Volume 2, Issue 10, October 2013
Page 318
ABSTRACT
The present work deals with the production of glucoamylase by Aspergillus niger, A. wentii, A. terreus, A. oryzae grown on potato dextrose agar medium in Solid-State fermentation. Solid-State fermentation was carried out using groundnut oil cake, rice bran, wheat bran and coconut oil cake which were purchased from the local market. During screening, it was found that wheat bran using A.oryzae was more productive. The optimization of the culture medium was carried out and the maximum glucoamylase activity 36.31 U/ml was obtained at optimum conditions of incubation time 5 days, temperature 35 °C, on wheat bran substrate at a pH of 6.0, inoculum age of 5 day old culture and inoculum level of 15%.
Keywords: Glucoamylase, Aspergillus oryzae, solid state fermentation, agro residues, optimization
1.INTRODUCTION
Glucoamylase (α-1, 4-glucan-glucohydrolases, EC 3.2.1.3) is an exoenzyme of great importance for saccharification of starchy materials and other related oligosaccharides. Glucoamylase (GA) consecutively hydrolyzes 1,4-alpha-glycosidic bonds from the non-reducing ends of starch and 1,6-alpha-glucsidic linkages in polysaccharides yielding glucose as the end-product, which in turn serves as a feedstock for biological fermentations[6,16]. Currently, GA’s have a great importance in biotechnology with a wide spectrum of applications, such as textile industry, cellulose, leather, detergents, liquor, bread, children cereals, ethanol production, high fructose syrups production and in various strategies in the pharmaceutical and chemical industries such as the synthesis of optically pure drugs and agrochemicals[8,6,16,29].GA’s are industrially important hydrolytic enzymes of biotechnological significance and are currently used in food and pharmaceutical industries. GA’s mainly used in the production of glucose syrup, high fructose corn syrup and alcohol. GA’s have got extensive biotechnological applications in food and fermentation industries, baking, and brewing and starch processing [1]. Various byproducts have been utilized as fermentation substrate for glucoamylase production by filamentous fungi [2,7,24]. The exclusive production of this enzyme is achieved by Aspergillus niger[28], A.oryze[4] and
A.terreus[3] in enzyme industry.
Traditionally, production of GA is performed mostly by two methods, Solid-Substrate Fermentation (SSF) and Submerged Fermentation (SMF) [1].In recent years, however, the SSF processes have been applied increasingly for the production of GA. SSF holds tremendous potential for the production of enzymes [18]. SSF is a low-cost technology having high productivity per reactor volume and easier downstream processes. Moreover, it can be of special interest in processes where the crude fermented product may be used directly as an enzyme source [13,19,27]. Studies show that agro-industrial residues are generally selected as an appropriate choice of medium in enzyme production by SSF [5]. Optimization of process parameters for amylase production using wheat bran has been already studied in SMF and SSF [10]. Moreover, utilizing combination of wheat bran, rice bran, and other rice components has been reported [25].Virtually all commercial interest in GA production is directed at making forms that will produce more glucose at the expense of byproducts without loss of activity. There are few reports on GA production in SSF systems [17,8,12,20,5,11]. In order to obtain higher enzyme titers, a number of factors need to be optimized, including a suitable microorganism and process parameters [8,21].
1.1 Sources and forms of Glucoamylase: GA’s may be derived from a wide variety of plants, animals and microorganisms, though most GA’s occur in fungi. The enzymes used commercially originate from strains of either A. niger or Rhizopus sp. where they are used for the conversion of malto-oligosaccharides into glucose. These enzymes are
Production and optimization of Glucoamylase
from wheat bran by Aspergillus oryzae NCIM
1212 under Solid State Fermentation
M. Siddhartha Kumar1, M.V.V. Chandana Lakshmi2* V.Sridevi3
1
M.Tech, Centre for Biotechnology, Department of Chemical Engineering, Andhra University, Visakhapatnam-03, Andhra Pradesh, India.
2*
Associate Professor, Centre for Biotechnology, Department of Chemical Engineering, Andhra University, Visakhapatnam-03, Andhra Pradesh, India.
Volume 2, Issue 10, October 2013
Page 319
generally regarded as safe (GRAS) by the Food and Drug Administration (FDA). The properties of GA have been reviewed comprehensively else where [22]. Since the discovery of two forms of GA from black koji mold in the 1950’s, many reports have appeared on the multiplicity of glucoamylase. The various forms of GA’s are thought to be the result of several mechanisms: mRNA modifications, limited proteolysis, variation in carbohydrate content or the presence of several structural genes.
