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Production of starch-gel digesting amyloglucosidase by Aspergillus oryzae HS-3 in solid state fermentation
Process Biochemistry 37 (2001) 453– 459 www.elsevier.com/locate/procbio
Production of starch-gel digesting amyloglucosidase by Aspergillus oryzae HS-3 in solid state fermentation Harpreet Singh, Sanjeev K. Soni * Department of Microbiology, Panjab Uni6ersity, Chandigarh 160014, India Received 19 February 2001; received in revised form 23 May 2001; accepted 7 June 2001
Abstract Aspergillus oryzae HS-3, isolated from local soil, produced very high levels of solid starch-gel digesting amyloglucosidase by solid state fermentation in Erlenmeyer flasks and enamel coated metallic trays. Productivity was affected by the nature of the solid substrate, nature of the moistening agent, level of moisture content, incubation temperature, presence or absence of carbon, nitrogen and mineral supplements. Maximum enzyme production of 5773 U/g fermented dry matter was obtained on wheat bran with distilled water at a ratio of 1:1.5 as the moistening agent after 96 h incubation at 30 – 40 °C. Enzyme production was stimulated by supplementing the wheat bran with 1% w/w each of lactose, soyabean meal and 1 mM each of CaCl2 and MgSO4. The enzyme showed optimum activity at 50 °C and pH 6.0. The thermal stability profile revealed a half-life of 6 h at 50 °C which improved significantly with the addition of Ca2 + . It could effectively digest the hard-gel of 15 – 20% corn starch without liquefying separately with a-amylase, exhibiting an overall liquefying efficiency of 98% and saccharifying efficiency of 85% after 24 h incubation at 40–60 °C. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Aspergillus oryzae; Amyloglucosidase; Glucoamylase; Solid-state fermentation; Starch-gel digestion
1. Introduction In conventional starch processing, a slurry containing about 15% starch is gelatinized where it is heated up to a temperature of 105 °C. This increases the viscosity of the slurry and poses problems with mixing and pumping. To overcome such viscosity-associated problems, thermostable a-amylase is added which can liquefy starch at higher temperature. Liquefied starch is then saccharified using fungal amyloglucosidase (AMG) at 50 – 60 °C. The process of starch saccharification, although currently used by starch processing industries, is energy intensive thus increasing the production cost of starch based products. Hence, with the view of reducing the energy cost of starch processing, there is an ongoing search for efficient amylases capable of digesting raw starch or hard starch gels. Such amylases have been
* Corresponding author. Tel.: + 91-172-534-143; fax: + 91-172541-409. E-mail address: [email protected] (S.K. Soni).
reported from different fungal [1,2] and bacterial [3–5] strains. Amyloglucosidases reported so far have been produced using submerged fermentations (SmF) [5,6] as well as solid state fermentations (SSF) [2,6,7]. However, the use of SSF has been found to be more advantageous than SmF and allows the cheaper production of enzymes [2,8,9]. In this study we report the production of very high levels of a hard starch-gel digesting amyloglucosidase under solid state fermentation and its properties. 2. Materials and methods
2.1. Microorganism Starch-gel hydrolysing fungal strains were isolated from soil in the vicinity of local flour mills. Based on the levels of the AMG produced by solid state cultures and the properties of the enzyme, one isolate designated as HS-3 was selected for further studies and was identified as a strain of Aspergillus oryzae.
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 2 3 8 - 2
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H. Singh, S.K. Soni / Process Biochemistry 37 (2001) 453–459
2.2. Solid state fermentation Five grams of wheat bran in 250 ml Erlenmeyer flask, moistened with 5 ml distilled water and autoclaved at 15 psi for 30 min, was taken as the basal medium for SSF studies and had a pH of 5.8– 6.0. This was inoculated with spore suspension of the organism (8× 104 spores/g of the basal medium) and incubated at 30 °C for 7 days. Visual observations regarding the growth were made on each day and the culture was extracted twice with 50 ml 0.1 M Citrate-Phosphate buffer, pH 5.0 and filtered. The pooled filtrate of the two extractions was centrifuged (10,000×g; 4 °C) and used as the source of enzymes.
2.3. Enzyme assay The activity of amyloglucosidase was determined at 50 °C by mixing 0.25 ml of appropriately diluted enzyme source with 0.25 ml of 1% (w/v) soluble starch dissolved in 0.1 M Citrate-Phosphate buffer, pH 5.0. The reducing sugar released after 10 min was measured following a dinitrosalicylic acid (DNS) method [10]. One Unit of AMG activity was defined as the amount of enzyme that released 1 mmol of reducing sugar equivalent to glucose, per min, under the assay conditions while the enzyme productivity has been expressed as U/g of fermented dry matter. All values given are averages of three determinations.
2.4. Optimization of fermentation conditions for enzyme production The amyloglucosidase production was optimized in SSF of wheat bran, unless otherwise stated, by altering various physicochemical and cultural conditions and observing the effect after 96 h of incubation at 30 °C, unless otherwise stated. 1. The effect of substrates in SSF for AMG production was studied by using a variety of solid substrates and their combinations (in equal proportions) in the basal media. 2. The effect of the nature of the moistening agent was studied by using various moistening agents including tap water, distilled water and different buffers (0.1 M) with variable pH. 3. The effect of the ratio of the moistening agent was studied by altering the level of distilled water in the basal medium for SSF. Since the substrate and distilled water ratio of 1:1.5 yielded the highest enzyme productivity, all the subsequent optimization studies for enzyme production were carried out with this moisture level in the basal medium 4. The effect of incubation temperature on enzyme production was studied by incubating the inoculated standard basal media at different temperatures in-
cluding 25, 30, 37, 50 and 60 °C. Since 37 °C produced better enzyme yields as compared to 30 °C, the subsequent optimization studies were carried out at this incubation temperature. 5. The effect of carbon supplementation on enzyme production was studied by adding different carbon sources in the basal media at a level of 1% w/w. 6. The effect of nitrogen supplementation was studied by adding different nitrogen sources in the basal media at a level of 1% w/w. 7. The effect of the supplementation of metal ions was studied by adding various metal salts in the moistening agent, used in the basal media, at a concentration of 1 mM.
