Article pubs.acs.org/JAFC
Characterization of Barley Serpin Z7 That Plays Multiple Roles in Malt and Beer Xiaomin Li,†,‡,# Zhao Jin,†,‡,# Fei Gao,†,‡,# Jian Lu,†,*,‡,#,§ Guolin Cai,†,‡,# Jianjun Dong,⊥ Junhong Yu,⊥ and Mei Yang⊥ †
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, People’s Republic of China ‡ National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, People’s Republic of China # School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, People’s Republic of China § Industrial Technology Research Institute of Jiangnan University in Suqian, 888 Renmin Road, Suqian 223800, People’s Republic of China ⊥ State Key Laboratory of Biological Fermentation Engineering of Beer, Tsingtao Brewery Company, Ltd., Qingdao 266100, People’s Republic of China S Supporting Information *
ABSTRACT: Barley protein Z7 (BSZ7) is a well-known serine protease inhibitor that was regarded as a major effector of beer foam stability. Moreover, it has also been suggested to participate in haze formation and affect wort filterability. The present study purified BSZ7 from barley malt and characterized its secondary structure and modification, as well as its relationship with peroxidase, to elucidate the molecular base of BSZ7 that supports its multiple roles in malt and beer. It was found that after 30 min of heating, the secondary structure was not affected. BSZ7 has no inhibiting effect on nonspecific protease originated from malt, suggesting its negative role in wort filterability was accomplished by other means. Furthermore, the glycation of BSZ7 by the Maillard reaction may make some contribution to its survival during wort boiling. The interaction of BSZ7 with polysaccharides and polyphenols found by adding experiment may explain how it acts as a negative factor on wort filterability. Greater understanding of BSZ7 and other proteins of malts will lead to better improvements in brewing quality. KEYWORDS: BSZ7, secondary structure, glycation, melanoidins, wort filterability, peroxidase
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INTRODUCTION Barley protein Z belongs to the serine protease inhibitor family (serpin). Due to the high lysine content, it acts as a storage protein in the endosperm. In addition, it is also responsible for defense against insect pathogens because of its proteaseinhibiting activity.1−3 Protein Z displays different isoforms, including protein Zx (BSZx), protein Z4 (BSZ4), and protein Z7 (BSZ7),4 the latter two being major beer proteins with the capacity to survive during brewing.5 The genes encoding BSZ4 and BSZ7 are present on chromosomes 4 and 7 of barley, respectively,6 and BSZ4 comprises 80% of total protein Z in either barley or malt and thus is the predominant one.7 Both of them are suggested to be positive effectors on foam stability.8,9 BSZ7 was also found to be included in the silica eluent part during beer brewing, indicating it is a haze-forming protein in beer.10 In our previous work, comparative proteomics was performed between Dan’er and Metcalfe malts showing different wort filterability, and multiple BSZ7 spots were found to be in more abundance in Dan’er malt, indicating its negative role in wort filterability.11 Addition of BSZ7 to the mashing process significantly decreased the separation rate of wort, which further confirmed its effect on filterability.11 Besides these, BSZ7 has been demonstrated to be an inhibitor of α-chymotrypsin.12 © 2014 American Chemical Society
Although multiple roles of BSZ7 have been explored, the biochemical characterization of the protein was not fully studied. By Evans and Hejgaard,7 the content of BSZ7 was observed to be 38−771 μg/g in barley seeds of 91 varieties and consisting of 397 amino acid residues with an apparent molecular mass of 39 kDa.12,13 The primary structure of BSZ7 has been elucidated. BSZ7 shares 69, 72, and 71% identity with wheat chymotrypsin inhibitor WSZ1, barley BSZ4, and BSZx, respectively. Furthermore, BSZ7 shares 69−72 and 25−32% positional identity with plant and mammalian serpins. In the primary structure of BSZ7, four N-glycosylation sequences AsnXxx-Ser/Thr were found at positions 154, 171, 205, and 316, respectively.13 The partial modification of lysine residues with sugars has been demonstrated by isoelectric focusing of protein Z.14 Dan’er and Metcalfe are typical malting malts in beer brewing. Dan’er is the most popular domestic barley in China. However, due to the deficiency in wort filterability of Dan’er malt, Metcalfe is imported from Canada every year and used for malting and brewing instead. In our previous study, proteomic Received: Revised: Accepted: Published: 5643
