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

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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

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  BioMed   Central Page 1 of 9 (page number not for citation purposes) BMC Biochemistry  Open Access Research article Characterization of a β -glucanase produced by Rhizopus microsporus var. microsporus , and its potential for application in the brewing industry KleciusR SilveiraCelestino 1 , RicardoBCunha 2  and CarlosRFelix* 1  Address: 1 Laboratório de Enzimologia, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, CEP 70910-900, Brazil and 2 Centro Brasileiro de Serviços e Pesquisas em Proteínas (LB QP)-Divisão de Química Analítica, Instituto de Química, Universidade de Brasília, Brasília, DF, CEP 70910-900, BrazilEmail: KleciusR SilveiraCelestino-klecius@unb.br; RicardoBCunha-rbcunha@unb.br; CarlosRFelix*-carlosrf@unb.br * Corresponding author 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 molecularweight 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 barleyis highly desirable. Results: The zygomycete microfungus Rhizopus microsporus var. microsporus secreted substantial amountsof β -glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gelfiltration 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 activityagainst 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 amolecular mass of 33.7 kDa, as determined by mass spectrometry. The optimal pH and temperature forhydrolysis of 1,3-1,4- β -glucan were in the ranges of 4–5, and 50–60°C, respectively. The Km and Vmaxvalues 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 comparisonto the values determined for the mash treated with two commercial glucanases, the relative viscosity valuefor the mash treated with the 1,3-1,4- β -glucanase produced by R. microsporus var. microsporus . wasdetermined to be consistently lower. Conclusion: The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4- β -D-glucan4-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 valuesin the range of 4–5, and temperatures in the range of 50–60°C. The enzyme was able to reduce both theviscosity of the brewer mash and the filtration time, indicating its potential value for the brewing industry. Published: 05 December 2006 BMC Biochemistry   2006, 7 :23doi:10.1186/1471-2091-7-23Received: 03 July 2006Accepted: 05 December 2006This article is available from: http://www.biomedcentral.com/1471-2091/7/23© 2006 Celestino et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  BMC Biochemistry   2006, 7 :23http://www.biomedcentral.com/1471-2091/7/23Page 2 of 9 (page number not for citation purposes) Background 1,3-1,4- β -Glucans are polysaccharides, components of thecell walls of higher members of the Poaceae family. They areparticularly abundant in the endosperm cell walls of commercially valuable cereals such as barley, rye, sor-ghum, oats and wheat [1]. Structurally, these polysaccha-rides are linear glucans of up to 1,200 β -D-glucosylresidues 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 seg-ments are currrently reported [2]. During malt produc-tion, partial hydrolysis of barley β -glucans begins withseed germination [13]. However, the endogenous 1,3-1,4- β -glucanases are heat inactivated, and the remaining highmolecular weight β -glucans may cause severe problemssuch as increased brewer mash viscosity and turbidity[14].Increased viscosity impairs pumping and filtration, caus-ing lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous pre-cipitates in the finished beer [2]. Thus, both the level of glucan-hydrolysing activities achieved during germina-tion and the amounts of their substrates, mainly 1,3-1,4- β -glucan, are important factors in the production of highquality malts. Addition of exogenous 1,3-1,4- β -glucanasesto the mash could therefore be an outstanding option for improving the