2.5. Enzyme production using SSF in different production 6essels Enzyme production was studied in various sized Erlenmeyer flasks as well as in enamel coated metallic tray using wheat bran, moistened with 1.1.5 parts of distilled water as the solid medium. The flask fermentations involved 250, 500, 1000 and 2000 ml Erlenmeyer flasks containing 5, 10, 20 and 40 g of wheat bran while the tray fermentations involved the use of enamel trays (27×22× 4 cm), covered with aluminium foil, containing 50, 100, 150 and 200 g of wheat bran as the substrate in the basal media. The flasks and trays were inoculated with the appropriate volume of the standard spore suspension (8× 104 spores/g of the basal media) and incubated at 37 °C for 96 h after which the enzyme was extracted in tap water.
2.6. Partial purification and characterization of the amylase preparation The enzyme was partially purified by ultra-filtration through Amicon assemblies of 10 and 30 KD Cut off. The enzyme activity in both the permeate as well as the retentate was determined and characterized for temperature and pH activity profiles and thermostability profile at 50 °C in the absence and presence of 10 mM CaCl2 2H2O.
2.7. Hydrolysis of hard corn starch-gel The substrate for the reaction mixture in 100 ml Erlenmeyer flask, was composed of a hard-gel made with 25 ml of 20% soluble starch in 0.1 M citrate phosphate buffer, pH 5.5 having 10 mM CaCl2 and sterilized at 10 psi for 30 min. To this, 10 ml of the enzyme preparation having 100 U/ml was added and the mixture was incubated at 50 °C in New Brunswick Gyrotary water bath shaker (150 rpm) and samples were withdrawn at various time intervals. The extent of liquefaction and saccharification was estimated by mea-
H. Singh, S.K. Soni / Process Biochemistry 37 (2001) 453–459
suring the decrease in starch content with iodine method [11] and an increase in glucose level, with DNS method [10]. All results quoted represent the mean of at least three independent experiments. For the enzymic assays, the residual starch and glucose determinations, the standard deviations were less than 5% with the samples withdrawn from the same experiment. For samples taken from different experiments, the standard deviations were less than 10%.
3. Results and discussion There are several factors, which affect SSF processes. Among these, selection of a suitable strain, substrate and selection of process parameters (physical, chemical and biochemical) are crucial [8]. The selected strain of A. oryzae HS-3, used in the present study, colonised on the solid media well. The visual observations every 24 h revealed spreading white mycelial web on the entire surface of the wheat bran with sporulation starting after 2 days. Enzyme synthesis started along with growth and increased up with sporulation showing maximum yield after 96 h of incubation (Fig. 1). Due to the potential usefulness of the amyloglucosidase in the saccharification of starch-gel, the development of methods for cheaper production of enzyme is very important. One alternative low cost production method is the use of SSF. In this study SSF has been found to be a cheap way of producing high levels of AMG by A. oryzae HS-3 where the productivity levels have been found to be quite high as compared to the published reports [2,9]. Enzyme yields have further been increased by optimizing various important parameters of SSF. The nature of solid substrate is the most important factor in SSF. This not only supplies the nutrients to the culture but also serves as an anchorage for the
Fig. 1. Effect of incubation period on amyloglucosidase production by A. oryzae HS-3 in solid state fermentation.
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Table 1 Effect of different substrates on amyloglucosidase production by A. oryzae HS-3 Solid substrate (5 g each)
Enzyme activity (U/g of original substrate)
Wheat bran Rice bran — fatted Rice bran — defatted Rice husk Wheat flour Corn flour Sorghum’s spent grain Wheat bran+Sorghum’s spent grain Wheat bran+rice husk Wheat bran+rice bran-fatted Wheat bran+rice bran-defatted Wheat bran+wheat flour Wheat bran+corn flour Rice bran-fatted+Sorghum’s spent grain Rice bran-defatted+Sorghum’s spent grain Rice bran-fatted+rice husk Rice bran-defatted+rice husk Rice bran-fatted+rice bran-defatted Rice husk+Sorghum’s spent grain
2713 2085 1800 350 1450 1833 1163 1495 2025 1782 1440 1766 2050 1905 1096
1636 953 1711 608
The fungus was grown at 30 °C for 96 h with 1:1 distilled water in the basal medium.
microbial cells. Therefore, the particle size and the chemical composition of substrate are of critical importance [12]. An ideal solid substrate provides all necessary nutrients to the microorganism for optimum function. Various substrates used by different workers for amylases include wheat bran [13,14], soyabean meal, potato meal & alfalfa meal [15], maize bran [14], corn meal base, defatted ground nut cake, defatted castor seed cake, rice bran, lucerne leaf powder, lucerne stalks, rice husk, sweet potato, maize gluten (free from starch) and soya residue obtained after extraction of milk [16]. Of the various solid substrates used in the present study, wheat bran proved to be most suitable for the colonisation of A. oryzae HS-3, as indicated by the maximum visible growth on the surface of substrate and the highest enzyme yield (Table 1) which is possibly due to the presence of various suitable nutrients in wheat bran and/or due to its most suitable particle size and consistency required for anchorage, colonisation and enzyme excretion by the present Aspergillus strain. Rice bran also supported good growth and the difference in the enzyme yields on fatted and defatted forms of the rice bran is possibly due to the presence of oil in the former as a positive effector for enzyme production.
