Use of remazol blue dyed avicel for the determination of cellulolytic activity in basidiomycetes

Uso de Avicel colorida com Remazol Blue para determinação da atividade celulolítica em Basidiomycetos

Marcos José Correia1^*, José Antônio de Sousa Pereira Junior¹, Jefferson Cunha dos Santos¹, Maria Auxiliadora de Queiroz Cavalcanti²

¹Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil. ²Departamento de Micologia, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Recife, PE, Brasil

Submitted: January 26, 1998; Returned to authors for corrections: April 09, 1998;

Approved: January 11, 1999.

ABSTRACT

A modified method for direct determination of cellulolytic activity using Avicel colored with Remazol Brilliant Blue R (RBBR) in Agar test tubes is discussed. Refinements were introduced in a simple method for quantitation of cellulase activity, based on the release of dye from Avicel-RBBR medium by the enzymatic hydrolysis. Modifications in Avicel-dye preparation were enhanced and a spectrophotometer for direct OD measurement in agar test tubes used. The use of a spectrophotometer improved the precision of the collected data, since absorbance measurements could be done at the maximum wavelenght for RBBR (595 nm).

Key words: Avicel, cellulase, Remazol Blue, Lentinus edodes, Pleurotus ostreatus.

INTRODUCTION The majority of the tests for detection of cellulolytic activity of microorganisms is based on the formation of a clear halo around a colony growing in opaque agar plate medium. Even when the medium is well dispersed, these halos are difficult to detect (1-4,8,10,11). The visualization of these halos can be improved by the use of Congo Red dye, due its capacity to interact with a wide range of polysaccharides (10). However, the use of this dye may be limited by the interference of hydrolysis products or by the colony diameter, which occasionally may coincide with the halo diameter. These limitations are solved using a colony growth inhibitor and Phosfon to increase contrast between halo and opaque agar (8). The methods for detection of cellulolytic activity in agar plate media are very laborious and need great handling, increasing the risk of contamination by other microorganisms.

Smith, (10) proposed a quantitative method for cellulase in cellulose-azure, using test tubes with 0.75% agar covered by a dye-cellulose agar medium. The dye was released to the basal medium during cellulose degradation by the cellulase produced in the medium. Poincelot and Day, (6) determined cellulase activity by means of solubilization of Remazol Brilliant Blue R (RBBR) from dyed chromatography paper. Schmidt and Kebernik, (7) described a methodology to quantify cellulase and hemicellulase activities in agar tube cultures, using cellulose and hemicellulose colored according procedures of Ng and Zeikus, (5) and detection in a photometer at 578 nm.

The present work reports results of the determination of cellulolytic colored by a modified Wood method (11) dispensed in test tubes, and a spectrophotometer at 595 nm for measurements of maximum RBBR.

MATERIALS AND METHODS

Chemicals. Microcrystaline cellulose (Avicel® - Merck), Remazol Brilliant Blue R (RBBR - Sigma), Malt Extract Agar (Difco).

Cellulase assay media. Basal medium was prepared with 0.75% agar in phosphate buffer pH 5.5 (10); upper medium contained 1% Avicel-RBBR (obtained according to procedure described bellow) plus 1.5% agar and nutrients (7).

Microorganisms. Strains from Laboratório de Bioquímica, Universidade Federal Rural de Pernambuco, included Pleurotus ostreatus (Jacq. et Fr.) Kummer, strains P015, P017 and P021; and Lentinus edodes (Berk.) Sing., strain L022.

Equipment. Spectrophotometer Meternik model SP850.

Procedure. The dyed cellulose was obtained using a modified method of Ng and Zeikus, (5) and Wood, (12). Ten grams of Avicel® were suspended in distilled water at 50°C, and then 100ml of 0.7% RBBR were added. The mixture was maintained under vigorous agitation at the same temperature by approximately 35 min. During this interval, 20g Na₂SO₄ were added in small portions. Afterward, 10 mg Na₃PO₄ were added to increase the pH around 12 and to fix RBBR to cellulose molecules as well. The suspension was maintained under agitation by one aditional hour. The dyed cellulose (Avicel-RBBR) was filtered and washed repeatedly with distilled water 60°C until the filtered material became showed colorless. The washed Avicel-RBBR was rinsed with acetone and ether, and dried in vacuum or in stove at 45°C. Three mililiters of basal medium were added into test tubes, previously standardized with distilled water in a spectrophotometer, and autoclaved at 121ºC for 20 min. The upper medium (Avicel-RBBR) was autoclaved separately in a 200 ml aspirator flask. A stirring bar was placed into aspirator flask before autoclaving. The test tubes with basal medium were placed into a ice cold bath, and the upper Avicel-RBBR medium was added until 0.7 mm above the basal medium using a magnetic stirring device.

