Characterization of a β-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry
© Celestino et al; licensee BioMed Central Ltd. 2006
Received: 03 July 2006
Accepted: 05 December 2006
Published: 05 December 2006
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 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 18.104.22.168 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.
The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-β-D-glucan 4-glucanhydrolase (EC 22.214.171.124) 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.
1,3-1,4-β-Glucans are polysaccharides, components of the cell walls of higher members of the Poaceae family. They areparticularly abundant in the endosperm cell walls of commercially valuable cereals such as barley, rye, sorghum, oats and wheat . Structurally, these polysaccharides are linear glucans of up to 1,200 β-D-glucosyl residues linked through β-1,3 and β-1,4 glycosyl bonds. Variations in the proportions of β-1,3-(25–30%) and β-1,4-linkages, and in the length of the mixed-linked segments are currrently reported . During malt production, partial hydrolysis of barley 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, causing lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer . Thus, both the level of glucan-hydrolysing activities achieved during germination and the amounts of their substrates, mainly 1,3-1,4-β-glucan, are important factors in the production of high quality malts. Addition of exogenous 1,3-1,4-β-glucanases to the mash could therefore be an outstanding option for improving the brewing process. However, the β-glucanases currently marketed do not really meet the brewing industry's needs, mainly due to economic factors. Novel 1,3-1,4-β-glucanases with uncommon features would be highly desirable. Here we report on the production, purification and partial characterization of a 1,3-1,4-β-glucanase produced by R. microsporus var. microsporus, considering as well its potential for use in the brewing industry.
Results and Discussion
Hydrolysis of glucan substrates by the R. microsporus purified β-glucanase. ΔAbs 550 nm represents the net absorbance of the reaction mixture after incubation for 0.5 h with the enzyme at 50°C.
Barley β-glucan (1,3-1,4-β-glucan)
Summary of the purification protocol of the 1,3-1,4-β-glucanase produced by Rhizopus microsporus var. microsporus.
Concentrated crude extract
Sephacryl S-100 eluate
SP – Sepharose eluate
The R. microsporus purified β-glucanase was tested for its ability to hydrolyze several other glucan substrates. As may be seen in table 2, only the barley β-glucan was efficiently hydrolyzed, as indicated by the much higher net absorbance. In comparison to the activity against the 1,3-1,4-β-glucan, very low or no activity at all was shown by the enzyme against the substrates laminarin (1,3-β-glucan) and CM-cellulose (soluble 1,4-β-glucan), indicating clearly that the enzyme may be taken as a member of the EC 126.96.36.199 enzyme category.
Effect of pH and temperature optima
Effect of metal ions
Effect of metal ions on the activity of the purified 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus.
Residual activity (%)
Cu+2 (12 mM)
Mg+2 (12 mM)
Fe+3 (12 mM)
Zn+2 (12 mM)
Mn+2 (12 mM)
Ca+2 (12 mM)
Al+3 (12 mM)
Capillary Viscosimetry and Filtration rate
Total protein in the enzyme samples, filtration time, filtration time reduction and specific filtration time reduction of the brewer's mash not treated or treated with enzymes.
Total Protein in the enzyme sample (μg)
Filtration time (seconds)
Filtration time reduction (%)
Specific filtration time reduction (%/μg)
R. microsporus var microsporus enzyme
1854.5 × 10-3
Commercial enzyme A
75.9 × 10-3
Commercial enzyme B
95.8 × 10-3
Total protein in the enzyme samples, viscosity, viscosity reduction and specific viscosity reduction of the brewer's mash not treated or treated with enzymes.