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The nature of the moistening agent and the level of moisture content in the fermentation medium are the other main factors in SSF which often determine the success of a process [12]. Various agents used for moistening solid substrates in SSF include water, dilute buffer or salt solution. The critical importance of moisture level in SSF media and its influence on the biosynthesis of enzymes has been attributed to the interference of moisture in the physical properties of solid particles. Higher moisture level decreases porosity, changes wheat bran particle structure, promotes development of stickiness, reduces gas volume and exchange and decreases diffusion, which results in lowered oxygen transfer and enhanced formation of aerial mycelium [12]. Lower moisture content reduces the solubility of nutrients provided to the organism by solid substrate, a lower degree of swelling and higher water tension [17]. The substrate-distilled water ratio of 1:1.5 (w/v) was found to be optimum for enzyme synthesis in the present study (Table 2). The incubation temperature also affects the level of enzyme production under SSF [2,18– 20]. A. oryzae Table 2 Effect of the nature of the moistening agent and level of moisture content on amyloglucosidase production in SSF of wheat bran Moistening agent/moisture content Moistening agent (1:1) Citrate phosphate buffer, pH 3.0 Citrate phosphate buffer, pH 4.0 Citrate phosphate buffer, pH 5.0 Citrate phosphate buffer, pH 6.0 Phosphate buffer, pH 6.0 Phosphate buffer, pH 7.0 Phosphate buffer, pH 8.0 Tris–HCl buffer, pH 7.0 Tris–HCl buffer, pH 8.0 Tap water (pH 6.0) Distilled water (pH 6.2)
Enzyme activity (U/g of original substrate)
353 2200 2803 831 746 1480 905 2073 1173 2736 2833
Ratio of wheat bran and distilled water 1:1.0 2658 1:1.5 3355 1:2.0 2825 1:2.5 2168 1:3.0 2155 1:3.5 2021 1:4.0 2116 1:4.5 2051 1:5.0 1728 The fungus was grown at 30 °C in wheat bran based medium for 96 h.
Table 3 Effect of incubation temperature on the level of amyloglucosidase production by A. oryzae HS-3 under solid state fermentation Incubation temperature (°C)
Enzyme activity (U/g of original substrate)
30 37 40 45 50 60
3346 3556 3708 1801 828 43
The ratio of the wheat bran and distilled water used was 1:1.5.
HS-3 showed optimum productivity in a temperature range of 30–40 °C (Table 3). Maximum production at lower temperatures may be advantageous as it can reduce the rate of evaporation during incubation. Commercial wheat bran contains 8.5% starch and 9.5% protein [21] in addition to various minerals. The solid substrates may not provide all the nutrients needed by the organism for maximum enzyme production during SSF or some of the vital nutrients necessary for optimum growth and product formation may be present at sub-optimal levels. Hence, the exogenous addition of various nutrients to the solid medium improves the growth of organism and thus the product yield [2,19,22,23]. Supplementation of the wheat bran with various carbon sources, nitrogen sources and trace minerals stimulated the AMG production by A. oryzae HS-3 (Table 4). When all the optimized conditions including the supplementation of C, N sources and trace minerals were used together the level of production was further improved. The optimized (supplemented) basal media produced 5773 U/g fermented dry matter of the enzyme showing 115% improvement as compared to unoptimized control basal media. The optimized (unsupplemented) basal media yielded (4266 U/g) 59.6% improvement in enzyme activity as compared to unoptimized control (Fig. 2). Different types of production vessels have been used for carrying out solid state fermentation [24] but most of the laboratory studies on the production of enzymes using SSF technique have employed Erlenmeyer flasks [14,19,22] and trays [19,20,25]. When solid state fermentation with A. oryzae HS-3 was carried out in Erlenmeyer flasks of various sizes with corresponding increase in the quantity of the wheat bran and in trays with different quantities of wheat bran moistened with appropriate amounts of distilled water, the time course of enzyme production was similar to that in 250 ml Erlenmeyer flasks containing 5 g of wheat bran (control), in most of the cases. The results were quite encouraging for the large scale production of the enzyme though the yields exhibited slight decline with the increase in substrate quantity which is probably due to lesser degree of aeration (Table 5).
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As compared to conventional starch processing, direct starch gel saccharification is considered to be cost effective and desirable. Since the efficiency of hard starch-gel digestion improves at higher temperatures above 50 °C, the thermostable glucoamylases are on great significance. In this regard the amyloglucosidase from Aspergillus spp. HS-3 which was optimally active at 50 °C (Fig. 3) and displayed 90 and 85% of its peak activity at 40 and 60 °C could be a good candidate for the efficient hydrolysis of starch-gels. Even for conventional starch saccharification these properties are highly desirable. In fact, the search for thermostable AMGs is one future goal of conventional starch saccharification [26]. The thermal stability profile of the enzyme revealed the half-life to be 6 h at 50 °C which improved significantly with the addition of Ca2 + (Fig. 4). Since the enzyme is quite active even at the room temperature Table 4 Effect of different supplements on amyloglucosidase production by A. oryzae HS-3 under solid state fermentation Supplement
Carbon sources Glucose Soluble starch Corn starch Potato starch Fructose Maltose Lactose Sucrose Cellobiose Inulin Tween 80 Molasses Nitrogen sources Peptone Soyabean meal Yeast extract Corn steep liquor Beef extract Ammonium chloride Ammonium molybdate Ferrous ammonium sulphate Ammonium sulphate
Enzyme activity (U/g of original substrate)
3175 4301 3695 4753 4673 4846 4933 3603 3668 2998 4690 4795 4851 5258 5058 5200 4665 2871 4333 4615 4065
Minerals CuSO4 ZnSO4 MgSO4 MnSO4 CaCl2 CoCl2 FeSO4
4006 5263 5636 4968 5630 5326 5378
None (control)
4393
The fungus was grown at 37 °C with 1:1.5 moisture content in the wheat bran as the basal medium.
457
Fig. 2. Effect of optimization of physicochemical and nutritional factors on amyloglucosidase production by A. oryzae HS-3 in solid state fermentation. Wheat bran based medium with 1:1 distilled water as the moistening agent incubated at 30 °C. a Wheat bran based medium with 1:1.5 distilled water as the moistening agent incubated at 37 °C. d Wheat bran based medium with 1:1.5 distilled water as the moistening agent and supplemented with 1% w/w each of lactose and soyabean meal, 1 mM each of CaCl2 and MgSO4 incubated at 37 °C.