1.2 Substrates commonly used: Commonly used carbon sources are dextrin, fructose, glucose, lactose, maltose and starch are very expensive for commercial production of these enzymes. Various agricultural byproducts like wheat bran (WB), groundnut oil cake (GOC), rice husk (RH), coconut oil cake (COC) sugarcane bagasee (SB), potato residue (PR), rice bran (RB), green gram bran (GGB), black gram bran (BGB) and maize bran (MB)etc are abundantly used.
2 Material and Methods
2.1 Microorganism and its Maintenance: A. niger NCIM 616, A. wentii MTCC 1901, A. terreus MTCC 1782and
A.oryzae NCIM 1212 were purchased from the National Collection of Industrial Microorganisms (NCIM) Pune and The Microbial Type Culture Collection and Gene Bank (MTCC) Chandigarh and were maintained on potato dextrose agar (PDA) medium. Slants were grown at 30 ºC for 5 days and stored at 4 ºC.
2.2 Inoculum Preparation: A piece of culture from 5 days old slant was used to inoculate in seed flask containing 5g substrate with 100% moisture and incubated for 5 days at 30°C. After incubation, fermented dough was mixed aseptically followed by addition of 50 ml of saline containing 0.1% Tween-80. After 20 min mixture was filtered off through sterile glass wool to get spores. Spore count was determined by serial dilution and spread plating method[15].
2.3 Enzyme Production in Solid-State Fermentation: 5 g of substrates were kept separately in a 250 ml Erlenmeyer flask and then moistened with 5ml of water and sterilized at 121 °C for 30 min. The fermentation process was started by adding 1 ml of spore suspension (5 x 107spores/ml) as prepared above. The whole content was mixed thoroughly and then incubated at 30 °C for 5 days in a stationary condition.
2.4 Effect of Various Substrates and Organisms: Commercial quality of GOC, RB, WB and COC were procured from the local market, Visakhapatnam and used as the solid substrate, to study their effect on the production of GA using various fungal organisms. The best combination of solid substrate and organism was chosen for optimization.
2.5 Effect of Incubation Period: PDA medium was used for time course optimization. The effect of incubation time on GA activity was carried from 1-7 days and the fermentation was carried out at 30 °C keeping all other conditions unaltered. The optimum fermentation time achieved by this step was kept constant for the consecutive experiments.
2.6 Effect of Incubation Temperature:For selection of optimum temperature for the production of GA the temperatures
varying from 25 -50 °C were selected by keeping the remaining parameters same.
2.7 Effect of Initial pH: To study the effect of pH, the GA activity was measured at various pH ranging from 3.0
to 8.0 whereas the other parameters were unaltered. The pH of the reaction mixture was varied using different buffers.
2.8 Effect of Inoculum age:To study the effect of Inoculum age, the GA activity was measured from 1-7 days, keeping all the parameters unaltered.
2.9 Effect of Inoculum level: The effect of inoculum level on GA activity was carried at various inoculum levels 5-30% keeping all the parameters constant. The optimum inoculum level achieved by this step was kept constant for the consecutive experiments.
2.10 Enzyme Extraction: To the fermented dough 50 mM citrate buffer (pH 5) (1:10) was added and homogenized for 2 hours with a constant stirring at room temperature. This suspension was filtered through Whatman filter paper number 1 and the filtrate was again centrifuged at 6000 rpm for 15 min. This solid-free supernatant was used as enzyme source for assaying glucoamylase activity.
Volume 2, Issue 10, October 2013
Page 320
measured with 3, 5-dinitrosalicyclic acid (DNSA) reagent[15] using glucose as a GA activity unit (U) was expressed as
the amount of enzyme releasing one μ mole of glucose equivalent per minute per ml.
3 Results and Discussion
3.1 Substrate and Microorganisms Screening: The readily available cheaper agro residues - RB, WB and oil cakes - GOC, COC were used as a whole substrate without adding any additives and screened for the maximum production of GA with A. niger NCIM 616, A. wentii MTCC 1901, A. terreus MTCC 1782and A. oryzae
NCIM 1212. Maximum activity of 20.21 U/ml was found using WB as shown in Fig. 1. Wheat bran as the most promising substrate for glucoamylase production has been reported by several researchers [29,5,26,14,9].
Figure1 Effect of various substrates and microorganisms on production of glucoamylase in SSF
3.2 Optimization of Incubation Period: Maximum GA activity of 24.65 U/ml was observed at 5 days and the activity gradually decreases after 5 days as shown in the Fig. 2.The decrease in the activity may be due to the reduced consumption of nutrient materials. Maximum amylase production for 5 days of incubation has been reported[5].