Total Protein in the enzyme sample (μg)
Viscosity reduction (%)
Specific viscosity reduction (%/μg)
R. microsporus var microsporus enzyme
763 × 10-3
Commercial enzyme A
14.2 × 10-3
Commercial enzyme B
17.3 × 10-3
The zygomycete Rhizopus microsporus var. microsporus produced a 1,3-1,4-β-D-glucan 4-glucanhydrolase (EC 188.8.131.52) which could hydrolyze β-D-glucan substrate containing both 1,3- and 1,4-bonds. Its molecular mass as determined by both electrophoresis and mass spectrometry (MALDI-TOF) was about 33.7 kDa. Its optimum pH and temperature were found to be in the ranges of 4–5 and 50–60°C, respectively. Kinetic analysis and its capacity to reduce both the viscosity of the brewer mash and the filtration time, indicate the possibility to use this enzyme in the brewing industry.
Barley 1,3-1,4-β-glucan, chitin, CM-cellulose, manan, xylan, laminarin, molecular mass standard proteins and sodium dodecyl sulfate (SDS) were from Sigma Chemical Co., USA. Sephacryl S-100 and SP-Sepharose were from Pharmacia-LKT, Sweden. All other chemicals were of analytical grade.
Organism and enzyme production
The aerobic zygomycete microfungus Rhizopus microsporus var. microsporus was isolated from a malt silo. The fungus was maintained at 4°C, after growing for 48 hours in TLE modified solid medium [(0.5% chitin, 0.2% KH2PO4, 0.14%(NH4)2SO4, 0.03% MgSO4.7H2O, 0.0152% CaCl2, 0.02% glucose, 1.0 mL of 0.01% trace elements solutions (Fe+2, Mn+2, Zn+2, Co+2), 0.003% bactopeptone, 0.003% urea, and 2% agar, pH 6.8)], at 40°C.
For enzyme production, one liter Erlemeyer flasks containing 250 mL of the liquid medium (TLE with no agar), were inoculated with 150 cm2 blocks of solid medium taken from 2-day old R. microsporus var. microsporus cultures. Liquid cultures were then incubated for 24 hours with agitation (120 rpm) at 40°C. The culture supernatants were then separated from the mycelium by filtration, using filter paper. The supernatants were then freeze-dried and used either for enzyme assay or enzyme purification as described in the following sections.
1,3-1,4-β-glucanase activity was assayed by the reducing-sugar method  with β-1,3-β-1,4-glucan as the substrate. The assay system consisted of 50 μL of 1% (wt/vol) β-glucan dissolved in 100 mM sodium acetate buffer, pH 5.0, and 50 μL enzyme sample. The reaction was allowed to proceed for 30 min at 50°C, and was then stopped by the addition of 300 μL dinitrosalicylate reagent , and 5 min of boiling. The absorbance of the reaction mixture was determined at 550 nm using a Perkin Elmer mod. Lambda 11/Bio spectrophotometer. The amount of reducing sugar produced was determined using a curve constructed with glucose as standard. One unit of enzyme was defined as the amount of protein necessary to produce one μmol of reducing sugars.min-1.
The assays for xylanase, cellulase, 1,3-β-glucanase and mananase were performed as for 1,3-1,4-β-glucanase, except for the use of the substrates carboximetilcellulose, laminarin and manan, respectively. For chitinase the enzyme system consisted of 100 μL of enzyme sample, regenerated chitin 0,5% in 50 mM sodium acetate buffer, pH 5.2 . The reaction was allowed to run for 12h at 37°C and stopped by addition of dinitrosalycilic reagent. The amount of reducing sugar produced was quantified using a standard curve constructed with glucose.
Purifications of the 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus
The supernatants from cultures of R. microsporus var. microsporus grown in liquid medium containing β-glucan were concentrated by ultrafiltration [(Amicon system; 10 k-Da cut-off membrane (PM10)]. Aliquots of concentrated β-glucanase were loaded on a Sephacryl S-100 gel column (2.5 × 40 cm), equilibrated and eluted with 50 mM sodium acetate buffer, pH 5.0. Elution was performed at a flow rate of 24 mL.h-1, and fractions of 4.0 mL were collected. Active fractions were pooled and applied on a SP-Sepharose ion-exchange column (3.0 × 15 cm), previously equilibrated and eluted with 50 mM sodium acetate buffer, pH 5.0, and further eluted with a linear gradient formed with 100 mL of the acetate buffer and 100 mL of the same buffer containing 1 M NaCl. Elution was carried out at a flow rate of 24 mL.h-1, and fractions of 4 mL were collected. The resulting active fractions were pooled and dialyzed overnight against distilled water at 4°C, concentrated by ultrafiltration as above, and stored at -20°C until their use.