(30–40 °C), this can also be a potential candidate for use in combination with the culture of Saccharomyces cere6isiae, in the conventional processes, taking place in the alcohol industries, for simultaneous saccharification and alcohol fermentation of starchy mashes where the temperature of the fermenters is in the range of 30– 40 °C. As the conventional process of starch hydrolysis involves the action of a-amylase and the glucoamylase, several workers have demonstrated the synergistic effect of these two enzymes on starch degradation [2]. To exploit the opportunity of synergism, the temperature and pH optima of the a-amylases and AMGs should be compatible. However, the pH optima of the most AMGs is at or near 4.0, a-amylases which are active and stable at this low pH are extremely rare [26]. The amyloglucosidase of A. oryzae HS-3 showed pH optima Table 5 Amyloglucosidase yields in solid state fermentation using different fermentation vessels Type of fermentation vessel and quantity of wheat bran (g)
Enzyme activity (U/g of original substrate)
Erlenmeyer flasks 250 ml (5 g) 500 ml (10 g) 1000 ml (20 g) 2000 ml (40 g)
4120 3906 3766 3691
Enamel coated metallic trays (27×22×4 cm) 50 g 4083 100 g 4133 150 g 3930 200 g 3683 The fungus was grown at 37 °C in the wheat bran basal media with 1:1.5 distilled water as the moistening agent.
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Fig. 3. Effect of temperature on activity of A. oryzae HS-3 amyloglucosidase.
Fig. 5. Effect of pH on activity of A. oryzae HS-3 amyloglucosidase at 50 °C.
at 6.0 (Fig. 5) and displayed 80% of its peak activity at pH 7.0 where many a-amylases are also active. This enzyme, thus, can also be a potential candidate for synergistic use along with a-amylases for the hydrolyisis of starchy mashes. The AMG preparation from the solid state cultures of A. oryzae HS-3 could effectively digest the solid gel of 15 –20% corn starch without liquefying first with the a-amylase. The solid gel was liquefied and converted into a solution with a significant decrease in the viscosity within 1 h of incubation with the enzyme at 40, 50 or 60 °C. The data on hydrolysis at 50 °C showed a decline of starch content from 144 to 42.9 mg/ml in the reaction mixture after 30 min of incubation revealing the liquefaction efficiency of 70%. The disappearance of starch continued gradually with the further progress in the enzyme reaction and the final level of the residual starch at the end of 7 h of incubation was just 8.7 mg/ml revealing the overall liquefaction efficiency of 94 and 98% respectively after 7 and 24 h which remained
constant thereafter (Fig. 6). The formation of glucose in the reaction mixture did not pick up immediately with the disappearance of the starch. The level of sugar produced at the end of 30 min was just 14.5 mg/ml exhibiting a saccharification efficiency of 13%. The
Fig. 4. Thermal stability of A. oryzae HS-3 amyloglucosidase at 50 °C in the absence () and presence ( ) of 10 mM CaCl2.
Fig. 6. Profiles of liquefaction and saccharification in 15% starch gel digestion by A. oryzae amyloglucosidase at 40, 50 and 60 °C. Inset show the pattern of starch degradation and sugar formation.
H. Singh, S.K. Soni / Process Biochemistry 37 (2001) 453–459
formation of sugar improved gradually with the progress in the reaction revealing 110 and 131 mg/ml and thus showing 73 and 84% saccharification efficiency after 7 and 24 h, respectively, and remained constant thereafter (Fig. 6). With the change in incubation temperature to 40 and 60 °C the overall parameters exhibited the similar behaviour with the slight difference in the reaction rates and the final values of various parameters (Fig. 6). The ability of the crude amylase preparation from A. oryzae HS-3 to bring about the considerable liquefaction and the saccharification in the hard gel of 15– 20% corn starch with an overall liquefaction efficiency of over 98% and saccharification efficiency of 85%, after 24 h, suggests that the enzyme can be employed for the direct hydrolysis of starch gels, without the separate liquefaction with the a-amylase preparations, being done in the conventional processes. Moreover, as the enzyme is also able to digest the starch gel at a lower temperature of 40 °C, and has also revealed 75% of its peak activity at 30 °C, this enzyme can also be used in combination with a culture of S. cere6isiae in the gels of the starchy mashes for the simultaneous saccharification and alcohol production and thus appears to be a potential candidate for application in alcohol industry.
References [1] Oates CG. Towards an understanding of starch granule structure and hydrolysis. Trends Food Sci Technol 1997;8:375 –9. [2] Mamo G, Gessesse A. Production of raw starch digesting amyloglucosidase by Aspergillus spp. GP-21 in solid state fermentation. J Ind Microbiol Biotechnol 1999;22:622 – 6. [3] Itkor P, Tsukagoshi N, Udaka S. Purification and properties of divalent carbon-depending raw-starch-digesting a-amylase from Bacillus spp. B1018. J Ferment Bioeng 1989;68:247 –51. [4] Kelly CT, McTigue MA, Doyle EM, Fogarty WM. The raw starch-degrading alkaline amylases of Bacillus spp. IMD 370. J Ind Microbiol 1995;15:446 –8. [5] Okalo BN, Ezeogu LI, Ebisike CO. Raw starch digesting amylase from Thermoactinomyces thalophilus F13. World J Microbiol Biotechnol 1996;12:637 –8. [6] Pandey A. Glucoamylase research — an overview. Starch 1995;47:305 – 9. [7] Pandey A. Production of starch saccharifying enzyme (glucoamylase) in solid cultures. Starch 1992;44:75 –7. [8] Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation: I-bioprocesses and products. Process Biochem 2000;35:1153 –69.