Figure 2 Effect of incubation time on production of glucoamylase by A. oryzae on wheat bran in SSF
3.3 Optimization of Incubation Temperature: Incubation temperature was varied from 25 to 50 °C, keeping all other parameters constant. Maximum GA activity of 27.78 U/ml was observed at 35 °C and the activity gradually decreases after 35 °C as shown in the Fig. 3 and further fermentation was carried out at 35 °C.
Volume 2, Issue 10, October 2013
Page 321
3.4 Optimization of pH: The amount of GA produced is measured at each pH. The maximum GA activity of 30.56 U/ml is observed at pH 6.0 as shown in the Fig 4. It was also reported that GA production by A. oryaze was found
maximum at pH 5[23].
Figure 4 Effect of pH on production of glucoamylase by A. oryzae on wheat bran in SSF
3.5 Optimization of Inoculum age: The effect of inoculums age on glucoamylase production was studied by conducting the fermentation with different inoculums ages. The substrate was inoculated with 1-day old culture to 7-day old day culture in different flasks. The substrate was incubated at 35 °C for 5 days. After the completion of fermentation, the enzyme was extracted and analyzed for the glucoamylase activity and maximum glucoamylase activity of 35.62 U/ml was obtained as shown in the Fig 5.
Figure 5 Effect of Inoculum age on production of glucoamylase by A. oryzae on wheat bran in SSF
3.6 Optimization of Inoculum level: The effect of inoculums level on glucoamylase production was studied by conducting the fermentation with different inoculum levels. The substrate was inoculated with culture of 5-30% inoculum levels in different flasks. The substrate was incubated at 35 ºC for 5 days. After completion of fermentation, the enzyme was extracted and analyzed for the glucoamylase activity. 15% inoculum level gave maximum production of glucoamylase whose activity is 36.31 U/ml as shown in the Fig 6.
Figure 6 Effect of Inoculum level on production of glucoamylase by
Volume 2, Issue 10, October 2013
Page 322
The maximum activity of glucoamylase, 36.31 U/ml was achieved with A.oryzae NCIM 1212 at 5 days, 35 ºC on wheat bran substrate at a pH of 6.0, inoculum age of 5 day old culture and inoculum level of 15% per 5 g substrate.
4 Conclusion
GA’s are the most widely used enzymes in industries. In the present work, four different substrates and microorganisms were screened. It was found that, maximum activity of glucoamylase 36.31U/ml was found using wheat bran with A. oryzae NCIM 1212. Laboratory scale optimization of some of fermentation conditions for GA production by A. oryzae
NCIM 1212in SSF was carried out. The optimized fermentation media composed of incubation time 5 days, temperature 35°C,on wheat bran substrate at a pH of 6.0, inoculum age of 5 day old culture and inoculum level of 15%. Based on this study, it could be concluded that A. oryzae NCIM 1212 demonstrated high capacity for the production of glucoamylase through agro/agro industrial residues based solid state fermentation. Thus, it can be industrially exploited for the synthesis of glucoamylase. As well as optimization and improvement of process parameters carried out in this study proved to be fruitful in enhancing programs for enzymes of biotechnological important.
References
[1] H. Anto, U.B. Trivedi, and K.C.Patel, ‘Glucoamylase production by solid-state fermentation using rice flake manufacturing waste products as substrate,’ Biores. Technol., (2006) 97, 1161-1166.
