Author:
Celestino Klecius R Silveira,Cunha Ricardo B,Felix Carlos R
Abstract
Abstract
Background
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.
Results
The zygomycete microfungus 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 activity-containing culture supernatant. This isolated protein hydrolyzed 1,3-1,4-β-glucan (barley β-glucan), but showed only residual activity against 1,3-β-glucan (laminarin), or no activity at all against 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 50–60°C, respectively. The Km and Vmax values for hydrolysis of β-glucan at pH 5.0 and 50°C were 22.39 mg.mL-1 and 16.46 mg.min-1, respectively. The purified enzyme was highly sensitive to Cu+2, but showed less or no sensitivity to other divalent ions, and was able to reduce both the viscosity and the filtration time of a sample of brewer mash. In comparison to the values determined for the mash treated with two commercial glucanases, the relative viscosity value for the mash treated with the 1,3-1,4-β-glucanase produced by R. microsporus var. microsporus. was determined to be consistently lower.
Conclusion
The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-β-D-glucan 4-glucanhydrolase (EC 3.2.1.73) which is able to hydrolyze β-D-glucan that contains both the 1,3- and 1,4-bonds (barley β-glucans). Its molecular mass was 33.7 kDa. Maximum activity was detected at pH values in the range of 4–5, and temperatures in the range of 50–60°C. The enzyme was able to reduce both the viscosity of the brewer mash and the filtration time, indicating its potential value for the brewing industry.
Publisher
Springer Science and Business Media LLC
Subject
Molecular Biology,Biochemistry
Reference35 articles.
1. Stone BA, Clarke AE: Chemistry and Biology of 1,3-β-Glucans. 1992, Bundoora: La Trobe University Press;
2. Planas A: Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochimica et Biophysica Acta. 2000, 1543: 361-382.
3. Bamforth CW: β-Glucan and β-glucanases in malting and brewing: practical aspects. Brewers Digest. 1994, 69: 12-16.
4. Fincher GB, Stone BA: Advances in cereal science and technology. 1986, American Association of Cereal Chemists. St. Paul, 7:
5. Godfrey T, West S: Industrial enzymology. 1996, New York : Stockton Press