Enzymatic assay. For enzymatic activity assay, plugs 3mm diameter of the culture on malt extract agar were transferred to tubes containing Avicel-RBBR. Tests, were done using four replicates and three non inoculated tubes as control. Readings were done every two days, up to 50 days.

RESULTS AND DISCUSSION The measurements of absorbance up to 50 days at 595 nm allowed us to distinguish between low, median and high cellulase active strains. Strains P017, P021 and L022 presented a peak of absorbance followed by decrease, while strain P015 increased continuously through the experiment (

Fig. 1). These results are similar to exocellobiohidrolase and endoglucanase activities (13) obtained using the same strains. Readings at 578 nm, done according to Schmidt and Kebernik (7), did not detect the same maximum absorbance, which was 38% lower than that at 595 nm (

Fig. 2).

Figure 1. Median values of cellulytic activity of

Pleurotus ostreatus

(Fr.) Kummer, strains P015, P017, P021 and

Lentinula edodes

(Berk.) Pegler, strain L022, on Avicel colored with Remazol Brilliant Blue R in agar in test tubes, against a blank non inoculated medium. Reads were obtained a 595 nm and maximum transmitance was considered with non inoculated blank.

Figure 2.
Cellulytic activity of Pleurotus ostreatus (Fr.) Kummer, strains P015, on Avicel colored with Remazol Brilliant Blue R, measured at 578 and 595 nm.

Besides cellulolytic enzymes, Pleurotus sp. produces also na extracelullar peroxidase, associated to lignolytic activity, that may cause RBBR discoloration. This oxidase (RBBR-DcO) is H2O2-dependent and has an optimal pH activity of 3.5 (9). Enzyme activity decreases rapidly above pH 4.0 (9). The use of buffered basal medium at pH 5.0 and absence of H₂O₂ prevent this type of interference.

The use of a spectrophotometer improved the precision of the method. In addition, the stirring of the upper dyed medium while it is added to the basal medium improves the homogeneity of the misture, avoiding unequal substrate concentration, which may interfere in the cellulase activity.

The use of Wood’s modified procedure (12), in stead of the one proposed by Ng and Zeikus, 1980 (5), for cellulase dying, is more rapid and less RBBR consuming and more adequate for determination of cellulolytic activity is fungi.

ACKNOWLEDGMENTS This research was support by a grant from BNB/ETENE/FUNCI of the Banco do Nordeste do Brasil - BNB.

RESUMO

Uso de Avicel colorida com Remazol Blue para determinação da atividade celulolítica em Basidiomycetos

Neste trabalho, é discutido um método modificado para determinação da atividade celulolítica em fungos utilizando Avicel colorida com Remazol Brilliant Blue R (RBBR), diretamente em tubos com ágar, a partir de refinamentos introduzidos em uma metodologia simples e baseada na liberação deste corante pela ação hidrolítica em Avicel colorida com o RBBR. Foram feitas modificações no preparo da Avicel colorida, bem como um espectrofotômetro para medição direta da absorbância nos tubos com ágar foi usado. O uso de espectrofotômetro aumentou a precisão dos dados coletados, pois permitiu leituras de absorbância no comprimento de onda máximo para RBBR (595 nm).

Palavras-chave: celulase, Avicel, Remazol Blue, Pleurotus ostreatus.

• ¹
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*

Corresponding author. Mailing address: Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, CEP 52171-900 , Recife, PE, Brasil.

Publication Dates

• Publication in this collection
  27 May 1999
• Date of issue
  Oct 1998

History

• Reviewed
  09 Apr 1998
• Received
  26 Jan 1998
• Accepted
  11 Jan 1999