[2] Arasaratnam V., Mylvaganam K. and Balasubramaniam K., Improvement of glucoamylase production by Aspergillus niger in solid-state fermentation with paddy husk as support, J. Food Sci.and Technol., 38, 334-338 (2001)
[3] Berka R.M., Dunn-coleman N. and Ward M., Industrial enzymes from Aspergillus species, In Aspergillus Biology and Applications, Bennets J.W. and Klich M.A. (Edn.). Oxford, Heinemann, pp., 155-202(1992)
[4] Biesebeke R., Record E., van Biezen N., Heerikhuisen M., Franken A., Punt P. J. and van den Hondel C.A., Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates, Appl. Microbial. Biotechnol., 69,44-50(2005)
[5] Ellaiah P., Adinarayana K., Bhavani Y., Padmaja P. and Srinivasulu B., Optimization of process parameters for glucoamylase production under solid state fermentation by a newly isolated Aspergillus species, Proc. Biochem., 38,615–620(2002)
[6] Gupta R., Gigras P., Mohapatra H., Goswami V.K. and Chauhan B., Microbial α-amylases a biotechnological perspective, Proc. Biochem., 38,1599-1616(2003)
[7] Joshi V.K., Pandey A. and Sandhu D.K., Waste treatments in fermentation technology, In Biotechnology food fermentation. Joshi V.K. and Pandey, A., (Edn.), Trivendrum, India Educational Publication and Distribution, pp., 2,1291-1338(1999)
[8] Kanlakrit W., Ishimatsu K., Nakao M. and Hayashida S., Characteristics of raw starch-digesting glucoamylase from thermophilic Rhizomucor pusilus, J. Ferment. Technol., 65,379–385(1987)
[9] Kaur P., Grewal H.S. and Kocher G.S., Production of α-amylase by Aspergillus niger using wheat bran in submerged and solid state fermentations, Ind. J. Microbiol., 43, 143–145(2003)
[10]Kocher G.S., Kaur P. and Grewal H.S., Production of Alfa-Amylase by Aspergillus niger using wheat bran in submerged and solid state fermentations, Ind. J. Microbiol.., 43, 143-145(2003)
[11]Kumar S. and Satyanarayana T., Statistical optimization of a thermostable and neutral glucoamylase production by a thermophilic mold Thermomucor indicae-seudaticae in solid-state fermentation, World J. Microbiol. Biotechnol., 20,895–902(2004)
[12]Kumar S. and Satyanarayana T., Medium optimization for glucoamylase production by a yeast Pichia subpelliculosa ABWF 64, in submerged cultivation, World J. Microbiol. Biotechnol., 17, 83–87(2001)
[13]Lonsane B.K., Ghildyal N.P., Budiatman S. and Ramakrishna S.V., Engineering aspects of solid state fermentation, Enz. Microbiol. Technol., 7, 258–265(1985)
[14]Mahmood A., Aurengzeb M., Baig M.A. and Ahmad R., Production of glucoamylase from agro-wastes by Rhizopus nigricans, Pak. J. Biochem. Mol. Biol., 30, 49–54(1997)
[15]Miller G.L., Use of dinitrosalicyclic acid reagent for determination of reducing sugar, Analytical Chemistry., 31,426-429(1959)
[16]Norouzian D., Akbarzadeh A., Scharer J.M. and Young M.M., Fungal glucoamylases, Res. Rev. Paper., 24,80-85(2006)
[17]Pandey A., Glucoamylase research, An overview, Starch/ Starke., 47 439–445(1995)
[18]Pandey A., Selvakumar P., Soccol C.R. and Nigham P., Solid-state fermentation for the production of industrial enzymes, Curr. Sci., 77,149–162(1999)
[19]Pandey A., Recent process developments in solid-state fermentation, Proc. Biochem., 27,109–117(1992)
Volume 2, Issue 10, October 2013
Page 323
[21]Park Y.S., Kang S.W., Lee J.S., Hong S.I. and Kim S.W., Xylanase production in solid-state fermentation by Aspergillus niger mutant using statistical experimental design, Appl. Microbiol. Biotechnol., 58, 761–776(2002)
[22]Pazur J.H., Knull H.R. And Cepure A., Glucoenzymes,structure and properties of the two forms of glucoamylase from Aspergillus niger. Carbohyd. Res., 20, 83-96(1971)
[23]Ritesh Parbat and Barkha Singhal., Production of Glucoamylase by Aspergillus oryzae Under Solid State Fermentation Using Agro Industrial Products. Intl. J. Microbiol. Res., 2 (3), 204-207 (2011)
[24]Shivaramakrishnam S., Ganghadharan D., Nampoothiri K.M., Soccol C.R. and Pandey A., Alpha amylase production by Aspergillus oryzae employing solid-state fermentation, J. Scientific Ind. Res., 66, 621-626(2007)
[25]Singh H. and Soni S.K., Production of starch-gel digesting amyloglucosidase by Aspergillus oryzae HS-3 in solid state fermentation, Proc. Biochem., 37, 453–459 (2001)
[26]Soni S.K., Sundhu I.K., Bath K.S., Banerjee U.C. and Patnaik P.R., Extracellular amylase production by Saccharomycopsis capsularis and its evaluation for starch saccharification, Folia Microbiol., 41 243–248(1996)
[27]Tengerdy R.P., Solid Substrate Fermentation for Enzyme Production, In Advances in Biotechnology., Educational Publishers and Distributors, New Delhi, India., 13–16(1998)
[28]Wang X.J., Bai J.G. and Liang Y.X., Optimization of multienzyme production by two mixed strains in solid-state fermentation. Appl. Microbiol. Biotechnol., 73, 533-540(2006)
