Produção e caracterização de uma [beta]-glicanase de Rhizopus microsporus var. microsporus, e seu potencial para aplicação na indústria da cerveja

AUTOR(ES)

Klecius Renato Silveira Celestino

DATA DE PUBLICAÇÃO

2007

RESUMO

In the barley malting process, partial hydrolysis of β-glucans begins with seed germination. However, the endogenous 1,3-1,4-β-glucanases are heat inactivated, and the remaining high molecular weight β-glucans may cause severe problems such as increased brewer mash viscosity and turbidity. Increased viscosity impairs pumping and filtration, resulting in lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer. Therefore, the use of exogenous β-glucanases to reduce the β-glucans already present in the malt barley is highly desirable. The fungus Rhizopus microsporus var. microsporus secreted substantial amounts of β-glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gel filtration and ion exchange chromatographies of the β-glucanase activitycontaining culture supernatant. This isolated protein hydrolyzed 1,3-1,4-β-glucan (barley β- glucan), and no activity against 1,3-β-glucan (laminarin) and 1,4-β-glucan (cellulose), indicating that the R. microsporus var. microsporus enzyme is a member of the EC 3.2.1.73 category. The purified protein had a molecular mass of 33.7 kDa, as determined by mass spectrometry. The optimal pH and temperature for hydrolysis of 1,3-1,4-β-glucan were in the ranges of 4-5, and 50-60C, respectively. The enzyme 1,3-1,4-β-glucanase showed high stability at 37C without loss of enzymatic activity for 15 days. However at 50, 60 and 70C, the enzyme was much less stable with half lives of approximately 72h, 10 min and 1 min, respectively. The Km, Kcat, Kcat.Km-1 and Vmax values for hydrolysis of β-glucan at pH 5.0 and 50C were 19.8 mg.mL-1, 12.67s-1, 0.64mL.mg-1.s-1 and 16.5 U.mL-1, respectively. The purified enzyme was highly sensitive to Cu+2, but showed less or no sensitivity to other divalent ions. The presence of carbohydrate was detected in the pure 1,3-1,4-β- glucanase . It was activated by DTT and L-tryptophan and inhibited by β-mercaptoethanol. The enzyme was able to reduce both the viscosity and the filtration time of a sample of brewer mash, indicating its potential value for the brewing industry. In comparison to the values determined for the mash treated with two commercial glucanases, the relatives viscosity and filtration time values for the mash treated with the 1,3-1,4-β-glucanase produced by R. microsporus var. microsporus. was determined to be consistently lower.

ASSUNTO(S)

fungos - aplicações industriais biologia molecular cerveja - microbiologia enzimas de fungos

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