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solution (8 M 8-anilino-1-naphthalenesulfonate (ANS) dissolved in 0.02 M PBS buffer (pH 7.0)) was added to one well, whereas the other sample well without substrate buffer was used as blank control. The fluorescence intensity (FI) was determined using a SmartSpec microplate spectrophotometer (Bio-Rad, Hercules, CA, USA) with excitation and emission wavelengths at 390 and 470 nm, respectively. The protein hydrophobic index was calculated by plotting the FI on protein concentration. The hydrophobic index of total proteins in malt and wort of Dan’er and Metcalfe was detected with the same method. Malt Nonspecific Protease Activity Assay. Protease was extracted with the following procedures. Three grams of finely ground malt was mixed with 30 mL of 0.1 M citric acid/Na2PO4 buffer (pH 5.2) and then incubated at 20 °C for 1 h in a water bath to extract the protease. The crude extracts were centrifuged at 8000g for 10 min and then filtered with Whatman no. 4 filter paper (Whatman PLC, Maidstone, UK). The supernatant was collected and stored for protease activity analysis at 680 nm by determining tyrosine produced. The reaction mixture for the hydrolysis reaction (2 mL) contained 1% (w/v) bovine hemoglobin in 0.1 M citric acid/Na2PO4 buffer (pH 5.2) and an appropriate amount of protease extract. Both the protease extract and bovine hemoglobin were preheated at 40 °C before being added into the assay mixture. Reactions were initiated by adding the protease extract and terminated by the addition of 0.4 M trichloroacetic acid (TCA) after incubation at 40 °C for 30 min. The mixture was then allowed to stand for 10 min to precipitate nonhydrolyzed proteins. For blank control, TCA was added together with the bovine hemoglobin before reaction initiation. All of the reactions were repeated in three parallels. After centrifugation, 1 mL of the supernatant was mixed with 5 mL of 0.4 M Na2CO3 and 1 mL of Folin’s reagent and incubated at 40 °C for 20 min. Optical density was read at 680 nm, representing the tyrosine production. One unit of enzyme was defined as the amount of protease that produced 1 μg of tyrosine per minute at pH 5.2 and 40 °C. Melanoidins Extraction and Detection. Malt melanoidins was extracted using an ethanol−water solution method.15 Thirty grams of ground malt was added to 100 mL of 10% ethanol (v/v) and allowed to stand for 20 h to extract melanoidins. The crude extract was then centrifuged at 5000g for 10 min to remove insoluble material, after which the supernatant was added to ethanol to a final concentration of 65% and allowed to stand for 12 h to dissolve melanoidins. The ethanol was evaporated with a rotary evaporator RV10 (IKA, Staufen, Germany) before centrifugation. The corresponding supernatant was then mixed with isometric acetone to precipitate melanoidins and collected by centrifugation. As the acetone volatilized, melanoidins were resuspended with distilled water and dialyzed as soon as possible. The extracted melanoidins were separated by SDS-PAGE and detected with glycoprotein gel staining kit Protein Stains R (Sangon Biotech, Shanghai, China) following the manufacturer’s instructions. Adding Experiment of Purified BSZ7 to Mashing Process. A series of amounts of purified BSZ7 (0, 200, 400, and 600 μg/g malt) was added to the EBC Congress Mashing process with Dan’er malt. Macromolecular content and protease activity were measured as follows. The contents of arabinoxylan (AX), β-glucan, and polyphenaol were determined using the Douglas method,16 CongoRed method,17 and Folin−Ciocalteu colorimetry,18 respectively. High molecular weight protein (HWP) was precipitated with tannin19 and then measured.
analysis and adding experiments confirmed the negative effect of BSZ7 on filterability.11 Therefore, the two malts were selected for further purification and characterization of BSZ7 in the present study, to elucidate the structural and biochemical bases that support the multiple roles of BSZ7 during malting and brewing. More detailed analyses and better comprehension of BSZ7 may help improve brewing quality using the domestic barley Dan’er.