brewing process. However, the β -gluca-nases currently marketed do not really meet the brewing industry's needs, mainly due to economic factors. Novel1,3-1,4- β -glucanases with uncommon features would behighly desirable. Here we report on the production, puri-fication and partial characterization of a 1,3-1,4- β -gluca-nase produced by R. microsporus var. microsporus ,considering as well its potential for use in the brewing industry. Results and Discussion Enzyme production  A 1,3-1,4- β -glucan-degrading filamentous fungus was iso-lated from a malt silo in a brewery. This zygomycetemicrofungus was identified as Rhizopus microsporus var.microsporus by rcDNA analysis. It grew strongly in liquidmedium containing chitin as the sole carbon source, andproduced substantial amounts of β -glucanase activity (Figure 1) which was able to fully hydrolyze barley β -glu-can (1,3-1,4- β -glucan). The specificity of substrate hydrol- ysis by this enzyme (Table 2) fully supports theassumption that it belongs to the 3.2.1.73 category. Theinducible nature of 1,3-1,4- β -glucanase production hasalready been reported for 1,3- β -glucanase from Trichode-rma sp . [27]. Although cellulose and xylan were alsoinducers, the levels of enzyme secreted in the presence of these carbohydrates were considered smaller than theactivity induced by chitin. In cultures grown under agita-tion (120 rpm) at 40°C, the enzyme activity increasedfrom a minimum to a maximum level within 24h of growth. It has been reported that several other microrgan-isms, including Bacillus sp . [15] Trichoderma sp . [16], Talaromyces emersonii [17], and Rhizobium sp . [2], produce1,3-1,4- β -glucanase enzymes which are currently used inthe brewing industry. However, depending on the sub-strate ( β -glucan) used as inducer, production of the gluca-nases for industrial application may be very costly, to thepoint of being considered economically prohibitive [28].Chitin, on the other hand, is a relatively cheap and readily available carbon source in comparison to barley β -glucanand laminarin. The ability, therefore, of R. microsporus var.microsporus to produce 1,3-1,4- β -glucanase in the presenceof chitin, favors its use on an industrial scale. Enzyme Purification  The culture supernatant of R. microsporus var. microsporus grown in liquid medium containing chitin was concen-trated 10-fold by ultrafiltration, using a 10 kDa cut-off membrane. No 1,3-1,4- β -glucanase activity was found inthe filtrate. Chromatography of the concentrate on aSephacryl S-100 gel filtration column (not shown) fol-lowed by chromatography on an SP-Sepharose ionexchange column resulted in elution of two peaks of pro-teins (PGI and PGII) (Figure 2). While the PGI proteins were inactive, the PGII protein (fractions 22–34) showedsubstantial activity against 1,3-1,4- β -glucan. A summary of the purification steps of the 1,3-1,4- β -glucanase pro-duced by the R. microsporus var. microsporus is shown intable 1. The enzyme was purified (Figure 3) 55.529-fold  with a yield of 114.912% and a specific activity of 12.596U.mg  -1 . The molecular mass of the PGII protein was 36.5kDa, as indicated by SDS-PAGE analysis (Figure 3). This value is comparable to that (33.7 kDa) determined by  Time course of production of 1,3-1,4- β -glucanase by Rhizo-pus 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 1 Time course of production of 1,3-1,4- β -glucanase by Rhizo-pus 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 01234567890 12 24 36 48 60 72 84 Time (h)    A  c   t   i  v   i   t  y   (   U .  