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[9] Ramdas M, Holst O, Mattiasson B. Production of amyloglucosidase by Aspergillus niger under different cultivation regimens. World J Microbiol Biotechnol 1996;12:267 – 71. [10] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426 – 8. [11] Gogoi BK, Bejbaruah RL, Pillai KR, Baruah JN. Production, purification and characterization of an alpha-amylase produced by Saccharomycopsis fibuligera. J Appl Bacteriol 1987;63:373 –6. [12] Lonsane BK, Ghildyal NP, Budiatman S, Ramakrishna SV. Engineering aspects of solid state fermentation. Enzyme Microb Technol 1985;7:258 – 65. [13] Tobey JF, Yosten AA. Factors affecting the production of amylase by Bacillus thuringiensis. Dev Ind Microbiol 1975;18:499 – 510. [14] Qadeer MA, Anjum JI, Akhtar R. Biosynthesis of enzymes by solid state fermentation. Part II: production of alpha-amylase by Baillus subtilis. Pak J Sci Ind Res 1980;23:25 – 9. [15] Beckord LD, Kneen E, Lewis KH. Bacterial amylase production on wheat bran. Ind Eng Chem 1945;37:692 – 6. [16] Lulla BS, Subrahmanyan V. Influence of culture media on development of bacterial amylases. J Sci Ind Res 1954;13:410 –2. [17] Zadrazil F, Brunnert H. Investigations on physical parameters important for the SSF of straw by white rot fungi. Eur J Appl Microbiol Biotechnol 1981;11:183 – 8. [18] Sani A, Awe FA, Akinyanju JA. Amylase synthesis in Aspergillus fla6us and Aspergillus niger grown on cassava peel. J Ind Microbiol 1992;10:55 – 9. [19] Babu KR, Satyanarayana T. a-amylase production by thermophilic Bacillus coagulans in solid state fermentation. Process Biochem 1995;30:305 – 9. [20] Kotwal SM, Gote MM, Sainkar SR, Khan MI, Khire JM. Production of a-galactosidase by thermophilic fungus Humicola spp. in solid state fermentation and its application in soyamilk hydrolysis. Process Biochem 1998;33:337 – 43. [21] Fisher N. Indigestible constituents of cereals and other food stuffs. In: Birch GG, Green LF, editors. Molecular structure and function of food carbohydrates, London: Applied Science Publishers Ltd., 1973. p. 275 – 295. [22] Ramesh MV, Lonsane BK. A novel bacterial thermostable alpha-amylase from bacterial bran produced under solid state fermentation. Biotechnol Lett 1987;9:505 – 8. [23] Ramesh MV, Lonsane BK. Ability of a solid state fermentation technique to significantly minimise catabolite repression of aamylase production by Bacillus licheniformis M27. Appl Microbiol Biotechnol 1991;35:591 – 3. [24] Mitchell DA, Krieger N, Stuart DM, Pandey A. New developments in solid-state fermentation II. Rational approaches to the design, operation and scale-up of bioreactors. Process Biochem 2000;35:1211 – 25. [25] Ghildyal NP, Ramakrishna M, Lonsane BK, Karanth NG. Temperature variations and amyloglucosidase levels at different bed depths in a solid state fermentation system. Chem Eng J 1993;51:17 – 23. [26] Crabb WD, Mitchinson C. Enzymes involved in the processing of starch to sugars. Trends Biotechnol 1997;15:349 – 52.
Production of starch-gel digesting amyloglucosidase by Aspergillus oryzae HS-3 in solid state fermentation Harpreet Singh, Sanjeev K. Soni * Department of Microbiology, Panjab Uni6ersity, Chandigarh 160014, India Received 19 February 2001; received in revised form 23 May 2001; accepted 7 June 2001
Abstract Aspergillus oryzae HS-3, isolated from local soil, produced very high levels of solid starch-gel digesting amyloglucosidase by solid state fermentation in Erlenmeyer flasks and enamel coated metallic trays. Productivity was affected by the nature of the solid substrate, nature of the moistening agent, level of moisture content, incubation temperature, presence or absence of carbon, nitrogen and mineral supplements. Maximum enzyme production of 5773 U/g fermented dry matter was obtained on wheat bran with distilled water at a ratio of 1:1.5 as the moistening agent after 96 h incubation at 30 – 40 °C. Enzyme production was stimulated by supplementing the wheat bran with 1% w/w each of lactose, soyabean meal and 1 mM each of CaCl2 and MgSO4. The enzyme showed optimum activity at 50 °C and pH 6.0. The thermal stability profile revealed a half-life of 6 h at 50 °C which improved significantly with the addition of Ca2 + . It could effectively digest the hard-gel of 15 – 20% corn starch without liquefying separately with a-amylase, exhibiting an overall liquefying efficiency of 98% and saccharifying efficiency of 85% after 24 h incubation at 40–60 °C. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Aspergillus oryzae; Amyloglucosidase; Glucoamylase; Solid-state fermentation; Starch-gel digestion
1. Introduction In conventional starch processing, a slurry containing about 15% starch is gelatinized where it is heated up to a temperature of 105 °C. This increases the viscosity of the slurry and poses problems with mixing and pumping. To overcome such viscosity-associated problems, thermostable a-amylase is added which can liquefy starch at higher temperature. Liquefied starch is then saccharified using fungal amyloglucosidase (AMG) at 50 – 60 °C. The process of starch saccharification, although currently used by starch processing industries, is energy intensive thus increasing the production cost of starch based products. Hence, with the view of reducing the energy cost of starch processing, there is an ongoing search for efficient amylases capable of digesting raw starch or hard starch gels. Such amylases have been
* Corresponding author. Tel.: + 91-172-534-143; fax: + 91-172541-409. E-mail address: [email protected] (S.K. Soni).
reported from different fungal [1,2] and bacterial [3–5] strains. Amyloglucosidases reported so far have been produced using submerged fermentations (SmF) [5,6] as well as solid state fermentations (SSF) [2,6,7]. However, the use of SSF has been found to be more advantageous than SmF and allows the cheaper production of enzymes [2,8,9]. In this study we report the production of very high levels of a hard starch-gel digesting amyloglucosidase under solid state fermentation and its properties. 2. Materials and methods
2.1. Microorganism Starch-gel hydrolysing fungal strains were isolated from soil in the vicinity of local flour mills. Based on the levels of the AMG produced by solid state cultures and the properties of the enzyme, one isolate designated as HS-3 was selected for further studies and was identified as a strain of Aspergillus oryzae.
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 2 3 8 - 2
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2.2. Solid state fermentation Five grams of wheat bran in 250 ml Erlenmeyer flask, moistened with 5 ml distilled water and autoclaved at 15 psi for 30 min, was taken as the basal medium for SSF studies and had a pH of 5.8– 6.0. This was inoculated with spore suspension of the organism (8× 104 spores/g of the basal medium) and incubated at 30 °C for 7 days. Visual observations regarding the growth were made on each day and the culture was extracted twice with 50 ml 0.1 M Citrate-Phosphate buffer, pH 5.0 and filtered. The pooled filtrate of the two extractions was centrifuged (10,000×g; 4 °C) and used as the source of enzymes.
