Analytical Biochemistry 421 (2012) 802–804
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Notes & Tips
Fast and reliable method for simultaneous zymographic detection of glucoamylase and a-amylase in fungal fermentation Biljana Dojnov a, Zoran Vujcˇic´ b,⇑ a b
Center for Chemistry, Institute of Chemistry, Technology, and Metallurgy, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia
a r t i c l e
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Article history: Received 19 October 2011 Received in revised form 29 November 2011 Accepted 30 November 2011 Available online 8 December 2011 Keywords: a-Amylase Glucoamylase Aspergillus niger Zymogram SSF
a b s t r a c t Detection of a-amylase and glucoamylase in crude fermentation extracts using a single native electrophoresis gel and zymogram is described in this article. Proteins were printed on substrate gel and simultaneously onto a membrane in a three-sandwich gel. a-Amylase was detected on the substrate gel with copolymerized b-limit dextrins and iodine reagent. Glucoamylases were detected on the membrane using a coupled assay for glucose detection. Both amylases were detected in native gel using starch and iodine reagent. The described technique can be a helpful tool for monitoring and control of fermentation processes because fungal amylase producers almost always synthesize both amylases. Ó 2011 Elsevier Inc. All rights reserved.
a-Amylases (1,4-a-D-glucan glucanohydrolase, glycogenase EC 3.2.1.1) and glucoamylases (amyloglucosidase, exo-1,4-a-glucosidase. 1,4-a-D-glucan glucohydrolase EC 3.2.1.3) are extracellular glycoproteins synthesized by several filamentous fungi. These enzymes are of industrial importance due to their capacity to release oligosaccharides (a-amylase) and D-glucose (glucoamylase) from starch. Aspergillus niger is most frequently used for production of glucoamylase, whereas Aspergillus oryze is most often used for production of a-amylase. Solid-state fermentation (SSF)1 is more suitable for fungal amylase production than submerged fermentation (SmF) due to the former’s lower cost, higher efficiency, reduced product repression, easier maintenance, and so forth [1,2]. In the fungal fermentation extracts, glucoamylases rarely occur without a-amylase [3]. Other amylolytic enzymes, such as a-glycosidase, are also likely to be produced concomitantly. This poses a challenge for accurate comparison of glucoamylase or a-amylase productivity between culture strains by means of a simple assay of the crude fermentation extract. Zymograms using nonspecific substrates such as starch (most frequently used) are prone to overestimation. Specific assays for determination of glucoamylase ⇑ Corresponding author. Fax: +381 11 2636061. E-mail address:
[email protected] (Z. Vujcˇic´). Abbreviations used: SSF, solid-state fermentation; SmF, submerged fermentation; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PAA gel, polyacrylamide gel; RH, relative humidity; EF gel, native electrophoresis gel; NC membrane, nitrocellulose membrane; HRPO, horseradish peroxidase. 1
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.11.039
activity are developed based on reactions with b-limit dextrins as substrate [4]. Because of the industrial importance of both major amylolytic enzymes (a-amylase and glucoamylase), there is an exigency for their simultaneous detection in crude fermentation extracts. Crude fermentation extracts should be cleared of starch degradation products incurring during fermentation to provide for an accurate enzymatic assay. All of these reasons justify development of new techniques that are able to detect and distinguish/ identify both enzymes in crude extracts without prior preparation. Zymographic detection of amylolytic enzymes can provide these results without the need for prior preparation of crude culture extracts. Zymographic detection of amylase activity by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with copolymerized starch [5] is widely used in fungal fermentations [6]. Only amylases capable of renaturation are detectable on this zymogram. In addition, electrophoretic separation of native proteins (enzymes) based on their charge, as well as on their size difference, is used to separate the different enzymes present in a sample. The main advantage is that the enzymes are maintained in their original conformation, allowing enzymatic activity to be detected directly after electrophoretic separation. Zymographic detection of amylase with copolymerized starch after native electrophoresis is in use [7] as well as after isoelectric focusing [8,9]. The main disadvantage of zymograms with copolymerized starch in gels during electrophoretic separation is the impact on the migration of proteins [10]. Use of this zymographic technique cannot provide rapid results and cannot detect different amylases at the same time. A universal zymogram with starch and iodine
Notes & Tips / Anal. Biochem. 421 (2012) 802–804
Fig.1. Scheme of printing sandwich for simultaneous zymographic detection of aamylase and glucoamylase after native PAGE.
