Biochemical properties of an extracellular β-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate

Cynthia Barbosa Rustiguel¹ · Arthur Henrique Cavalcanti de Oliveira² · Héctor Francisco Terenzi¹ · João Atílio Jorge¹ · Luis Henrique Souza Guimarães*¹

¹Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras, Ribeirão Preto, Universidade de São Paulo, São Paulo, Brasil
²Departamento de Química, Faculdade de Filosofia, Ciências e Letras,Ribeirão Preto, Universidade de São Paulo, São Paulo, Brasil

*Corresponding author: lhguimaraes@ffclrp.usp.br

Financial support: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Keywords: agroindustrial substrate, Aspergillus, β-D-fructofuranosidase, solid state fermentation, sucrose.

The filamentous fungus A. phoenicis produced high levels of β-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14% recovery. The native molecular mass of the glycoprotein (12% of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55ºC and 4.5. The enzyme was stable for more than 1 hr at 50ºC and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80% of activity after storage at 4ºC by 200 hrs. Dichroism analysis showed the presence of random and β-sheet structure. A. phoenicis FFase II was activated by Mn²⁺, Mg²⁺ and Co²⁺, and inhibited by Cu²⁺, Hg²⁺ and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. K_d and V_max values were 18 mM and 189 U/mg protein using sucrose as substrate.