2.3. Enzyme assay The activity of amyloglucosidase was determined at 50 °C by mixing 0.25 ml of appropriately diluted enzyme source with 0.25 ml of 1% (w/v) soluble starch dissolved in 0.1 M Citrate-Phosphate buffer, pH 5.0. The reducing sugar released after 10 min was measured following a dinitrosalicylic acid (DNS) method [10]. One Unit of AMG activity was defined as the amount of enzyme that released 1 mmol of reducing sugar equivalent to glucose, per min, under the assay conditions while the enzyme productivity has been expressed as U/g of fermented dry matter. All values given are averages of three determinations.
2.4. Optimization of fermentation conditions for enzyme production The amyloglucosidase production was optimized in SSF of wheat bran, unless otherwise stated, by altering various physicochemical and cultural conditions and observing the effect after 96 h of incubation at 30 °C, unless otherwise stated. 1. The effect of substrates in SSF for AMG production was studied by using a variety of solid substrates and their combinations (in equal proportions) in the basal media. 2. The effect of the nature of the moistening agent was studied by using various moistening agents including tap water, distilled water and different buffers (0.1 M) with variable pH. 3. The effect of the ratio of the moistening agent was studied by altering the level of distilled water in the basal medium for SSF. Since the substrate and distilled water ratio of 1:1.5 yielded the highest enzyme productivity, all the subsequent optimization studies for enzyme production were carried out with this moisture level in the basal medium 4. The effect of incubation temperature on enzyme production was studied by incubating the inoculated standard basal media at different temperatures in-
cluding 25, 30, 37, 50 and 60 °C. Since 37 °C produced better enzyme yields as compared to 30 °C, the subsequent optimization studies were carried out at this incubation temperature. 5. The effect of carbon supplementation on enzyme production was studied by adding different carbon sources in the basal media at a level of 1% w/w. 6. The effect of nitrogen supplementation was studied by adding different nitrogen sources in the basal media at a level of 1% w/w. 7. The effect of the supplementation of metal ions was studied by adding various metal salts in the moistening agent, used in the basal media, at a concentration of 1 mM.
2.5. Enzyme production using SSF in different production 6essels Enzyme production was studied in various sized Erlenmeyer flasks as well as in enamel coated metallic tray using wheat bran, moistened with 1.1.5 parts of distilled water as the solid medium. The flask fermentations involved 250, 500, 1000 and 2000 ml Erlenmeyer flasks containing 5, 10, 20 and 40 g of wheat bran while the tray fermentations involved the use of enamel trays (27×22× 4 cm), covered with aluminium foil, containing 50, 100, 150 and 200 g of wheat bran as the substrate in the basal media. The flasks and trays were inoculated with the appropriate volume of the standard spore suspension (8× 104 spores/g of the basal media) and incubated at 37 °C for 96 h after which the enzyme was extracted in tap water.
2.6. Partial purification and characterization of the amylase preparation The enzyme was partially purified by ultra-filtration through Amicon assemblies of 10 and 30 KD Cut off. The enzyme activity in both the permeate as well as the retentate was determined and characterized for temperature and pH activity profiles and thermostability profile at 50 °C in the absence and presence of 10 mM CaCl2 2H2O.
2.7. Hydrolysis of hard corn starch-gel The substrate for the reaction mixture in 100 ml Erlenmeyer flask, was composed of a hard-gel made with 25 ml of 20% soluble starch in 0.1 M citrate phosphate buffer, pH 5.5 having 10 mM CaCl2 and sterilized at 10 psi for 30 min. To this, 10 ml of the enzyme preparation having 100 U/ml was added and the mixture was incubated at 50 °C in New Brunswick Gyrotary water bath shaker (150 rpm) and samples were withdrawn at various time intervals. The extent of liquefaction and saccharification was estimated by mea-
H. Singh, S.K. Soni / Process Biochemistry 37 (2001) 453–459
suring the decrease in starch content with iodine method [11] and an increase in glucose level, with DNS method [10]. All results quoted represent the mean of at least three independent experiments. For the enzymic assays, the residual starch and glucose determinations, the standard deviations were less than 5% with the samples withdrawn from the same experiment. For samples taken from different experiments, the standard deviations were less than 10%.
3. Results and discussion There are several factors, which affect SSF processes. Among these, selection of a suitable strain, substrate and selection of process parameters (physical, chemical and biochemical) are crucial [8]. The selected strain of A. oryzae HS-3, used in the present study, colonised on the solid media well. The visual observations every 24 h revealed spreading white mycelial web on the entire surface of the wheat bran with sporulation starting after 2 days. Enzyme synthesis started along with growth and increased up with sporulation showing maximum yield after 96 h of incubation (Fig. 1). Due to the potential usefulness of the amyloglucosidase in the saccharification of starch-gel, the development of methods for cheaper production of enzyme is very important. One alternative low cost production method is the use of SSF. In this study SSF has been found to be a cheap way of producing high levels of AMG by A. oryzae HS-3 where the productivity levels have been found to be quite high as compared to the published reports [2,9]. Enzyme yields have further been increased by optimizing various important parameters of SSF. The nature of solid substrate is the most important factor in SSF. This not only supplies the nutrients to the culture but also serves as an anchorage for the
Fig. 1. Effect of incubation period on amyloglucosidase production by A. oryzae HS-3 in solid state fermentation.