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MATERIALS AND METHODS
Commercial Barley Malt. Samples of barley malts, the domestic Dan’er and the imprted Metcalfe, were collected from commercial maltsters in China and stored at 4 °C in our laboratory. Each maltster has its specialized malting conditions for these two barley cultivars. The Dan’er malt was produced with Dan’er barley that was harvested in 2011 in Jiangsu province of China. Barley of Metcalfe was harvested in 2011 in western Canada and imported to China for malting. Congress wort was obtained by the European Brewery Convention (EBC) Congress mash. Total Protein Extraction from Malt and Wort of Dan’er and Metcalfe. Malt sample was ground into flour and then added into 5 times volume of 0.02 M PBS buffer (pH 5.5) supplemented with 0.1 M NaCl, to extract proteins at 4 °C overnight. The soluble proteins were obtained by centrifugation of crude extract at 4 °C (5000 g, 30 min) and precipitated with 100% saturation of ammonia sulfate. For wort sample, proteins were precipitated with 100% saturation of ammonia sulfate directly. All of the pellet was then resuspended in 0.02 M PBS buffer (pH 5.5) appending 0.1 M NaCl and then dialyzed against 2 L of the same buffer five times (4 h each time). Purification of BSZ7 from Dan’er Malt. The soluble protein of Dan’er malt was fractionally precipitated with 20% and then 70% saturation of ammonia sulfate. The precipitant was collected and resuspended in 0.02 M PBS buffer (pH 5.5) containing 0.1 M NaCl and then dialyzed against 2 L of the same buffer five times (4 h each time). After that, the crude BSZ7 solution was incubated at 75 °C for 15 min to precipitate the hot unstable proteins, which were then removed by centrifugation at 10000 g for 15 min and filtered through a 0.22 μm membrane. The filtration was then loaded on a size exclusion column Hiload 26/60 Superdex 75 pg and then washed with 0.02 M PBS buffer (pH 5.5) containing 0.1 M NaCl at a flow rate of 2 mL/ min. The eluted solutions were collected every 5 mL per tube. To discriminate the fraction containing BSZ7, 20 μL of sample in each tube was displayed on SDS-PAGE, and finally fractions containing the 39 kDa band corresponding to molecular weight of BSZ7 were pooled and concentrated by ultrafiltration for a second size exlusion column Hiload 26/60 Superdex 75 pg purification. After the two rounds of size exclusion purification, BSZ7 was collected and the protein concentration was analyzed using RC-DC quantity kit (GE Healthcare, Pittsburgh, PA, USA). To test the purity of collected BSZ7, 20 μL of sample was displayed on SDS-PAGE, and the corresponding band was then cut for MALDI-TOF/TOF identification. In detail, the 39 kDa band was manually excised from the SDS-PAGE gel and washed with the destaining buffer (25 mM ammonia sulfate, 50% (v/v) acetonitrile), followed by dehydration with 100% acetonitrile. The protein was in-gel digested by trypsin and then submitted to the Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker-Daltonics Inc., Billerica, MA, USA) as described.11 Circular Dichroism (CD) Spectroscopy. BSZ7 was diluted with deionized water to a final concentration of 0.5 mg/mL and then submitted on the MOS-450 AF-CD (Biologic, France) for secondary structure determination. Far-ultraviolet (far-UV, 190−250 nm) and quartz cell with 0.2 mm path length were used. The secondary structure was analyzed with CONTIN software, and data were expressed as mean-residue ellipticity. Hydrophobic Index Determination. A series of concentrations of BSZ7 (0.10, 0.15, 0.20, 0.25, 0.30, 0.35, and 0.40 mg/mL) was obtained by diluting the purified BSZ7 with 0.02 M PBS buffer (pH 7.0). Two hundred microliters of each sample was added to two separate wells of a 96-well plate, after which 2.5 μL of substrate
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RESULTS AND DISCUSSION Purification of BSZ7 from Dan’er Malt. BSZ7 is one of the thermally stable beer proteins that can survive during boiling. Therefore, the crude extracts were heated at 75 °C for 15 min prior to chromatographic separation, so that most of the hot unstable proteins were denatured and precipitated during the heating process. A few of the surviving proteins, including BSZ7, were then submitted to the Superdex G75 size exclusion column. All of the fractions collected were exhibited on SDSPAGE gel (Figure 1a), and the one containing a single 39 kDa 5644
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BSZ7 at different degrees for 30 min. There was no degraded band displayed in the SDS-PAGE gel (Supporting Information supplementary Figure 1), and there was only a decrease in band intensity of the heated BSZ7s spectra. No significant peak shifts were revealed (Figure 2). With increasing temperature the fraction of α-helix decreases, whereas the fraction of β-sheet and β-turn increases (Table 1). Despite these changes in secondary structure unit contents, the content of α-helix remains high, giving BSZ7 thermal stability. Hydrophobicity of BSZ7. Compared with proteins in malt and wort, BSZ7 was the most hydrophobic one (Figure 3).