m   L   -   1    ) Xylan Cellulose Chitin Figure 1  BMC Biochemistry   2006, 7 :23http://www.biomedcentral.com/1471-2091/7/23Page 3 of 9 (page number not for citation purposes) mass spectrometry analysis for this enzyme (Figure 3). While 1,3-1,4- β -glucanases from Bacillus sp . have smaller molecular masses varying in the range of 25–30 kDa [2],the enzymes from Clostridium thermocellum (38 kDa) [34], Bacteroides succinogenes (37 kDa) [33] and Talaromycesemersonii (40.7 kDa) [2] showed comparable molecular mass values. Enzyme specificity   The R. microsporus purified β -glucanase was tested for itsability to hydrolyze several other glucan substrates. Asmay be seen in table 2, only the barley β -glucan was effi-ciently 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 -glu-can) and CM-cellulose (soluble 1,4- β -glucan), indicating clearly that the enzyme may be taken as a member of theEC 3.2.1.73 enzyme category. Effect of pH and temperature optima  The effect of pH and temperature on the activity of thepurified 1,3-1,4- β -glucanase from Rhizopus microsporusvar. microsporus is shown in figures 4 and 5, respectively.  At 50°C, the enzyme showed substantial activity in thepH range of from 2 to 6. Maximal activity was recorded inthe range of from 4 to 5. No enzyme activity was detectedat pH higher than 6 (Figure 4). At pH 5.0, the purifiedenzyme was substantially active in the temperature range Table 2: Hydrolysis of glucan substrates by the R. microsporus purified β -glucanase. ∆ Abs 550 nm represents the net absorbance of the reaction mixture after incubation for 0.5 h with the enzyme at 50°C. Substrate  ∆ Abs 550 nm Laminarin (1,3- β -glucan)0.029Chitin0.001CM-cellulose (1,4- β -glucan)0.001Xylan0.026Manan0.000Barley β -glucan (1,3-1,4- β -glucan)0.826 Ion exchange (SP-Sepharose column) chromatography of the concentrated culture filtrate of Rhizopus microsporus var. micro-sporus grown in liquid medium containing 0.5% chitin Figure 2 Ion exchange (SP-Sepharose column) chromatography of the concentrated culture filtrate of Rhizopus microsporus var. micro-sporus grown in liquid medium containing 0.5% chitin. 00,010,020,030,040,050,060,070,080,090,11 8 15 22 29 36 43 50 57 64 71 78 85 92Fractions    A   b  s   (   2   8   0  n  m   ) 00,511,522,5    A  c   t   i  v   i   t  y   (   U .  m   L   -   1    )   N  a   C   l   (   M   )  Abs (280nm) Activity NaCl(M) PGI PGII  Figure 2  BMC Biochemistry   2006, 7 :23http://www.biomedcentral.com/1471-2091/7/23Page 4 of 9 (page number not for citation purposes) from 20°C to 65°C. Maximal activity was detected at 50°C and 60°C, indicating that the optimal temperaturefor glucan hydrolysis is 55°C (Figure 5). The optima pHand temperature values determined for the purified 1,3-1,4- β -glucanase from R. microsporus var. microsporus  weresimilar to those determined for 1,3-1,4- β -glucanases fromseveral other fungi and bacteria [2]. In addition, these val-ues are comparable to those presented by enzymes cur-rently being used in the brewing industry [11,2]. The purified 1,3-1,4- β -glucanase retained 100% and 87% of its activity after incubation for 2 h and 24 h, respectively,at 50°C. The half-lives of the enzyme at the temperaturesof 60°C and 70°C were found to be 10 min and 1 min,respectively. At 50°C, the half-life was 72 h (data not shown). For hydrolysis of β -glucan by a novel 1,3-1,4- β -glucanase produced by Bacillus halodurans C-125 , the pHoptimum was between 6 and 8, and the temperature opti-mum was 60°C. After 2 h incubation at 50°C and 60°C,the residual activity remained 100% and 50%, respec-tively. The enzymatic activity was abolished after 3 minincubation at 70°C. The optimum temperature for hydrolysis of lichenan by a 1,3-1,4- β -glucanase from Bacteroides succinogenes at ph 6.0 was 50°C [33]. Effect of metal ions  The effect of several ions on the activity of the purified 1,3-1,4- β -glucanase produced by R. microsporus var. micro- sporus is shown in Table 3. The enzyme was sensitive tocopper and fairly sensitive to zinc and manganese, but insensitive to magnesium, calcium and aluminum (Table3). Glucanases produced by Rhizopus oryzae [29], Bacillusclausii [30], Bacillus halodurans [32] and Trichoderma har- zianum [31] show similar sensitivity to the divalent metalion copper. 