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Table 1 Effect of different substrates on amyloglucosidase production by A. oryzae HS-3 Solid substrate (5 g each)
Enzyme activity (U/g of original substrate)
Wheat bran Rice bran — fatted Rice bran — defatted Rice husk Wheat flour Corn flour Sorghum’s spent grain Wheat bran+Sorghum’s spent grain Wheat bran+rice husk Wheat bran+rice bran-fatted Wheat bran+rice bran-defatted Wheat bran+wheat flour Wheat bran+corn flour Rice bran-fatted+Sorghum’s spent grain Rice bran-defatted+Sorghum’s spent grain Rice bran-fatted+rice husk Rice bran-defatted+rice husk Rice bran-fatted+rice bran-defatted Rice husk+Sorghum’s spent grain
2713 2085 1800 350 1450 1833 1163 1495 2025 1782 1440 1766 2050 1905 1096
1636 953 1711 608
The fungus was grown at 30 °C for 96 h with 1:1 distilled water in the basal medium.
microbial cells. Therefore, the particle size and the chemical composition of substrate are of critical importance [12]. An ideal solid substrate provides all necessary nutrients to the microorganism for optimum function. Various substrates used by different workers for amylases include wheat bran [13,14], soyabean meal, potato meal & alfalfa meal [15], maize bran [14], corn meal base, defatted ground nut cake, defatted castor seed cake, rice bran, lucerne leaf powder, lucerne stalks, rice husk, sweet potato, maize gluten (free from starch) and soya residue obtained after extraction of milk [16]. Of the various solid substrates used in the present study, wheat bran proved to be most suitable for the colonisation of A. oryzae HS-3, as indicated by the maximum visible growth on the surface of substrate and the highest enzyme yield (Table 1) which is possibly due to the presence of various suitable nutrients in wheat bran and/or due to its most suitable particle size and consistency required for anchorage, colonisation and enzyme excretion by the present Aspergillus strain. Rice bran also supported good growth and the difference in the enzyme yields on fatted and defatted forms of the rice bran is possibly due to the presence of oil in the former as a positive effector for enzyme production.
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The nature of the moistening agent and the level of moisture content in the fermentation medium are the other main factors in SSF which often determine the success of a process [12]. Various agents used for moistening solid substrates in SSF include water, dilute buffer or salt solution. The critical importance of moisture level in SSF media and its influence on the biosynthesis of enzymes has been attributed to the interference of moisture in the physical properties of solid particles. Higher moisture level decreases porosity, changes wheat bran particle structure, promotes development of stickiness, reduces gas volume and exchange and decreases diffusion, which results in lowered oxygen transfer and enhanced formation of aerial mycelium [12]. Lower moisture content reduces the solubility of nutrients provided to the organism by solid substrate, a lower degree of swelling and higher water tension [17]. The substrate-distilled water ratio of 1:1.5 (w/v) was found to be optimum for enzyme synthesis in the present study (Table 2). The incubation temperature also affects the level of enzyme production under SSF [2,18– 20]. A. oryzae Table 2 Effect of the nature of the moistening agent and level of moisture content on amyloglucosidase production in SSF of wheat bran Moistening agent/moisture content Moistening agent (1:1) Citrate phosphate buffer, pH 3.0 Citrate phosphate buffer, pH 4.0 Citrate phosphate buffer, pH 5.0 Citrate phosphate buffer, pH 6.0 Phosphate buffer, pH 6.0 Phosphate buffer, pH 7.0 Phosphate buffer, pH 8.0 Tris–HCl buffer, pH 7.0 Tris–HCl buffer, pH 8.0 Tap water (pH 6.0) Distilled water (pH 6.2)
Enzyme activity (U/g of original substrate)
353 2200 2803 831 746 1480 905 2073 1173 2736 2833
Ratio of wheat bran and distilled water 1:1.0 2658 1:1.5 3355 1:2.0 2825 1:2.5 2168 1:3.0 2155 1:3.5 2021 1:4.0 2116 1:4.5 2051 1:5.0 1728 The fungus was grown at 30 °C in wheat bran based medium for 96 h.
Table 3 Effect of incubation temperature on the level of amyloglucosidase production by A. oryzae HS-3 under solid state fermentation Incubation temperature (°C)
Enzyme activity (U/g of original substrate)
30 37 40 45 50 60
3346 3556 3708 1801 828 43
The ratio of the wheat bran and distilled water used was 1:1.5.
HS-3 showed optimum productivity in a temperature range of 30–40 °C (Table 3). Maximum production at lower temperatures may be advantageous as it can reduce the rate of evaporation during incubation. Commercial wheat bran contains 8.5% starch and 9.5% protein [21] in addition to various minerals. The solid substrates may not provide all the nutrients needed by the organism for maximum enzyme production during SSF or some of the vital nutrients necessary for optimum growth and product formation may be present at sub-optimal levels. Hence, the exogenous addition of various nutrients to the solid medium improves the growth of organism and thus the product yield [2,19,22,23]. Supplementation of the wheat bran with various carbon sources, nitrogen sources and trace minerals stimulated the AMG production by A. oryzae HS-3 (Table 4). When all the optimized conditions including the supplementation of C, N sources and trace minerals were used together the level of production was further improved. The optimized (supplemented) basal media produced 5773 U/g fermented dry matter of the enzyme showing 115% improvement as compared to unoptimized control basal media. The optimized (unsupplemented) basal media yielded (4266 U/g) 59.6% improvement in enzyme activity as compared to unoptimized control (Fig. 2). Different types of production vessels have been used for carrying out solid state fermentation [24] but most of the laboratory studies on the production of enzymes using SSF technique have employed Erlenmeyer flasks [14,19,22] and trays [19,20,25]. When solid state fermentation with A. oryzae HS-3 was carried out in Erlenmeyer flasks of various sizes with corresponding increase in the quantity of the wheat bran and in trays with different quantities of wheat bran moistened with appropriate amounts of distilled water, the time course of enzyme production was similar to that in 250 ml Erlenmeyer flasks containing 5 g of wheat bran (control), in most of the cases. The results were quite encouraging for the large scale production of the enzyme though the yields exhibited slight decline with the increase in substrate quantity which is probably due to lesser degree of aeration (Table 5).
