African Journal of Biotechnology Vol. 5 (12), pp. 1239-1245, 16 June 2006   

ISSN 1684–5315 © 2006 Academic Journals        

Full Length Research Paper

Glucoamylase isoform (GAII) purified from a thermophilic fungus Scytalidium thermophilum 15.8 with biotechnological potential

Mariana Cereia¹, Luis Henrique S. Guimarães¹, Simone C. Peixoto-Nogueira², João A. Jorge¹, Héctor F. Terenzi¹, Lewis J. Greene³ and Maria de Lourdes T.M. Polizeli¹*

¹Depto. de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto – USP, Brazil.

²Depto. de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto – USP, Brazil.

³Centro de Química de Proteínas, Faculdade de Medicina de Ribeirão Preto – USP, Brazil.

*Corresponding author. E-mail: polizeli@ffclrp.usp.br; Phone: +55 16 36024680. Fax: +55 16 36331758.

Accepted 26 April, 2006

Scytalidium thermophilum 15.8 produced two extracellular glucoamylases. Using a DEAE-Cellulose chromatographic column glucoamylases form II (GAII) was separated and purified from glucoamylases form I (GAI) that was previously purified and characterised (Cereia et al., 2000) when the filtrate of the culture medium was applied to a DEAE-Cellulose chromatographic column.  GAII bound to the DEAE-Cellulose and was eluted with a NaCl gradient, while GAI did not bind to the resin. GAII presented electrophoretic homogeneity in 6% denaturing and non-denaturing PAGE, separately, with a molecular mass of 83 kDa, after the second round DEAE-Cellulose purification step. The enzyme pI was 7.2. Optima pH and activity temperature were 5.5 and 55ºC respectively for starch and maltose as substrates, with a termostability of 2.5 min at 60ºC.  Enzymatic activities were activated by 1 mM Na⁺, Mn²⁺ and Mg²⁺ or 10 mM NH⁴⁺, Ba²⁺ and Mg²⁺. The carbohydrate content was 10%. The kinetic parameters Km and Vmax with starch and maltose as substrate were 0.2 and 1.5 mg/ml, and 22.3 and 4.39 U/mg of protein, respectively. The amino acid sequence of GAII had 92% homology with the glucoamylase of Humicola grisea var. thermoidea after 13 cycles.  Generally, GAII had different properties compared with GAI (Cereia et al., 2000).

Key words: Glucoamylase, Scytalidium thermophilum, starch hydrolysis, amylase, fungus.