band corresponding to BSZ7 was considered to be purified BSZ7, which was further confirmed by MALDI-TOF/TOF.
Figure 1. (a) SDS-PAGE of BSZ7 samples during the purification process: lane 1, crude extract; lane 2, salt fractionation; lane 3, heat treating; lane 4, size exclusion column. (b) SDS-PAGE of melanoidins and BSZ7 stained with the glycoprotein gel staining kit; the band corresponding to BSZ7 in melanoidins is indicated by the arrow.
Secondary Structure of BSZ7. CD was used to study the thermal and optical properties of BSZ7. There are two negative minima (280 and 222 nm) and a maximum (192−195 nm), representing a typical α-helix structure, in the far-UV spectra of purified BSZ7 (Figure 2). As shown in Table 1, a significant
Figure 3. Surface hydrophobic index of BSZ7 and proteins from Dan’er malts and worts.
Previous studies have suggested that the hydrophobicity of a protein would increase its positive effect on beer foam.20,21 Another well-known foam-promoting protein, LTP1, was found to decrease rapidly during boiling, thus reducing beer foam stability,22 wheereas, after heating for 30 min, BSZ7 showed no degradation or secondary structure alteration (Supporting Information Supplementary Figures 1 and 2). This result suggested a greater contribution of BSZ7 than LTP1 in beer foam promotion. However, the high hydrophobicity of BSZ7 will also lead to its greater tendency to be left in the spent grain and precipitate to form beer haze, because it was suggested to be able to bind and precipitate with small fragments during wort boiling.23 No Inhibiting Effect of BSZ7 on Malt Nonspecific Protease. In the previous comparative proteomic research, it was found that the deficiency on filterability of Dan’er malt was partially caused by BSZ7.11 The barley serpins were reported to be responsible for protecting the barley grain against insects and fungal infection by inhibiting the activity of pest proteases that digest the contents of grains.24 However, none of these inhibitors has been shown to have inhibiting activity on the endogenous protease of barley or malt.24 As a negative effecter on malt filterability, more BSZ7 in Dan’er malt was first thought to hinder the wort filtering through inhibiting protease activity and leaving more proteins in wort. Unexpectedly, the purified BSZ7 from malt showed no effect on nonspecific protease activity of malt (Suppporting Information supple-
Figure 2. CD spectra of primary and heat-treated BSZ7.
Table 1. Secondary Structure Elements of Heat-Treated BSZ7 secondary structure elements (%) heat treatment
α-helix
β-sheet
β-turn
random coil
untreated control 80 °C, 30 min 90 °C, 30 min 100 °C, 30 min
75.4 75.2 72.6 63.8
0.0 0.1 0.8 2.9
4.4 4.4 4.6 10.8
20.2 20.3 21.9 22.5
fraction of ordered secondary structural elements was found. There was a large amount of α-helix with a small proportion of random coil in the BSZ7 molecular, and no β-sheet or β-turn was found. To observe the influence of wort boiling on BSZ7, SDSPAGE and CD were performed following heat treatment of 5645
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Figure 4. MALDI-TOF MS spectra of purified BSZ7. The marked signals correspond to the unmodified peptides and their glycated form with 162 Da increment of BSZ7.