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As compared to conventional starch processing, direct starch gel saccharification is considered to be cost effective and desirable. Since the efficiency of hard starch-gel digestion improves at higher temperatures above 50 °C, the thermostable glucoamylases are on great significance. In this regard the amyloglucosidase from Aspergillus spp. HS-3 which was optimally active at 50 °C (Fig. 3) and displayed 90 and 85% of its peak activity at 40 and 60 °C could be a good candidate for the efficient hydrolysis of starch-gels. Even for conventional starch saccharification these properties are highly desirable. In fact, the search for thermostable AMGs is one future goal of conventional starch saccharification [26]. The thermal stability profile of the enzyme revealed the half-life to be 6 h at 50 °C which improved significantly with the addition of Ca2 + (Fig. 4). Since the enzyme is quite active even at the room temperature Table 4 Effect of different supplements on amyloglucosidase production by A. oryzae HS-3 under solid state fermentation Supplement
Carbon sources Glucose Soluble starch Corn starch Potato starch Fructose Maltose Lactose Sucrose Cellobiose Inulin Tween 80 Molasses Nitrogen sources Peptone Soyabean meal Yeast extract Corn steep liquor Beef extract Ammonium chloride Ammonium molybdate Ferrous ammonium sulphate Ammonium sulphate
Enzyme activity (U/g of original substrate)
3175 4301 3695 4753 4673 4846 4933 3603 3668 2998 4690 4795 4851 5258 5058 5200 4665 2871 4333 4615 4065
Minerals CuSO4 ZnSO4 MgSO4 MnSO4 CaCl2 CoCl2 FeSO4
4006 5263 5636 4968 5630 5326 5378
None (control)
4393
The fungus was grown at 37 °C with 1:1.5 moisture content in the wheat bran as the basal medium.
457
Fig. 2. Effect of optimization of physicochemical and nutritional factors on amyloglucosidase production by A. oryzae HS-3 in solid state fermentation. Wheat bran based medium with 1:1 distilled water as the moistening agent incubated at 30 °C. a Wheat bran based medium with 1:1.5 distilled water as the moistening agent incubated at 37 °C. d Wheat bran based medium with 1:1.5 distilled water as the moistening agent and supplemented with 1% w/w each of lactose and soyabean meal, 1 mM each of CaCl2 and MgSO4 incubated at 37 °C.
(30–40 °C), this can also be a potential candidate for use in combination with the culture of Saccharomyces cere6isiae, in the conventional processes, taking place in the alcohol industries, for simultaneous saccharification and alcohol fermentation of starchy mashes where the temperature of the fermenters is in the range of 30– 40 °C. As the conventional process of starch hydrolysis involves the action of a-amylase and the glucoamylase, several workers have demonstrated the synergistic effect of these two enzymes on starch degradation [2]. To exploit the opportunity of synergism, the temperature and pH optima of the a-amylases and AMGs should be compatible. However, the pH optima of the most AMGs is at or near 4.0, a-amylases which are active and stable at this low pH are extremely rare [26]. The amyloglucosidase of A. oryzae HS-3 showed pH optima Table 5 Amyloglucosidase yields in solid state fermentation using different fermentation vessels Type of fermentation vessel and quantity of wheat bran (g)
Enzyme activity (U/g of original substrate)
Erlenmeyer flasks 250 ml (5 g) 500 ml (10 g) 1000 ml (20 g) 2000 ml (40 g)
4120 3906 3766 3691
Enamel coated metallic trays (27×22×4 cm) 50 g 4083 100 g 4133 150 g 3930 200 g 3683 The fungus was grown at 37 °C in the wheat bran basal media with 1:1.5 distilled water as the moistening agent.
458
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Fig. 3. Effect of temperature on activity of A. oryzae HS-3 amyloglucosidase.
Fig. 5. Effect of pH on activity of A. oryzae HS-3 amyloglucosidase at 50 °C.
at 6.0 (Fig. 5) and displayed 80% of its peak activity at pH 7.0 where many a-amylases are also active. This enzyme, thus, can also be a potential candidate for synergistic use along with a-amylases for the hydrolyisis of starchy mashes. The AMG preparation from the solid state cultures of A. oryzae HS-3 could effectively digest the solid gel of 15 –20% corn starch without liquefying first with the a-amylase. The solid gel was liquefied and converted into a solution with a significant decrease in the viscosity within 1 h of incubation with the enzyme at 40, 50 or 60 °C. The data on hydrolysis at 50 °C showed a decline of starch content from 144 to 42.9 mg/ml in the reaction mixture after 30 min of incubation revealing the liquefaction efficiency of 70%. The disappearance of starch continued gradually with the further progress in the enzyme reaction and the final level of the residual starch at the end of 7 h of incubation was just 8.7 mg/ml revealing the overall liquefaction efficiency of 94 and 98% respectively after 7 and 24 h which remained
constant thereafter (Fig. 6). The formation of glucose in the reaction mixture did not pick up immediately with the disappearance of the starch. The level of sugar produced at the end of 30 min was just 14.5 mg/ml exhibiting a saccharification efficiency of 13%. The
Fig. 4. Thermal stability of A. oryzae HS-3 amyloglucosidase at 50 °C in the absence () and presence ( ) of 10 mM CaCl2.
Fig. 6. Profiles of liquefaction and saccharification in 15% starch gel digestion by A. oryzae amyloglucosidase at 40, 50 and 60 °C. Inset show the pattern of starch degradation and sugar formation.
H. Singh, S.K. Soni / Process Biochemistry 37 (2001) 453–459
formation of sugar improved gradually with the progress in the reaction revealing 110 and 131 mg/ml and thus showing 73 and 84% saccharification efficiency after 7 and 24 h, respectively, and remained constant thereafter (Fig. 6). With the change in incubation temperature to 40 and 60 °C the overall parameters exhibited the similar behaviour with the slight difference in the reaction rates and the final values of various parameters (Fig. 6). The ability of the crude amylase preparation from A. oryzae HS-3 to bring about the considerable liquefaction and the saccharification in the hard gel of 15– 20% corn starch with an overall liquefaction efficiency of over 98% and saccharification efficiency of 85%, after 24 h, suggests that the enzyme can be employed for the direct hydrolysis of starch gels, without the separate liquefaction with the a-amylase preparations, being done in the conventional processes. Moreover, as the enzyme is also able to digest the starch gel at a lower temperature of 40 °C, and has also revealed 75% of its peak activity at 30 °C, this enzyme can also be used in combination with a culture of S. cere6isiae in the gels of the starchy mashes for the simultaneous saccharification and alcohol production and thus appears to be a potential candidate for application in alcohol industry.
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