reagent is commonly used to detect both amylase and glucoamylase; however, this does not actually provide accurate information about a specific enzyme [6,11,12]. Simultaneous measurement of a-amylase and glucoamylase activities in a specific extract (sake rice koji) has been achieved by means of capillary electrophoresis of the SDS–protein complex [13]. However, this method requires specific apparatus that makes it complicated to work with. In this article, we describe a new fast zymographic method for identification of a-amylase and glucoamylase after native electrophoresis. After only one electrophoretic separation of crude fermentation extracts, it is possible to identify and distinguish two prime industrial amylolytic enzymes. A. niger ATCC 10864 strain (spore concentration of 6.2 105) was used for SSF. Equal quantities of granulated triticale (Triticosecale sp.) and water (48 g each) were mixed and used as substrate for fermentation. SSF was carried out at 28 °C and 60% relative humidity (RH) for 120 h. Samples were taken during the fermentation process at 24, 29, 33, 57, 81, 96, 105, and 120 h. Fermentation was terminated by adding buffer solution (0.05 M acetate buffer [pH 4.5] and 0.1% Tween 20) in a 1:5 ratio (w/v) and homogenized with an IKA Turex homogenizer. Extraction was carried out at room temperature for 1 h. The obtained mixture was centrifuged at 5000g for 15 min. Native electrophoresis was performed in 10% polyacrylamide gel (PAA gel) according to Davis [14]. The native electrophoresis gel (EF gel) was printed simultaneously in a sandwich consisting of PAA gel with copolymerized b-limit dextrins and of nitrocellulose membrane (NC membrane), as represented schematically in Fig. 1. The EF gel was placed on the gel with b-limit dextrins, and subsequently NC membrane and two pieces of filter paper were carefully placed on the other side of the EF gel. The sandwich was covered with paper wadding, and a weight unit was placed on top. Enzymes from the EF gel were transferred onto NC membrane by capillary force and on dextrin gel at the same time. Printing onto NC membrane was stopped after 30 min, whereas printing on dextrin gel was carried out by incubation at 37 °C for 80 min.
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a-Amylase was detected in PAA gel with copolymerized b-limit dextrins (PAA [7.5%], b-limit dextrins [0.8%, w/v], 50 mM acetate buffer [pH 5.0], 2 mM NaCl, and 0.1 mM CaCl2). b-Limit dextrin was prepared from wheat starch using soybean b-amylase [15]. After printing, the gel was incubated at 30 °C for 15 min, briefly washed with distilled water (1 min), and subsequently stained with iodine solution (0.04% [w/v] I2 and 0.4% [w/v] KI). a-Amylase activity appeared as clear bands on purple background (Fig. 2A). All amylases were detected as clear bands on blue background using soluble starch as substrate (Fig. 2B). After printing, the EF gel was transferred into buffered substrate solution (1.0% [w/v] starch, 50 mM acetate [pH 5.0], 2.0 mM NaCl, and 0.1 mM CaCl2) for 30 min at 30 oC. Thereafter, the gel was incubated in the same buffer without starch for 30 min at 30 °C. After rinsing in water (5 min), amylolytic activity was terminated by the addition of staining solution (1.3% [w/v] I2 and 3% [w/v] KI). Glucoamylases were detected on NC membrane. After printing, NC membrane was washed with distilled water for 5 min. Thereafter, NC membrane was incubated in buffered substrate solution (1.0% [w/v] starch, 50 mM acetate [pH 5.0], 2.0 mM NaCl, and 0.1 mM CaCl2) and reaction mix for glucose detection in a 9:1 ratio for 30 min at 30 °C. Glucose (the final product of this reaction) was detected in the reaction mix by coupled reaction with glucose oxidase and horseradish peroxidase (HRPO) [16] using 4-Cl-a-naphthol as substrate. Product-specific reaction was colored purple and insoluble and appears on the NC membrane in bands corresponding to glucoamylase (Fig. 2C). During the first days of SSF (from 29 to 57 h), A. niger produced only a-amylase under fermentation conditions used in this study (Fig. 2A). After 57 h, A. niger started to produce one of two glucoamylase isoforms (Fig. 2C). At 81 h of fermentation, A. niger produced the second glucoamylase isoform (with lower mobility) (Fig. 2C). Maximum production of both enzymes was achieved at 96 h (Fig. 2B). After 96 h, production of a-amylase was lower than production of glucoamylase (Fig. 2B). Arrows indicate the position of each amylase isoform, and it is clear that all bands visible on the zymogram gel with starch as substrate (Fig. 2B) are also visible in one of the two other zymograms (Fig. 2A or C). The described zymographic technique can be used for all crude fermentation extracts of amylase producers. It gives a fast and reliable answer to the question of amylase production (a-amylase or glucoamylase) and the extent of production. This zymographic technique facilitates clear distinction of a-amylase and glucoamylase, thereby eliminating errors that are introduced by the use of
Fig.2. Simultaneous zymographic detection of a-amylase and glucoamylase after native PAGE. (A) Zymogram for a-amylase in PAA gel with copolymerized b-limit dextrins. (B) Zymogram for a-amylase and glucoamylase using starch as substrate. (C) Zymogram for glucoamylase on NC membrane, based on coupled reaction with glucose oxidase and HRPO, using 4-Cl-a-naphthol as substrate for glucose detection (final product of starch hydrolysis). Arrows indicate the position of amylase isoforms (a-amylase and glucoamylase).
