase production by S. viridosporus T7A, which might be related to the stabilization of the enzyme molecule and/or a higher enzyme release due to the reduction in pellet size.

ACKNOWLEDGMENTS

This work was financially supported by FAPERJ (Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior) and FUJB (Fundação Universitária José Bonifácio).

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Publication Dates

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

History

• Received
  19 Jan 1999
• Accepted
  09 Apr 1999