the oxidation−reduction processes,25 which indicated another role of BSZ7 in malt and beer. No peaks with increment of 162 Da were found in the MALDI-TOF MS spectra of the two BSZ7 spots in 2DE maps of Dan’er malt. As shown in Figure 5, panels a and b, the MS of one BSZ7 spot contained one cluster of peaks and the other showed two clusters, in which the major signals were m/z 1486.783 and 2421.329 and m/z 3052.489, respectively. Four peaks in the second cluster (m/z 3052.495, 3442.772, 3459.765, and 3456.799) were calculated to be glycopeptides analyzed by the oligosaccharide structure prediction tool GlycanMod (http://web.expasy.org/glycomod/). They were thought to be the glycation products of peptide NVTAGLIEEILPAGSIDNTTR (154−174, m/z 2148.143), which contains two of the four N-glycosylation sequences Asn-Xxx-Ser/Thr (NXS or NXT) in BSZ7.13 The N-oligosaccharide components were HexHexNAc1−NeuAc−PentPhos (m/z 550.13), Hex2HexNAc−Deoxyhexose2NeuGc−Pent (m/z 1258.433), Hex 2HexNAc 2 −DeoxyhexosePent 3 (m/z 1272.449), and Hex4HexNAc−Deoxyhexose2Pent (m/z 1275.449), respectively. The latter three structures are paucimannose-type (MannGlcNAc-based structure), and the Pent is probably core xylosylation universally existing in plant glycoproteins.30 BSZ7 purified from barley flour was shown to be nonglycosylated;12 however, this does not mean that BSZ7 in malt was N-terminally blocked. As previously suggested, glycation can stabilize the protein conformation through hydrogen bonds
mentary Figure 2), suggesting it plays its negative role in wort filterability through other actions. BSZ7 Is a Component of Melanoidins. Melanoidins are complicated compound products of the Maillard reaction from sugars and proteins or amino acids during food processing and preservation.25 BSZ7 was detected to display multiple spots in the 2DE maps (Supporting Information supplementary Figure 3).26 Moreover, it has been suggested that protein Z was glycated during processing through the Maillard reaction in the previous study.14,27 Therefore, BSZ7 was proposed to be one component of the melanoidin compounds. Stained with a glycoprotein gel staining kit, a band in the melanoidins lane showed a migration rate similar to that of the BSZ7 one (Figure 1b), indicating BSZ7 is a component of melanoidins, which was further identified by MALDI-TOF/TOF as described under Materials and Methods. In agreement with studies on the nonenzymatic glycated LTP1 and protein Z during malting and brewing processes,27,28 several peaks in the MALDI-TOF MS spectra of BSZ7 were found to have 162 Da increments (Figure 4). Two peptides, m/z 1658.787 and 1686.860, were observed to have an increase of ∼162 Da compared to the unmodified peptides m/z 1496.725 and 1524.802, both of which contained the glycation site lysine (K).13 The glycation of protein Z has been proved to protect it from precipitation in the wort boiling process and improve foam stability.29 In addition, the melanoidins participate in beer color formation and stabilize 5646
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Figure 5. MALDI-TOF MS spectra of the two different spots corresponding to BSZ7 in the 2DE map of Dan’er barley malt. The putative glycopeptides are indicated by arrows.