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soluble starch in common zymographic practice [6,12]. A simultaneous zymogram of a-amylase and glucoamylase can be a helpful tool for monitoring and controlling fermentation processes because fungal amylase producers almost always produce both amylases. From the results described in this article for SSF of A. niger, it is possible to define the appropriate fermentation time in order to obtain only one amylase isoform or both of them, depending on the fermentation goal or fungal strain. Acknowledgments This study was supported by a grant from the Serbian Ministry of Education and Science (project grant 172048). References [1] M.P. Nandakumar, M.S. Thakur, K.S.M.S. Raghavarao, N.P. Ghildyal, Studies on catabolite repression in solid state fermentation for biosynthesis of fungal amylases, Lett. Appl. Microbiol. 29 (1999) 380–384. [2] A. Pndey, C.R. Soccol, C. Larroche, Current Developments in Solid State Fermentations, Springer, New Delhi, 2008. [3] A. Yuhki, T. Watanabe, K. Matsuda, Purification and properties of saccharogenic amylase from Piricularia oryzae, Starch 29 (1977) 265–272. [4] C. Kuek, D.K. Kidby, Determination of glucoamylase in culture filtrates containing other amylolytic enzymes, Starch 37 (1985) 161–162. [5] T.F. Martinez, F.J. Alarcon, M. Diaz-Lopez, F.J. Moyano, Improved detection of amylase activity by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with copolymerized starch, Electrophoresis 21 (2000) 2940– 2943.
[6] M.K. Upadhyay, R. Sharma, A.K. Pandey, R.C. Rajak, An improved zymographic method for detection of amylolytic enzymes of fungi on polyacrylamide gels, Mycologist 19 (2005) 138–140. [7] G.P. Manchenko, Handbook of Detection of Enzymes on Electrophoretic Gels, second ed., CRC Press, Boca Raton, FL, 2003. [8] C. Suresh, A.K. Dubey, R. Kini, S. Umesh-Kumar, N.G. Karanth, Separation and direction of raw and gelatinized starch hydrolyzing activities of glucoamylase on isoelectric focusing gels, Electrophoresis 20 (1999) 483–485. [9] A. Valencia-Jimenez, J.W. Arboleda, M.F. Grossi-de-Sa, Detection of a-amylase inhibitors by a zymography method, performed in isoelectric focusing electrophoretic PhastGels, Anal. Biochem. 366 (2007) 113–115. [10] T.F. Martinez, C.J. Newbold, R.J. Wallace, F.J. Moyano, Effects of highmolecular-mass substrates on protein migration during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Electrophoresis 23 (2002) 1–7. [11] S. Negi, R. Banerjee, Optimization of extraction and purification of glucoamylase produced by Aspergillus awamori in solid state fermentation, Biotechnol. Bioproc. Eng. 14 (2009) 60–66. [12] M. Riaz, M.H. Rashid, L. Sawyer, S. Akhtar, M.R. Javed, H. Nadeem, M. Wear, Physiochemical properties and kinetics of glucoamylase produced from deoxyD-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis, Food Chem. 130 (2012) 24–30. [13] T. Watanabe, A. Yamamato, S. Nagai, S. Terabe, Simultaneous measurement of a-amylase and glucoamylase activities in sake rice koji by capillary electrophoresis of sodium dodecyl sulfate–protein complexes and activity measurement of glucoamylase by in-capillary enzyme reaction method, Electrophoresis 19 (1998) 2331–2337. [14] B.J. Davis, Disc electrophoresis: II. Method and application to human serum proteins, Ann. N. Y. Acad. Sci. 121 (1964) 404–427. [15] N. Bozˇic´, I. Vukic´evic´, B. Dojnov, Z. Vujcˇic´, Production of b-limited dextrins by soybean a-amylase and their use in zymography, 1st FCUB ERA Workshop, Food Safety and Health Effects of Food, Belgrade, 2011. [16] L. Faye, A new enzymatic staining method for the detection of radish âfructosidase in gel electrophoresis, Anal. Biochem. 112 (1981) 90–95.
