Characterization of a beta-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry

Abstract

Background: In the barley malting process, partial hydrolysis of beta-glucans begins with seed germination. However, the endogenous 1,3-1,4-beta-glucanases are heat inactivated, and the remaining high molecular weight beta-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 beta-glucanases to reduce the beta-glucans already present in the malt barley is highly desirable.

Results: The zygomycete microfungus Rhizopus microsporus var. microsporus secreted substantial amounts of beta-glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gel filtration and ion exchange chromatographies of the beta-glucanase activity-containing culture supernatant. This isolated protein hydrolyzed 1,3-1,4-beta-glucan (barley beta-glucan), but showed only residual activity against 1,3-beta-glucan (laminarin), or no activity at all against 1,4-beta-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-beta-glucan were in the ranges of 4-5, and 50-60 degrees C, respectively. The Km and Vmax values for hydrolysis of beta-glucan at pH 5.0 and 50 degrees 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-beta-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-beta-D-glucan 4-glucanhydrolase (EC 3.2.1.73) which is able to hydrolyze beta-D-glucan that contains both the 1,3- and 1,4-bonds (barley beta-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 degrees 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.

Figure 1

Time course of production of 1,3-1,4-β-glucanase by Rhizopus microsporus var. microsporus in the presence of 0.5% of either xylan, cellulose or chitin at a temperature of 40°C and at 120 rpm

Figure 2

Ion exchange (SP-Sepharose column) chromatography of the concentrated culture filtrate of Rhizopus microsporus var. microsporus grown in liquid medium containing 0.5% chitin.

Figure 3

SDS-PAGE (A) and MALDI-TOF mass spectrometry (B) analysis of the purified 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus. A: line 1, molecular weight markers; line 2, PGI protein fraction; line 3, PGII protein fraction.

Figure 4

Effect of pH on the activity of the purified 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus, at 50°C.

Figure 5

Effect of temperature on the activity of the purified 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus, at pH 5.0.

Figure 7

Hydrolysis (μmol·min^-1·mL^-1) of β-glucan by the purified 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus, in the presence of different concentrations of 1,3-1,4-β-glucan.

Figure 6

Thermostability of the purified 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus, at temperatures of 50°C (●), 60°C (○) and 70°C (▲), at pH 5.0.