or van der Waals contacts and thus endows the protein with resistance against wort boiling,31 which further explains the superior thermal stability of BSZ7. Effect of BSZ7 on Wort Filterability. According to BSZ7 content in various barley cultures, a series of amounts of purified BSZ7 was added as EBC Congress mashing with Dan’er malt to observe the effect of excess BSZ7 on wort filterability. Our previous research has shown that extra BSZ7 will seriously decrease the wort separation rate and the turbidity, but have little effect on viscosity.11 To explain this effect, macromolecular contents in the wort with distinct added BSZ7 were analyzed (Figure 6a−c). With increasing added BSZ7, the concentrations of polysaccharides including β-glucan and AX were decreased, and no difference in HWP among the wort was found. However, β-glucan and AX were degraded by their corresponding hydrolysis enzymes, which have not been
reported to be affected by BSZ7. Therefore, we propose the hypothesis that the decreased contents of β-glucan and AX were due to their interaction with BSZ7 by the Maillard reaction, which well explained the unchanged viscosity of wort but decreased turbidity. Due to the high viscosity of polysaccharides, the strong hydrophobicity, and huge size of compounds, they cannot easily pass the filter membrane and therefore caused the decreased wort separation rate. Besides these, the contents of polyphenols were also decreased significantly with increased extra BSZ7 (Figure 6d). It has been proved that the cross-link or oxidative polymerization between protein and polyphenols is an important negative factor in wort filterability.32 However, there was no significant change of polyphenol concentrations in the wort using Metcalfe malt by adding a similar amount of BSZ7 (Supporting Information supplementary Figure 4), indicating there is an enzyme promoting the cross-link, and this enzyme 5647
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Figure 6. Effect of added BSZ7 during mashing on the contents of HWP (a), β-glucan (b), AX (c), and polyphenols (d) in wort.
was in a different redundancy or exhibited distinct activity between these two malts. Peroxidases (POD) from barley are heat-stable enzymes responsible for phenolic substrate oxidative polymerization, which caused the increased color and turbidity of wort.33,34 They have been found to be in more abundance in Dan’er malt compared with Metcalfe malt.11 Furthermore, the activity of POD was higher in Dan’er malt than in Metcalfe (Supporting Information supplementary Figure 5). Therefore, a series of amounts of horeadish peroxidase (0, 200, and 400 U/g malt, Sino-American Biotech., China) instead of malt POD was mixed with purified BSZ7 (600 μg/g malt) and then added simultaneously to test their synergetic effect on wort filterability. As peroxidase increased, the concentration of free polyphenols decreased significantly (Figure 7). At the same time, the wort separation rate was negatively hindered by them. In conclusion, the present study has characterized BSZ7, a well-known serpin Z of barley playing multiple roles in barley, malt, and wort. The secondary structure and glycation facilitate its survival during wort boiling and improvement of foam stability in beer. Moreover, its existence in melanoidins indicated its potential role in the oxidation−reduction process. However, the high hydrophobicity of BSZ7 and its interaction with polysaccharides in the wort may hinder the compound’s passing through the filter membrane, although it may decrease the viscosity to some degree. Finally, our data indicated that the cross-links induced by POD between polyphenols and BSZ7 may also create a negative effect on wort filterability. The glycation of BSZ7 and interaction between polysaccharides and BSZ7 still need more evidence. At the same time, the catalytic mechanism of POD is under study by another project.
Figure 7. Effect of simultaneous addition of BSZ7 and POD on the content of polyphenols in wort.
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ASSOCIATED CONTENT
S Supporting Information *
SDS-PAGE of heat-treated BSZ7; effect of distinct amount of added BSZ7 on the activity of protease from Dan’er malt; two BSZ7 spots in 2DE map of Dan’er malt; effect of distinct amount of added BSZ7 on the concentration of free polyphenols in Metcalfe malt; activity of POD in malts of different cultivars. This material is available free of charge via the Internet at http://pubs.acs.org. 5648
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AUTHOR INFORMATION
Corresponding Author
*(J.L.) E-mail:
[email protected]. Phone: +86 510 8591 8191. Fax: +86 510 8591 8191. Funding
This research was supported by the Major State Basic Research Development Program o f China (973 Pr ogram, 2012CB720802 and 2013CB733602), the National High Technology Research and Development Program of China (863 Program, 2013AA102109), the National Natural Science Foundation of China (31171736), the Key Program of National Natural Science Foundation of China (31130043), the Key Project of Educational Commission of Jiangsu Province of China (JHB2012-26), the Fundamental Research Funds for the Central Universities (JUDCF10047, JUSRP1015, and JUSRP51302A), the Key Technologies R&D Program of Jiangsu Province of China (BE2012397), and the Program of Introducing Talents of Discipline to Universities (111 Project, 111-2-06). Notes
The authors declare no competing financial interest.
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REFERENCES
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