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 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 3 Table 1: Summary of the purification protocol of the 1,3-1,4- β -glucanase produced by Rhizopus microsporus var. microsporus . Steps Total Protein(mg)Total Activity(U)Specific activity(U.mg -1 )Purification (-fold)Yield(%) Concentrated crude extract1.5740.2280.1971100.000Sephacryl S-100 eluate0.2350.2270.9664.91599.561SP – Sepharose eluate0.0210.26212.59655.529114.912  BMC Biochemistry   2006, 7 :23http://www.biomedcentral.com/1471-2091/7/23Page 5 of 9 (page number not for citation purposes) Kinetic Parameters  The purified 1,3-1,4- β -glucanase produced by R. micro- sporus var. microsporus hydrolyzed 1,3 – 1,4- β -glucan in aMichaelis-Menten fashion (Figure 7). Kinetic parameters were calculated using a Michaelis-Menten plot with a non-linear regression data analysis program [10]. Values of 19.8 mg.mL  -1 , 12.7s -1 and 16.5 U.mL  -1  were determinedfor Km, Kcat and Vmax, respectively. Km values of 1.2 –1.5 mg.mL  -1 for hydrolysis of barley β -glucan and 0.8 – 2mg.mL  -1 for lichenan were reported for the 1,3-1,4- β -glu-canase produced by Bacillus sp [2]. Values of 1,296 ± 51,2.50 ± 0.09, and 518 were reported for Kcat (s -1 ), Km(mg.mL  -1 ) and Kcat/Km (s -1 .M -1 ) respectively, for hydrol- ysis of lichenan by a 1,3-1,4- β -glucanase produced by  Bacteroides succinogenes . [33]. Capillary Viscosimetry and Filtration rate  The specific filtration rate and specific viscosity rate of themash after incubation with the 1,3-1,4- β -glucanase from R. microsporus var microsporus  were compared with those values calculated for two commercial β -glucanases cur-rently used in the brewing industry. The results are shownin Tables 4 and 5. Even at lower enzyme concentration, the 1,3-1,4- β -glucanase from R. microsporus var microsporus caused a higher reduction in the filtration rate (20.4%) of the mash (table 4). Similar results were obtained for thespecific viscosity of the brewer's mash after treatment withthe three β -glucanases (table 5). Conclusion  The zygomycete Rhizopus microsporus var. microsporus pro-duced a 1,3-1,4- β -D-glucan 4-glucanhydrolase (EC3.2.1.73) which could hydrolyze β -D-glucan substratecontaining both 1,3- and 1,4-bonds. Its molecular mass asdetermined by both electrophoresis and mass spectrome-try (MALDI-TOF) was about 33.7 kDa. Its optimum pHand temperature were found to be in the ranges of 4–5and 50–60°C, respectively. Kinetic analysis and its capac-ity to reduce both the viscosity of the brewer mash and the Thermostability of the purified 1,3-1,4- β -glucanase from Rhiz-opus microsporus var. microsporus , at temperatures of 50°C ( ● ), 60°C ( ❍ ) and 70°C ( ▲ ), at pH 5.0 Figure 6 Thermostability of the purified 1,3-1,4- β -glucanase from Rhiz-opus microsporus var. microsporus , at temperatures of 50°C ( ● ), 60°C ( ❍ ) and 70°C ( ▲ ), at pH 5.0. 01020304050607080901000 5 10 15 20 25 30 35 40 45 50 55Time (minutes)    R   e   l   a   t   i  v   e   a   c   t   i  v   i   t  y   (   %   ) Figure 6 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 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. 0204060801000 10 20 30 40 50 60 70 80 Temperature (ºC)    R  e   l  a   t   i  v  e  a  c   t   i  v   i   t  y   (   %   ) Figure 5 Effect of pH on the activity of the purified 1,3-1,4- β -glucanase from Rhizopus microsporus var.microsporus , at 50°C 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. 0204060801000 1 2 3 4 5 6 7 8 9 10pH    R  e   l  a   t   i  v  e  a  c   t   i  v   i   t  y   (   %   ) Figure 4 Table 3: Effect of metal ions on the activity of the purified 1,3-1,4- β -glucanase from Rhizopus microsporus var. microsporus . Ion Residual activity (%) Control100Cu +2 (12 mM)0.3Mg +2 (12 mM)95.2Fe +3 (12 mM)89.6Zn +2 (12 mM)65.0Mn +2 (12 mM)62.3Ca +2 (12 mM)105.9Al +3 (12 mM)109.8
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