Protein was determined by the Bradford method , with bovine serum albumin as standard.
Enzyme samples were examined by electrophoresis under denaturing conditions in polyacrylamide slab gels (SDS-PAGE) as described by Laemmli . Protein bands in the gel were visualized by the silver staining method .
1,3-1,4-β-glucanase was analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry with a Reflex IV mass spectrometer (Bruker Daltonik, Bremen, Germany) in linear positive mode. The purified enzyme sample (50 μg) was dissolved in 50 μL of 0.1 % (v/v) TFA, from which 1 μL was mixed with 1 μL of a saturated matrix solution of sinapinic acid dissolved in 50% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid, and applied to the MALDI plate. BSA was used for external mass calibration.
Effects of ions
The effects of several metallic ions (Cu+2, Mg+2, Fe+3, Zn+2, Ca+2, and Al+3) on the purified 1,3-1,4-β-glucanase were tested measuring the activity of the enzyme at 50°C (see 1,3-1,4–β-glucanase assay) in the presence of the ions.
pH and temperature optima
The effect of temperature on the enzyme was carried out at the temperature range of from 4° to 70°C, at pH 5.0 (in 50 mM sodium acetate buffer). The optimum pH value was determined by monitoring the enzyme activity at 50°C at pH values from 3.0 to 9.0. The following buffers were used: pH 3.0 – 6.0, 50 mM sodium acetate; pH 7.0, 50 mM sodium phosphate; and pH 8.0 – 9.0, 50 mM tris-HCl.
For determination of kinetic parameters, the enzyme assays were performed at 50°C, using 1,3-1,4-β-glucan at concentrations varying from 0.05 to 2.0 % dissolved in 50 mM sodium acetate buffer, pH 5.0. Km, Kcat and Vmax values were obtained using a Michaelis-Menten plot with a non-linear regression data analysis program .
Preparation of the mash
12.5 g of malt was triturated in a hammer mill (MAROTEC), drizzled into a sieve of 0.2 mm spacing, and dissolved in 50 mL of sodium acetate buffer (100 mM, pH 5.5), pre-heated to 45°C. Reaction started with 1.0 mL of enzyme sample taken from chromatography on a Sephacryl S-100 column, and allowed to proceed for 30 min at 45°C, followed by other periods of 10 min at 50°C, 15 min at 60°C, 60 min at 70°C, and 5 min of boiling. The reaction was then stopped by the addition of 100 mL of cold water and immediate cooling in an ice-water bath at about 20°C.
Decrease in mash viscosity was measured by capillary viscosimetry using an Oswald viscosimeter [12, 20]. Samples of 30 mL of mash were filtered using filter paper and placed in a viscosimeter at 20°C. Mash viscosity in the absence of enzyme was used as a control. The specific viscosity rate was calculated using the following equations:
μmash = (μwater × Tmash × ρmash)/(Twater × ρwater) (1)
Δμ = (μmash control – μmash) × 100/(μmash control) (2)
Δμφ = Δμ/δ (3)
Where μ is the viscosity, T is the flow time, Δμ is viscosity reduction, δ is total protein, Δμφ is specific viscosity rate and ρ is the density.
The filtration rate was determined by filtration of 50 mL of mash through a filter paper . Filtration rate in the absence of enzyme was used as a control. The specific filtration rate was calculated using the following equations:
Δψ = (ψmash control – ψmash) × 100/(ψmash control) (4)
Δψφ = Δψ/δ (5)
CRF acknowledge the scholarship awarded by CNPq (process: 305123/2005-0).
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